G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
tenascin C
G00001158 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000021010 (Vega human gene)
ENSG00000041982 (Ensembl human gene)
3371 (Entrez Gene)
339 (G2Cdb plasticity & disease)
TNC (GeneCards)
187380 (OMIM)
Marker Symbol
HGNC:5318 (HGNC)
Protein Expression
4592 (human protein atlas)
Protein Sequence
P24821 (UniProt)

Synonyms (2)

  • MGC167029
  • TN

Literature (111)

Pubmed - other

  • Quantitative analysis of three-dimensional human mammary epithelial tissue architecture reveals a role for tenascin-C in regulating c-met function.

    Taraseviciute A, Vincent BT, Schedin P and Jones PL

    Department of Cell Biology, Stem Cells, and Development, University of Colorado Denver, Aurora, Colorado, USA.

    Remodeling of the stromal extracellular matrix and elevated expression of specific proto-oncogenes within the adjacent epithelium represent cardinal features of breast cancer, yet how these events become integrated is not fully understood. To address this question, we focused on tenascin-C (TN-C), a stromal extracellular matrix glycoprotein whose expression increases with disease severity. Initially, nonmalignant human mammary epithelial cells (MCF-10A) were cultured within a reconstituted basement membrane (BM) where they formed three-dimensional (3-D) polarized, growth-attenuated, multicellular acini, enveloped by a continuous endogenous BM. In the presence of TN-C, however, acini failed to generate a normal BM, and net epithelial cell proliferation increased. To quantify how TN-C alters 3-D tissue architecture and function, we developed a computational image analysis algorithm, which showed that although TN-C disrupted acinar surface structure, it had no effect on their volume. Thus, TN-C promoted epithelial cell proliferation leading to luminal filling, a process that we hypothesized involved c-met, a proto-oncogene amplified in breast tumors that promotes intraluminal filling. Indeed, TN-C increased epithelial c-met expression and promoted luminal filling, whereas blockade of c-met function reversed this phenotype, resulting in normal BM deposition, proper lumen formation, and decreased cell proliferation. Collectively, these studies, combining a novel quantitative image analysis tool with 3-D organotypic cultures, demonstrate that stromal changes associated with breast cancer can control proto-oncogene function.

    The American journal of pathology 2010;176;2;827-38

  • Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture.

    Kii I, Nishiyama T, Li M, Matsumoto K, Saito M, Amizuka N and Kudo A

    Department of Biological Information, Tokyo Institute of Technology, 4259 Midori-ku, Nagatsuta, Yokohama 226-8501.

    Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.

    The Journal of biological chemistry 2010;285;3;2028-39

  • B and C domain containing tenascin-C: urinary markers for invasiveness of urothelial carcinoma of the urinary bladder?

    Richter P, Tost M, Franz M, Altendorf-Hofmann A, Junker K, Borsi L, Neri D, Kosmehl H, Wunderlich H and Berndt A

    Institute of Pathology, University Hospital Jena, Ziegelmühlenweg 1, 07743 Jena, Germany.

    Purpose: Surveillance of urothelial carcinoma of the urinary bladder (UBC) patients with respect to tumour recurrence and invasiveness is crucial for therapy and prognosis. Therefore, evaluation of non-invasive methods to monitor tumour progression is of high clinical interest. The study was aimed at investigating urinary concentrations of tenascin-C splicing domains for their value as tumour surveillance markers.

    Methods: Urinary concentration of B and C domain containing tenascin-C (Tn-C) was analysed by ELISA technology in 104 UBC patients, 11 patients with cystitis and 15 healthy donors as control. The investigation was supplemented by Tn-C immunohistochemistry and Western blotting.

    Results: A statistically significant increase in urinary concentrations of both Tn-C B and C domain with tumour progression could be evidenced. A concordant tumour-associated enhanced protein deposition in the carcinoma stroma could be demonstrated by immunohistochemistry in invasive UBC. Western blotting reveals proteolytic fragmentation of urinary Tn-C.

    Conclusions: In summary, detection of Tn-C splicing domains in urine is suggested as a marker for the surveillance of UBC recurrence and invasiveness.

    Journal of cancer research and clinical oncology 2009;135;10;1351-8

  • Tenascin-C and type I and III collagen expression in total Achilles tendon rupture. An immunohistochemical study.

    Pajala A, Melkko J, Leppilahti J, Ohtonen P, Soini Y and Risteli J

    Division of Orthopaedics, Oulu University Hospital, Oulu, Finland. ari_pajala@mail.suomi.net

    Unlabelled: Tendon tissue degeneration and changes in collagen composition play a role in spontaneous rupture of the human Achilles tendon. Tenascin-C has been shown to be present in the tissue pathology and changes in tissue loading. We made an immunohistological study of the expression of tenascin-C and type I and III collagens in ruptured human Achilles tendons.

    Methods: Three tissue samples in ten individuals, one from the Achilles tendon rupture and two control samples from four and sixteen centimeters proximal in same tendon were collected at surgery. The specimen were fixed and labelled with specific antibodies to type I and III procollagens (PICP, PINP and PIIINP), mature type III collagen (IIINTP) and tenascin-C. The amount of reacting tissue was evaluated visually and graded on a semiquantitative scale.

    Results: No difference in the expression of tenascin-C was found between the sites. Instead, mature type III collagen content (p=0.008) and type III collagen synthesis (p=0.016) were significantly increased at the rupture site relative to the control site 2. The amount of newly synthesized type I collagen (PINP, PICP) was relatively high at all sites, as expected.

    Conclusion: The expression of type III collagen is increased at the rupture site in the human Achilles tendon, but that of tenascin-C remains unchanged. This finding supports a tissue composition alteration background for Achilles tendon rupture, while the role of mechanical loading at the rupture site remains controversial.

    Histology and histopathology 2009;24;10;1207-11

  • Endogenous tenascin-C enhances glioblastoma invasion with reactive change of surrounding brain tissue.

    Hirata E, Arakawa Y, Shirahata M, Yamaguchi M, Kishi Y, Okada T, Takahashi JA, Matsuda M and Hashimoto N

    Department of Neurosurgery, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan.

    Tenascin-C is an extracellular matrix glycoprotein implicated in embryogenesis, wound healing and tumor progression. We previously revealed that tenascin-C expression is correlated with the prognosis of patients with glioblastoma. However, the exact role of endogenous tenascin-C in regulation of glioblastoma proliferation and invasion remains to be established. We show here that endogenous tenascin-C facilitates glioblastoma invasion, followed by reactive change of the surrounding brain tissue. Although shRNA-mediated knockdown of endogenous tenascin-C does not affect proliferation of glioblastoma cells, it abolishes cell migration on a two-dimensional substrate and tumor invasion with brain tissue changes in a xenograft model. The tyrosine phosphorylation of focal adhesion kinase, a cytoplasmic tyrosine kinase that associates with integrins, was decreased in tenascin-C-knockdown cells. In the analysis of clinical samples, tenascin-C expression correlates with the volume of peritumoral reactive change detected by magnetic resonance imaging. Interestingly, glioblastoma cells with high tenascin-C expression infiltrate brain tissue in an autocrine manner. Our results suggest that endogenous tenascin-C contributes the invasive nature of glioblastoma and the compositional change of brain tissue, which renders tenascin-C as a prime candidate for anti-invasion therapy for glioblastoma.

    Cancer science 2009;100;8;1451-9

  • Syndecan-1 and tenascin expression in cystic tumors of the pancreas.

    Kylänpää L, Hagström J, Lepistö A, Linjama T, Kärkkäinen P, Kiviluoto T and Haglund C

    Department of General Surgery, Helsinki University Central Hospital, Helsinki, Finland.

    Context: Since benign and malignant mucin-producing tumors of the pancreas may be difficult to distinguish from each other; preoperative methods for differential diagnosis would reduce unnecessary surgery.

    Objective: To compare syndecan-1 and tenascin immunoexpression in benign and malignant cystic pancreatic tumors.

    Design: We used immunohistochemical staining for syndecan-1 and tenascin antibodies in tumor tissue samples.

    Setting: Helsinki University Central Hospital.

    Patients: Tissue material came from 33 patients undergoing surgery from 1979 to 2005 for cystic pancreatic tumors.

    Results: A statistically significant difference appeared in syndecan-1 expression between benign (mucinous cystic neoplasms and intraductal papillary mucinous neoplasms) and mucinous carcinomas, but there was no significant difference in tenascin immunoexpression between these tumor groups.

    Conclusion: Our findings suggest that low syndecan-1 expression might serve as a predictive factor for malignancy in cystic tumors of the pancreas.

    JOP : Journal of the pancreas 2009;10;4;378-82

  • Tenascin C in medullary thyroid microcarcinoma and C-cell hyperplasia.

    Koperek O, Prinz A, Scheuba C, Niederle B and Kaserer K

    Department of Clinical Pathology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.

    Tenascin C (Tn-C) is an extracellular matrix glycoprotein that is expressed early in carcinogenesis including intraepithelial neoplastic lesions of different organs. In this study, we analyze whether stroma reaction seen by Tn-C expression is detected early in tumorigenesis of medullary thyroid carcinoma (MTC) including medullary microcarcinoma and C-cell hyperplasia (CCH), which is accepted to be a precursor lesion of MTC in the setting of RET oncogene germ-line mutation. Tn-C was expressed in the stroma of all medullary microcarcinoma and in the stroma next to CCH. Stromal Tn-C expression was significantly more often seen in CCH with concomitant MTC than in isolated CCH of hereditary as well as nonhereditary cases (p = 0.001 and p = 0.016, respectively). We conclude that Tn-C expression and thus early stroma remodeling is seen in medullary microcarcinoma and CCH. Stromal Tn-C expression seems to be an indicator of a further step in carcinogenesis of MTC irrespective of a RET oncogene germ-line mutation.

    Virchows Archiv : an international journal of pathology 2009;455;1;43-8

  • Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease.

    Midwood K, Sacre S, Piccinini AM, Inglis J, Trebaul A, Chan E, Drexler S, Sofat N, Kashiwagi M, Orend G, Brennan F and Foxwell B

    Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, UK. k.midwood@imperial.ac.uk

    Although there have been major advances in the treatment of rheumatoid arthritis with the advent of biological agents, the mechanisms that drive cytokine production and sustain disease chronicity remain unknown. Tenascin-C (encoded by Tnc) is an extracellular matrix glycoprotein specifically expressed at areas of inflammation and tissue damage in inflamed rheumatoid joints. Here we show that mice that do not express tenascin-C show rapid resolution of acute joint inflammation and are protected from erosive arthritis. Intra-articular injection of tenascin-C promotes joint inflammation in vivo in mice, and addition of exogenous tenascin-C induces cytokine synthesis in explant cultures from inflamed synovia of individuals with rheumatoid arthritis. Moreover, in human macrophages and fibroblasts from synovia of individuals with rheumatoid arthritis, tenascin-C induces synthesis of proinflammatory cytokines via activation of Toll-like receptor 4 (TLR4). Thus, we have identified tenascin-C as a novel endogenous activator of TLR4-mediated immunity that mediates persistent synovial inflammation and tissue destruction in arthritic joint disease.

    Funded by: Arthritis Research UK; Medical Research Council: G0700108

    Nature medicine 2009;15;7;774-80

  • Expression of tenascin-c and CD44 receptors in cardiac myxomas.

    Donato G, Conforti F, Zuccalà V, Russo E, Maltese L, Perrotta I and Amorosi A

    Department of Pathology, School of Medicine, University Magna Graecia, Catanzaro, Italy. gdonato@unicz.it

    Background: Myxomas are the most frequent primary cardiac neoplasms. They have an abundant extracellular matrix rich in proteoglycans. Interactions between cells and matrix are very important in the development of tumors, but data about myxomas in this setting are scarce because of the rarity of such neoplasms. The expression of tenascin-c and hyaluran receptors in cardiac myxoma has never been investigated. Moreover, it is now well recognized that cells of cardiac myxoma differentiate along endothelial lines.

    Methods: We have analyzed left atrial myxomas from 13 consecutive patients (six male and seven female, surgically treated), via immunohistochemical methods for the expression of molecules also implicated in angiogenesis in normal and pathological conditions, like tenascin-c and hyaluran receptors CD44s, CD44v5 and CD44v6.

    Results: Our data suggest that tenascin-c and CD44s play a synergic and perhaps complementary role in development of cardiac myxomas. In particular, tenascin-c seems to promote aggregation of cells and differentiation in vascular structures, whereas CD44s receptors might be important for cellular motility. Cell proliferation rate in such tumors was very low (MIB-1 labeling index <1%) and uniform in all the areas of the neoplasms regardless of the presence of characteristic structures such as cords and rings of multinucleated cells or the expression of tenascin-c and CD44 receptors.

    Conclusions: This study shows that cardiac myxomas express in the extracellular matrix tenascin-c and on the cellular membranes of neoplastic cells the hyaluran receptor CD44s. Such molecules take part in the mechanism of development of the myxomas and might be in the future the target of nonsurgical treatments.

    Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology 2009;18;3;173-7

  • Role of fibrillar Tenascin-C in metastatic pancreatic cancer.

    Chen J, Chen Z, Chen M, Li D, Li Z, Xiong Y, Dong J and Li X

    Hepatobiliary Surgery Institute, Southwest Hospital, Third Military Medical University, Chongqing, P.R. China.

    Interaction of cancer cells with stroma cells facilitates tumor progression by rebuilding the existing extracellular matrix (ECM) microenvironment. In the tumor, upregulation of Tenascin-C (Tn-C) expression potentially can alter tumor behavior. However, the molecular mechanisms by which tumor-stroma interactions affect the tumor microenvironment have not been well characterized. In this study, we analyzed the expression of fibrillar Tn-C (fTn-C) in human metastatic pancreatic cancers. After co-culturing two pancreatic cancer cell lines, highly metastatic BxPc3 cells and non-metastatic PaCa2 cells, with stromal fibroblasts (SF), we evaluated the roles of matrix metalloproteinase 2 (MMP-2) activation and SF in promoting Tn-C organization. Next, we evaluated whether fibrillar Tn-C promotes pancreatic cancer cell movement using cell adhesion and migration assays. Finally, we observed the relationship between MMP-2 activation and fTn-C formation in vivo by injecting the BxPc3 and PaCa2 cells into nude mice. We found that fTn-C was increased in metastatic pancreatic cancer. The fTn-C expression correlated with MMP-2 activity. In the in vitro co-culture, fTn-C organization was found only in BxPc3/SF co-cultures, and required the participation of active MMP-2. The fTn-C reduced cell adhesion and promote pancreatic cancer cell migration by decreasing the adhesive interactions between integrin alpha6beta1 and the ECM. The in vivo tumorigenesis analysis showed that the fTn-C formation and active MMP-2 were significantly increased in the BxPc3 tumors, compared to the PaCa2 tumors. These results demonstrate that Tn-C deposition into the ECM requires participation of active MMP-2 and SF. The deposited Tn-C could promote pancreatic cancer progression.

    International journal of oncology 2009;34;4;1029-36

  • Tenascin C: a defensive role in sentinel lymph nodes of melanoma patients?

    Gazzaniga P, Cigna E, Vincenzi B, Bottoni U, Vasaturo F, Alfano C, Calvieri S, Frati L, Scuderi N, Aglianò AM and Gradilone A

    Journal of dermatological science 2009;53;3;239-41

  • Tenascin-C is a novel RBPJkappa-induced target gene for Notch signaling in gliomas.

    Sivasankaran B, Degen M, Ghaffari A, Hegi ME, Hamou MF, Ionescu MC, Zweifel C, Tolnay M, Wasner M, Mergenthaler S, Miserez AR, Kiss R, Lino MM, Merlo A, Chiquet-Ehrismann R and Boulay JL

    Department of Research, Laboratory of Molecular Neuro-Oncology, University Hospital, Basel, Switzerland.

    Tenascin-C (TNC) expression is known to correlate with malignancy in glioblastoma (GBM), a highly invasive and aggressive brain tumor that shows limited response to conventional therapies. In these malignant gliomas as well as in GBM cell lines, we found Notch2 protein to be strongly expressed. In a GBM tumor tissue microarray, RBPJk protein, a Notch2 cofactor for transcription, was found to be significantly coexpressed with TNC. We show that the TNC gene is transactivated by Notch2 in an RBPJk-dependent manner mediated by an RBPJk binding element in the TNC promoter. The transactivation is abrogated by a Notch2 mutation, which we detected in the glioma cell line Hs683 that does not express TNC. This L1711M mutation resides in the RAM domain, the site of interaction between Notch2 and RBPJk. In addition, transfection of constructs encoding activated Notch2 or Notch1 increased endogenous TNC expression identifying TNC as a novel Notch target gene. Overexpression of a dominant negative form of the transcriptional coactivator MAML1 or knocking down RBPJk in LN319 cells led to a dramatic decrease in TNC protein levels accompanied by a significant reduction of cell migration. Because addition of purified TNC stimulated glioma cell migration, this represents a mechanism for the invasive properties of glioma cells controlled by Notch signaling and defines a novel oncogenic pathway in gliomagenesis that may be targeted for therapeutic intervention in GBM patients.

    Cancer research 2009;69;2;458-65

  • Investigation of the Sp1-binding site polymorphism within the COL1A1 gene in participants with Achilles tendon injuries and controls.

    Posthumus M, September AV, Schwellnus MP and Collins M

    UCT/MRC Research Unit for Exercise Science and Sports Medicine of the Department of Human Biology, Faculty of Health Sciences, University of Cape Town, South Africa.

    Sequence variants within the type V collagen (COL5A1) and tenascin C (TNC) genes have to date been shown to be associated with chronic Achilles tendinopathies and/or spontaneous Achilles tendon ruptures. Type V collagen and tenascin C are quantitatively minor components of tendon, while type I collagen is the major structural component. There is increased expression of the COL1A1 gene, which encodes for the alpha1 chain of type I collagen, in the painful Achilles tendon. A functional Sp1-binding site polymorphism (SNP rs1800012; IVS1+1023G>T) within this gene has been shown to be associated with several connective tissue disorders. The aim of this study was to determine whether the Sp1-binding site polymorphism within the COL1A1 gene is associated with chronic Achilles tendinopathies and/or spontaneous Achilles tendon ruptures. Achilles tendinopathy (n=85), Achilles rupture (n=41) and asymptomatic control (n=125) participants were genotyped for the COL1A1 Sp1-binding site polymorphism. There were no observed statistical differences in the genotype (p=0.602) or allele (p=0.694) distributions between the groups. In conclusion, this study has shown that there is no association between the Sp1-binding site polymorphism within the first intron of COL1A1 and Achilles tendinopathy or Achilles tendon rupture within the population studied.

    Journal of science and medicine in sport 2009;12;1;184-9

  • Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms.

    Hancox RA, Allen MD, Holliday DL, Edwards DR, Pennington CJ, Guttery DS, Shaw JA, Walker RA, Pringle JH and Jones JL

    Department of Cancer Studies and Molecular Medicine, Infirmary Close, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester, UK. raa9@le.ac.uk

    Introduction: The stromal microenvironment has a profound influence on tumour cell behaviour. In tumours, the extracellular matrix (ECM) composition differs from normal tissue and allows novel interactions to influence tumour cell function. The ECM protein tenascin-C (TNC) is frequently up-regulated in breast cancer and we have previously identified two novel isoforms - one containing exon 16 (TNC-16) and one containing exons 14 plus 16 (TNC-14/16).

    Methods: The present study has analysed the functional significance of this altered TNC isoform profile in breast cancer. TNC-16 and TNC-14/16 splice variants were generated using PCR-ligation and over-expressed in breast cancer cells (MCF-7, T47D, MDA-MD-231, MDA-MB-468, GI101) and human fibroblasts. The effects of these variants on tumour cell invasion and proliferation were measured and compared with the effects of the large (TNC-L) and fully spliced small (TNC-S) isoforms.

    Results: TNC-16 and TNC-14/16 significantly enhanced tumour cell proliferation (P < 0.05) and invasion, both directly (P < 0.01) and as a response to transfected fibroblast expression (P < 0.05) with this effect being dependent on tumour cell interaction with TNC, because TNC-blocking antibodies abrogated these responses. An analysis of 19 matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases 1 to 4 (TIMP 1 to 4) revealed that TNC up-regulated expression of MMP-13 and TIMP-3 two to four fold relative to vector, and invasion was reduced in the presence of MMP inhibitor GM6001. However, this effect was not isoform-specific but was elicited equally by all TNC isoforms.

    Conclusions: These results demonstrate a dual requirement for TNC and MMP in enhancing breast cancer cell invasion, and identify a significant role for the tumour-associated TNC-16 and TNC-14/16 in promoting tumour invasion, although these isoform-specific effects appear to be mediated through MMP-independent mechanisms.

    Funded by: Breast Cancer Now: 2001:232

    Breast cancer research : BCR 2009;11;2;R24

  • Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation.

    Fiorilli P, Partridge D, Staniszewska I, Wang JY, Grabacka M, So K, Marcinkiewicz C, Reiss K, Khalili K and Croul SE

    Department of Neuroscience and Center for Neurovirology, Temple University School of Medicine, Philadelphia, PA, USA.

