G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
G00000293 (Mus musculus)

Databases (6)

ENSG00000038427 (Ensembl human gene)
1462 (Entrez Gene)
629 (G2Cdb plasticity & disease)
118661 (OMIM)
Marker Symbol
HGNC:2464 (HGNC)
Protein Sequence
P13611 (UniProt)

Synonyms (1)

  • PG-M

Literature (81)

Pubmed - other

  • The intracranial aneurysm susceptibility genes HSPG2 and CSPG2 are not associated with abdominal aortic aneurysm.

    Baas AF, Medic J, van't Slot R, de Vries JP, van Sambeek MR, Verhoeven EL, Boll BP, Grobbee DE, Wijmenga C, Blankensteijn JD and Ruigrok YM

    Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, the Netherlands. a.f.baas@umcutrecht.nl

    Background: A genetic variant on chromosome 9p21 associates with abdominal aortic aneurysm (AAA) and intracranial aneurysm (IA), indicating that despite the differences in pathology there are shared genetic risk factors. We investigated whether the IA susceptibility genes heparan sulfate proteoglycan 2 (HSPG2) and chondroitin sulfate proteoglycan 2 (CSPG2) associate with AAA as well.

    Methods: Using tag single nucleotide polymorphisms (SNPs), all common variants were analyzed in a Dutch AAA case-control population in a 2-stage genotyping approach. In stage 1, 12 tag SNPs in HSPG2 and 22 tag SNPs in CSPG2 were genotyped in 376 patients and 648 controls. Genotyping of significantly associated SNPs was replicated in a second independent cohort of 360 cases and 376 controls.

    Results: In stage 1, no HSPG2 SNPs and 1 CSPG2 SNP associated with AAA (rs2652106, P = .019). Association of this SNP was not replicated (P = .342).

    Conclusions: Our findings demonstrate that, in contrast to IA, HSPG2 and CSPG2 do not associate with AAA.

    Angiology 2010;61;3;238-42

  • Versican overexpression in human breast cancer lesions: known and new isoforms for stromal tumor targeting.

    Kischel P, Waltregny D, Dumont B, Turtoi A, Greffe Y, Kirsch S, De Pauw E and Castronovo V

    Department of Biology, Metastasis Research Laboratory, GIGA Cancer, University of Liege, Liège, Belgium. philippe.kischel@ulg.ac.be

    Proteoglycans play a key role in cancer development and progression by participating in the constitution of a specific fertile tumor microenvironment. As they are largely overexpressed in the malignant stroma, proteoglycans provide a reservoir of potential new targets for anticancer therapies, because they can serve to convey toxic payloads in the close proximity of cancer cells and subsequently destroy them. In this context, versican, a proteoglycan largely overexpressed in several solid cancers, bears the potential to be such an ideal target. As 4 main versican isoforms have been characterized, we sought to determine which isoform could represent the best target in human breast cancer. We used a series of 10 primary breast cancer lesions that were characterized as overexpressing the versican protein, when compared with matched normal breast tissues, using shotgun mass spectrometry and immunohistochemistry experiments. Quantitative polymerase chain reaction and western-blotting experiments were used to evaluate versican isoform expression in breast cancer/normal tissue pairs for which ARN quality was excellent. All known isoforms were significantly overexpressed in the malignant lesions, both at the mRNA and at the protein levels. In the course of this study, we also identified and cloned a new alternatively spliced versican isoform, referred to as V4, which was also found to be upregulated in human breast cancer. This study provides for the first time a comprehensive mRNA and protein analysis of versican isoforms expression in human breast tissues, and offers insights into which therapeutic strategy would be best suited to target versican in human breast cancer lesions.

    International journal of cancer 2010;126;3;640-50

  • Genetic variants A1826H and D2937Y in GAG-beta domain of versican influence susceptibility to intestinal-type gastric cancer.

    Ju H, Lim B, Kim M, Noh SM, Han DS, Yu HJ, Choi BY, Kim YS, Kim WH, Ihm C and Kang C

    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-gu, Daejeon, 305-701, Korea.

    Purpose: Versican regulates adhesion, migration, proliferation, and survival of cells, and plays an important role in cancer development. A case-control association study was performed to test genetic association of versican polymorphisms with susceptibility to gastric cancer.

    Methods: In this study, 1,101 unrelated Korean subjects including 612 gastric cancer patients and 489 healthy controls were genotyped for all 21 exonic polymorphisms in the versican gene (VCAN) encoding amino acid changes in versican. Cancer susceptibility associations with the polymorphisms were assessed using multivariate logistic regression analysis with adjustment for age and gender and with control for multiple testing.

    Results: Two amino acid changes in GAG-beta domain of versican encoded by two almost fully correlated (r (2) = 0.97) nonsynonymous single-nucleotide polymorphisms in VCAN were associated with gastric cancer. The association was evident in intestinal-type but not in diffuse-type gastric cancer. The minor-allele homozygote of rs188703 (G > A, R1826H) or rs160277 (G > T, D2937Y) was significantly associated with a twofold decreased susceptibility to intestinal-type gastric cancer when compared with the other genotypes (adjusted odds ratio = 0.52 or 0.51, P = 0.0098 or 0.0087, respectively).

    Conclusions: The intestinal-type gastric cancer susceptibility is associated with two amino acid changes of versican in the GAG-beta domain, which is critical for enhancement of cell proliferation and activation of EGFR signal pathway by versican, and changes from the major to minor alleles may impair the function to decrease susceptibility to cancer.