    Medulloblastoma spreads by leptomeningeal dissemination rather than by infiltration that characterizes other CNS tumors, eg, gliomas. This study represents an initial attempt to identify both the molecules that mediate medulloblastoma adhesion to leptomeninges and the pathways that are key to survival and proliferation of tumor following adhesion. As a first step in molecule identification, we produced adhesion of D283 medulloblastoma cells to the extracellular matrix (ECM) of H4 glioma cells in vitro. Within this context, D283 cells preferentially expressed the alpha9 and beta1 integrin subunits; antibody and disintegrin blockade of alpha9 and beta1 binding eliminated the adhesion. The H4 ECM was enriched in tenascin, a binding partner for the alpha9beta1 integrin heterodimer. Purified tenascin-C supported D283 cell adhesion. The adhesion was blocked by antibodies to alpha9 and beta1 integrin. In vivo data were similar; immunohistochemistry of primary human medulloblastomas with leptomeningeal extension demonstrated increased expression of alpha9 and beta1 integrins as well as tenascin at the interface of brain and leptomeningeal tumor. These data suggest that tumor-cell expressions of alpha9 and beta1 integrins in combination with extracellular tenascin are necessary for medulloblastoma adhesion to the leptomeninges. As a first step in the identification of pathways that mediate survival and proliferation of tumor following adhesion, we demonstrated that adhesion to H4 ECM was associated with survival and proliferation of D283 cells as well as activation of the MAPK pathway in a growth factor deficient environment. Antibody blockade of alpha9 and beta1 integrin binding that eliminated adhesion also eliminated the in vitro survival benefit. These data suggest that adhesion of medulloblastoma to the meninges is necessary for the survival and proliferation of these tumor cells at the secondary site.

    Funded by: NCI NIH HHS: R01 CA095518, R01 CA095518-06A2; NINDS NIH HHS: 5P01NS036466-11, P01 NS036466

    Laboratory investigation; a journal of technical methods and pathology 2008;88;11;1143-56

  • Lack of association between Tenascin-C gene and spondyloarthritis.

    Zinovieva E, Lebrun N, Letourneur F, Laurent FX, Said-Nahal R, Chiocchia G and Breban M

    Institut Cochin, INSERM U567, CNRS UMR8104, Paris, France.

    Objectives: We previously identified a new susceptibility region linked to SpA in 9q31-34. Tenascin-C (TNC) appears as one of the best positional and functional candidate genes lying within this SPA2 locus. The objectives of the present study were to identify TNC polymorphisms, and to examine their putative association with SpA.

    Methods: We first performed variants screening in 20 independent SpA patients from families with high linkage score to the SPA2 locus, and three unrelated controls: TNCs coding regions (28 exons), intron-exon boundaries and 5'- and 3'-flank regions were fully re-sequenced to identify polymorphisms. Then we genotyped selected variants in 183 independent trios, and assessed their intrafamilial association with SpA by transmission disequilibrium test.

    Results: Variants screening allowed us to identify 26 polymorphisms, 7 of which were selected for further study, in addition to an intronic polymorphism previously reported as associated with Achilles tendon injuries. In intrafamilial association test, none of the variants showed significant transmission disequilibrium. Results from analysis restricted to AS were not different from those obtained on the whole SpA group.

    Conclusions: TNC was one of the best positional and functional candidate genes within the SPA2 locus. Nevertheless, we found no association between polymorphisms in this gene and SpA. However, we cannot exclude that variants located in intronic regions or in the vicinity of TNC, which were not tested in the present study, could be implicated in the predisposition to SpA.

    Rheumatology (Oxford, England) 2008;47;11;1655-8

  • Identification of VEGF-regulated genes associated with increased lung metastatic potential: functional involvement of tenascin-C in tumor growth and lung metastasis.

    Calvo A, Catena R, Noble MS, Carbott D, Gil-Bazo I, Gonzalez-Moreno O, Huh JI, Sharp R, Qiu TH, Anver MR, Merlino G, Dickson RB, Johnson MD and Green JE

    Laboratory of Cancer Biology and Genetics, NCI, NIH, Bethesda, MD 20892, USA.

    Metastasis is the primary cause of death in patients with breast cancer. Overexpression of c-myc in humans correlates with metastases, but transgenic mice only show low rates of micrometastases. We have generated transgenic mice that overexpress both c-myc and vascular endothelial growth factor (VEGF) (Myc/VEGF) in the mammary gland, which develop high rates of pulmonary macrometastases. Gene expression profiling revealed a set of deregulated genes in Myc/VEGF tumors compared to Myc tumors associated with the increased metastatic phenotype. Cross-comparisons between this set of genes with a human breast cancer lung metastasis gene signature identified five common targets: tenascin-C(TNC), matrix metalloprotease-2, collagen-6-A1, mannosidase-alpha-1A and HLA-DPA1. Signaling blockade or knockdown of TNC in MDA-MB-435 cells resulted in a significant impairment of cell migration and anchorage-independent cell proliferation. Mice injected with clonal MDA-MB-435 cells with reduced expression of TNC demonstrated a significant decrease (P<0.05) in (1) primary tumor growth; (2) tumor relapse after surgical removal of the primary tumor and (3) incidence of lung metastasis. Our results demonstrate that VEGF induces complex alterations in tissue architecture and gene expression. The TNC signaling pathway plays an important role in mammary tumor growth and metastases, suggesting that TNC may be a relevant target for therapy against metastatic breast cancer.

    Funded by: Intramural NIH HHS: Z01 BC005740-15; NCI NIH HHS: 2R01 CA104963, N01-CO-12400, N01CO12400, R01 CA072460, R01 CA72460; NIA NIH HHS: 2 R01 AG14963-06, R01 AG014963

    Oncogene 2008;27;40;5373-84

  • Combined lysophosphatidic acid/platelet-derived growth factor signaling triggers glioma cell migration in a tenascin-C microenvironment.

    Lange K, Kammerer M, Saupe F, Hegi ME, Grotegut S, Fluri E and Orend G

    Institute of Biochemistry and Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland.

    The antiadhesive extracellular matrix molecule tenascin-C abrogates cell spreading on fibronectin through competitive inhibition of syndecan-4, thereby preventing focal adhesion kinase (FAK) activation and triggering enhanced proteolytic degradation of both RhoA and tropomyosin 1 (TM1). Here, we show that simultaneous signaling by lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) initiates glioma cell spreading and migration through syndecan-4-independent activation of paxillin and FAK and by stabilizing expression of RhoA, TM1, TM2, and TM3. By using gene silencing methods, we show that paxillin, TM1, TM2, and TM3 are essential for LPA/PDGF-induced cell spreading on a fibronectin/tenascin-C (FN/TN) substratum. LPA/PDGF-induced cell spreading and migration on FN/TN depends on phosphatidylinositol 3-kinase, RhoKinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 but is independent of phospholipase C and Jun kinase. RNA microarray data reveal expression of tenascin-C, PDGFs, LPA, and the respective receptors in several types of cancer, suggesting that the TN/LPA/PDGF axis exists in malignant tumors. These findings may in turn be relevant for diagnostic or therapeutic applications targeting cancer.

    Funded by: Worldwide Cancer Research: 06-0610

    Cancer research 2008;68;17;6942-52

  • Prognostic significance of tenascin-C expression in clear cell renal cell carcinoma.

    Ohno Y, Izumi M, Yoshioka K, Ohori M, Yonou H and Tachibana M

    Department of Urology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo, Japan. yoshio-o@tokyo-med.ac.jp

    Tenascin-C is an extracellular matrix protein that plays an important role in cell proliferation, migration and tumor invasion in various types of cancer. However, few reports exist on tenascin-C expression in renal cell carcinoma (RCC). This study aimed to assess the prognostic significance of tenascin-C in clear cell RCC. Using immunohistochemistry, 137 formalin-fixed, paraffin-embedded tissue sections obtained from patients with clear cell RCC were examined for tenascin-C expression. Tenascin-C expression was observed in 55 (40.1%) of the 137 clear cell RCC sections. Tumor cells displayed membranous and/or cytoplasmic staining for tenascin-C. Tenascin-C expression was more prominent near the pseudocapsule of the tumor and around the tumor vessels. Tenascin-C expression was significantly associated with a higher stage (P=0.0065) and higher nuclear grade (P=0.0001). However, there was no correlation between the tenascin-C expression and venous involvement. The cancer-specific survival rate in patients with a tenascin-C-positive primary tumor was significantly lower than that in those with a tenascin-C-negative primary tumor in univariate analysis (P=0.0017). However, tenascin-C expression did not exhibit a significant value for cancer-related death in the Cox regression analysis. In patients with stage 1-3 disease, the 5-year metastasis-free rate in patients with the tenascin-C-positive primary tumor was significantly lower than that in those with the tenascin-C-negative primary tumor (67.8 vs. 88.5%, respectively; P=0.0038). The Cox regression analysis showed that tenascin-C expression is a significant predictor of metastasis (P=0.0345). The tenascin-C expression was strongly related to the stage, nuclear grade and 5-year metastasis-free rate. Therefore, tenascin-C expression may be a possible marker for the metastatic potential of clear cell RCC.

    Oncology reports 2008;20;3;511-6

  • EMMPRIN modulates migration and deposition of TN-C in oral squamous carcinoma.

    Dang D, Atakilit A and Ramos DM

    Department of Orofacial Sciences, University of California at San Francisco, San Francisco, CA 94143-0512, USA.

    The extracellular matrix metalloproteinase inducer (EMMPRIN), found on the surface of many tumor cells, stimulates the production of matrix metalloproteinases (MMPs) by both fibroblasts and the tumor cells themselves. To evaluate its possible role as a tumor promoter, we first overexpressed EMMPRIN, by retroviral transduction, into poorly invasive squamous cell carcinoma (SCC) cells. Secondly, we knocked down its expression using small interfering RNA (siRNA) in invasive SCC cells. The cell lines were then re-evaluated for migration on fibronectin (FN). Overexpression of EMMPRIN, promoted motility, whereas the siRNA decreased migration. The MMP expression by these variant SCC cell lines was also manipulated by EMMPRIN. The expression of MMP-2, -3, and -9 coincided with the expression of EMMPRIN. Cocultures of SCC/peritumor fibroblasts (PTF) were used to investigate tenascin-C (TN-C) matrix deposition. The cocultures overexpressing EMMPRIN, deposited several fold greater levels of TN-C compared to the control cocultures. In addition, the siRNA cocultures deposited minimal amounts of TN-C. In the presence of the broad spectrum MMP inhibitor, GM6001, TN-C deposition by the EMMPRIN overexpressing cocultures was suppressed. Thus EMMPRIN regulates migration, MMP production by SCC cells and deposition of the TN-C matrix.

    Funded by: NIDCR NIH HHS: P01DE13904, R01 DE11930, R01 DE12856

    Anticancer research 2008;28;4B;2049-54

  • Biological and genetic interaction between tenascin C and neuropeptide S receptor 1 in allergic diseases.

    Orsmark-Pietras C, Melén E, Vendelin J, Bruce S, Laitinen A, Laitinen LA, Lauener R, Riedler J, von Mutius E, Doekes G, Wickman M, van Hage M, Pershagen G, Scheynius A, Nyberg F, Kere J and PARSIFAL Genetics Study Group

    Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.

    Neuropeptide S receptor 1 (NPSR1, GPRA 154, GPRA) has been verified as a susceptibility gene for asthma and related phenotypes. The ligand for NPSR1, Neuropeptide S (NPS), activates signalling through NPSR1 and microarray analysis has identified Tenascin C (TNC) as a target gene of NPS-NPSR1 signalling. TNC has previously been implicated as a risk gene for asthma. We aimed therefore to study the genetic association of TNC in asthma- and allergy-related disorders as well as the biological and genetic interactions between NPSR1 and TNC. Regulation of TNC was investigated using NPS stimulated NPSR1 transfected cells. We genotyped 12 TNC SNPs in the cross-sectional PARSIFAL study (3113 children) and performed single SNP association, haplotype association and TNC and NPSR1 gene-gene interaction analyses. Our experimental results show NPS-dependent upregulation of TNC-mRNA. The genotyping results indicate single SNP and haplotype associations for several SNPs in TNC with the most significant association to rhinoconjunctivitis for a haplotype, with a frequency of 29% in cases (P = 0.0005). In asthma and atopic sensitization significant gene-gene interactions were found between TNC and NPSR1 SNPs, indicating that depending on the NPSR1 genotype, TNC can be associated with either an increased or a decreased risk of disease. We conclude that variations in TNC modifies, not only risk for asthma, but also for rhinoconjunctivitis. Furthermore, we show epistasis based on both a direct suggested regulatory effect and a genetic interaction between NPSR1 and TNC. These results suggest merging of previously independent pathways of importance in the development of asthma- and allergy-related traits.

    Human molecular genetics 2008;17;11;1673-82

  • Tenascin-C expression relates to clinicopathological features in pilocytic and diffuse astrocytomas.

    Maris C, Rorive S, Sandras F, D'Haene N, Sadeghi N, Bièche I, Vidaud M, Decaestecker C and Salmon I

    Department of Pathology, Erasme University Hospital, Université Libre de Bruxelles, Brussels, Belgium.

    Aims: Tenascin-C (TN-C) is an extracellular matrix brain glycoprotein for which conflicting in vitro and in vivo results are reported in the literature dealing with its involvement in astrocytoma aggressiveness, in particular astrocytoma invasion. In view of these conflicting results and the lack of data available on low-grade astrocytomas, the present study focuses on pilocytic World Health Organization (WHO) grade I, and diffuse WHO grade II astrocytomas, that is, two histological entities associated with very different invasive abilities.

    Methods: Using real-time reverse transcription polymerase chain reaction and immunohistochemistry, we analysed the TN-C expression in normal brain tissue as well as in a series of 54 pilocytic and 53 grade II astrocytomas.

    Conclusions: Our data on normal brain showed that while TN-C is largely expressed in supratentorial white matter, it was largely absent in infratentorial white matter. Paralleling these observations, we showed that TN-C expression in low-grade astrocytomas similarly varies according to tumour site. Cox regression analysis evidenced that TN-C provided an independent prognostic value which is enhanced in the case of grade II astrocytomas for which positive TN-C expression is associated with a higher risk of recurrence. We also analysed TN-C expression specifically in vascular areas of low-grade astrocytomas without demonstrating any prognostic value for this additional feature.

    Results: Similarly to normal brain, low-grade astrocytomas exhibit variations in TN-C expression with site, and this expression is associated with an independent prognostic value in terms of recurrence.

    Neuropathology and applied neurobiology 2008;34;3;316-29

  • Tenascin is highly expressed in endometriosis and its expression is upregulated by estrogen.

    Tan O, Ornek T, Seval Y, Sati L and Arici A

    Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut; Department of Obstetrics and Gynecology, Bridgeport Hospital, Bridgeport, Connecticut.

    Objective: To investigate the localization of tenascin expression in the endometrium of women without endometriosis and in endometriotic implants, and to determine the in vitro regulation of tenascin by E(2) in these tissues.

    Design: Experimental laboratory study.

    Setting: University medical center.

    Reproductive age women with or without endometriosis.

    Proliferative (n = 14), and secretory (n = 14) endometrium from women without endometriosis and endometriosis implants (n = 14) were used for immunohistochemical analysis. Endometrial and endometriotic stromal cells were grown in culture and treated with E(2), the estrogen receptor antagonist ICI 182 780 (ICI) alone, E(2) in combination with ICI, or vehicle (control) for 24 hours, and tenascin expression was analyzed by Western blotting.

    Expression levels of tenascin in normal endometrium and endometriotic implants and its regulation by E(2).

    Tenascin immunostaining revealed an increasing intensity in the stromal cells, starting from normal secretory endometrium, then normal proliferative endometrium, and reaching the highest expression in endometriotic implants. Estradiol induced a significant increase in tenascin protein levels in the endometriotic stromal cells in culture.

    The modulation of tenascin as an extracellular matrix protein by E(2) in endometriotic stromal cells may be one of the factors playing a role in the development of endometriosis.

    Fertility and sterility 2008;89;5;1082-1089

  • GATA-6 is a novel transcriptional repressor of the human Tenascin-C gene expression in fibroblasts.

    Ghatnekar A and Trojanowska M

    Division of Rheumatology and Immunology, Medical University of South Carolina, CSB 912, SC 29425-2229, USA.

    In this study we show that GATA-6 is a novel repressor of TN-C gene expression. We demonstrated that overexpression of GATA-6 in fibroblasts inhibited basal levels, as well as markedly decreased IL-4- and TGF-beta-induced TN-C mRNA and protein levels. A GATA-6 response element was mapped to position -467 to -460 of the TN-C promoter. In addition, we showed that GATA-6 binds this site both in vitro and in vivo.

    Funded by: NIAMS NIH HHS: AR42334, R01 AR042334, R01 AR042334-13, R01 AR044883

    Biochimica et biophysica acta 2008;1779;3;145-51

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Effect of hypoxia and interleukin-1beta on expression of tenascin-C in temporomandibular joint.

    Tojyo I, Yamaguchi A, Nitta T, Yoshida H, Fujita S and Yoshida T

    Department of Oral and Maxillofacial Surgery, Wakayama Medical University, Wakayama, Japan. kagoky@wakayama-med.ac.jp

    Objective: The expression of tenascin-C in the synovial membrane of the internal derangement (ID) of the temporomandibular joint (TMJ) has been reported. Hypoxia of the synovial membrane in TMJ is considered to be a cause for the pathophysiology of ID. In this study, we clarify the contribution of hypoxia and interleukin-1beta in the expression of tenascin-C in ID of TMJ.

    Synovial fibroblasts and disk cells obtained from ID of TMJs were cultured and treated with interleukin-1beta under normoxia and hypoxia. A Western blot analysis and reverse transcription-polymerase chain reaction analysis were used to identify tenascin-C in cultured synovial fibroblasts and disk cells. In addition, the immunohistochemical staining of tenascin-C was carried out for the specimens of ID of TMJs and normal.

    Results: The combination of hypoxia and interleukin-1beta caused a significant increase in tenascin-C protein and mRNA of synovial fibroblasts. In contrast, the combination caused no increase in tenascin-C in disk cells. However, the immunohistochemical staining demonstrated tenascin-C to be significantly detected in both the synovial tissue and disks in ID of TMJ.

    Conclusions: These results indicate that hypoxic conditions with inflammation modulate the tenascin-C expression in synovial fibroblasts, but not in disk cells.

    Oral diseases 2008;14;1;45-50

  • Regulation of tenascin-C expression by tumor necrosis factor-alpha in cultured human osteoarthritis chondrocytes.

    Nakoshi Y, Hasegawa M, Sudo A, Yoshida T and Uchida A

    Department of Orthopedic Surgery and Department of Pathology and Matrix Biology, Mie University Graduate School of Medicine, Mie, Japan.

    Unlabelled: Expression of tenascin-C reappears in articular cartilage of persons with osteoarthritis (OA), while it is almost abolished in normal mature cartilage. Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, is upregulated in OA cartilage and is involved in the progression of OA, and stimulates tenascin-C expression in other types of cells. We investigated regulation of tenascin-C expression by TNF-alpha through nuclear factor-alphaB (NF-kappaB) in OA cartilage in vivo and in vitro.

    Methods: Human articular cartilages were obtained from patients with OA and immunofluorescence examination of tenascin-C and the activated RelA subunit was performed. Cultured chondrocytes isolated from human OA cartilage were treated with TNF-alpha and with SN50. Activation of RelA subunit of NF-kappaB was examined by immunolabeling. Changes in tenascin-C protein concentrations were determined by immunofluorescence of cells after monensin treatment and Western blot analysis of the cell lysates, and mRNA levels were analyzed by quantitative real-time polymerase chain reaction.

    Results: Increased intensity of tenascin-C staining was observed in the damaged cartilage compared with normal cartilage. Activated RelA staining in chondrocyte nuclei was prominent in tenascin-C-positive areas of OA cartilage. Treatment of cultured chondrocytes by TNF-alpha induced translocation of activated RelA to the nuclei, followed by upregulation of tenascin-C expression in both mRNA and protein. Treatment with SN50 inhibited increases of RelA and tenascin-C expression in chondrocytes.

    Conclusion: TNF-alpha stimulated tenascin-C expression through NF-kappaB signaling with RelA activation in cultured OA chondrocytes, suggesting involvement of tenascin-C in OA cartilage remodeling.

    The Journal of rheumatology 2008;35;1;147-52

  • Tenascin C interacts with ecto-5'-nucleotidase (eN) and regulates adenosine generation in cancer cells.

    Sadej R, Inai K, Rajfur Z, Ostapkowicz A, Kohler J, Skladanowski AC, Mitchell BS and Spychala J

    Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA.

    Tenascin C is expressed in invasive human solid tumors; however its specific role in cancer biology remains obscure. Previously, we have found that ecto-5'-nucleotidase (eN) is a marker of ER (-) breast carcinoma and elevated expression correlates with invasive mesenchymal cell phenotype. To investigate for the potential relationship between eN and protein components of the extracellular matrix (ECM) we measured adenosine generation from AMP in cells incubated with soluble ECM proteins. We found that tenascin C was the only ECM component that strongly inhibited ecto-5'-nucleotidase (eN) activity in situ and adenosine generation from AMP (75% inhibition, p < 0.01). The inhibition was comparable to that induced by concanavalin A, a well-defined and strong inhibitor of eN. Resin immobilized tenascin C, but not collagen, and only weakly fibronectin, specifically and quantitatively bound cell-extracted eN. We further developed breast cancer cell line with reduced eN expression and tested changes in cell adhesion on different ECM. Breast cancer cells expressing reduced eN attached 56% weaker (p < 0.05) to immobilized tenascin C. This difference was not detected with other ECM proteins. Finally, control breast cancer cells migrated slower on tenascin C when compared with clone with reduced eN expression. These data suggest that eN is a novel and specific receptor for tenascin C and that the interaction between these proteins may influence cell adhesion and migration and also lead to decreased generation of local adenosine.