    Journal of cancer research and clinical oncology 2010;136;2;195-201

  • Transforming growth factor beta3 regulates the versican variants in the extracellular matrix-rich uterine leiomyomas.

    Norian JM, Malik M, Parker CY, Joseph D, Leppert PC, Segars JH and Catherino WH

    Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.

    Uterine leiomyoma are common, benign tumors that are enriched in extracellular matrix. The tumors are characterized by a disoriented and loosely packed collagen fibril structure similar to other diseases with disrupted Transforming growth factor beta (TGF-beta) signaling. Here we characterized TGF-beta3 signaling and the expression patterns of the critical extracellular matrix component versican in leiomyoma and myometrial tissue and cell culture. We also demonstrate the regulation of the versican variants by TGF-beta3. Using leiomyoma and matched myometrium from 15 patients, messenger RNA (mRNA) from leiomyoma and myometrium was analyzed by semiquantitative real time reverse transcription-polymerase chain reaction (RT-PCR), while protein analysis was done by western blot. Transforming growth factor beta3 transcripts were increased 4-fold in leiomyoma versus matched myometrium. Phosphorylated-TGF-beta RII and phosphorylated-Smad 2/3 complex were greater in leiomyoma as documented by Western blot. The inhibitor Smad7 transcripts were decreased 0.44-fold. The glycosaminoglycan (GAG)-rich versican variants were elevated in leiomyoma versus myometrial tissue: specifically V0 (4.27 +/- 1.12) and V1 (2.01 +/- 0.27). Treatment of leiomyoma and myometrial cells with TGF-beta3 increased GAG-rich versican variant expression 7 to 12 fold. Neutralizing TGF-beta3 antibody decreased the expression of the GAG-rich versican variants 2 to 8 fold in leiomyoma cells. Taken together, the aberrant production of excessive and disorganized extracellular matrix that defines the leiomyoma phenotype involves the activation of the TGF-beta signaling pathway and excessive production of GAG-rich versican variants.

    Funded by: Intramural NIH HHS: Z01 HD008737-07

    Reproductive sciences (Thousand Oaks, Calif.) 2009;16;12;1153-64

  • Mutational hot spot potential of a novel base pair mutation of the CSPG2 gene in a family with Wagner syndrome.

    Ronan SM, Tran-Viet KN, Burner EL, Metlapally R, Toth CA and Young TL

    Duke University Eye Center, Durham, NC 27710, USA.

    Objective: To report a 3-generation white family clinically diagnosed variably with Wagner, Stickler, and Jansen syndromes and screened for sequence variants in the COL2A1 and CSPG2 genes. Wagner syndrome is an autosomal dominant vitreoretinopathy with a predisposition to retinal detachment and cataracts. It has significant phenotypic overlap with allelic Jansen syndrome and ocular Stickler syndrome type 1. Sticker syndrome type 1 maps to chromosome 12q13.11-q13.2, with associated COL2A1 gene mutations. Wagner syndrome maps to chromosome 5q13-q14 and is associated with mutations in CSPG2 encoding versican, a proteoglycan present in human vitreous.

    Methods: Genomic DNA samples derived from venous blood were collected from all family members. Complete sequencing of COL2A1 was performed on a proband. Primers for polymerase chain reaction and sequencing were designed to cover all exon and intron-exon boundaries. Direct sequencing of CSPG2 was performed on all family member samples.

    Results: No detectable COL2A1 mutations were noted, making the diagnosis of ocular Stickler syndrome highly unlikely for this family. A unique base pair substitution (c.9265 + 1G>T) in intron 8 of the CSPG2 gene cosegregating with disease status was identified. This mutation occurred in a highly conserved previously reported splice site with a similar base pair substitution (G>A). Direct sequencing of this splice site mutation in 107 unrelated external controls revealed no variants, supporting the rarity of this base pair change and its causation in Wagner syndrome. This novel base pair substitution is thought to cause the deletion of exon 8 and formation of a truncated protein product.

    Conclusion: Mutation screening of CSPG2 in autosomal dominant vitreoretinopathy families is important for accurate diagnosis.

    This study underscores the importance of obtaining extensive pedigree information and comparative ophthalmologic clinical information, as the phenotypic findings may vary greatly among independent family members. The study also affirms the paradigm shift from diagnosis assignment based on eponyms to that based on gene mutation type.

    Funded by: NEI NIH HHS: EY014685, R01 EY014685

    Archives of ophthalmology (Chicago, Ill. : 1960) 2009;127;11;1511-9

  • Topographical variation in the distributions of versican, aggrecan and perlecan in the foetal human spine reflects their diverse functional roles in spinal development.

    Smith SM, Whitelock JM, Iozzo RV, Little CB and Melrose J

    Raymond Purves Bone and Joint Research Laboratories, Institute of Bone and Joint Research, Kolling Institute of Medical Research, The Royal North Shore Hospital, University of Sydney, Level 10, Building B6, St. Leonards, NSW, 2065, Australia.

    We evaluated the immunohistochemical distribution of three major proteoglycans of cartilage, i.e., aggrecan, versican and perlecan vis-a-vis collagens I and II in the developing human spine of first-trimester foetuses. Aggrecan and perlecan were prominently immunolocalised in the cartilaginous vertebral body rudiments and to a lesser extent within the foetal intervertebral disc. In contrast, versican was only expressed in the developing intervertebral disc interspace. Using domain-specific monoclonal antibodies against the various modules of versican, we discovered the V0 isoform as the predominant form present. Versican immunolocalisations conducted with antibodies directed to epitopes in its N and C termini and GAG-alpha and GAG-beta core protein domains provided evidence that versican in the nucleus pulposus was either synthesised devoid of a G3 domain or this domain was proteolytically removed in situ. The V0 versican isoform was localised with prominent fibrillar components in the annular lamellae of the outer annulus fibrosus. Perlecan was a notable pericellular proteoglycan in the annulus fibrosus and nucleus pulposus but poorly immunolocalised in the marginal tissues of the developing intervertebral disc, apparently delineating the intervertebral disc-vertebral body interface region destined to become the cartilaginous endplate in the mature intervertebral disc. The distribution of collagens I and II in the foetal spine was mutually exclusive with type I present in the outer annulus fibrosus, marginal tissues around the vertebral body rudiment and throughout the developing intervertebral disc, and type II prominent in the vertebral rudiment, absent in the outer annulus fibrosus and diffusely distributed in the inner annulus fibrosus and nucleus pulposus. Collectively, our findings suggest the existence of an intricate and finely balanced interplay between various proteoglycans and collagens and the spinal cell populations which synthesise and assemble these components during spinal development.

    Histochemistry and cell biology 2009;132;5;491-503

  • Abundance and location of proteoglycans and hyaluronan within normal and myxomatous mitral valves.

    Gupta V, Barzilla JE, Mendez JS, Stephens EH, Lee EL, Collard CD, Laucirica R, Weigel PH and Grande-Allen KJ

    Department of Bioengineering, Rice University, 6100 Main Street, Houston, TX 77251, USA.

    Introduction: Extracellular matrix changes occur in many heart valve pathologies. For example, myxomatous mitral valves are reported to contain excess proteoglycans and hyaluronan. However, it is unknown which specific proteoglycans are altered in myxomatous valves. Because proteoglycans perform varied functions in connective tissues, this study was designed to identify and localize three matrix-associated proteoglycans, as well as hyaluronan and the hyaluronan receptor for endocytosis, within myxomatous and normal mitral valves.

    Methods: Human mitral posterior leaflets (control, n=6-9; myxomatous, n=14-21; mean age, 61 years for all groups) were histochemically stained for proteoglycan core proteins, hyaluronan, and the hyaluronan receptor for endocytosis. Stain intensity was semiquantitatively graded to determine differences in marker abundance between normal and myxomatous valves. The proteoglycans were localized to different regions of the leaflet by correspondence to parallel Movat-stained sections.

    Results: The proteoglycans decorin, biglycan, and versican were more abundant in myxomatous valves than in normal controls (P<.03). There was a gender effect on proteoglycan presence, but no age-related trends were observed. Hyaluronan and the hyaluronan receptor for endocytosis were distributed throughout all valves. There was no significant difference in hyaluronan between groups, but expression of the hyaluronan receptor for endocytosis was reduced in myxomatous valves compared to normal controls (P<.002).

    Conclusion: Excess decorin, biglycan, and versican may be associated with the remodeling of other matrix components in myxomatous mitral valves. Decreased expression of the hyaluronan receptor for endocytosis in myxomatous valves suggests that hyaluronan metabolism could be altered in myxomatous mitral valve disease. These findings contribute towards elucidating the pathogenesis of myxomatous mitral valve disease and developing potential new therapies.

    Funded by: NHLBI NIH HHS: F30 HL094019, HL 081558, R21 HL081558; NIGMS NIH HHS: GM 69961, R01 GM069961, R01 GM069961-01A1, R01 GM069961-02, R01 GM069961-03, R01 GM069961-04, T32 GM008362

    Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology 2009;18;4;191-7

  • The biological role and regulation of versican levels in cancer.

    Ricciardelli C, Sakko AJ, Ween MP, Russell DL and Horsfall DJ

    Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA, 5005, Australia. carmela.ricciardelli@adelaide.edu.au

    Increased expression of the proteoglycan, versican is strongly associated with poor outcome for many different cancers. Depending on the cancer type, versican is expressed by either the cancer cells themselves or by stromal cells surrounding the tumor. Versican plays diverse roles in cell adhesion, proliferation, migration and angiogenesis, all features of invasion and metastasis. These wide ranging functions have been attributed to the central glycosaminoglycan-binding region of versican, and to the N-(G1) and C-(G3) terminal globular domains which collectively interact with a large number of extracellular matrix and cell surface structural components. Here we review the recently identified mechanisms responsible for the regulation of versican expression and the biological roles that versican plays in cancer invasion and metastasis. The regulation of versican expression may represent one mechanism whereby cancer cells alter their surrounding microenvironment to facilitate the malignant growth and invasion of several tumor types. A greater understanding of the regulation of versican expression may contribute to the development of therapeutic methods to inhibit versican function and tumor invasion.

    Cancer metastasis reviews 2009;28;1-2;233-45

  • Structure and regulation of the versican promoter: the versican promoter is regulated by AP-1 and TCF transcription factors in invasive human melanoma cells.

    Domenzain-Reyna C, Hernández D, Miquel-Serra L, Docampo MJ, Badenas C, Fabra A and Bassols A

    Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.