    Funded by: NCI NIH HHS: R01-CA34085

    Biochimica et biophysica acta 2008;1782;1;35-40

  • A peptide derived from tenascin-C induces beta1 integrin activation through syndecan-4.

    Saito Y, Imazeki H, Miura S, Yoshimura T, Okutsu H, Harada Y, Ohwaki T, Nagao O, Kamiya S, Hayashi R, Kodama H, Handa H, Yoshida T and Fukai F

    Department of Molecular Patho-Physiology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan.

    Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated beta1 integrin-mediated cell adhesion of nonadherent and adherent cell types, by inducing activation of beta1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced beta1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated beta1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced beta1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of beta1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase Calpha inhibitor Gö6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in beta1 integrin necessary for its functional activation. A lateral interaction of beta1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change.

    The Journal of biological chemistry 2007;282;48;34929-37

  • Myofibre damage in human skeletal muscle: effects of electrical stimulation versus voluntary contraction.

    Crameri RM, Aagaard P, Qvortrup K, Langberg H, Olesen J and Kjaer M

    Department of Exercise Science, Concordia University, Montreal, Canada, and Institute of Sports Medicine, Copenhagen, Bispebjerg Hospital, Denmark. regina.crameri@dsto.defence.gov.au

    Disruption to proteins within the myofibre after a single bout of unaccustomed eccentric exercise is hypothesized to induce delayed onset of muscle soreness and to be associated with an activation of satellite cells. This has been shown in animal models using electrical stimulation but not in humans using voluntary exercise. Untrained males (n=8, range 22-27 years) performed 210 maximal eccentric contractions with each leg on an isokinetic dynamometer, voluntarily (VOL) with one leg and electrically induced (ES) with the other leg. Assessments from the skeletal muscle were obtained prior to exercise and at 5, 24, 96 and 192 h postexercise. Muscle tenderness rose in VOL and ES after 24 h, and did not differ between groups. Maximal isometric contraction strength, rate of force development and impulse declined in the VOL leg from 4 h after exercise, but not in ES (except at 24 h). In contrast, a significant disruption of cytoskeletal proteins (desmin) and a rise of myogenic growth factors (myogenin) occurred only in ES. Intracellular disruption and destroyed Z-lines were markedly more pronounced in ES (40%) compared with VOL (10%). Likewise, the increase in satellite cell markers [neural cell adhesion molecule (N-CAM) and paired-box transcription factor (Pax-7)] was more pronounced in ES versus VOL. Finally, staining of the intramuscular connective tissue (tenascin C) was increased equally in ES and VOL after exercise. The present study demonstrates that in human muscle, the delayed onset of muscle soreness was not significantly different between the two treatments despite marked differences in intramuscular histological markers, in particular myofibre proteins and satellite cell markers. An increase in tenascin C expression in the midbelly of the skeletal muscle in both legs provides further evidence of a potential role for the extracellular matrix in the phenomenon of delayed onset of muscle soreness.

    The Journal of physiology 2007;583;Pt 1;365-80

  • Endothelin receptor type B counteracts tenascin-C-induced endothelin receptor type A-dependent focal adhesion and actin stress fiber disorganization.

    Lange K, Kammerer M, Hegi ME, Grotegut S, Dittmann A, Huang W, Fluri E, Yip GW, Götte M, Ruiz C and Orend G

    Center for Biomedicine, Department of Clinical and Biological Sciences, University of Basel, Basel, Switzerland.

    Tenascin-C, an extracellular matrix molecule of the tumor-specific microenvironment, counteracts the tumor cell proliferation-suppressing effect of fibronectin by blocking the integrin alpha(5)beta(1)/syndecan-4 complex. This causes cell rounding and stimulates tumor cell proliferation. Tenascin-C also stimulates endothelin receptor type A (EDNRA) expression. Here, we investigated whether signaling through endothelin receptors affects tenascin-C-induced cell rounding. We observed that endothelin receptor type B (EDNRB) activation inhibited cell rounding by tenascin-C and induced spreading by restoring expression and function of focal adhesion kinase (FAK), paxillin, RhoA, and tropomyosin-1 (TM1) via activation of epidermal growth factor receptor, phospholipase C, c-Jun NH(2)-terminal kinase, and the phosphatidylinositol 3-kinase pathway. In contrast to EDNRB, signaling through EDNRA induced cell rounding, which correlated with FAK inhibition and TM1 and RhoA protein destabilization in the presence of tenascin-C. This occurred in a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-dependent manner. Thus, tumorigenesis might be enhanced by tenascin-C involving EDNRA signaling. Inhibition of tenascin-C in combination with blocking both endothelin receptors could present a strategy for sensitization of cancer and endothelial cells toward anoikis.

    Funded by: Worldwide Cancer Research: 06-0610

    Cancer research 2007;67;13;6163-73

  • Tenascin cytotactin epidermal growth factor-like repeat binds epidermal growth factor receptor with low affinity.

    Iyer AK, Tran KT, Borysenko CW, Cascio M, Camacho CJ, Blair HC, Bahar I and Wells A

    Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

    Select epidermal growth factor (EGF)-like (EGFL) repeats of human tenascin cytotactin (tenascin C) can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of classical growth factors such as EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14th EGFL repeat (Ten14). Ten14 assumes a tight EGF-like fold with truncated loops, consistent with circular dichroism studies. We generated bound structures for Ten14 with EGFR using two different approaches, resulting in two distinctly different conformations. Normal mode analysis of both structures indicated that the binding pocket of EGFR exhibits a significantly higher mobility in Ten14-EGFR complex compared to that of the EGF-EGFR complex; we hypothesized this may be attributed to loss of key high-affinity interactions within the Ten14-EGFR complex. We proved the efficacy of our in silico models by in vitro experiments. Surface plasmon resonance measurements yielded equilibrium constant K(D) of 74 microM for Ten14, approximately three orders of magnitude weaker than that of EGF. In accordance with our predicted bound models, Ten14 in monomeric form does not bind EGFR with sufficient stability so as to induce degradation of receptor, or undergo EGFR-mediated internalization over either the short (20 min) or long (48 h) term. This transient interaction with the receptor on the cell surface is in marked contrast to other EGFR ligands which cause EGFR transit through, and signaling from intracellular locales in addition to cell surface signaling.

    Journal of cellular physiology 2007;211;3;748-58

  • Expression of large tenascin-C splice variants by hepatic stellate cells/myofibroblasts in chronic hepatitis C.

    El-Karef A, Kaito M, Tanaka H, Ikeda K, Nishioka T, Fujita N, Inada H, Adachi Y, Kawada N, Nakajima Y, Imanaka-Yoshida K and Yoshida T

    Department of Pathology and Matrix Biology, Mie University Graduate School of Medicine, Mie, Japan.

    Earlier studies have suggested involvement of tenascin-C (TN-C) in liver fibrosis. Here, we examined expression of TN-C variants and types of alternatively spliced fibronectin-type III (FNIII) repeats in chronic hepatitis.

    Methods: Using three monoclonal antibodies against TN-C variants, immunohistochemical staining was performed and the correlation with histological parameters of chronic hepatitis C was examined. The cellular source was also determined and variant expression and their types were tested using isolated rat hepatic stellate cells (HSCs), liver myofibroblasts, and/or LI90 cells.

    Results: Large variants were not expressed in normal liver, but were up-regulated in chronic hepatitis, especially at sites of interface hepatitis and confluent necrosis, showing stronger correlations between staining intensity and these than with other parameters or fibrosis. TN-C deposition was closely correlated with increase in the number of alpha-smooth muscle actin-positive cells, i.e. activated HSCs/myofibroblasts, and in situ hybridization showed TN-C mRNA signals in the cells. Activated HSCs and myofibroblasts in culture highly expressed large variants of TN-C. In LI90 cells, sequencing of large variants revealed that the FNIII repeats D and A1/A4, followed by B, were preferentially included.

    Conclusions: TN-C and its variants are produced by HSCs/myofibroblasts, suggesting important roles in liver fibrogenesis.

    Journal of hepatology 2007;46;4;664-73

  • Microarray-based identification of tenascin C and tenascin XB, genes possibly involved in tumorigenesis associated with neurofibromatosis type 1.

    Lévy P, Ripoche H, Laurendeau I, Lazar V, Ortonne N, Parfait B, Leroy K, Wechsler J, Salmon I, Wolkenstein P, Dessen P, Vidaud M, Vidaud D and Bièche I

    Laboratoire de Génétique Moléculaire-Institut National de la Sante et de la Recherche Medicale U745, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris V, 4 avenue de l'Observatoire, Paris, France.

    Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex variety of clinical manifestations. The hallmark of NF1 is the onset of heterogeneous (dermal or plexiform) benign neurofibromas. Plexiform neurofibromas can give rise to malignant peripheral nerve sheath tumors, which are resistant to conventional therapies.

    To identify new signaling pathways involved in the malignant transformation of plexiform neurofibromas, we applied a 22,000-oligonucleotide microarray approach to a series of plexiform neurofibromas and malignant peripheral nerve sheath tumors. Changes in the expression of selected genes were then confirmed by real-time quantitative reverse transcription-PCR.

    Results: We identified two tenascin gene family members that were significantly deregulated in both human NF1-associated tumors and NF1-deficient primary cells: Tenascin C (TNC) was up-regulated whereas tenascin XB (TNXB) was down-regulated during tumor progression. TNC activation is mainly due to the up-regulation of large TNC splice variants. Immunohistochemical studies showed that TNC transcripts are translated into TNC protein in TNC-overexpressing tumors. Aberrant transcriptional activation of TNC seems to be principally mediated by activator protein transcription factor complexes.

    Conclusion: TNXB and TNC may be involved in the malignant transformation of plexiform neurofibromas. Anti-TNC antibodies, already used successfully in clinical trials to treat malignant human gliomas, may be an appropriate new therapeutic strategy for NF1.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2007;13;2 Pt 1;398-407

  • Extracellular matrix proteins and myofibroblasts in granulomas of sarcoidosis, atypical mycobacteriosis, and tuberculosis of the lung.

    Kaarteenaho-Wiik R, Sademies O, Pääkkö P, Risteli J and Soini Y

    Department of Internal Medicine, University of Oulu and Oulu University Hospital, PO Box 5000, FIN-90014 Oulu, Finland. riitta.kaarteenaho-wiik@oulu.fi

    Sarcoidosis, atypical mycobacteriosis, and tuberculosis are common diseases of human lung with a typical feature of formation of granulomas. The structure of granulomas has not been elucidated completely. We studied the expression of tenascin-C, precursor proteins of collagens I and III, and the presence of myofibroblasts in granulomas of sarcoidosis, atypical mycobacteriosis, and tuberculosis of human lung. Twenty-five histologic samples of lung were analyzed by immunohistochemistry using antibodies to tenascin-C and aminoterminal propeptides of collagens I and III. To identify the myofibroblast-type cells in granulomas, the sections were also stained with antibodies against alpha-smooth muscle actin, vimentin, and desmin. In every case, tenascin-C and precursor proteins of collagens I and III were expressed around granulomas. Precursor protein of collagen I was expressed also within them. In tuberculosis and atypical mycobacteriosis, expression of tenascin-C and precursor protein of collagen I was stronger than in sarcoidosis. The cells demarcating granulomas and, thus, colocalizing with tenascin-C and both collagen precursors were positive for alpha-smooth muscle actin and vimentin, which suggests that these cells are myofibroblasts. They were also more abundantly present in tuberculosis and atypical mycobacteriosis, as suggested by alpha-smooth muscle actin staining. We concluded that tenascin-C and precursor proteins of collagens I and III are expressed around granulomas in sarcoidosis, atypical mycobacteriosis, and tuberculosis of the lung; and furthermore, their expression colocalize with the expression of myofibroblasts. Our results further point to the fact that fibrogenesis and matrix turnover is stronger in tuberculosis and atypical mycobacteriosis than in sarcoidosis.

    Human pathology 2007;38;1;147-53

  • Plasma large Tenascin-C spliced variant as a possible biomarker for the prediction of hepatic recurrence in colorectal cancer.

    Takeda A, Otani Y, Hirooka E, Okada K, Torii T, Shinozuka N and Koyama I

    Surgery 2007;141;1;124-5

  • Epithelial-derived TGF-beta2 modulates basal and wound-healing subepithelial matrix homeostasis.

    Thompson HG, Mih JD, Krasieva TB, Tromberg BJ and George SC

    Department of Biomedical Engineering, University of California, Irvine, Irvine, California 92697-2715, USA.

    The epithelium influences the mesenchyme during dynamic processes such as embryogenesis, wound healing, fibrosis, and carcinogenesis. Since transforming growth factor-beta (TGF-beta) modulates these processes, we hypothesized that epithelial-derived TGF-beta also plays a critical role in maintaining the extracellular matrix at basal conditions. We utilized an in vitro model of the epithelial-mesenchymal trophic unit in the human airways to determine the role of epithelial-derived TGF-beta in modulating the extracellular matrix under basal and wound-healing conditions. When differentiated at an air-liquid interface, the human bronchial epithelium produces active TGF-beta2 at a concentration of 50-70 pg/ml, whereas TGF-beta1 is undetectable. TGF-beta2 increases two- to threefold following scrape injury in a dose-dependent fashion and significantly enhances both alpha-smooth muscle actin expression in the underlying collagen-embedded fibroblasts and secretion of tenascin-C into the matrix. Multiphoton microscopy demonstrates substantially enhanced second harmonic generation from fibrillar collagen in the matrix. Pretreatment of the matrix with either sirolimus (2.5 nM) or paclitaxel (10 nM) abolishes the increases in both TGF-beta2 and second harmonic generation in response to epithelial injury. In the absence of the epithelium, exogenous active TGF-beta2 (0-400 pg/ml) produces a biphasic response in the second harmonic signal with a minimum occurring at the epithelial-derived basal level. We conclude that epithelial-derived TGF-beta2 is secreted in response to injury, significantly alters the bulk optical properties of the extracellular matrix, and its tight regulation may be required for normal collagen homeostasis.

    Funded by: NCRR NIH HHS: P41RR001192; NHLBI NIH HHS: HL-067954

    American journal of physiology. Lung cellular and molecular physiology 2006;291;6;L1277-85

  • The up-regulated expression of tenascin C in human nasal polyp tissues is related to eosinophil-derived transforming growth factor beta1.

    Liu Z, Lu X, Wang H, Gao Q and Cui Y

    Department of Otolaryngology-Head and Neck Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. zhengliuent@hotmail.com

    Background: Tissue remodeling is an important characteristic of nasal polyps (NPs). However, the mechanisms underlying the remodeling processes are poorly defined. This study investigated the role of transforming growth factor (TGF) beta1 and eosinophils in the expression of tenascin C (Tn-C), an extracellular matrix glycoprotein, in NPs.

    Methods: The protein expression of Tn-C and TGF-beta1 was examined by means of immunohistochemistry in NPs and normal control inferior turbinate tissues. Furthermore, cell culture, quantitative RT-PCR, and in situ immunocytofluorescence techniques were used to investigate the direct effect of TGF-beta1 and eosinophils on Tn-C production in primary nasal epithelial cells.

    Results: Tn-C protein expression was significantly up-regulated in NP tissues and correlated with TGF-beta1+ eosinophils. TGF-beta1 and eosinophils dramatically induced Tn-C mRNA and protein expression in nasal epithelial cells. The effect of eosinophils could be inhibited partly by a neutralizing antibody to TGF-beta1.

    Conclusion: Eosinophil-derived TGF-beta1 may contribute, at least in part, to the tissue remodeling in NPs.

    American journal of rhinology 2006;20;6;629-33

  • Adenoviral-mediated expression and local deposition of recombinant tenascin-C perturbs cell-dependent matrix contraction.

    Hsia HC and Schwarzbauer JE

    Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA. hsiahc@umdnj.edu

    Background: To mimic the wound environment, we have developed a three-dimensional (3-D) fibrin-fibronectin (FN) matrix model that is formed in vitro from purified proteins and approximates the provisional matrix. Tenascin-C, a large extracellular matrix (ECM) glycoprotein, is expressed transiently in tissue adjacent to areas of injury and contacts the provisional matrix in vivo. We have constructed a novel recombinant adenovirus vector (Ad-70Ten) to up-regulate local expression and secretion of a recombinant form of tenascin-C.

    Methods: Ad-70Ten and a control vector were constructed and used to infect cultured mammalian cells. Post-infection monitoring of expression was accomplished by immunoblot and immunohistochemical techniques. Local protein deposition was examined by immunofluorescence. Cell contractility was assessed by ability of infected cells to contract 3-D fibrin-FN matrices. Some matrices also contained lysophosphatidic acid (LPA), an activator of Rho GTPase.

    Results: Adenovirus-infected cells demonstrated high recombinant tenascin-C expression and deposited protein at sites of cell-matrix contacts resulting in significantly reduced contractility with 2.5-fold lower contraction of the matrix compared with control cells. Matrix contraction could be restored by treatment with LPA.

    Conclusion: These results show that endogenous expression of tenascin-C down-regulates cell contractility and strongly suggest that it exerts its effects via a Rho GTPase signaling pathway. Taken with previous findings, these results suggest that tenascin-C acts in both a paracrine and autocrine manner via Rho GTPase pathways. This report demonstrates that recombinant adenovirus infection is a feasible method to induce high expression of large matrix proteins in mammalian cells, allowing better approximation of in vivo circumstances for investigations of locally secreted matrix protein. While the current vector has been constructed for research purposes, it also represents a proof in principle that adenoviral vectors encoding large proteins may have potential benefit in clinical applications.

    Funded by: NCI NIH HHS: CA44627, R01 CA044627; NIGMS NIH HHS: K08 GM072546

    The Journal of surgical research 2006;136;1;92-7

  • Tenascin expression in actinic keratosis.

    Lentini M, Schepis C, Cuppari DA and Batolo D

    Dipartimento di Patologia Umana, Policlinico Universitario Pad.D, 98124 Messina, Italia. maria.lentini@unime.it

    Background: Tenascin is an extracellular matrix protein frequently expressed around neoplastic and non-neoplastic lesions of the skin. Actinic keratoses (AKs) are intraepidermal neoplastic lesions of the sun-exposed skin. They are classified according to the extension of dysplasia in four stages; they also present different histological varieties.

    Methods: We performed an immunohistochemical study using tenascin monoclonal antibody diluted 1 : 50 on 150 cases of AKs classified, respectively, in histotypes (38 hypertrophic, 18 atrophic, 21 bowenoid, 19 acantolytic, and 40 mixed) and in stages (27 stage I, 46 stage II, 42 stage III, and 35 stage IV; 14 in tumoral progression).

    Results: Tenascin positivity was observed in all cases at the dermal level close to the epithelial lesion. The intensity of reaction increased from stage I to stage IV and, of course, also in tumoral progression. Its expression was not related to the histotypes. In very few cases, the atypical keratinocytes were positive.

    Conclusions: Tenascin expression in AKs is related to the stages of dysplasia. In fact, the immunostaining intensity corresponds to the degree of the dysplasia rather than the thickness of the involved epidermis. Tenascin plays a role in neoplastic progression working as an anti-adhesive factor.

    Journal of cutaneous pathology 2006;33;11;716-20

  • Contact stimulation of fibroblasts for tenascin production by melanoma cells.

    Adám B, Tóth L, Pásti G, Balázs M and Adány R

    Department of Preventive Medicine, School of Public Health, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.

    Tenascin, an extracellular matrix glycoprotein, is widely expressed in the stroma of almost all types of solid tumours including malignant melanomas. On the basis of its antiadhesive character, it has been supposed that tenascin accumulation facilitates tumour cell invasion and consequent metastasis formation. We aimed to investigate the mechanism by which melanoma cells can modulate the production of tenascin by host stromal cells. The expression of tenascin in cocultures of fibroblasts and five melanoma cell lines, as well as in fibroblast monocultures treated with melanoma conditioned media, was analysed by immunofluorescent staining and image analysis. Tenascin production could not be observed in control fibroblasts or in melanoma cell monocultures. Faint labelling for tenascin could be detected in fibroblast monocultures treated with melanoma cell conditioned media while a very intense staining for tenascin could be seen in melanoma cell-fibroblast cocultures. The tenascin staining in the cocultures was associated with the fibroblasts that were in close contact with melanoma cells. The level of tenascin production around the fibroblasts in different areas of the cocultures correlated well with the density of melanoma cells. Our results indicate that tenascin production of fibroblasts in the tumour stroma is directly modulated by melanoma cells mainly through cell-to-cell contact signalling.

    Melanoma research 2006;16;5;385-91

  • Tenascin-C is induced by mutated BMP type II receptors in familial forms of pulmonary arterial hypertension.

    Ihida-Stansbury K, McKean DM, Lane KB, Loyd JE, Wheeler LA, Morrell NW and Jones PL

    University of Pennsylvania, Institute for Medicine & Engineering, Department of Pathology and Laboratory Medicine, Philadelphia, PA 19104-6383, USA.