    Versican is a large chondroitin sulfate proteoglycan of the extracellular matrix that is involved in a variety of cellular processes. We showed previously that versican, which is overexpressed in cutaneous melanomas as well as in premalignant lesions, contributes to melanoma progression, favoring the detachment of cells and the metastatic dissemination. Here, we investigated the transcriptional regulation of the versican promoter in melanoma cell lines with different levels of biological aggressiveness and stages of differentiation. We show that versican promoter up-regulation accounts for the differential expression levels of mRNA and protein detected in the invasive SK-mel-131 human melanoma cells. The activity of the versican promoter increased 5-fold in these cells in comparison with that measured in non-invasive MeWo melanoma cells. Several transcriptional regulatory elements were identified in the proximal promoter, including AP-1, Sp1, AP-2, and two TCF-4 sites. We show that promoter activation is mediated by the ERK/MAPK and JNK signaling pathways acting on the AP-1 site, suggesting that BRAF mutation present in SK-mel-131 cells impinge upon the up-regulation of the versican gene through signaling elicited by the ERK/MAPK pathway. This is the first time the AP-1 transcription factor family has been shown to be related to the regulation of versican expression. Furthermore, deletion of the TCF-4 binding sites caused a 60% decrease in the promoter activity in SK-mel-131 cells. These results showing that AP-1 and TCF-4 binding sites are the main regulatory regions directing versican production provide new insights into versican promoter regulation during melanoma progression.

    The Journal of biological chemistry 2009;284;18;12306-17

  • Deregulation of versican and elastin binding protein in solar elastosis.

    Knott A, Reuschlein K, Lucius R, Stäb F, Wenck H and Gallinat S

    Beiersdorf AG, Paul Gerson Unna Skin Research Center, P.O. Box 519, Unnastrasse 48, 20245, Hamburg, Germany. Anja.Knott@Beiersdorf.com

    Several changes in skin appearance including loss of elasticity and wrinkle formation are associated with alterations in the composition of the dermal extracellular matrix. They are induced by intrinsic aging or by environmental factors such as UV light referred to as photoaging. A general characteristic in the histology of photoaged skin is the accumulation of elastotic material suggesting impaired formation and/or massive breakdown of elastic fibres. In order to shed light on some of the underlying mechanisms we tracked two of the major players in elastic fibre formation in different skin conditions: EBP (elastin binding protein), a regulator of elastic fibre assembly and VER (versican), a component of functional elastic fibres as well as non-functional elastotic material. Using quantitative RT-PCR on skin biopsies we found that the expression levels of VER and EBP were unaltered during intrinsic skin aging. Upon acute UV stress however, VER and EBP showed different regulation patterns: VER mRNA increased after 6 h and was further up-regulated until 24 h. The EBP mRNA by contrast was reduced after 6 h but showed massive induction at 24 h after acute UV stress. In chronically sun-exposed skin, VER protein was accumulated similar to elastotic material in the extracellular space, whereas its mRNA level was consistently reduced compared to sun-protected skin. The EBP mRNA by contrast showed slightly increased expression levels in the sun-exposed area compared to its sun-protected counterpart. Based on these data we propose a model which may help to explain parts of the mechanisms leading to the formation of elastotic masses. We further hypothesize that the presence of elastotic material triggers some yet unknown feedback mechanism(s) resulting in altered expression patterns of VER and EBP in chronically sun-exposed skin.

    Biogerontology 2009;10;2;181-90

  • The accumulation of versican in the nodules of benign prostatic hyperplasia.

    True LD, Hawley S, Norwood TH, Braun KR, Evanko SP, Chan CK, LeBaron RC and Wight TN

    Department of Pathology, University of Washington, Seattle, Washington 98195-6100, USA. ltrue@u.washington.edu

    Background: Proteoglycans, a complex group of extracellular matrix (ECM) molecules, are elevated in benign prostatic hyperplasia (BPH). Versican is a stromal proteoglycan present in prostate tissue. Versican expression is elevated in tissues with increased proliferation. Based on these observations, we determined the extent and distribution of versican expression in prostates with BPH.

    Methods: The involvement of versican in BPH nodules was compared with levels in non-nodular transition (TZ) and peripheral zone (PZ) tissues from 18 human prostate glands using immunohistochemistry, Northern blots and/or QRTPCR to localize versican and quantify versican mRNA transcript levels, and Western blots to assess gene product levels.

    Results: Increased versican immunoreactivity was observed in the stroma of BPH nodules. Higher steady state levels of versican variants V0, V1, and V3 mRNA transcript and gene product were detected in the nodular tissues than in the non-nodular TZ or PZ parenchyma.

    Conclusions: These results suggest that versican may play a role in nodule formation in BPH.

    Funded by: NIA NIH HHS: R03 AG019492-01, R03 AG19492-01; NIDA NIH HHS: R03 DA019492

    The Prostate 2009;69;2;149-58

  • Association analysis of genes involved in the maintenance of the integrity of the extracellular matrix with intracranial aneurysms in a Japanese cohort.

    Ruigrok YM, Rinkel GJ, Wijmenga C, Kasuya H, Tajima A, Takahashi T, Hata A, Inoue I and Krischek B

    Department of Neurology, Rudolf Magnus Institute of Neuroscience, Utrecht, The Netherlands.

    Background: An association between versican (CSPG2), perlecan (HSPG2), fibrillin 2 (FBN2) and collagen 4A1 (COL4A1) gene variants and intracranial aneurysms (IA) has been reported in 2 studies analyzing Dutch IA patients. The aim of this study was to verify these associations in a Japanese IA population. In addition, a meta-analysis on the association of these genes and IA for the combined Dutch and Japanese populations was performed.

    Methods: The associated single nucleotide polymorphisms (SNPs) in these genes identified in the Dutch study were genotyped in 632 Japanese IA patients and 808 healthy control subjects using TaqMan SNP genotyping assays.

    Results: A similar association to that previously found in the Dutch population was found for the CSPG2 (rs251124) and HSPG2 (rs3767137) SNPs, although both associations were not statistically significant in the Japanese population (CSPG2 OR 1.18, 95% CI 0.98-1.41, p = 0.08; HSPG2 OR 1.09, 95% CI 0.90-1.32). Combining the Dutch and Japanese data for a meta-analysis showed an overall association between the CSPG2 SNP and IA (OR 1.29, 95% CI 1.12-1.48, p = 0.0005) and the HSPG2 SNP and IA (OR 1.22, 95% CI 1.08-1.39, p = 0.002). No differences in SNP frequency were observed for FBN2 and COL4A1 between Japanese patients and controls.

    Conclusions: By analyzing HSPG2, CSPG2, FBN2 and COL4A1, we were able to replicate the association of CSPG2 and show that there is a trend for HSPG2 towards association in the Japanese IA population by means of a meta-analysis combining the Dutch and Japanese results. The association of FBN2 and COL4A1 could not be replicated in the Japanese IA population.

    Cerebrovascular diseases (Basel, Switzerland) 2009;28;2;131-4

  • Versican overexpression in cutaneous malignant melanoma.

    Gambichler T, Kreuter A, Grothe S, Altmeyer P, Brockmeyer NH and Rotterdam S

    Department of Dermatology and Allergology, Ruhr-University Bochum, Gudrunstr. 56, 44791 Bochum, Germany. t.gambichler@klinikum-bochum.de

    Objective: Tumor growth regulation by extracellular matrix components has been one of the main topics on tumor biology in the last years. We aimed to investigate the protein expression pattern of decorin and versican in superficial spreading melanoma (SSM) and its precursors.

    Paraffin-embedded sections of benign nevi (BN), dysplastic nevi (DN), and primary SSM were assessed. Immunohistochemistry was performed for decorin and versican antibodies.

    Results: We investigated 64 patients with BN (n = 29), DN (n = 15), and SSM (n = 20) with a median Breslow thickness of 0.8 mm (0.2 - 4.6 mm). We did not observe decorin or versican immunoreactivity in melanocytes but in peritumoral stroma. Kruskal-Wallis ANOVA did not reveal significant differences of decorin expression between the groups investigated (P = 0.19). However, compared to BN and DN median expression of versican was significantly increased in SSM (P = 0.016 and P = 0.019, respectively). Decorin as well versican expression of SSM did not significantly correlate with Breslow tumor thickness or Clark level.

    Conclusion: Our data indicate that decorin is not differentially expressed in peritumoral stroma of SSM, DN, BN, and thus unlikely of pathogenetic significance in melanoma transformation and/or progression. By contrast, we have demonstrated that SSM is associated with a significant overexpression of peritumoral versican suggesting a role for versican in the pathogenesis of melanoma.

    European journal of medical research 2008;13;11;500-4

  • Androgen receptor regulation of the versican gene through an androgen response element in the proximal promoter.

    Read JT, Rahmani M, Boroomand S, Allahverdian S, McManus BM and Rennie PS

    Prostate Center, Vancouver General Hospital, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada.

    Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor (AR)-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and dihydrotestosterone stimulate expression of a versican promoter-driven luciferase reporter vector (versican-Luc). Further, both basal and androgen-stimulated versican-Luc activities were significantly diminished in LNCaP cells, when AR gene expression was knocked down using a short hairpin RNA. Methylation-protection footprinting analysis revealed an AR-protected element between positions +75 and +102 of the proximal versican promoter, which strongly resembled a consensus steroid receptor element. Electrophoretic mobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated versican-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce versican promoter activity and mRNA levels in AR-positive prostate stromal fibroblasts. Interestingly, overexpression of beta-catenin in the presence of androgen augmented versican promoter activity 10- and 30-fold and enhanced versican mRNA levels 2.8-fold in fibroblasts. In conclusion, we demonstrate that AR transactivates versican expression, which may augment tumor-stromal interactions and may contribute to prostate cancer progression.

    The Journal of biological chemistry 2007;282;44;31954-63

  • Pre-eclampsia-associated alterations in decorin, biglycan and versican of the umbilical cord vein wall.

    Gogiel T, Galewska Z, Romanowicz L, Jaworski S and Bańkowski E

    Department of Medical Biochemistry, Medical Academy of Białystok, ul. Mickiewicza 2, 15-089 Białystok-1, Poland. tgogiel@amb.edu.pl

    Objective: The role of proteoglycans in the rearrangement of the extracellular matrix of the umbilical cord vein wall in pre-eclampsia is not known. Decorin, biglycan and versican are the main proteoglycans of the umbilical cord vein wall. We decided to test whether the amounts of these proteoglycans alter in pre-eclampsia.

    Study was performed on the umbilical cord veins taken from 10 newborns delivered by healthy mothers (control group) and from 10 newborns delivered by mothers with pre-eclampsia. Proteoglycans were extracted in dissociative conditions, purified by Q-Sepharose anion exchange chromatography and lyophilised. Decorin, biglycan and versican were analysed by SDS-PAGE followed by Western blotting before and after treatment with chondroitinase ABC. The amounts of decorin, biglycan and versican core proteins were assessed by ELISA method.

    Results: We found that both control and pre-eclamptic umbilical cord vein wall contained all the three proteoglycans. ELISA assay showed the amounts of the core proteins of decorin, biglycan and versican were distinctly higher in pre-eclamptic material in comparison to control vessel. Western blotting confirmed that the expression of all these proteoglycan core proteins increased in pre-eclampsia. They featured in the same electrophoretic mobility-45 and 47 kDa for decorin, 45 kDa for biglycan, and 300 and 320 kDa for versican.