    Familial forms of human pulmonary arterial hypertension (FPAH) have been linked to mutations in bone morphogenetic protein (BMP) type II receptors (BMPR2s), yet the downstream targets of these receptors remain obscure. Here we show that pulmonary vascular lesions from patients harboring BMPR2 mutations express high levels of tenascin-C (TN-C), an extracellular matrix glycoprotein that promotes pulmonary artery (PA) smooth muscle cell (SMC) proliferation. To begin to define how TN-C is regulated, PA SMCs were cultured from normal subjects and from those with FPAH due to BMPR2 mutations. FPAH SMCs expressed higher levels of TN-C than normal SMCs. Similarly, expression of Prx1, a factor that drives TN-C transcription, was elevated in FPAH vascular lesions and SMCs derived thereof. Furthermore, Prx1 and TN-C promoter activities were significantly higher in FPAH vs. normal SMCs. To delineate how BMPR2s control TN-C, we focused on receptor (R)-Smads, downstream effectors activated by wild-type BMPR2s. Nuclear localization and phosphorylation of R-Smads was greater in normal vs. FPAH SMCs. As well, indirect blockade of R-Smad signaling with a kinase-deficient BMP receptor Ib upregulated TN-C in normal SMCs. Because ERK1/2 MAPKs inhibit the transcriptional activity of R-Smads, and because ERK1/2 promotes TN-C transcription, we determined whether ERK1/2 inhibits R-Smad signaling in FPAH SMCs and whether this activity is required for TN-C transcription. Indeed, ERK1/2 activity was greater in FPAH SMCs, and inhibition of ERK1/2 resulted in nuclear localization of R-Smads and inhibition of TN-C. These studies define a novel signaling network relevant to PAH underscored by BMPR2 mutations.

    Funded by: NHLBI NIH HHS: 1 R01 HL-68798-01, P50 HL-57144-06

    American journal of physiology. Lung cellular and molecular physiology 2006;291;4;L694-702

  • Involvement of tenascin-C and PG-M/versican in flexor tenosynovial pathology of idiopathic carpal tunnel syndrome.

    Tsujii M, Hirata H, Yoshida T, Imanaka-Yoshida K, Morita A and Uchida A

    Department of Orthopaedic Surgery, Faculty of Medicine, Mie University, Tsu, Mie, Japan.

    Increased intra-carpal-tunnel pressure due to swelling of the flexor tenosynovium is the most probable pathological mechanism of idiopathic carpal tunnel syndrome (CTS). To clarify the role of tenascin-C and PG-M/versican, which have often been found to be involved in tissue remodeling and vascular stenosis in the pathogenesis of CTS, we histologically and biochemically examined the production of extracellular matrix in the flexor tenosynovium from 40 idiopathic CTS patients. Tenascin-C was temporarily expressed in the vessel wall, synovial lining and fibrous tissue, with expression regulated differently in each tissue. Tenascin-C expression by vessels correlated with disease duration and appeared to be involved in vascular lesion pathology. Morphometric analysis showed that tenascin-C expression by small arteries is correlated with PG-M/versican expression in surrounding connective tissue. PG-M/versican was also present at the neointima of severely narrowed vessels. Although tenascin-C expression by synovial lining and connective tissue shows marked regional variation and seems inconsistent, in vitro examination suggested that tenascin-C production by these tissues is regulated in response to mechanical strain on the flexor tenosynovium.

    Histology and histopathology 2006;21;5;511-8

  • Tenascin-C protein expression and mRNA splice variants in thyroid carcinoma.

    Tseleni-Balafouta S, Gakiopoulou H, Fanourakis G, Voutsinas G, Balafoutas D and Patsouris E

    Department of Pathology, Medical School, University of Athens, 75 Mikras Asias Str., GR-11527 Athens, Greece. stseleni@cc.uoa.gr

    Tenascin-C (Tn-C) is a matricellular protein involved in the initial and intermediate stages of cell adhesion. The present study is the first undertaken to comparatively investigate Tn-C in neoplastic, non-neoplastic thyroid lesions and normal thyroid tissues. Forty-eight thyroid specimens were studied immunohistochemically using a monoclonal antibody against Tn-C. Immunohistochemistry was supplemented by RT-PCR analysis of the two Tn-C mRNA splice variants in 13 thyroid cancer cell lines. Normal and non-neoplastic tissues were devoid of Tn-C, as well as follicular neoplasms, Huerthle-cell and anaplastic carcinomas. Most papillary carcinomas showed a focally intensive extracellular staining, localized in the connective tissue stroma, whereas most medullary carcinomas showed a staining in the connective tissue but also in intracellular location mainly. RT-PCR analysis detected Tn-C mRNA in all thyroid cancer cell lines with prevalence of the large splice variant in all but the medullary line, characterized by a higher Tn-Csmall:Tn-Clarge ratio. In conclusion, Tn-C re-expression has been observed in papillary and medullary thyroid carcinomas with different staining patterns accompanied by the prevalence of different mRNA splice variants in cell cultures. It seems possible that Tn-C is rather synthesized by tumor cells than by activated stromal cells.

    Experimental and molecular pathology 2006;80;2;177-82

  • Vascular tenascin-C regulates cardiac endothelial phenotype and neovascularization.

    Ballard VL, Sharma A, Duignan I, Holm JM, Chin A, Choi R, Hajjar KA, Wong SC and Edelberg JM

    Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021, USA.

    Microenvironmental cues mediate postnatal neovascularization via modulation of endothelial cell and bone marrow-derived endothelial progenitor cell (EPC) activity. Numerous signals regulate the activity of both of these cell types in response to vascular injury, which suggests that parallel mechanisms regulate angiogenesis in the vascular beds of both the heart and bone marrow. To identify mediators of such shared pathways, in vivo bone marrow/cardiac phage display biopanning was performed and led to the identification of tenascin-C as a candidate protein. Functionally, tenascin-C inhibits cardiac endothelial cell spreading and enhances migration in response to angiogenic growth factors. Analysis of human coronary thrombi revealed tenascin-C protein expression colocalized with the endothelial cell/EPC marker Tie-2 in intrathrombi vascular channels. Immunostains in the rodent heart demonstrated that tenascin-C also colocalizes with EPCs homing to sites of cardiac angiogenic induction. To determine the importance of tenascin-C in cardiac neovascularization, we used an established cardiac transplantation model and showed that unlike wild-type mice, tenascin-C-/- mice fail to vascularize cardiac allografts. This demonstrates for the first time that tenascin-C is essential for postnatal cardiac angiogenic function. Together, our data highlight the role of tenascin-C as a microenvironmental regulator of cardiac endothelial/EPC activity.

    Funded by: NHLBI NIH HHS: HL67839; NIA NIH HHS: AG19738, AG20320, AG20918

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;6;717-9

  • Platelet derived growth factor induced tenascin-C transcription is phosphoinositide 3-kinase/Akt-dependent and mediated by Ets family transcription factors.

    Jinnin M, Ihn H, Asano Y, Yamane K, Trojanowska M and Tamaki K

    Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan.

    Previous studies have identified several cytokines as inducers of tenascin-C (TN-C) expression in various tissue culture systems. However, the signaling pathways of the regulation of TN-C expression are almost unknown. In this study, we clarified the molecular mechanism(s) underlying the regulation of the TN-C gene by platelet derived growth factor (PDGF) in cultured human dermal fibroblasts. PDGF induced the expression of TN-C protein as well as mRNA in a dose-dependent manner. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the PDGF-mediated upregulation of TN-C mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not. The PDGF-mediated induction of TN-C expression was inhibited by the treatment of fibroblasts with a selective phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, or LY294002. These results suggest that PDGF induced the expression of TN-C at a transcriptional level via phosphoinositide3-kinase/Akt signaling pathways. We performed serial 5' deletions and a transient transfection analysis to define the region in the TN-C promoter mediating the responsiveness to PDGF. Overexpression of Sp1, Ets1, or Ets2 activated the TN-C promoter and superinduced TN-C promoter activity stimulated by PDGF, whereas overexpression of Fli1 inhibited the effects of PDGF on TN-C expression. Mutation of the Sp1/3 binding sites or Ets binding sites in the TN-C promoter region responsible to PDGF abrogated the PDGF-inducible promoter activity. Immunoprecipitation analysis revealed that Sp1, Ets1, and Ets2 form a transcriptionally active complex. On the other hand, the interaction of Fli1 with Sp1 decreased after PDGF treatment. These results suggest that the upregulation of TN-C expression by PDGF involves Ets family transcription factors, co-operating with Sp1.

    Journal of cellular physiology 2006;206;3;718-27

  • Tenascin-C protein is induced by transforming growth factor-beta1 but does not correlate with time to tumor progression in high-grade gliomas.

    Hau P, Kunz-Schughart LA, Rümmele P, Arslan F, Dörfelt A, Koch H, Lohmeier A, Hirschmann B, Müller A, Bogdahn U and Bosserhoff AK

    Department of Neurology, University of Regensburg, Universitätsstrasse 84, 93053, Regensburg, Germany. peter.hau@medbo.de

    Background: Tenascin-C is an extracellular matrix protein known to correlate with prognosis in patients with glioblastoma, probably by stimulation of invasion and neoangiogenesis. Transforming Growth Factor-beta1 (TGF-beta1) plays an important role in the biology of high-grade gliomas, partly by regulating invasion of these tumors into parenchyma. This study was designed to evaluate if TGF-beta1 induces the expression and deposition of Tenascin-C in the extracellular matrix of high-grade gliomas which may be pivotal for the invasion of these tumors into healthy parenchyma.

    Methods: A series of 20 high-grade gliomas was stained immunohistochemically with Tenascin-C- and TGF-beta1- specific antibodies. Expression levels of both proteins were evaluated and correlated with each other, time to progression and molecular and morphological markers of invasion. A quantitative PCR assay was performed evaluating the induction of Tenascin-C mRNA by treatment with TGF-beta1 in vitro.

    Results: Tenascin-C was expressed in 18 of 19 (95%) evaluable tumors, whereas 14 of 20 tumors (70%) expressed TGF-beta1 in a significant percentage of cells. Treatment with TGF-beta1 did induce the expression of Tenascin-C at the mRNA and protein level in vitro. The expression of Tenascin-C and TGF-beta1 did neighter statistically correlate with each other nor with time to progression.

    Conclusion: In our series, Tenascin-C and TGF-beta1 were expressed in the vast majority of high-grade gliomas. We could not detect a correlation of one of the proteins with time to progression. Nevertheless, we describe induction of Tenascin-C by TGF-beta1, possibly providing a mechanism for the invasion of high-grade gliomas into healthy parenchyma.

    Journal of neuro-oncology 2006;77;1;1-7

  • beta-Catenin regulates the expression of tenascin-C in human colorectal tumors.

    Beiter K, Hiendlmeyer E, Brabletz T, Hlubek F, Haynl A, Knoll C, Kirchner T and Jung A

    Pathologisch-Anatomisches Institut, Friedrich-Alexander-Universität Erlangen-Nürnberg, Krankenhausstr. 8-10, D-91054 Erlangen, Germany.

    Tenascin-C (TN-C) is a component of the extracellular matrix (ECM). It is expressed during development and re-expressed in many types of cancers, where it is involved in the modulation of adhesion and proliferation. TN-C expression is especially high at sites of epithelial mesenchymal transition (EMT), which are found frequently at the invasion front of well-differentiated human colorectal adenocarcinomas. Tumor cells in this compartment are characterized by a strong nuclear expression of the oncogenic transcription factor beta-catenin. Here, we demonstrate that TN-C is a beta-catenin target gene in human colorectal tumors. Thus, by far the most common mutations in colorectal tumors, found in the Wnt-signaling pathway and leading to the stabilizing of beta-catenin, might influence invasion by altering adhesive properties and EMT of tumor cells.

    Oncogene 2005;24;55;8200-4

  • Gene expression changes during the development of acute lung injury: role of transforming growth factor beta.

    Wesselkamper SC, Case LM, Henning LN, Borchers MT, Tichelaar JW, Mason JM, Dragin N, Medvedovic M, Sartor MA, Tomlinson CR and Leikauf GD

    Department of Environmental Health, P.O. Box 670056, University of Cincinnati, Cincinnati, OH 45267-0056, USA. george.leikauf@uc.edu

    Rationale: Acute lung injury can occur from multiple causes, resulting in high mortality. The pathophysiology of nickel-induced acute lung injury in mice is remarkably complex, and the molecular mechanisms are uncertain.

    Objectives: To integrate molecular pathways and investigate the role of transforming growth factor beta (TGF-beta) in acute lung injury in mice.

    Methods: cDNA microarray analyses were used to identify lung gene expression changes after nickel exposure. MAPPFinder analysis of the microarray data was used to determine significantly altered molecular pathways. TGF-beta1 protein in bronchoalveolar lavage fluid, as well as the effect of inhibition of TGF-beta, was assessed in nickel-exposed mice. The effect of TGF-beta on surfactant-associated protein B (Sftpb) promoter activity was measured in mouse lung epithelial cells.

    Genes that decreased the most after nickel exposure play important roles in lung fluid absorption or surfactant and phospholipid synthesis, and genes that increased the most were involved in TGF-beta signaling. MAPPFinder analysis further established TGF-beta signaling to be significantly altered. TGF-beta-inducible genes involved in the regulation of extracellular matrix function and fibrinolysis were significantly increased after nickel exposure, and TGF-beta1 protein was also increased in the lavage fluid. Pharmacologic inhibition of TGF-beta attenuated nickel-induced protein in bronchoalveolar lavage. In addition, treatment with TGF-beta1 dose-dependently repressed Sftpb promoter activity in vitro, and a novel TGF-beta-responsive region in the Sftpb promoter was identified.

    Conclusions: These data suggest that TGF-beta acts as a central mediator of acute lung injury through the alteration of several different molecular pathways.

    Funded by: NHLBI NIH HHS: HL65612; NIEHS NIH HHS: ES06096, ES07250, ES10562, U01 ES015675

    American journal of respiratory and critical care medicine 2005;172;11;1399-411

  • Tenascin-C promotes cell survival by activation of Akt in human chondrosarcoma cell.

    Jang JH and Chung CP

    Department of Biochemistry, Inha University College of Medicine, Jung-gu, Incheon 400-712, South Korea. juhjang@inha.ac.kr

    Tenascin-C (TnC) is an extracellular matrix protein that is highly expressed in tumor stroma. In this report, we examined the roles of TnC-mediated cell adhesion in the modulation of chondrosarcoma cell survival. We found that hTnC-mediated adhesion could confer a significant (P<0.05) survival advantage to human chondrosarcoma cell line, JJ012, following serum-deprivation compared with the same cells grown on poly-lysine. This pro-survival signal was due to the activation of the Akt upon adhesion to hTnC. Moreover, hTnC-induced Akt activation was blocked by LY294002 and the expression of dominant-negative Akt. Taken together, these studies support that the TnC-mediated adhesion can promote cell survival through Akt in human chondrosarcoma cells.

    Cancer letters 2005;229;1;101-5

  • Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.

    Liu T, Qian WJ, Gritsenko MA, Camp DG, Monroe ME, Moore RJ and Smith RD

    Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, USA.

    The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

    Funded by: NCRR NIH HHS: P41 RR018522, RR18522; NIGMS NIH HHS: U54 GM-62119-02, U54 GM062119

    Journal of proteome research 2005;4;6;2070-80

  • Coding SNP in tenascin-C Fn-III-D domain associates with adult asthma.

    Matsuda A, Hirota T, Akahoshi M, Shimizu M, Tamari M, Miyatake A, Takahashi A, Nakashima K, Takahashi N, Obara K, Yuyama N, Doi S, Kamogawa Y, Enomoto T, Ohshima K, Tsunoda T, Miyatake S, Fujita K, Kusakabe M, Izuhara K, Nakamura Y, Hopkin J and Shirakawa T

    Laboratory for Genetics of Allergic Diseases, SNP Research Center, RIKEN, Yokohama, Japan. akimatsu@src.riken.go.jp

    The extracellular matrix glycoprotein tenascin-C (TNC) has been accepted as a valuable histopathological subepithelial marker for evaluating the severity of asthmatic disease and the therapeutic response to drugs. We found an association between an adult asthma and an SNP encoding TNC fibronectin type III-D (Fn-III-D) domain in a case-control study between a Japanese population including 446 adult asthmatic patients and 658 normal healthy controls. The SNP (44513A/T in exon 17) strongly associates with adult bronchial asthma (chi2 test, P=0.00019, Odds ratio=1.76, 95% confidence interval=1.31-2.36). This coding SNP induces an amino acid substitution (Leu1677Ile) within the Fn-III-D domain of the alternative splicing region. Computer-assisted protein structure modeling suggests that the substituted amino acid locates at the outer edge of the beta-sheet in Fn-III-D domain and causes instability of this beta-sheet. As the TNC fibronectin-III domain has molecular elasticity, the structural change may affect the integrity and stiffness of asthmatic airways. In addition, TNC expression in lung fibroblasts increases with Th2 immune cytokine stimulation. Thus, Leu1677Ile may be valuable marker for evaluating the risk for developing asthma and plays a role in its pathogenesis.

    Human molecular genetics 2005;14;19;2779-86

  • Tenascin C induces a quiescent phenotype in cultured adult human astrocytes.

    Holley JE, Gveric D, Whatmore JL and Gutowski NJ

    Institute of Biomedical and Clinical Sciences, Peninsula Medical School (Exeter), Department of Neurology, Royal Devon and Exeter Hospital, Exeter, United Kingdom. janet.holley@pms.ac.uk

    Astrocytic scar formation occurs subsequent to brain and spinal cord injury and impedes repair. The exact mechanisms of scar formation have yet to be elucidated but it is known that astrocytes within the scar have a different antigenic phenotype from normal or reactive astrocytes. Astrocyte cell culture offers a suitable system to identify factors that induce the scar phenotype as well as factors that reverse this process and that may help identify therapeutic strategies to treat astrogliosis. However, when placed in standard culture conditions, astrocytes become activated/reactive and express molecules characteristic of scar tissue in vivo. In the present study, we made use of this phenomenon to identify culture conditions that change the activated phenotype of cultured astrocytes into one characteristic of normal quiescent astrocytes. In particular, we examined the effect of extracellular matrix (ECM) proteins found in the human brain, on the phenotype of human adult astrocytes. Significantly fewer astrocytes expressed scar properties when grown on tenascin-C (TN-C) than those cultured on other ECM proteins or poly-L-lysine-coated dishes. TN-C also significantly reduced the proliferation rate of the astrocytes in vitro. In addition, further manipulation of culture conditions induced partial astrocyte reactivation. Our findings suggest that astrocytes grown on TN-C revert to a quiescent, nonactivated state that is partially reversible. This raises the possibility that therapeutic strategies aimed at manipulating TN-C levels during CNS injury may help reduce astrocytic scarring.

    Funded by: Multiple Sclerosis Society: 651

    Glia 2005;52;1;53-8

  • The guanine-thymine dinucleotide repeat polymorphism within the tenascin-C gene is associated with achilles tendon injuries.

    Mokone GG, Gajjar M, September AV, Schwellnus MP, Greenberg J, Noakes TD and Collins M

    UCT/MRC Research Unit for Exercise Science and Sports Medicine, University of Cape Town, PO Box 115, Newlands 7725, South Africa.

    Background: Although there is a high incidence of tendon injury as a result of participation in physical activity, the mechanisms responsible for such injuries are poorly understood. Investigators have suggested that some people may have a genetic predisposition to develop tendon injuries; in particular, genes on the tip of the long arm of chromosome 9 might, at least in part, be associated with this condition. The tenascin-C gene, which has been mapped to chromosome 9q32-q34, encodes for a structural component of tendons.

    Hypothesis: The tenascin-C gene is associated with Achilles tendon injury.

    Case control study; Level of evidence, 3.

    Methods: A total of 114 physically active white subjects with symptoms of Achilles tendon injury and 127 asymptomatic, physically active white control subjects were genotyped for the guanine-thymine dinucleotide repeat polymorphism within the tenascin-C gene.

    Results: A significant difference in the allele frequencies of this polymorphism existed between the 2 groups of subjects (chi(2) = 51.0, P = .001). The frequencies of the alleles containing 12 repeats (symptomatic group, 18.9% vs control group, 10.2%) and 14 repeats (symptomatic group, 9.2% vs control group, 0.8%) were significantly higher in the symptomatic group, while the frequencies of the alleles containing 13 repeats (symptomatic group, 8.8% vs control group, 24.0%) and 17 repeats (symptomatic group, 7.5% vs control group, 20.1%) were significantly lower in this same group. Subjects who were homozygous or heterozygous for the underrepresented alleles (13 and 17 repeats) but who did not possess an overrepresented allele (12 and 14 repeats) may have a lower risk of developing Achilles tendon injuries (odds ratio, 6.2; 95% confidence interval, 3.5-11.0; P < .001).

    Conclusions: The guanine-thymine dinucleotide repeat polymorphism within the tenascin-C gene is associated with Achilles tendon injury. Alleles containing 12 and 14 guanine-thymine repeats were overrepresented in subjects with tendon injuries, while the alleles containing 13 and 17 repeats were underrepresented.

    Persons who have variants of the tenascin-C gene with 12 and 14 guanine-thymine repeats appear to have a 6-fold risk of developing Achilles tendon injuries.

    The American journal of sports medicine 2005;33;7;1016-21

  • Transplantation of reconstructed human skin on nude mice: a model system to study expression of human tenascin-X and elastic fiber components.

    Zweers MC, Schalkwijk J, van Kuppevelt TH, van Vlijmen-Willems IM, Bergers M, Lethias C and Lamme EN

    Department of Dermatology, University Medical Centre Nijmegen, Nijmegen, The Netherlands. m.zweers@derma.umcn.nl

    Tenascin-X is a large extracellular matrix protein that is widely expressed in connective tissues during development and in the adult. Genetically determined deficiency of tenascin-X causes the connective tissue disease Ehlers-Danlos syndrome. These patients show reduced collagen density and fragmentation of elastic fibers in their skin. In vitro studies on the role of tenascin-X in elastic fiber biology are hampered because monolayers of fibroblasts do not deposit tenascin-X and elastic fibers into the extracellular matrix. Here, we applied an organotypic culture model of fibroblasts and keratinocytes to address this issue. We investigated the deposition of tenascin-X and elastin into skin-equivalent in vitro and also in vivo after transplantation onto immunodeficient mice. Whereas tenascin-C and fibrillin-1 were readily expressed in the skin-equivalents before transplantation, tenascin-X and elastin were not present. Three weeks post-grafting, a network of elastin was observed that coincided with the appearance of tenascin-X. At the ultrastructural level, microfibrils were observed, some of which were associated with elastin. Transplanted skin-equivalents containing tenascin-X-deficient fibroblasts showed deposition of immunoreactive elastin in similar quantities and distribution as those containing control fibroblasts. This suggests that tenascin-X is important for the stability and maintenance of established elastin fibers, rather than for the initial phase of elastogenesis. Thus, the transplantation of reconstructed skin on nude mice allows the study of tenascin-X and elastin expression and could be used as a model system to study the potential role of tenascin-X in matrix assembly and stability.

    Cell and tissue research 2005;319;2;279-87

  • [The expression of tenascin-C mRNA in keloids and hypertrophic scars].

    Han CM, He XJ and Ma Q

    Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China.

    Objective: To investigate the expression of Tenascin-C mRNA in keloids and hypertrophic Total RNA was isolated from normal adult skin. A cDNA fragment (base 5941-6481bp) of the scars.

    Methods: full-length human Tenascin-C cDNA was synthesized by polymerase chain reaction and subcloned in pGEM-T-easy. Dioxygen-labeled anti-sense and sense probes were synthesized by using a Sp6/T7 RNA synthesis kit in the present of Dig-UTP in vitro. The samples were taken from keloids in 10, hypertrophic scars in 10 and normal adult skin in 5. The hybridization was performed with 4% paraformaldehyde-fixed and wax-embedded sections to detect the Tenascin-C mRNA.

    Results: The Tenascin-C mRNA was negative in the normal adult epidermis and weakly located in the fibroblasts of the papillary dermis and the epidermal adnexa. In all of the 10 keloid specimens, the Tenascin-C mRNA was positive throughout the epidermis and widely distributed in the dermis included in the fibroblasts, endothelial cells and epidermal adnexa. In the specimens of the 3 hypertrophic scars,the Tenascin-C mRNA was also positive in the epidermis, but in the other 7 cases, it became negative. In the dermis of the hypertrophic scar,the Tenascin-C mRNA was weaker than that in the keloid, but stronger than that in the normal skin.

    Conclusions: The expression of Tenascin-C mRNA is markedly enhanced in the keloids.

    Zhonghua zheng xing wai ke za zhi = Zhonghua zhengxing waike zazhi = Chinese journal of plastic surgery 2005;21;1;40-3

  • Serum tenascin-C as a potential predictive marker of angiogenesis in non-small cell lung cancer.

    Ishiwata T, Takahashi K, Shimanuki Y, Ohashi R, Cui R, Takahashi F, Shimizu K, Miura K and Fukuchi Y

    Department of Respiratory Medicine, Juntendo University, School of Medicine, Bunkyo-Ku, Tokyo 113-8421, Japan.

    Background: Tenascin (Tn)-C is an extracellular matrix protein that is involved in tissue interactions during fetal development and oncogenesis. However, the role of serum Tn-C in non-small cell lung cancer (NSCLC) has not been clarified.

    In this study, we determined the serum levels of Tn-C among NSCLC patients who underwent surgery, as well as other factors implicated for angiogenesis, to address the clinical implications in NSCLC.

    The median concentration of serum Tn-C in NSCLC patients was slightly higher than that of normal controls, but this difference was not statistically significant. There was a positive correlation between serum Tn-C levels and microvessel density (MVD), serum osteopontin (OPN) and vascular endothelial growth factor (VEGF). In contrast, there was no correlation between serum Tn-C levels and serum carcinoembryonic antigen (CEA) and sialyl lewis-X (SLX) levels. The overall survival of patients with low Tn-C levels (<96 ng/ml) was significantly greater than that of patients with high Tn-C levels (> or =96 ng/ml). Intratumoral Tn-C expression was co-localized with expression of microvessels in the stroma of the cancer cells by immunohistochemical analysis. Moreover, enhanced in vitro migration of human umbilical vascular endothelial cells (HUVEC) was induced by recombinant Tn-C. Collectively, Tn-C may play an important role in angiogenesis of patients with NSCLC, and the determination of serum Tn-C may be useful in predicting intratumoral vasculature and patients' prognosis.

    Anticancer research 2005;25;1B;489-95

  • Distribution pattern of tenascin-C in glioblastoma: correlation with angiogenesis and tumor cell proliferation.

    Behrem S, Zarković K, Eskinja N and Jonjić N

    Department of Pathology, Medical Faculty, University of Rijeka, Rijeka, Croatia.

    Tenascin-C (TN-C) is an extracellular matrix protein which participates in different processes like normal fetal development, wound healing, inflammation, keloids and rheumatoid arthritis. Furthermore, the immunostaining for TN-C is seen in the stroma of various malignant tumors as in glioblastoma multiforme (GBM), however, the significance of these findings is still not clear. In this study 62 GBM samples were analyzed immunohistochemically for distribution patterns of TN-C and correlated with angiogenesis and tumor cell proliferation. Tenascin-C in GBM localizes in two compartments, perivascular and intercellular space. Intercellular tenascin-C (TN-C ic) showed focal distribution in 66%, and diffuse one in 34% of cases. Perivascular tenascin-C (TN-C pv) showed strong correlation with microvascular density (MVD) and vascular endothelial growth factor (VEGF) expression. Moreover, it seems that TN-C pv enhanced the effect of VEGF. Intercellular TN-C did not correlate with MVD and VEGF expression, but showed strong correlation with proliferation index. Furthermore, tumors with diffuse TN-C ic expression had higher proliferation indices than tumors with focal TN-C expression. Our results indicate that TN-C plays a role in angiogenesis and tumor cell proliferation, but beside the intensity of expression, the distribution patterns are also important in these processes. This study also suggests that perivascular and intercellular TN-C compartments have probably different sources and different roles in GBM.

    Pathology oncology research : POR 2005;11;4;229-35

  • Adhesion-mediated signal transduction in human articular chondrocytes: the influence of biomaterial chemistry and tenascin-C.

    Mahmood TA, de Jong R, Riesle J, Langer R and van Blitterswijk CA

    IsoTis SA, Prof. Bronkhorstlaan 10, 3723 MB Bilthoven, The Netherlands. tmahmood@amgen.com

    Chondrocyte 'dedifferentiation' involves the switching of the cell phenotype to one that no longer secretes extracellular matrix found in normal cartilage and occurs frequently during chondrocyte expansion in culture. It is also characterized by the differential expression of receptors and intracellular proteins that are involved in signal transduction pathways, including those associated with cell shape and actin microfilament organization. The objective of this study was to examine the modulation of chondrocyte phenotype by cultivation on polymer substrates containing poly(ethylene glycol) (PEG). We observed differential arrangement of actin organization in articular chondrocytes, depending on PEG length. When cultivated on 300 g/mol PEG substrates at day 19, chondrocytes had lost intracellular markers characteristic of the differentiated phenotype, including type II collagen and protein kinase C (PKC). On these surfaces, chondrocytes also expressed focal adhesion and signaling proteins indicative of cell attachment, spreading, and FA turnover, including RhoA, focal adhesion kinase, and vinculin. The switch to a dedifferentiated chondrocyte phenotype correlated with integrin expression. Conversely, the expression of CD44 receptors coincided with chondrogenic characteristics, suggesting that binding via these receptors could play a role in maintaining the differentiated phenotype on such substrates. These effects can be similar to those of compounds that interfere in intracellular signaling pathways and can be utilized to engineer cellular response.

    Experimental cell research 2004;301;2;179-88

  • Folding of epidermal growth factor-like repeats from human tenascin studied through a sequence frame-shift approach.

    Zanuttin F, Guarnaccia C, Pintar A and Pongor S

    International Centre for Genetic Engineering and Biotechnology, Protein Structure and Bioinformatics Group, Trieste, Italy.

    In order to investigate the factors that determine the correct folding of epidermal growth factor-like (EGF) repeats within a multidomain protein, we prepared a series of six peptides that, taken together, span the sequence of two EGF repeats of human tenascin, a large protein from the extracellular matrix. The peptides were selected by sliding a window of the average length of tenascin EGF repeats over the sequence of EGF repeats 13 and 14. We thus obtained six peptides, EGF-f1 to EGF-f6, that are 33 residues long, contain six cysteines each, and bear a partial overlap in the sequence. While EGF-f1 corresponds to the native EGF-14 repeat, the others are frame-shifted EGF repeats. We carried out the oxidative folding of these peptides in vitro, analyzed the reaction mixtures by acid trapping followed by LC-MS, and isolated some of the resulting products. The oxidative folding of the native EGF-14 peptide is fast, produces a single three-disulfide species with an EGF-like disulfide topology and a marked difference in the RP-HPLC retention time compared with the starting product. On the contrary, frame-shifted peptides fold more slowly and give mixtures of three-disulfide species displaying RP-HPLC retention times that are closer to those of the reduced peptides. In contrast to the native EGF-14, the three-disulfide products that could be isolated are mainly unstructured, as determined by CD and NMR spectroscopy. We conclude that both kinetics and thermodynamics drive the correct pairing of cysteines, and speculate about how cysteine mispairing could trigger disulfide reshuffling in vivo.

    European journal of biochemistry 2004;271;21;4229-40

  • Tenascin-C in primary malignant melanoma of the skin.

    Ilmonen S, Jahkola T, Turunen JP, Muhonen T and Asko-Seljavaara S

    Department of Plastic Surgery, Helsinki University Hospital, Helsinki, Finland. suvi.ilmonen@hus.fi

    Aims: To investigate the expression and the prognostic role of glycoprotein Tenascin-C (Tn-C) in primary melanoma of the skin.

    The immunohistochemical expression of Tn-C was studied in 98 primary melanomas and related to inflammation, invasion, and patient outcome. Patients were followed up for disease recurrence for 0.04-7.4 years (median 3.9) and for survival for 0.5 to 12.1 years (median 9.3). The expression of Tn-C was evaluated for each tumour invasion border; the stromal and intracytoplasmic Tn-C of the melanoma islets were also recorded. Tn-C is widely expressed in primary melanoma samples, the staining pattern varying from focal to diffuse in different parts of the tumour. No correlation existed between intensity of Tn-C staining and inflammation. No stromal Tn-C was detected at the upper dermal lateral border in 12 patients, nor at the deep, dermal or subcutaneous border in 14 patients. These patients showed better disease-free survival (DFS) than did those cases with focal or diffuse staining (P = 0.06, P = 0.05). Also, absence of intracytoplasmic Tn-C was a beneficial prognostic factor for DFS (P = 0.04). In multivariate analysis, tumour ulceration and intracytoplasmic Tn-C expression of melanoma cells were independent adverse prognostic factors for DFS.

    Conclusions: In primary melanoma of the skin, absence of Tn-C in the stroma of invasion fronts and within tumour cells seems to be related to a more benign disease behaviour with a lower risk of developing metastases.

    Histopathology 2004;45;4;405-11

  • Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.

    Suzuki Y, Yamashita R, Shirota M, Sakakibara Y, Chiba J, Mizushima-Sugano J, Nakai K and Sugano S

    Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan. ysuzuki@ims.u-tokyo.ac.jp

    Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a "block" structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5' ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

    Genome research 2004;14;9;1711-8

  • Distribution of tenascin-C and -X, and soft X-ray analysis of the mandibular symphysis during mandible formation in the human fetus.

    Kurihara K and Sato I

    Department of Anatomy, School of Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan.

    In the development of the human mandible, the process of bone calcification, distribution and expression of tenascin-C and -X in the mental symphyseal region are unknown. The purpose of this study was to determine the distribution of these extracellular matrices in the connective tissue around calcified tissues located on the mental symphyseal region of the human fetus during development through histological and radiographical studies. The radiographic density increased from 16 weeks to 24 weeks gestation in all examined regions; in contrast, the diameter of muscle fiber in the suprahyoid muscles (digastric anterior and geniohyoid muscles) inserted into the inner mental symphyseal region increased from 24 weeks gestation. The extracellular matrices (tenascin) were shown to have a different distribution in the mental symphyseal region of the human fetus at each stage. These different distributions of tenascin-C and -X were found around the epithelium and the endomysium of the mental symphyseal region, and affect the specific formation of the mandible during ossification with hyoid muscle development in human fetus.

    Okajimas folia anatomica Japonica 2004;81;2-3;49-55

  • EGF-Like domain of tenascin-C is proapoptotic for cultured smooth muscle cells.

    Wallner K, Li C, Shah PK, Wu KJ, Schwartz SM and Sharifi BG

    Atherosclerosis Research Center, Division of Cardiology, and Burns and Allen Research Institute, Cedars-Sinai Medical Center, and UCLA School of Medicine, Los Angeles, Calif 90048, USA.

    Objective: Based on our previous observations on the expression of Tenascin-C (Tn-C) in human atherosclerotic plaques and its colocalization with macrophages, we explored whether Tn-C undergoes fragmentation and the potential pathobiological significance of this fragmentation.

    Using cultured human smooth muscle cells (SMCs), we found that Tn-C upregulates expression of matrix metalloproteinases (MMPs). Western blot analysis revealed that Tn-C substrate is fragmented and most of the cleavage products have fibronectin-like and epidermal growth factor-like (EGF-like) domains of Tn-C. One fragment that contains an EGF-like domain was found in some human atherosclerotic plaques. Cell culture studies revealed that the recombinant EGF-like domain inhibits growth, induces apoptosis of SMCs in a dose-dependent, time-dependent, and caspase-dependent manner, and activates caspase-3 before SMC detachment. Conversely, the caspase inhibitor z-YVAD.cmk, serum, and protease inhibitors blocked cell apoptosis conferred by the EGF-like domain. In addition, these inhibitors blocked EGF-like domain-induced caspase-3 activation. In contrast to this EGF-like domain, intact Tn-C, its fibronectin-like, and its fibrinogen-like domains were inactive.

    Conclusions: Together with our previous observations, our data suggest that Tn-C upregulates MMP expression that cleaves Tn-C into fragments containing the EGF-like domain. This domain has proapoptotic activity for SMCs.

    Funded by: NHLBI NIH HHS: HL50566, R01 HL090653, R01 HL104068

    Arteriosclerosis, thrombosis, and vascular biology 2004;24;8;1416-21

  • Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac.

    De Wever O, Nguyen QD, Van Hoorde L, Bracke M, Bruyneel E, Gespach C and Mareel M

    Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, Gent, Belgium.

    Myofibroblasts are present at the invasion front in colon cancer. In an attempt to understand their putative proinvasive activity, we have developed an in vitro model. Myofibroblasts isolated from colon cancer tissue or obtained through transdifferentiation of colon fibroblasts by transforming growth factor (TGF)-beta stimulate invasion of colon cancer cells into collagen type I and Matrigel. We identified two convergent proinvasive agents secreted by myofibroblasts: namely scatter factor/hepatocyte growth factor (SF/HGF) and the TGF-beta-upregulated extracellular matrix glycoprotein tenascin-C (TNC), each of which is necessary though not sufficient for invasion. Myofibroblast-stimulated invasion into collagen type I is characterized by a change from a round, nonmigratory morphotype with high RhoA and low Rac activity to an elongated, migratory morphotype with low RhoA and high Rac activity. RhoA inactivation is determined by the epidermal growth factor (EGF)-like repeats of TNC through EGF-receptor signaling that confers a permissive and priming signal for the proinvasive activity of SF/HGF that activates Rac via c-Met. We confirmed the validity of this mechanism by using pharmacological modulators and dominant negative or constitutive active mutants that interfere with RhoA-Rho kinase and Rac signaling. Our in vitro results point to a new putative proinvasive signal for colon cancer cells provided by myofibroblasts in the tumor stroma.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2004;18;9;1016-8

  • Expression of extracellular matrix components versican, chondroitin sulfate, tenascin, and hyaluronan, and their association with disease outcome in node-negative breast cancer.

    Suwiwat S, Ricciardelli C, Tammi R, Tammi M, Auvinen P, Kosma VM, LeBaron RG, Raymond WA, Tilley WD and Horsfall DJ

    Dame Roma Mitchell Cancer Research Laboratory, Hanson Institute, Adelaide University, Adelaide, South Australia.

    Purpose: The purpose is to determine whether the levels of expression of extracellular matrix components in peritumoral stroma are predictive of disease outcome for women with node-negative breast cancer.

    Tumor tissue from 86 patients with node-negative breast cancer was examined by immunohistochemical staining for the expression of versican, chondroitin sulfate (CS), tenascin, and hyaluronan (HA). With the exception of HA, the expression of the extracellular matrix components was measured by video image analysis. Statistical correlation of the immunohistochemical data with clinicopathological characteristics and disease outcome was performed.

    Results: All of the extracellular matrix components were present in the peritumoral stroma of the entire study cohort. In contrast, immunoreactivity within the cancer cell was observed in 82% of tumors for HA, 12% for CS, and 4% for tenascin; no immunostaining of cancer cells for versican was observed for any of the tumors. Cox regression and Kaplan-Meier analyses indicated that elevated expression of stromal versican predicted increased risk and rate of relapse in this cohort. Elevated expression of tenascin was predictive of increased risk and rate of death only. Although neither CS nor HA were predictive of disease outcome in this cohort, tumor size was predictive of increased risk and rate of both relapse and survival.

    Conclusions: Elevated expression within peritumoral stromal matrix of versican and tenascin was predictive of relapse-free and overall survival, respectively, in women with node-negative breast cancer.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2004;10;7;2491-8

  • Interaction between cell and extracellular matrix in heart disease: multiple roles of tenascin-C in tissue remodeling.

    Imanaka-Yoshida K, Hiroe M and Yoshida T

    Department of Pathology, Mie University School of Medicine, Tsu, Japan. imanaka@doc.medic.mie.u.ac.jp

    The heart remodels myocardial tissue in physiological and pathological response. The cell-extracellular matrix (ECM) interaction provides not only structural and mechanical support but also important biological signaling during tissue remodeling. Among various ECM molecules, tenascin-C (TNC) is well known as a regulator of multiple cellular functions during embryogenesis, wound healing or cancer progression. In the heart, TNC appears in several important steps of embryonic development such as the initial differentiation of cardiomyocytes or coronary vasculo/angiogenesis, but it is not detected in a normal adult myocardium. However, TNC is found to re-express after myocardial injury and may regulate cellular behavior during tissue remodeling by modulating the attachment of cardiomyocytes to connective tissue, by enhancing migration and differentiation of myofibroblasts, and by inducing matrix metallo-proteinases. TNC also interacts with other ECM molecules and may modulate progression of fibrosis. Furthermore, transient and site specific expression of TNC closely associated with myocardial injury and inflammation suggests not only its key roles during tissue remodeling but also that TNC can be a marker for myocardial disease activity.

    Histology and histopathology 2004;19;2;517-25

  • Tenascin-C upregulation by transforming growth factor-beta in human dermal fibroblasts involves Smad3, Sp1, and Ets1.

    Jinnin M, Ihn H, Asano Y, Yamane K, Trojanowska M and Tamaki K

    Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

    In cultured human dermal fibroblasts, transforming growth factor (TGF)-beta induced the mRNA expression of tenascin-C (TN-C). The molecular mechanism(s) underlying this process is not presently understood. In this study, we performed serial 5' deletion and a transient transfection analysis to define a region in the TN-C promoter mediating the inducible responsiveness to TGF-beta. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. A DNA affinity precipitation assay revealed that Smad2/Smad3 bound to this site in a transient and specific manner. Overexpression of Smad3 or Smad4 activated the TN-C promoter activity and superinduced the TN-C promoter activity stimulated by TGF-beta. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the TN-C promoter activity in a synergistic manner. Mutation of the Smad-binding sites, the Ets-binding sites, or Sp1/3-binding sites in the TN-C promoter abrogated the TGF-beta/Smad-inducible promoter activity. Immunoprecipitation analysis revealed that Smad3, Sp1, and Ets1 form a transcriptionally active complex. Furthermore, the interaction between Smads and CBP/p300 in TGF-beta signaling was confirmed. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the TN-C promoter mediating responsiveness to TGF-beta involving Smad3/4, Sp1, Ets1, and CBP/p300.

    Oncogene 2004;23;9;1656-67

  • Tenascin-C regulates angiogenesis in tumor through the regulation of vascular endothelial growth factor expression.

    Tanaka K, Hiraiwa N, Hashimoto H, Yamazaki Y and Kusakabe M

    Department of Surgery, Jikei University School of Medicine, Tokyo, Japan.