    Conclusion: The content of decorin, biglycan and versican in the umbilical cord vein wall is elevated in pre-eclampsia in comparison to the corresponding control vessel. These alterations may affect the mechanical properties of this vessel and disturb foetal blood circulation.

    European journal of obstetrics, gynecology, and reproductive biology 2007;134;1;51-6

  • Versican, a major hyaluronan-binding component in the dermis, loses its hyaluronan-binding ability in solar elastosis.

    Hasegawa K, Yoneda M, Kuwabara H, Miyaishi O, Itano N, Ohno A, Zako M and Isogai Z

    Department of Biochemistry and Molecular Biology, Aichi Prefectural College of Nursing and Health, Nagoya, Aichi, Japan.

    Versican interacts with hyaluronan (HA) at its N-terminus and with fibrillin-1 at its C terminus. As versican in the dermis connects microfibrils to the HA-rich matrix for viscoelasticity, dermal diseases may involve destruction of these complexes. A recombinant versican protein, rVN, covering the HA binding region (HABR) of human versican and a polyclonal antibody, 6084, against rVN were prepared and characterized. Blotting analyses of skin extracts with 6084 and biotin-conjugated HA revealed that versican was a major HA-binding component in the dermis. Matrix metalloprotease-12, which is expressed in areas of solar elastosis, degraded versican and abrogated its HA-binding ability. Immunohistochemical analyses revealed that the elastic materials in solar elastosis lesions were negative for 6084, but positive for 2B1, an antibody recognizing the C-terminus of versican, indicating loss of the HABR in the aggregated elastic fibers. This loss of the HA-binding ability of versican followed by HA exclusion may be responsible for the pathological and phenotypical changes observed in solar elastosis.

    The Journal of investigative dermatology 2007;127;7;1657-63

  • The role of versican isoforms V0/V1 in glioma migration mediated by transforming growth factor-beta2.

    Arslan F, Bosserhoff AK, Nickl-Jockschat T, Doerfelt A, Bogdahn U and Hau P

    Department of Neurology, University of Regensburg, Universitaetsstrasse 84, Regensburg 93053, Germany.

    Versican is a large chondroitin sulphate proteoglycan produced by several tumour cell types, including high-grade glioma. The increased expression of certain versican isoforms in the extracellular matrix (ECM) plays a role in tumour cell growth, adhesion and migration. Transforming growth factor-beta2 (TGF-beta2) is an important modulator of glioma invasion, partially by remodeling the ECM. However, it is unknown whether it interacts with versican during malignant progression of glioma cells. Here, we analysed the effect of TGF-beta2 on the expression of versican isoforms. The expression of versican V0/V1 was upregulated by TGF-beta2 detected by quantitative polymerase chain reaction and immunoprecipitation, whereas V2 was not induced. Using time-lapse scratch and spheroid migration assays, we observed that the glioma migration rate is significantly increased by exogenous TGF-beta2 and inhibited by TGF-beta2-specific antisense oligonucleotides. Interestingly, an antibody specific for the DPEAAE region of glycosaminoglycan-beta domain of versican was able to reverse the effect of TGF-beta2 on glioma migration in a dose-dependent manner. Taken together, we report here that TGF-beta2 triggers the malignant phenotype of high-grade gliomas by induction of migration, and that this effect is, at least in part, mediated by versican V0/V1.

    British journal of cancer 2007;96;10;1560-8

  • Prognostic significance of stromal versican expression in human endometrial cancer.

    Kodama J, Hasengaowa, Kusumoto T, Seki N, Matsuo T, Ojima Y, Nakamura K, Hongo A and Hiramatsu Y

    Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. kodama@cc.okayama-u.ac.jp

    Background: Versican expression may enhance tumor invasion and metastasis. However, the expression of versican in human endometrial cancer has seldom been characterized. The aim of this study was to investigate versican expression in endometrial cancers.

    We immunohistochemically investigated the expression of versican protein in 167 endometrial cancers and analyzed the correlation with various observed clinicopathological features, including patient outcome.

    Results: Stromal versican expression was significantly higher in the advanced-stage (P = 0.010) and high-grade (P = 0.049) cancers, lymph node metastasis (P = 0.012), and ovarian metastasis (P = 0.024). Epithelial versican expression was significantly higher in patients with lymph node metastasis (P = 0.014) and lymph-vascular space involvement (P = 0.014). The disease-free survival (DFS) and overall survival (OS) rates of patients exhibiting high stromal versican expression were significantly lower than those of patients exhibiting low stromal versican expression (P < 0.0001). Multivariate analysis showed that high stromal versican expression was an independent prognostic factor for DFS and OS.

    Conclusions: Versican enrichment of the stroma may be associated with tumor progression in endometrial cancer. Stromal versican expression can serve as an indicator of poor prognosis for patients with endometrial cancer.

    Annals of oncology : official journal of the European Society for Medical Oncology 2007;18;2;269-74

  • Chondroitin sulfate proteoglycan 2 (CSPG2) gene polymorphisms rs173686 and rs251124 are not associated with intracranial aneurysms in Chinese Han nationality.

    Sun H, Zhang D and Zhao J

    Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.

    Background: There is evidence suggesting that genetic variants in the chondroitin sulfate proteoglycan2 (CSPG2, also known as versican) gene are involved in the pathogenesis of intracranial aneurysms (IAs). Some authors have demonstrated that single nucleotide polymorphisms (SNPs) rs173686 and rs251124 in the promoter region of the CSPG2 gene are associated with IAs. We performed a case-control study to investigate whether these SNPs might affect the development of IAs in Chinese Han nationality.