    In order to verify whether tenascin-C (TN-C) is involved in angiogenesis as an extracellular signal molecule during tumorigenesis, cancerous cell transplantation experiments and coculture experiments were carried out, focusing on the regulation of vascular endothelial growth factor (VEGF). The A375 human melanoma cells introduced the GFP gene (A375-GFP), implanted subcutaneously into BALB/cA nude (WT) and TN-C knockout BALB/cA nude (TNKO) congenic mice. Furthermore, coculture experiments between A375-GFP and embryonic mesenchyme, which was prepared from both genotypes, were carried out to investigate the molecular mechanism in the cell-cell interactions. Both the content of TN-C and that of VEGF in the tumor and the conditioned medium were analyzed by the sandwich ELISA method. Seven days after transplantation of the A375-GFP, capillary nets became far more abundant in the tumors grown in WT mice than those in TNKO mice. Interestingly, VEGF and TN-C expressions showed antithetical expression patterns between the tumors in WT mice and those in TNKO mice. This peculiar phenomenon seems to be caused by a time lag prior to the onset of the mesenchymal regulation for the TN-C expression of A375-GFP. The coculture experiments revealed that WT mesenchyme had a much stronger effect than TNKO mesenchyme on both TN-C and VEGF expression. However, the defects of TNKO mesenchyme were restored in all cases by additional TN-C. These results clearly indicated that the expressions of both TN-C and VEGF depend on the surrounding mesenchyme, and that the function of mesenchyme is regulated by its own mesenchymal TN-C. In conclusion, the present data suggest that the matrix microenvironment organized by the host mesenchyme is very important for angiogenesis in tumor development.

    International journal of cancer 2004;108;1;31-40

  • Prognostic significance of matrix metalloproteinase-2, cathepsin D, and tenascin-C expression in colorectal carcinoma.

    Sis B, Sağol O, Küpelioğlu A, Sokmen S, Terzi C, Fuzun M, Ozer E and Bishop P

    Department of Pathology, School of Medicine, Dokuz Eylül University, Inciralti, Izmir 35340, Turkey. banu.sis@deu.edu.tr

    Matrix metalloproteinase-2 (MMP-2) and cathepsin D (CD) play a significant role in degrading the components of basement membrane and extracellular matrix (ECM), whereas tenascin-C (TN-C) is a glycoprotein of the ECM related to cell adhesion and detachment. These proteins have been implicated in tumor invasion and metastasis. Therefore, we aimed at investigating the prognostic significance of MMP-2, CD, and TN-C expressions in primary colorectal cancer. Overall, 112 colorectal adenocarcinomas were included in the present study. MMP-2, CD, and TN-C expressions were evaluated by immunohistochemistry and correlated with clinicopathologic prognostic parameters and survival. Diffuse stromal TN-C immunostaining was found to be significantly correlated with advanced stage and shorter survival time (p = 0.002 and 0.02, respectively). MMP-2 expression was found to correlate with lymph vessel invasion (p = 0.006) and stage (p = 0.03). CD expression was related to depth of invasion (p = 0.005). No significant relationship was found between survival and MMP-2 and CD expression (p > 0.05). In multivariate analysis, stage and vascular invasion were independent prognostic factors, whereas TN-C did not retain a clear independent relationship to survival (p > 0.05). Our findings suggest that TN-C expression may be a potential prognostic marker in colorectal carcinoma. However, MMP-2 and CD do not appear to be significant indicators of survival.

    Pathology, research and practice 2004;200;5;379-87

  • Tenascin in preinvasive lesions of the vulva and vulvar cancer.

    Goepel C, Stoerer S and Koelbl H

    Department of Gynecology, Martin Luther University, 06097 Halle Saale, Germany.

    Objective: Vulvar cancer is a rare malignancy, representing approximately 5% of all female genital tract cancers and 1% of all malignancies in women. The incidence of vulvar carcinoma in situ (VIN III) has nearly doubled from 1.1 to 2.1 per 100,000 women-years between 1973 and 1987. It is more frequent among older women, but the incidence is increasing among younger women. Different factors have been associated with an increased risk for vulvar cancers, for example HPV and genital infection. In this study we investigated normal, preinvasive and invasive vulvar tissues to look for relevant changes of Tenascin, a glycoprotein, already detected in pre-invasive and invasive lesions of the breast.

    Paraffin-embedded slides from 15 normal vulvar tissue, 14 cases of vulvar cancer, 51 cases of VIN (12 VIN I, 18 VIN II, 21 VIN III) and 5 cases of vulvar vestibulitis were investigated with immunofluorescent microscopical methods.

    Results: Tenascin was detectable and clearly enhanced in the stroma underlying the basement membrane in all samples of patients with VIN. The stroma of all specimens of patients with vulvar cancers showed intensive and strong reactivity. No Tenascin expression was observed in normal human vulvar skin. Tenascin expression also markedly increased in tissue lesions with skin inflammation. Tissue samples presenting vulvar intraepithelial neoplasia showed different intensity of staining, with a higher grade of VIN resulting in more immunohistochemical reactivity.

    Conclusion: Our findings indicate the potential role of Tenascin staining as a marker of malignancy and a predictor of invasiveness in vulvar intraepithelial neoplasia. The preponderance of these lesions has been noted in an increasingly younger population (median age: 50 years). Tenascin was detectable in inflammatory vulvar disease, preinvasive and invasive vulvar lesions, but not in normal vulvar tissue. The enhancement of Tenascin in vulvar premalignant and malignant disease seems to be of inflammatory origin.

    Anticancer research 2003;23;6C;4587-91

  • Involvement of large tenascin-C splice variants in breast cancer progression.

    Tsunoda T, Inada H, Kalembeyi I, Imanaka-Yoshida K, Sakakibara M, Okada R, Katsuta K, Sakakura T, Majima Y and Yoshida T

    Department of Pathology, Mie University School of Medicine, Mie, Japan.

    Alternative splicing of fibronectin-like type III (FNIII) repeats of tenascin-C (Tn-C) generates a number of splice variants. The distribution of large variants, typical components of provisional extracellular matrices that are up-regulated during tumor stroma remodeling, was here studied by immunoblotting and immunohistochemistry using a monoclonal antibody against the FNIII B domain (named 4C8MS) in a series of human breast cancers. Large Tn-C variants were found at only low levels in normal breast tissues, but were highly expressed at invading sites of intraductal cancers and in the stroma of invasive ductal cancers, especially at invasion fronts. There was a positive correlation between the expression of large Tn-C variants and the cell proliferation rate determined by immunolabeling of the Ki-67 antigen. Of the Tn-C recombinant fragments (all FNIII repeats or mFNIII FL, the conserved FNIII domain only, the epidermal growth factor-like domain, and the fibrinogen-like domain) which were expressed by CHO-K1 cells transfected with mouse Tn-C cDNAs, only the mFNIII FL enhanced in vitro migration and mitotic activity of mammary cancer cells derived from a Tn-C-null mouse. Addition of 4C8MS blocked the function of mFNIII FL. These findings provide strong evidence that the FNIII alternatively spliced region has important roles in tumor progression of breast cancer.

    The American journal of pathology 2003;162;6;1857-67

  • Induction of tenascin-C by tumor-specific EWS-ETS fusion genes.

    Watanabe G, Nishimori H, Irifune H, Sasaki Y, Ishida S, Zembutsu H, Tanaka T, Kawaguchi S, Wada T, Hata J, Kusakabe M, Yoshida K, Nakamura Y and Tokino T

    Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo, Japan.

    Ewing sarcoma (ES) and peripheral primitive neuroectodermal tumors (PNETs) are associated with a chromosomal translocation resulting in a fusion of the amino-terminus of EWS with the DNA-binding domain of an ETS transcription factor (most commonly FLI1 or ERG). Although previous reports suggested that these chimera proteins would act as aberrant transcription factors, their downstream targets have not been fully elucidated. To identify downstream targets of these EWS-ETS fusion proteins, we introduced EWS-ETS fusion constructs into a human fibrosarcoma cell line, HT-1080, by retroviral transduction. Here we report that Tenascin-C (TNC) is induced to a significantly higher level in cells expressing EWS-ETSs than in cells expressing normal ETSs. Furthermore, through use of an antisense cDNA expression vector we show that expression of endogenous TNC mRNA and protein were reduced coordinately with attenuation of EWS-FLI1 fusion protein expression. A chromatin immunoprecipitation assay showed direct interaction between the TNC promoter and the EWS-FLI1 fusion protein in vivo. In addition, a luciferase reporter assay revealed that EWS-ETSs upregulated the TNC gene through four ETS binding sites in the TNC promoter. High levels of TNC expression were observed in a subset of ES cell lines (3 of 6) and primary tumors (4 of 6). Together with previous studies showing that TNC expression is involved in the invasive and malignant phenotype of several tumor types, our data suggest that the oncogenic effect of EWS-ETS may be mediated in part by upregulating of TNC expression.

    Genes, chromosomes & cancer 2003;36;3;224-32

  • Tenascin-C levels in the vitreous of patients with proliferative diabetic retinopathy.

    Mitamura Y, Takeuchi S, Ohtsuka K, Matsuda A, Hiraiwa N and Kusakabe M

    Diabetes care 2002;25;10;1899

  • Tenascin-C modulates matrix contraction via focal adhesion kinase- and Rho-mediated signaling pathways.

    Midwood KS and Schwarzbauer JE

    Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.

    A provisional matrix consisting of fibrin and fibronectin (FN) is deposited at sites of tissue damage and repair. This matrix serves as a scaffold for fibroblast migration into the wound where these cells deposit new matrix to replace lost or damaged tissue and eventually contract the matrix to bring the margins of the wound together. Tenascin-C is expressed transiently during wound repair in tissue adjacent to areas of injury and contacts the provisional matrix in vivo. Using a synthetic model of the provisional matrix, we have found that tenascin-C regulates cell responses to a fibrin-FN matrix through modulation of focal adhesion kinase (FAK) and RhoA activation. Cells on fibrin-FN+tenascin-C redistribute their actin to the cell cortex, downregulate focal adhesion formation, and do not assemble a FN matrix. Cells surrounded by a fibrin-FN+tenascin-C matrix are unable to induce matrix contraction. The inhibitory effect of tenascin-C is circumvented by downstream activation of RhoA. FAK is also required for matrix contraction and the absence of FAK cannot be overcome by activation of RhoA. These observations show dual requirements for both FAK and RhoA activities during contraction of a fibrin-FN matrix. The effects of tenascin-C combined with its location around the wound bed suggest that this protein regulates fundamental processes of tissue repair by limiting the extent of matrix deposition and contraction to fibrin-FN-rich matrix in the primary wound area.

    Molecular biology of the cell 2002;13;10;3601-13

  • Value of tenascin-C content and association with clinicopathological parameters in uterine cervical lesions.

    Buyukbayram H and Arslan A

    Faculty of Medicine, Department of Pathology, Dicle University, 21280 Diyarbakir, Turkey. husram@dicle.edu.tr

    To determine whether the content of the matrix protein tenascin-C (Tn-C) is of diagnostic or prognostic value in cervical lesions, we evaluated increases in Tn-C immunoreactivity in 80 formalin-fixed, paraffin-embedded biopsies and surgical specimens of the uterine cervix. Tn-C content in the basement membrane zone and in the stroma was graded and compared to some prognostic parameters. In the normal cervix, Tn-C formed a thin continuous band. In cervicitis, Tn-C bands thickened in the basement membrane zone and the adjacent stroma in the form of thin filaments. In 30 squamous intraepithelial lesions (SILs) of various grades, Tn-C bands were either slightly (1+) or moderately (2+) thickened in the basement membrane zone, while slight stromal Tn-C immunoreactivity in the form of thin bands was observed in 12 cases, regardless of grade and inflammatory stromal reaction. In invasive carcinoma, Tn-C content was markedly increased in the stroma and around the invasive nests of tumors. The intensity of Tn-C immunoreactivity was significantly higher in grade I tumors than in others (p < 0.04). The intensity of increase in Tn-C immunoreactivity was 10.5-fold (95% CI 3.39-32.5) higher in invasive cervical carcinomas than in others (cervicitis, low-grade SIL and high-grade SIL) (p = 0.0001). A significant correlation was found between weak Tn-C immunoreactivity and lymphatic space invasion (p = 0.001), lymph node metastasis (p = 0.01), desmoplastic stromal component (p = 0.0001) and stromal inflammation (p = 0.002). In conclusion, increase in Tn-C immunoreactivity may be of value in the assessment of noninvasive and invasive cervical lesions and the appearance of Tn-C may be an indicator of adequate biologic defense in cervical cancer patients.

    International journal of cancer 2002;100;6;719-22

  • Tenascin-C expression and distribution in cultured human chondrocytes and chondrosarcoma cells.

    Ghert MA, Qi WN, Erickson HP, Block JA and Scully SP

    Division of Orthopaedic Surgery, Duke University Medical Center, Durham, NC 27710, USA.

    Tenascin-C (TNC) is an oligomeric glycoprotein of the extracellular matrix with several distinct isoforms variably expressed during embryogenesis, tumorogenesis, angiogenesis and wound healing. In the normal human adult, TNC is found in large concentrations in articular cartilage, suggesting tissue-specific function. The purpose of this study was to determine the specific in vitro TNC splicing patterns of articular chondrocytes and a human chondrosarcoma cell line. Cells were cultured in a three-dimensional bead system and TNC splice variant expression and distribution were examined with the use of Western blotting techniques, semi-quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. At both the transcriptional and post-translational levels, the chondrocytes were found to express significantly higher levels of the smaller 220 kDa isoform (P < 0.01), which was predominantly incorporated into the matrix. The splicing pattern of the malignant cells was characterized by a higher proportion of the larger 320 kDa isoform which was extruded into the media. In vivo studies are necessary to verify the expression of the large TNC isoform in chondrosarcoma and the production and integration of the smaller isoform in normal chondroid matrix. In addition, elucidation of the biologic functions of the two major TNC isoforms may lead to the development of novel diagnostic and therapeutic approaches to chondrosarcoma.

    Funded by: NIAMS NIH HHS: AR42863

    Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2002;20;4;834-41

  • Tenascin in meningioma: expression is correlated with anaplasia, vascular endothelial growth factor expression, and peritumoral edema but not with tumor border shape.

    Kiliç T, Bayri Y, Ozduman K, Acar M, Diren S, Kurtkaya O, Ekinci G, Buğra K, Sav A, Ozek MM and Pamir MN

    Neurooncology Laboratories, Institute of Neurological Sciences, Mamara University, Istanbul, Turkey. turkilic@turk.net

    Objective: Tenascin is an extracellular matrix glycoprotein that is expressed during embryogenesis, inflammation, angiogenesis, and carcinogenesis. The aim of this study was to investigate how tenascin expression relates to histological grade, angiogenesis, and radiological findings in meningiomas.

    Methods: Twenty typical, 20 atypical, and 5 malignant meningiomas were studied retrospectively. Tenascin expression and vascular endothelial growth factor (VEGF) expression in the tumor tissue were investigated by immunohistochemistry. Tenascin messenger ribonucleic acid expression was also studied by comparative reverse transcriptase-polymerase chain reaction. Magnetic resonance images from each case were assessed for peritumoral edema and tumor border shape.

    Results: The atypical and malignant meningiomas showed higher levels of tenascin expression than the typical meningiomas. The more sensitive messenger ribonucleic acid-based methods confirmed this finding. Tenascin expression was correlated with peritumoral edema and VEGF expression but not with tumor border shape. In the 13 tumors with marked tenascin expression, peritumoral edema was Grade 0 in one, Grade 1 in three, and Grade 2 in nine specimens. In the same 13 tumors, VEGF expression was Grade 1 in five and Grade 2 in eight specimens, and the findings for tumor border shape were Grade 0 in seven, Grade 1 in four, and Grade 2 in two specimens.

    Conclusion: In meningiomas, tenascin expression is correlated with anaplasia, tumor-associated edema, and VEGF expression but not with tumor border shape. This protein may play a role in the neoplastic and/or angiogenic processes in atypical and malignant meningiomas and may thus be a potential target for meningioma therapy.

    Neurosurgery 2002;51;1;183-92; discussion 192-3

  • Degradation of tenascin-C and activity of matrix metalloproteinase-2 are associated with tumor recurrence in early stage non-small cell lung cancer.

    Cai M, Onoda K, Takao M, Kyoko IY, Shimpo H, Yoshida T and Yada I

    Department of Thoracic and Cardiovascular Surgery, Mie University School of Medicine, Tsu, Mie, Japan 514-8507.

    Purpose: To find out an effective prognostic factor for early stage non-small cell lung cancer (NSCLC), we examined the relationship of the degree of tenascin-C (TN-C) degradation in relapsed NSCLC tumors with the prognosis of the patients. The molecular mechanism of TN-C degradation was also evaluated.

    In 63 stage-1 NSCLC patients, TN-C protein was analyzed by Western blotting, and the activity of matrix metalloproteinase (MMP)-2 was examined by gelatin zymography in 23 stage-1 NSCLC patients.

    Results: Degradation of TN-C was detected in 12 of 63 patients. TN-C degradation was detected in 9 of 17 patients (52.9%) that showed local and distant cancer recurrences. In short, in 9 of 12 patients (75%) showing TN-C degradation, lung cancer recurrence was recognized. The actual frequency of free-from-recurrence at 4 years was 28.1% in patients with tumors showing TN-C degradation, and actual frequency of free-from-recurrence at 4 years and 10 years was 82.1% and 76.6% in patients without TN-C degradation (P < 0.001). In 23 stage-1 NSCLC patients, in tumors with or without degraded TN-C, the mean ratio of tumor:normal-tissue of activated MMP-2 was 3.5 +/- 0.4 or 1.54 +/- 0.4, respectively. Significantly increased activity of MMP-2 was recognized in tumors showing TN-C degradation (P < 0.001).

    Conclusions: These results suggest that TN-C degradation is a reliable marker for recurrence potential of stage-1 NSCLC and that MMP-2 may be a protease breaking down TN-C in lung cancer.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2002;8;4;1152-6

  • Clinical impact and functional aspects of tenascin-C expression during glioma progression.

    Herold-Mende C, Mueller MM, Bonsanto MM, Schmitt HP, Kunze S and Steiner HH

    Molecular Biology Laboratory, Neurosurgery Hospital, University of Heidelberg, Germany. Christel_Herold-Mende@med.uni-heidelberg.de

    The extracellular matrix protein tenascin-C is expressed in processes like embryogenesis and wound healing and in neoplasia. Tenascin-C expression in gliomas has been described previously; however, the relation to clinical data remains inconsistent. Generally, analysis of tenascin-C function is difficult due to different alternatively spliced isoforms. Our studies focus on changes in tenascin-C expression in human gliomas, correlating these changes with tumor progression and elucidating the functional role of the glioma cell-specific tenascin-C isoform pool. Eighty-six glioma tissues of different World Health Organization (WHO) grades were analyzed immunohistochemically for tenascin-C expression. The influence of the specific tenascin-C isoforms produced by glioblastoma cells on proliferation and migration was examined in vitro using blocking antibodies recognizing all isoforms. In general, tenascin-C expression increased with tumor malignancy. Perivascular staining of tenascin-C around tumor-supplying blood vessels was observed in all glioblastoma tissues, whereas in WHO II and III gliomas, perivascular tenascin-C staining appeared less frequently. The appearance of perivascular tenascin-C correlated significantly with a shorter disease-free time. Analysis of proliferation and migration in the presence of blocking antibodies revealed an inhibition of proliferation by around 30% in all 3 glioblastoma cell cultures, as well as a decrease in migration of 30.6-46.7%. Thus we conclude that the endogenous pool of tenascin-C isoforms in gliomas supports both tumor cell proliferation and tumor cell migration. In addition, our data on the perivascular staining of tenascin-C in WHO II and III gliomas and its correlation with a shorter disease-free time suggest that tenascin-C may be a new and potent prognostic marker for an earlier tumor recurrence.

    International journal of cancer 2002;98;3;362-9

  • Tenascin-C is highly expressed in respiratory distress syndrome and bronchopulmonary dysplasia.

    Kaarteenaho-Wiik R, Kinnula VL, Herva R, Soini Y, Pöllänen R and Pääkkö P

    Department of Internal Medicine, University of Oulu and Oulu University Hospital, Oulu, Finland.

    Tenascin-C is an extracellular matrix (ECM) glycoprotein expressed in human tissues during organogenesis and in fibrotic and neoplastic processes. We hypothesized that its expression would increase in human lung in neonatal disorders such as infant respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Tenascin-C expression was studied by immunohistochemistry (IHC) and mRNA in situ hybridization (ISH). The extent of tenascin-C immunoreactivity was scored as absent (0), low (+), moderate (++), strong (+++), or very strong (++++) separately in different types of pulmonary cells in controls (seven cases), RDS (19 cases), and BPD (12 cases). In controls, tenascin-C expression was low (+) underneath alveolar and bronchiolar epithelium, moderate (++) in intima of veins, and strong (+++) around chondrocytes. In RDS, tenascin-C expression was moderate (++) or strong (+++) underneath both bronchiolar and often detached alveolar epithelium underlying hyaline membranes in the walls of dilated alveoli. In particular, the patients with RDS who survived for 1 day or more had strong expression of tenascin-C within alveolar walls. In patients with BPD, tenascin-C was very strongly (++++) expressed in the remodeled fibrotic alveolar walls underneath regenerative epithelium. Increased expression of tenascin-C mRNA was seen below the alveolar and bronchiolar epithelia in RDS and BPD. The cells in these locations showed alpha-smooth muscle actin immunoreactivity, suggesting a myofibroblast phenotype. In conclusion, tenascin-C is highly expressed in the walls of alveoli and bronchioli in RDS and BPD, suggesting an association between the expression of this protein and the presence of these disorders.

    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 2002;50;3;423-31

  • The alternatively spliced domain TnFnIII A1A2 of the extracellular matrix protein tenascin-C suppresses activation-induced T lymphocyte proliferation and cytokine production.

    Puente Navazo MD, Valmori D and Rüegg C

    Centre Pluridisciplinaire d'Oncologie, University of Lausanne Medical School, Lausanne, Switzerland.

    Several lines of evidences have suggested that T cell activation could be impaired in the tumor environment, a condition referred to as tumor-induced immunosuppression. We have previously shown that tenascin-C, an extracellular matrix protein highly expressed in the tumor stroma, inhibits T lymphocyte activation in vitro, raising the possibility that this molecule might contribute to tumor-induced immunosuppression in vivo. However, the region of the protein mediating this effect has remained elusive. Here we report the identification of the minimal region of tenascin-C that can inhibit T cell activation. Recombinant fragments corresponding to defined regions of the molecule were tested for their ability to inhibit in vitro activation of human peripheral blood T cells induced by anti-CD3 mAbs in combination with fibronectin or IL-2. A recombinant protein encompassing the alternatively spliced fibronectin type III domains of tenascin-C (TnFnIII A-D) vigorously inhibited both early and late lymphocyte activation events including activation-induced TCR/CD8 down-modulation, cytokine production, and DNA synthesis. In agreement with this, full length recombinant tenascin-C containing the alternatively spliced region suppressed T cell activation, whereas tenascin-C lacking this region did not. Using a series of smaller fragments and deletion mutants issued from this region, we have identified the TnFnIII A1A2 domain as the minimal region suppressing T cell activation. Single TnFnIII A1 or A2 domains were no longer inhibitory, while maximal inhibition required the presence of the TnFnIII A3 domain. Altogether, these data demonstrate that the TnFnIII A1A2 domain mediate the ability of tenascin-C to inhibit in vitro T cell activation and provide insights into the immunosuppressive activity of tenascin-C in vivo.

    Journal of immunology (Baltimore, Md. : 1950) 2001;167;11;6431-40

  • Increased expression of tenascin-C-binding epithelial integrins in human bullous keratopathy corneas.

    Ljubimov AV, Saghizadeh M, Pytela R, Sheppard D and Kenney MC

    Ophthalmology Research Laboratories, Burns & Allen Research Institute, Cedars-Sinai Medical Center, University of California Los Angeles School of Medicine, Los Angeles, California 90048, USA. ljubimov@cshs.org

    We previously found an abnormal deposition of an extracellular matrix glycoprotein, tenascin-C (TN-C), in human corneas with pseudophakic/aphakic bullous keratopathy (PBK/ABK). In this work, we studied cellular TN-C receptors in normal and PBK/ABK corneas. Cryostat sections of normal and PBK/ABK corneas were stained by immuno-fluorescence for TN-C receptors: alpha2, alpha8, alpha9, alphaVbeta3, beta1, and beta6 integrins, and annexin II. Beta6 integrin mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using beta2-microglobulin gene to normalize the samples. In PBK/ABK compared to normal corneas, relatively minor changes were observed for alpha2 and beta1 integrins, and for annexin II. Alpha8, alpha9, and beta6 subunits of TN-C receptors, alpha8beta1 alpha9beta1, and alphaVbeta6, respectively, were absent from normal central corneas but were found in the central epithelium of PBK/ABK corneas. Beta6 integrin showed the most significant accumulation. It correlated best with the expression of TN-C rather than with the expression of other alphaVbeta6 ligands, fibronectin, and vitronectin. RT-PCR analysis also showed elevated levels of beta6 mRNA in PBK/ABK compared to normal corneas. Therefore, accumulation of TN-C in PBK/ABK corneas was accompanied by an increased expression of its three binding integrins, especially alphaVbeta6 in the corneal epithelium. The interaction of tenascin-C with these integrins may contribute to the fibrotic process that occurs in PBK/ABK corneas.

    Funded by: NEI NIH HHS: EY10836

    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 2001;49;11;1341-50

  • Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor.

    Swindle CS, Tran KT, Johnson TD, Banerjee P, Mayes AM, Griffith L and Wells A

    Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

    Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal-regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.

    The Journal of cell biology 2001;154;2;459-68

  • Glial tumor cell adhesion is mediated by binding of the FNIII domain of receptor protein tyrosine phosphatase beta (RPTPbeta) to tenascin C.

    Adamsky K, Schilling J, Garwood J, Faissner A and Peles E

    Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

    The extracellular domain of receptor protein tyrosine phosphatase beta (RPTPbeta) is composed of several domains which mediate its interactions with distinct ligands present on the surface of either neurons or glial cells. Here, we demonstrate that the fibronectin type III domain (FNIII) of RPTPbeta binds to glial tumor-derived cell lines and primary astrocytes. We used affinity purification to isolate several proteins that specifically bind to the FNIII domain of RPTPbeta. One of these, a 240 kDa protein that was purified from U118MG glioblastoma cell, was identified as tenascin C based on the amino acid sequence of several tryptic peptides. The interaction of RPTPbeta with tenascin C was found to mediate cell adhesion. Adhesion and spreading of SF763T astrocytoma cells expressing RPTPbeta on tenascin C was specifically abolished by the addition of a soluble fragment containing the FNIII domain of the receptor. RPTPbeta-dependent cell adhesion was mediated by binding to the alternatively spliced FNIII repeats A1,2,4 (TnfnA1,2,4) of tenascin C. Furthermore, COS cells expressing RPTPbeta adhere to TnfnA1,2,4, while the parental cells did not. These results demonstrate that the FNIII domain of RPTPbeta binds to tenascin C and suggest that RPTPbeta present on glial tumor cells is a primary adhesion receptor system to the extracellular matrix.

    Oncogene 2001;20;5;609-18

  • Tenascin-C suppresses Rho activation.

    Wenk MB, Midwood KS and Schwarzbauer JE

    Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.

    Cell binding to extracellular matrix (ECM) components changes cytoskeletal organization by the activation of Rho family GTPases. Tenascin-C, a developmentally regulated matrix protein, modulates cellular responses to other matrix proteins, such as fibronectin (FN). Here, we report that tenascin-C markedly altered cell phenotype on a three-dimensional fibrin matrix containing FN, resulting in suppression of actin stress fibers and induction of actin-rich filopodia. This distinct morphology was associated with complete suppression of the activation of RhoA, a small GTPase that induces actin stress fiber formation. Enforced activation of RhoA circumvented the effects of tenascin. Effects of active Rho were reversed by a Rho inhibitor C3 transferase. Suppression of GTPase activation allows tenascin-C expression to act as a regulatory switch to reverse the effects of adhesive proteins on Rho function. This represents a novel paradigm for the regulation of cytoskeletal organization by ECM.

    The Journal of cell biology 2000;150;4;913-20

  • Significance of alpha 9 beta 1 and alpha v beta 6 integrin expression in breast carcinoma.

    Arihiro K, Kaneko M, Fujii S, Inai K and Yokosaki Y

    Second Department of Pathology, Hiroshima University School of Medicine, Japan.

    Background: Both alpha 9 beta 1 and alpha v beta 6 integrins have been newly identified from the tracheal epithelium of guinea pig. It has been pointed out that alpha 9 beta 1 functions as a receptor for tenascin-C and osteopontin. As for the ligands of alpha v beta 6, fibronectin and tenascin-C have been identified. It has not been ascertained whether alpha 9 beta 1 and alpha v beta 6 are expressed in normal breast tissue, benign breast lesion or breast carcinoma.

    Methods: Immunohistochemical staining for alpha 9 beta 1 and alpha v beta 6 was performed in benign breast lesion and breast carcinoma specimens. Western blotting was carried out on 11 breast carcinoma cases.

    Results: alpha 9 beta 1 was expressed in the cytoplasm of carcinoma cells in 23 of 90 cases (26%) and alpha v beta 6 in the membrane of carcinoma cells in 16 of 90 cases (18%). However, these findings of alpha 9 beta 1 and alpha v beta 6 did not correlate with any clinicopathological factors including the patients' age, tumor size, histological type of carcinoma, location of carcinoma cells and hormone receptor status. With regard to the histological grade of carcinoma, alpha v beta 6 and alpha 9 beta 1 expression did not statistically correlate, although no expression of alpha v beta 6 was observed in 14 cases of Grade I. On Western-blott analysis strong and weak bands consistent with alpha v beta 6 were noted in the membrane fraction extracted from breast carcinoma cells. On the other hand weak bands consistent with alpha 9 subunit were noted in the whole cell lysates of breast carcinoma cells and very weak or no bands consistent with alpha 9 subunit were noted in the membrane fraction extracted from the breast carcinoma cells.

    Conclusions: Significance of alpha 9 beta 1 and alpha v beta 6 integrins expression in breast carcinoma was still unknown on clinicopathological examination. The findings of Western blot analysis may indicate that the transportation system of glycoproteins such as integrins to the cell membrane of carcinoma cells is disturbed, although these integrins can be produced.

    Breast cancer (Tokyo, Japan) 2000;7;1;19-26

  • The interaction between F3 immunoglobulin domains and protein tyrosine phosphatases zeta/beta triggers bidirectional signalling between neurons and glial cells.

    Revest JM, Faivre-Sarrailh C, Maeda N, Noda M, Schachner M and Rougon G

    Laboratoire de Génétique et Physiologie du Développement, CNRS 6545 Parc Scientifique de Luminy, Marseille, France.

    F3, a mouse glycosyl-phosphatidylinositol anchored molecule of the immunoglobulin superfamily, is known to influence axonal growth and fasciculation via multiple interactions of its modular immunoglobulin-like domains. We prepared an Fc chimeric molecule (F3IgFc) to identify molecules interacting with these domains and characterize the functional impact of the interactions. We affinity-isolated tenascin-C and isoforms of the proteoglycan-type protein tyrosine phosphatases zeta/beta (PTPzeta/RPTPbeta) from extracts of developing mouse brain. We showed that both PTPzeta/RPTPbeta and tenascin-C can bind directly to F3, possibly in an exclusive manner, with the highest affinity for the F3-PTPzeta/RPTPbeta interaction. We observed a strong binding of F3IgFc-coated fluorospheres to astrocytes in neural primary cultures and to C6 astrocytoma cells, and demonstrated, in antibody perturbation experiments, that F3-Ig binding on astrocytes depends on its interaction with PTPzeta/RPTPbeta. We also found by confocal analysis that tenascin-C and PTPzeta/RPTPbeta were colocalized on astrocytes which suggests a complex interplay of interactions between PTPzeta/RPTPbeta, tenascin-C and F3. We showed that the interaction between PTPzeta/RPTPbeta and F3-Ig-like domains can trigger bidirectional signalling. C6 glia-expressed PTPzeta/RPTPbeta stimulated neurite outgrowth by cortical and cerebellar neurons, whereas preclustered F3IgFc specifically modified the distribution of phosphotyrosine labelling in these glial cells. Both effects could be prevented and/or mimicked by anti-F3 and anti-6B4PG antibodies. These results identify F3 and PTPzeta/RPTPbeta as potential mediators of a reciprocal exchange of information between glia and neurons.

    The European journal of neuroscience 1999;11;4;1134-47

  • Identification of the ligand binding site for the integrin alpha9 beta1 in the third fibronectin type III repeat of tenascin-C.

    Yokosaki Y, Matsuura N, Higashiyama S, Murakami I, Obara M, Yamakido M, Shigeto N, Chen J and Sheppard D

    Department of Internal Medicine, National Hiroshima Hospital, 513 Jike, Saijoh Higashi-Hiroshima 739-0041, Japan. yokosaki@hirosima.hosp.go.jp

    The integrin alpha9 subunit forms a single heterodimer, alpha9 beta1 that mediates cell adhesion to a site within the third fibronectin type III repeat of tenascin-C (TNfn3). In contrast to at least 3 other integrins that bind to this region of tenascin-C, alpha9 beta1 does not recognize the common integrin recognition motif, Arg-Gly-Asp (RGD). In this report, we have used substitution mutagenesis to identify a unique ligand recognition sequence in TNfn3. We introduced mutations substituting alanine for each of the acidic residues in or adjacent to each of the exposed loops predicted from the solved crystal structure. Most of these mutations had little or no effect on adhesion of alpha9-transfected SW480 colon carcinoma cells, but mutations of either of two acidic residues in the B-C loop region markedly reduced attachment of these cells. In contrast, cells expressing the integrin alphav beta3, previously reported to bind to the RGD sequence in the adjacent F-G loop, attached to all mutant fragments except one in which the RGD site was mutated to RAA. The peptide, AEIDGIEL, based on the sequence of human tenascin-C in this region blocked the binding of alpha9-transfected cells, but not beta3-transfected cells to wild type TNfn3. This sequence contains a tripeptide, IDG, homologous to the sequences LDV, IDA, and LDA in fibronectin and IDS in VCAM-1 recognized by the closely related integrin alpha4 beta1. These findings support the idea that this tripeptide motif serves as a ligand binding site for the alpha4/alpha9 subfamily of integrins.

    Funded by: NHLBI NIH HHS: HL/AI33259

    The Journal of biological chemistry 1998;273;19;11423-8

  • Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C.

    Denda S, Müller U, Crossin KL, Erickson HP and Reichardt LF

    Neuroscience Program, Department of Physiology, University of California San Francisco 94143-0724, USA.

    The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine alpha 8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine alpha 8 and beta1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified alpha 8 beta 1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface alpha 8 beta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands.

    Funded by: NINDS NIH HHS: R01 NS019090, R01 NS019090-14; PHS HHS: P01-16033

    Biochemistry 1998;37;16;5464-74

  • Mapping of a defined neurocan binding site to distinct domains of tenascin-C.

    Rauch U, Clement A, Retzler C, Fröhlich L, Fässler R, Göhring W and Faissner A

    Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany. rauch@biochem.mpg.de

    Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH2-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH2-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein was designed to contain the last downstream glycosaminoglycan attachment site and was expressed as a proteoglycan. In ligand overlay blots carried out with brain extracts, it recognized tenascin-C. The interaction was abolished by the addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module. The fusion protein directly reacted with this fragment in ligand blot and enzyme-linked immunosorbent assay procedures. Both tenascin-C and TNfn4,5 were retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded a KD value of 17 nM for purified tenascin-C. We conclude that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.

    The Journal of biological chemistry 1997;272;43;26905-12

  • Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alpha v beta 3 integrin to promote epidermal growth factor receptor phosphorylation and growth.

    Jones PL, Crack J and Rabinovitch M

    Division of Cardiovascular Research, Research Institute, The Hospital for Sick Children, University of Toronto, Ontario, Canada.

    Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matrix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta 3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with alpha v beta 3 integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta 3 integrin ligands in type I collagen. In turn, alpha v beta 3 interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.

    The Journal of cell biology 1997;139;1;279-93

  • Binding of the NG2 proteoglycan to type VI collagen and other extracellular matrix molecules.

    Burg MA, Tillet E, Timpl R and Stallcup WB

    La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.

    Previous studies have suggested that the NG2 proteoglycan interacts with type VI collagen. We have further characterized this interaction using a solid phase binding assay in which purified NG2 was shown to bind to pepsin-solubilized type VI collagen. In addition, NG2 bound a recombinant alpha2 (VI) collagen chain but did not appreciably bind to the recombinant alpha1 (VI) chain or the N-terminal domain of alpha3 (VI) (N9-N2). Binding of NG2 to type VI collagen was shown to be concentration-dependent and saturable and to depend mainly on the NG2 core protein, since chondroitinase-treated NG2 bound the collagen as well as undigested samples. In addition, the binding studies revealed several other possible ligands for NG2, including type II collagen, type V collagen, tenascin, and laminin. Binding of the proteoglycan to these molecules was also shown to be mediated by domains contained within the NG2 core protein. The ability of NG2 to bind to these extracellular matrix molecules was compared with that of the chondroitin sulfate proteoglycan decorin, revealing an almost identical binding pattern of the two proteoglycans to the different collagen types. In addition, decorin was found to effectively inhibit the ability of NG2 to bind to collagen, thus suggesting that the two proteoglycans may bind to some of the same regions on the collagen substrates. In contrast, decorin did not bind tenascin and was ineffective in inhibiting the binding of NG2 to tenascin or laminin, indicating that NG2 may bind these two molecules using a separate domain that is distinct from its collagen binding region.

    Funded by: NINDS NIH HHS: R01 NS21990

    The Journal of biological chemistry 1996;271;42;26110-6

  • Differential effects of the integrins alpha9beta1, alphavbeta3, and alphavbeta6 on cell proliferative responses to tenascin. Roles of the beta subunit extracellular and cytoplasmic domains.

    Yokosaki Y, Monis H, Chen J and Sheppard D

    Lung Biology Center, Center for Occupational and Environmental Health, Cardiovascular Research Institute, Department of Medicine, University of California, San Francisco, San Francisco, California 94143, USA.

    Members of the integrin family manifest considerable overlap in ligand specificity, and many cells have the capacity to express multiple integrin receptors for the same ligand. For example, at least 5 different integrins recognize tenascin as a ligand, and 4 of these bind to the same region of the protein, the third fibronectin type III repeat (TNfn3). We utilized colon carcinoma cells (SW480) that do not normally attach to TNfn3 to examine the possibility that ligation of different integrin receptors for this ligand would induce different effects on cell behavior and intracellular signaling. Heterologous expression of the tenascin receptors alphavbeta3 and alpha9beta1 produced comparable effects on cell adhesion and spreading on TNfn3, but alphavbeta3-transfectants proliferated considerably better on each concentration examined. alphavbeta6-transfectants attached (although less avidly), but completely failed to spread or proliferate. Expression of a chimeric beta subunit composed of the beta3 extracellular domain fused to the beta6 transmembrane and cytoplasmic domains resulted in adhesion and spreading similar to that seen with beta3-transfectants, but considerably less proliferation. When the same cell lines were plated on fibronectin, alphavbeta6-transfectants spread and proliferated as well as cells transfected with the chimeric beta3/beta6 subunit, but, again, neither cell line proliferated as well as cells expressing alphavbeta3. Cell proliferation was always associated with spreading and with phosphorylation of the focal adhesion kinase, paxillin, and the mitogen-activated kinase, Erk2, but cell attachment in the absence of spreading or proliferation was not associated with phosphorylation of any of these proteins. These data suggest that different integrin receptors for a single ligand can produce markedly different effects on cell proliferation, and that both the extracellular and cytoplasmic domains of integrin beta subunits contribute to these differences.

    Funded by: NHLBI NIH HHS: HL/A133259, HL47412, HL53949

    The Journal of biological chemistry 1996;271;39;24144-50

  • Tenascin-C expression by angiogenic vessels in human astrocytomas and by human brain endothelial cells in vitro.

    Zagzag D, Friedlander DR, Dosik J, Chikramane S, Chan W, Greco MA, Allen JC, Dorovini-Zis K and Grumet M

    Department of Pathology, New York University Medical Center, New York 10016, USA.

    The expression of the extracellular matrix glycoprotein tenascin-C (TN) is enhanced in human astrocytomas and correlates with angiogenesis. To determine whether vascular cells are able to synthesize TN, we investigated the expression of TN protein and mRNA in nine astrocytomas. Immunogold electron microscopy in two glioblastomas multiforme detected the presence of TN in an extracellular perivascular location and to a lesser extent among tumor cells, confirming light microscopy immunohistochemical findings. In situ hybridization of astrocytomas using a digoxigenin-labeled antisense riboprobe detected strong staining for TN mRNA in vascular cells, especially in hyperplastic vessels, including those at the invasive edge of the tumors but not in vessels of normal brains. We observed weaker staining in tumor cells indicating a higher level of TN mRNA in vascular than in tumor cells. No staining was detected with the sense probe. Moreover, we investigated the ability of human brain microvessel endothelial cells (HBMECs) in primary culture to synthesize TN in vitro. Western blot analysis of the culture supernatants from HBMECs detected large amounts of TN. Immunogold silver staining demonstrated the presence of TN on the surface of HBMECs and in the subendothelial matrix. The distribution of TN mRNA in vascular cells of astrocytomas and the ability of HBMECs to synthesize TN in vitro demonstrate that vascular cells, including endothelial cells, are a major source of TN associated with angiogenesis. Furthermore, our results suggest that TN expression may be associated with endothelial cell activation and may play an important role in angiogenesis.

    Funded by: NCI NIH HHS: CA 16087-17; NINDS NIH HHS: NS 31088

    Cancer research 1996;56;1;182-9

  • Binding of tenascin-C to soluble fibronectin and matrix fibrils.

    Chung CY, Zardi L and Erickson HP

    Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    The small splice variant of tenascin-C (TN) has eight fibronectin type III (FN3) domains. The major large splice variant has three (in chicken) or seven (in human) additional FN3 domains inserted between domains five and six. Chiquet-Ehrismann et al. (Chiquet-Ehrismann, R., Matsuoka, Y., Hofer, U., Spring, J., Bernasconi, C., and Chiquet, M. (1991, Cell Regul. 2, 927-938) demonstrated that the small variant bound preferentially to fibronectin in enzyme-linked immunosorbent assay, and only the small variant was incorporated into the matrix by cultures of chicken fibroblasts. Here we have studied human TN, and confirmed that the small variant binds preferentially to purified fibronectin and to fibronectin-containing extracellular matrix. Thus this differential binding appears to be conserved across vertebrate species. Using bacterial expression proteins, we mapped the major binding site to the third FN3 domain of TN. Consistent with this mapping, a monoclonal antibody against an epitope in this domain did not stain TN segments bound to cell culture matrix fibrils. The enhanced binding of the small TN variant suggests the existence of another, weak binding site probably in FN3 domains 6-8, which is only positioned to bind fibronectin in the small splice variant. This binding of domains 6-8 may involve a third molecule present in matrix fibrils, as the enhanced binding of small TN was much more prominent to matrix fibrils than to purified fibronectin.