    Methods: The study group comprised 240 Chinese Han nationality patients with at least one intracranial aneurysm and 240 healthy Han nationality controls. Genomic DNA was isolated from blood leukocytes. The SNPs rs173686 and rs251124 were genotyped by PCR amplification and DNA sequencing. Differences in genotype and allele frequencies between patients and controls were tested by the chi-square method.

    Results: Genotype and allele frequencies of the SNPs rs173686 and rs251124 were both demonstrated to be in Hardy-Weinberg equilibrium. No significant difference in genotype or allele frequencies between case and control groups was detected at either of the two SNPs.

    Conclusions: The data do not support the hypothesis that the two SNPs (rs173686 and rs251124) in the promoter region of the CSPG2 gene influence the development of intracranial aneurysms in Chinese Han nationality.

    Upsala journal of medical sciences 2007;112;3;289-95

  • Identification of genes related to Parkinson's disease using expressed sequence tags.

    Kim JM, Lee KH, Jeon YJ, Oh JH, Jeong SY, Song IS, Kim JM, Lee DS and Kim NS

    Laboratory of Human Genomics, Genome Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) Daejeon, Korea.

    In a search for novel target genes related to Parkinson's disease (PD), two full-length cDNA libraries were constructed from a human normal substantia nigra (SN) and a PD patient's SN. An analysis of the gene expression profiles between them was done using the expressed sequence tags (ESTs) frequency. Data for the differently expressed genes were verified by quantitative real-time RT-PCR, immunohistochemical analysis and a cell death assay. Among the 76 genes identified with a significant difference (P > 0.9), 21 upregulated genes and 13 downregulated genes were confirmed to be differentially expressed in human PD tissues and/or in an MPTP-treated mice model by quantitative real-time RT-PCR. Among those genes, an immunohistochemical analysis using an MPTP mice model for alpha-tubulin including TUBA3 and TUBA6 showed that the protein levels are downregulated, as well as the RNA levels. In addition, MBP, PBP and GNAS were confirmed to accelerate cell death activity, whereas SPP1 and TUBA3 to retard this process. Using an analysis of ESTs frequency, it was possible to identify a large number of genes related to human PD. These new genes, MBP, PBP, GNAS, SPP1 and TUBA3 in particular, represent potential biomarkers for PD and could serve as useful targets for elucidating the molecular mechanisms associated with PD.

    DNA research : an international journal for rapid publication of reports on genes and genomes 2006;13;6;275-86

  • The versican gene and the risk of intracranial aneurysms.

    Ruigrok YM, Rinkel GJ and Wijmenga C

    Department of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Utrecht, The Netherlands. ij.m.ruigrok@neuro.azu.nl

    The proteoglycan versican is an excellent candidate gene for intracranial aneurysms (IAs) because it plays an important role in extracellular matrix assembly and is localized in a previously implicated locus for IAs on chromosome 5q.

    Methods: We analyzed all the common variations using 16-tag single nucleotide polymorphisms (SNPs) and haplotypes in the versican gene using a 2-stage genotyping approach. For stage 1, 16 SNPs were genotyped in 307 cases and 639 controls. For stage 2, the two SNPs yielding the most significant associations (P<0.01) were genotyped in a second independent cohort of 310 cases for confirmation of the associations.

    Results: In stage 1, we found several SNPs in strong linkage disequilibrium and haplotypes constituting these SNPs associated with IAs in the Dutch population (strongest SNP association for rs173686 with odds ratio=1.34, 95% CI=1.09 to 1.65, P=0.004). In stage 2, we confirmed association for the 2 SNPs with the most significant associations (strongest SNP association for rs173686 with odds ratio=1.36, 95% CI=1.11 to 1.67, P=0.003).

    Conclusions: SNPs in strong linkage disequilibrium and haplotypes constituting these SNPs in the versican gene are associated with IAs suggesting that variation in or near the versican gene plays a role in susceptibility to IAs.

    Stroke 2006;37;9;2372-4

  • Erosive vitreoretinopathy and wagner disease are caused by intronic mutations in CSPG2/Versican that result in an imbalance of splice variants.

    Mukhopadhyay A, Nikopoulos K, Maugeri A, de Brouwer AP, van Nouhuys CE, Boon CJ, Perveen R, Zegers HA, Wittebol-Post D, van den Biesen PR, van der Velde-Visser SD, Brunner HG, Black GC, Hoyng CB and Cremers FP

    Department of Human Genetics, Radboud University Nijmegen Medical Centre, The Netherlands.

    Purpose: Linkage intervals for erosive vitreoretinopathy (ERVR) and Wagner disease previously were found to overlap at 5q14.3. In a Japanese family with Wagner disease, a CSPG2/Versican splice site mutation (c.4004-2A-->G) was recently reported that resulted in a 39-nucleotide exon 8 in-frame deletion. We investigated whether CSPG2/Versican was mutated in six Dutch families and one Chinese family with Wagner disease and in a family with ERVR.

    Methods: In all families, extensive ophthalmic examinations, haplotype analysis of the 5q14.3 region, and sequence analysis of CSPG2/Versican were performed. The effects of splice site mutations were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (QPCR).