    Funded by: NCI NIH HHS: R37 CA47056

    The Journal of biological chemistry 1995;270;48;29012-7

  • The human integrin alpha 8 beta 1 functions as a receptor for tenascin, fibronectin, and vitronectin.

    Schnapp LM, Hatch N, Ramos DM, Klimanskaya IV, Sheppard D and Pytela R

    Department of Medicine, University of California, San Francisco 94143, USA.

    The integrin family of adhesion receptors consists of at least 21 heterodimeric transmembrane proteins that differ in their tissue distribution and ligand specificity. The recently identified alpha 8 integrin subunit associates with beta 1 and is predominantly expressed in smooth muscle and other contractile cells in adult tissues, and in mesenchymal and neural cells during development. We now show that alpha 8 beta 1 specifically localizes to focal contacts in cells plated on the extracellular matrix proteins fibronectin or vitronectin. In addition we show that human embryonic kidney cells (293), transfected with alpha 8 cDNA, express alpha 8 beta 1 on their surface and use this receptor for adhesion to fibronectin and vitronectin. Furthermore, alpha 8 beta 1 binds to both fibronectin- and vitronectin-Sepharose and can be specifically eluted from either matrix protein by the arginine-glycine-aspartic acid (RGD)-containing peptide, GRGDSP. Because fibronectin and vitronectin adhesion appeared to be mediated by RGD, we examined additional RGD-containing proteins, including tenascin, fibrinogen, thrombospondin, osteopontin, and denatured collagen type I. We found that only tenascin was able to mediate adhesion of alpha 8-transfected 293 cells. By using recombinant fragments of tenascin in adhesion assays, we were able to localize the alpha 8 beta 1 binding domain of tenascin to the RGD-containing third fibronectin type III repeat. These data strongly suggest that tenascin, fibronectin, and vitronectin are ligands for alpha 8 beta 1 and that this integrin binds to the RGD site in each of these ligands through mechanisms that are distinct and separate from alpha 5- and alpha v-containing integrins.

    Funded by: NCI NIH HHS: CA53259; NHLBI NIH HHS: HL/A133259, HL191551; ...

    The Journal of biological chemistry 1995;270;39;23196-202

  • The integrin receptor alpha 8 beta 1 mediates interactions of embryonic chick motor and sensory neurons with tenascin-C.

    Varnum-Finney B, Venstrom K, Muller U, Kypta R, Backus C, Chiquet M and Reichardt LF

    Department of Physiology, University of California, San Francisco 94143-0724, USA.

    This paper identifies a neuronal receptor for tenascin-C (tenascin/cytotactin), an extracellular matrix protein that has previously been detected in developing sensory and motor neuron pathways and has been shown to regulate cell migration in the developing CNS. Antibodies specific for each subunit of the integrin alpha 8 beta 1 are used to demonstrate that alpha 8 beta 1 mediates neurite outgrowth of embryonic sensory and motor neurons on this extracellular matrix protein. In addition, expression of alpha 8 in K562 cells results in surface expression of alpha 8 beta 1 heterodimers that are shown to promote attachment of this cell line to tenascin. The major domain in tenascin that mediates neurite outgrowth is shown to be localized to fibronectin type III repeats 6-8.

    Funded by: NINDS NIH HHS: P01 NS016033, P01 NS016033-19; PHS HHS: P01-16033

    Neuron 1995;14;6;1213-22

  • Distribution of integrins alpha v beta 6 and alpha 9 beta 1 and their known ligands, fibronectin and tenascin, in human airways.

    Weinacker A, Ferrando R, Elliott M, Hogg J, Balmes J and Sheppard D

    Lung Biology Center, University of California, San Francisco 94143, USA.

    We have previously identified two integrins, alpha 9 beta 1 and alpha v beta 6, from guinea pig airway epithelium. The extracellular matrix protein tenascin is a ligand for both of these receptors, and fibronectin is also a ligand for alpha v beta 6. In the present study, we used immunohistochemistry to examine the expression and spatial distribution of the alpha 9 subunit, alpha v beta 6, tenascin, and fibronectin in the proximal airways of 10 normal nonsmoking subjects and eight patients undergoing lung resection for cancer. We also performed the same analyses on sections of peripheral lung obtained from an additional seven subjects undergoing lung resection. alpha 9 was highly expressed throughout the airway epithelium (but not on alveolar epithelium) irrespective of clinical status. In contrast, alpha v beta 6 was expressed on proximal airway epithelial cells in four of eight smokers undergoing lung resection, but in none of the normal subjects and none of the distal airways examined. On bronchial epithelial cells cultured from resected airways, alpha v beta 6 was highly expressed on cells grown from patients who did not appear to express the receptor in vivo, as well as from subjects who did, suggesting that some component of the in vitro environment can induce expression. Although both tenascin and fibronectin were present below the proximal airway epithelium of both normal nonsmoking subjects and smokers, the spatial patterns of integrin and ligand expression were not congruent, because the integrins were present diffusely on the cell surface and on some cells that were not in contact with the basement membrane, whereas the ligands were present principally in the subepithelial layer. These findings are compatible with the existence of as-yet unidentified ligands for each of these integrins--for example, ligands involved in homotypic cell-cell interactions within the epithelium.

    Funded by: NHLBI NIH HHS: HL/AI33259, HL07155, HL47412

    American journal of respiratory cell and molecular biology 1995;12;5;547-56

  • Human tenascin gene. Structure of the 5'-region, identification, and characterization of the transcription regulatory sequences.

    Gherzi R, Carnemolla B, Siri A, Ponassi M, Balza E and Zardi L

    Laboratory of Cell Biology, Instituto Nazionale per la Ricerca sul Cancro, Genova, Italy.

    This report describes the genomic organization of the 5'-region of the human tenascin-C (TN) gene and the functional characterization of its promoter. Approximately 2300 base pairs of the TN gene 5'-flanking region have been cloned and sequenced. This genomic region contains several potential binding sites for transcription factors. By primer extension and S1 nuclease analysis we have localized the transcription start site. The first exon of the TN gene (179 base pairs long) is present in the two major TN transcripts, showing that the expression of these two mRNAs is regulated by a single promoter. The 220 bases upstream to the transcription start site are equally active in directing the expression of chloramphenicol acetyltransferase (CAT) reporter gene in TN producer and nonproducer cells. Using deletion fragments of the human 5'-flanking region we have shown the presence of putative "silencer" elements in the -220 to -2300 region active in both TN producer and nonproducer cell lines. Furthermore, we have demonstrated that the selective transcription in TN producing cells requires the presence of a 1.3-kilobase portion of the TN gene intron 1 in the CAT expression vectors. These findings indicate that complex mechanisms control the transcriptional regulation of TN gene.

    The Journal of biological chemistry 1995;270;7;3429-34

  • Analysis of aggrecan and tenascin gene expression in mouse skeletal tissues by northern and in situ hybridization using species specific cDNA probes.

    Glumoff V, Savontaus M, Vehanen J and Vuorio E

    University of Turku, Department of Molecular Biology and Medical Biochemistry, Finland.

    Cartilage matrix is an interacting multicomponent system of collagen fibrils, fibril-associated small proteoglycans, and large proteoglycans and glycoproteins entrapped within the fibrillar network. In order to better understand the relationships between these different components we have constructed short cDNA clones for detection of mRNAs for two major noncollagenous macromolecules of cartilage matrix, aggrecan and tenascin. We subsequently determined their corresponding mRNA levels by Northern analysis in a panel of total RNAs isolated from several newborn mouse tissues. The expression of aggrecan was strictly restricted to cartilages while tenascin mRNA was present at variable levels in most of the tissues studied. The cDNA clones were also used to identify the cells responsible for aggrecan and tenascin production in newborn mouse tissues by in situ hybridization. With this technique aggrecan mRNA was detected in chondrocytes throughout the developing skeleton in a pattern very similar but not identical to those of type II and IX collagen mRNAs. In the newborn mouse skeleton tenascin and aggrecan mRNAs were expressed essentially in a mutually exclusive manner, tenascin transcripts being present in osteoblasts, periosteal and perichondrial cells, and in cells at articular surfaces. None of these cells expressed the cartilage specific collagen or aggrecan genes. The results further suggest different patterns of gene expression in chondrocytes based on their location in the different cartilages.

    Biochimica et biophysica acta 1994;1219;3;613-22

  • The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin.

    Yokosaki Y, Palmer EL, Prieto AL, Crossin KL, Bourdon MA, Pytela R and Sheppard D

    Department of Medicine, University of California, San Francisco 94143.

    We have previously reported the sequence of the integrin alpha 9 subunit, a partner of the beta 1 subunit that is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. In the present study, we have stably expressed alpha 9 beta 1 on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480 and used these transfected cells lines to identify ligand(s) for this integrin. Transfected cells did not appear to utilize alpha 9 beta 1 for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing alpha 9 beta 1 adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, we were able to localize the binding site for alpha 9 beta 1 to the third type III repeat. This repeat contains the arginine-glycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for alpha v-integrins. However, the RGD site does not appear to be the binding site for alpha 9 beta 1, as the attachment of alpha 9 transfectants to this fragment was not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA.

    Funded by: NHLBI NIH HHS: HL25816, HL47412, HLA133259; ...

    The Journal of biological chemistry 1994;269;43;26691-6

  • Multiple integrins mediate cell attachment to cytotactin/tenascin.

    Prieto AL, Edelman GM and Crossin KL

    Scripps Research Institute, Department of Neurobiology, La Jolla, CA 92037.

    To identify potential cell surface receptors for chicken cytotactin (CT), we have characterized the ability of recombinant fusion proteins spanning the proximal fibronectin (FN) type III repeats of the molecule to support attachment of glioma and carcinoma cell lines. The third FN type III repeat, which contains the RGD tripeptide, supported cell attachment and cell spreading; however, mutation of RGD to RAD did not result in significant loss of either activity. In addition, the same repeat of mouse CT, which contains a natural mutant, RVD, also supported cell attachment and spreading, although at a lower level; both activities were increased by mutation of the RVD sequence to RGD. Studies utilizing RGD-containing peptides and well-characterized antibodies to integrins indicated that cell attachment to the third FN type III repeat was mediated by at least two different integrin receptors of the alpha v subtype. Additional cellular receptors may also be involved in cell attachment to CT. For example, an antibody to the beta 1 subfamily of integrins partially inhibited binding of cells to intact CT but did not inhibit cell binding to the third FN type III repeat. These findings suggest that the RGD site in CT is able to mediate cell attachment to integrins and thus is not a cryptic adhesion site. They also open the possibility that the functions of CT in processes such as counteradhesion, cell migration, cell proliferation, and cell differentiation may be mediated in part by interaction with multiple integrins.

    Funded by: NIDDK NIH HHS: DK-04256

    Proceedings of the National Academy of Sciences of the United States of America 1993;90;21;10154-8

  • Endothelial cell attachment and spreading on human tenascin is mediated by alpha 2 beta 1 and alpha v beta 3 integrins.

    Sriramarao P, Mendler M and Bourdon MA

    La Jolla Institute for Experimental Medicine, CA 92037.

    Human umbilical vein endothelial cells were found to attach and partially spread on human tenascin. The attachment of endothelial cells to tenascin results in elongated cells with interconnecting processes and is distinct from the flattened appearance of endothelial cells on fibronectin, collagen, vitronectin or laminin substrata, suggesting a role for tenascin in modulating cell adhesion and motility. Endothelial attachment to tenascin was partially inhibitable by the SRRGDMS peptide derived from human tenascin and completely inhibitable by anti-integrin antibodies to alpha 2 beta 1 and alpha v beta 3. Endothelial cell attachment to tenascin could be inhibited up to 80% with anti-alpha 2 and anti-beta 1 monoclonal antibodies P1E6 and P4C10, respectively, and this was associated with a complete loss in cell spreading. In contrast, pretreatment of endothelial cells with the anti-alpha v beta 3 monoclonal antibody LM609, resulted in a 35% inhibition in cell attachment but did not alter cell spreading. In combination the anti-alpha 2 and anti-alpha v beta 3 antibodies, could completely abrogate cell spreading and attachment to tenascin-coated surfaces. Affinity purification of 125I-labeled endothelial cell extract on a tenascin matrix column followed by immunoprecipitation with monoclonal antibodies to different integrin alpha and beta subunits resulted in the identification of alpha 2 beta 1 and alpha v beta 3 integrins, respectively, as tenascin binding receptors. Collagen affinity-purified alpha 2 beta 1 receptor from endothelial cells bound not only to collagen and laminin but also to tenascin in a radio receptor binding assay. The results demonstrate that alpha 2 beta 1 and alpha v beta 3 mediate distinct endothelial cell interactions with tenascin; cell spreading and cell binding, respectively. Binding by alpha v beta 3 is mediated by the SRRGDMS site on tenascin, whereas the alpha 2 beta 1 binding site remains undefined. The interaction of alpha 2 beta 1 and alpha v beta 3 with tenascin may be regulated in a cell type-specific manner as evidenced by the binding of endothelial cell alpha 2 beta 1 and alpha v beta 3 to tenascin, and the lack of binding by the same receptors on osteosarcoma MG63 to tenascin.

    Funded by: NCI NIH HHS: CA 52879

    Journal of cell science 1993;105 ( Pt 4);1001-12

  • Structure and chromosomal localization of the human gene for a brain form of prostaglandin D2 synthase.

    White DM, Mikol DD, Espinosa R, Weimer B, Le Beau MM and Stefansson K

    Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.

    We have cloned and characterized the human gene for the 21-kDa brain form of prostaglandin D2 synthase. The gene was isolated from a human genomic lambda library and spans 3600 base pairs. It consists of seven exons and six introns. Southern blot analysis indicates that there is a single copy of the gene in the haploid genome. The transcriptional start site was mapped to a G residue 74 base pairs 5' of the ATG initiation codon. A TATA box-like element (ATAAATA) is situated 21 base pairs upstream of the mRNA start site. The gene was mapped to chromosome 9 bands q34.2-q34.3. The gene bears close resemblance to the genes for murine major urinary protein and ovine beta-lactoglobulin.

    Funded by: NCI NIH HHS: CA 42557, CA40046; NHLBI NIH HHS: HL 07237

    The Journal of biological chemistry 1992;267;32;23202-8

  • Structure of a fibronectin type III domain from tenascin phased by MAD analysis of the selenomethionyl protein.

    Leahy DJ, Hendrickson WA, Aukhil I and Erickson HP

    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032.

    Fibronectin type III domains are found in many different proteins including cell surface receptors and cell adhesion molecules. The crystal structure of one such domain from the extracellular matrix protein tenascin was determined. The structure was solved by multiwavelength anomalous diffraction (MAD) phasing of the selenomethionyl protein and has been refined to 1.8 angstrom resolution. The folding topology of this domain is identical to that of the extracellular domains of the human growth hormone receptor, the second domain of CD4, and PapD. Although distinct, this topology is similar to that of immunoglobulin constant domains. An Arg-Gly-Asp (RGD) sequence that can function for cell adhesion is found in a tight turn on an exposed loop.

    Funded by: NCI NIH HHS: CA-47056; NIDCR NIH HHS: DE-07801; NIGMS NIH HHS: GM-34102

    Science (New York, N.Y.) 1992;258;5084;987-91

  • Structure of the human hexabrachion (tenascin) gene.

    Gulcher JR, Nies DE, Alexakos MJ, Ravikant NA, Sturgill ME, Marton LS and Stefansson K

    Department of Neurology, University of Chicago, IL 60637.

    The structure of the gene encoding human hexabrachion (tenascin) has been determined from overlapping clones isolated from a human genomic bacteriophage library. The genomic inserts were characterized by restriction mapping, Southern blot analysis, PCR, and DNA sequencing. The coding region of the hexabrachion gene spans approximately 80 kilobases of DNA and consists of 27 exons separated by 26 introns. The exon-intron structure supports a hypothesis based on the cDNA sequence that the hexabrachion gene is an assembly of DNA modules that are also found elsewhere in the genome. Single exons may encode a module, a portion of a module, or a group of modules. The 15 type III units similar to those found in fibronectin are each encoded either by a single exon or by two exons interrupted by an intron. All type III units known to be spliced out of the smaller forms of the protein are encoded by one exon. The fibrinogen-like domain of 210 amino acids is encoded by five exons. The 14.5 epidermal growth factor-like repeats are all encoded by a single exon.

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;21;9438-42

  • The complete cDNA sequence of human hexabrachion (Tenascin). A multidomain protein containing unique epidermal growth factor repeats.

    Nies DE, Hemesath TJ, Kim JH, Gulcher JR and Stefansson K

    Department of Neurology, University of Chicago, Illinois 60637.

    Hexabrachion (Tenascin) is a large glycoprotein that appears in extracellular matrices as a disulfide-linked multimer. It is synthesized in an ordered fashion at particular sites during development, is made in large amounts by certain tumors, and is found in restricted tissue locations in the adult. In this report, we describe the sequence of a full length cDNA of human hexabrachion. The encoded protein contains a total of 2203 amino acids and is a linear array of discrete reiterated domains. At the 5' end are encoded hydrophobic residues and 8 flanking cysteines predicted to be responsible for assembly of hexabrachion polypeptides into a radially arranged, six-armed complex. Following this region are 14 1/2 contiguous 31-amino acid epidermal growth factor-like repeats that have a unique structure with respect to the known examples of this type of domain. Immediately adjacent to these repeats lie 15 uninterrupted segments of approximately 90 amino acids which are similar to the Type III units found in fibronectin. At the carboxyl terminus of the protein is a 210-amino acid domain that is similar to fibrinogen. The domain structure of this protein is consistent with the potential for interaction with multiple ligands and for roles in cell adhesion and/or signaling.

    Funded by: AHRQ HHS: 5 P01 HS-21442-03; NINDS NIH HHS: 1 P01 NS-24575-01

    The Journal of biological chemistry 1991;266;5;2818-23

  • Human tenascin: primary structure, pre-mRNA splicing patterns and localization of the epitopes recognized by two monoclonal antibodies.

    Siri A, Carnemolla B, Saginati M, Leprini A, Casari G, Baralle F and Zardi L

    Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.

    By sequencing cDNA clones which cover the complete coding region of human tenascin (TN), we have established its primary structure. This confirms that, as in the case of chicken, TN is mainly made up of three groups of sequences with a high homology to Epidermal Growth Factor (EGF) fibronectin (FN) type III repeat and fibrinogen. Furthermore, we have determined the amino-terminal sequence of the mature peptide directly on purified TN. The main differences with respect to the chicken TN molecule are that in the human there are 14 and half EGF-like repeats compared to 13 and half in the chicken and that, as previously reported, there are 15 FN-like repeats compared to 11 in the chicken. By Polymerase Chain Reaction (PCR) amplification we have also studied the different splicing patterns of the TN pre-mRNA in cultured cells. The results show the presence of at least four different isoforms containing different numbers of FN-like type III repeats. Using purified human TN as immunogen, we have obtained numerous monoclonal antibodies (Mabs) to TN. By screening a human melanoma cDNA library in the expression vector lambda gt11 with these Mabs and subsequently sequencing the insert of the positive clones, we have been able to localize, within the TN molecule, the epitopes recognized by two of these Mabs: BC-4, which recognizes an epitope within the EGF-like sequence and BC-2 which recognizes an epitope within the FN like type III repeats whose expression is regulated by alternative splicing of the TN pre-mRNA. Thus, while the Mab BC-4 may be useful in studies on TN distribution (since it recognizes all different TN isoforms) BC-2 may be useful in the study of the expression of particular TN isoforms generated by the alternative splicing of the TN primary transcript.

    Nucleic acids research 1991;19;3;525-31

  • An alternatively spliced region of the human hexabrachion contains a repeat of potential N-glycosylation sites.

    Gulcher JR, Nies DE, Marton LS and Stefansson K

    Department of Neurology, University of Chicago, IL 60637.

    We have cloned and sequenced two cDNA molecules that code for parts of two forms of human hexabrachion. The smaller clone has a sequence that corresponds to the previously published sequence of a cDNA clone coding for a part of chicken hexabrachion [Jones, F. S., Burgoon, M. P., Hoffman, S., Crossin, K. L., Cunningham, B. A. & Edelman, G. M. (1988) Proc. Natl. Acad. Sci. USA 85, 2186-2190]. It has eight consecutive domains similar to the type III homology units from fibronectin, several epidermal growth factor repeats, and a domain similar to the beta and gamma chains of fibrinogen. The larger clone has 5' and 3' ends that are identical to the smaller clone but also has an alternatively spliced 1.9-kilobase internal segment. The unique segment contains remarkable repeats of potential glycosylation sites and an additional seven type III homology units.

    Funded by: AHRQ HHS: 5P01 HS-21442-03

    Proceedings of the National Academy of Sciences of the United States of America 1989;86;5;1588-92

Gene lists (4)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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