    Results: Three novel intron 7 sequence variants (c.4004-5T-->C, c.4004-5T-->A, c.4004-1G-->A) were identified in seven families. The c.4004-5T-->C variant was identified in four families with Wagner disease and a family with ERVR. The families were shown to carry the same 5q14.3 haplotype, strongly suggesting that this is a common Dutch founder variant. All three changes segregated with the disease in the respective families and were absent in 250 healthy individuals. In patients with the c.4004-5T-->A and c.4004-1G-->A variants, RT-PCR analysis of CSPG2/Versican showed activation of a cryptic splice site resulting in a 39-nt exon 8 in-frame deletion in splice variant V0. QPCR revealed a highly significant (P < 0.0001) and consistent increase of the V2 (>38-fold) and V3 (>12-fold) splice variants in all patients with intron 7 nucleotide changes and in a Chinese Wagner disease family, in which the genetic defect remains to be found.

    Conclusions: Wagner disease and ERVR are allelic disorders. Seven of the eight families exhibit a variant in intron 7 of CSPG2/Versican. The conspicuous clustering of sequence variants in the splice acceptor site of intron 7 and the consistent upregulation of the V2 and V3 isoforms strongly suggest that Wagner disease and ERVR may belong to a largely overlooked group of diseases that are caused by mRNA isoform balance shifts, representing a novel disease mechanism.

    Investigative ophthalmology & visual science 2006;47;8;3565-72

  • Versican degradation and vascular disease.

    Kenagy RD, Plaas AH and Wight TN

    Center for Cardiovascular Biology and Regenerative Medicine, University of Washington, Department of Surgery, Seattle, WA 98109-4714, USA. rkenagy@u.washington.edu

    Versican is an abundant proteoglycan in the blood vessel wall that is increased after vascular injury and accumulates in advanced atherosclerotic plaques. Versican is a large molecule with domains that mediate binding to cytokines, enzymes, lipoproteins, other extracellular matrix molecules, and signaling receptors. There is evidence that versican exists in the normal, as well as the diseased, vessel wall as discrete fragments, which represent these functional domains. We review the literature on versican degradation in vascular tissue and the function of versican domains, all of which suggest that proteolytic modification of versican may have physiologic as well as pathologic implications for the vascular system.

    Funded by: NHLBI NIH HHS: HL18645, HL30946, P01 HL018645-30, R01 HL030946, R01 HL030946-25

    Trends in cardiovascular medicine 2006;16;6;209-15

  • Versican G3 domain regulates neurite growth and synaptic transmission of hippocampal neurons by activation of epidermal growth factor receptor.

    Xiang YY, Dong H, Wan Y, Li J, Yee A, Yang BB and Lu WY

    Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario M4N 3M5, Canada.

    Versican is one of the major extracellular matrix (ECM) proteins in the brain. ECM molecules and their cleavage products critically regulate the growth and arborization of neurites, hence adjusting the formation of neural networks. Recent findings have revealed that peptide fragments containing the versican C terminus (G3 domain) are present in human brain astrocytoma. The present study demonstrated that a versican G3 domain enhanced cell attachment, neurite growth, and glutamate receptor-mediated currents in cultured embryonic hippocampal neurons. In addition, the G3 domain intensified dendritic spines, increased the clustering of both synaptophysin and the glutamate receptor subunit GluR2, and augmented excitatory synaptic activity. In contrast, a mutated G3 domain lacking the epidermal growth factor (EGF)-like repeats (G3deltaEGF) had little effect on neurite growth and glutamatergic function. Treating the neurons with the G3-conditioned medium rapidly increased the levels of phosphorylated EGF receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase (pERK), indicating an activation of EGFR-mediated signaling pathways. Blockade of EGFR prevented the G3-induced ERK activation and suppressed the G3-provoked enhancement of neurite growth and glutamatergic function but failed to block the G3-mediated enhancement of cell attachment. These combined results indicate that the versican G3 domain regulates neuronal attachment, neurite outgrowth, and synaptic function of hippocampal neurons via EGFR-dependent and -independent signaling pathway(s). Our findings suggest a role for ECM proteolytic products in neural development and regeneration.

    The Journal of biological chemistry 2006;281;28;19358-68

  • 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

  • Identification of the genetic defect in the original Wagner syndrome family.

    Kloeckener-Gruissem B, Bartholdi D, Abdou MT, Zimmermann DR and Berger W

    Division of Medical Molecular Genetics and Gene Diagnostics, University of Zurich, Zurich, Switzerland. kloeckener@medgen.unizh.ch

    Purpose: The aim of the present study was to determine the genetic defect in Wagner syndrome, a rare disorder belonging to the group of hereditary vitreoretinal degenerations. This disease has been genetically mapped to chromosome 5q14.3.

    Methods: Molecular analysis was performed in the progeny of the original pedigree described by Wagner in 1938. We searched for pathogenic mutations and their effects in two candidate genes, CSPG2 and EDIL3, which locate to the critical chromosomal interval. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was used to investigate potential splice defects of CSPG2 transcripts.

    Results: While no alterations were detected in the exons of EDIL3, several changes were identified in the CSPG2 gene. Only one of the novel changes, a heterozygous G to A substitution of the first nucleotide in intron 8, cosegregates with the disease phenotype. This change disrupts the highly conserved splice donor sequence. In blood cells of an index patient, we found CSPG2 transcripts with normally spliced exon 8/9 junction but also two additional CSPG2 transcripts, which were not detected in the control. One lacks the entire exon 8, while the other is missing only the last 21 bp of exon 8.

    Conclusions: CSPG2 encodes versican, a large proteoglycan, which is an extracellular matrix component of the human vitreous and participates in the formation of the vitreous gel. The splice site mutation described here may lead to a complete lack of exon 8 in CSPG2 transcripts, which shortens the predicted protein by 1754 amino acids and leads to severe reduction of glycosaminoglycan attachment sites.

    Molecular vision 2006;12;350-5

  • Versican G3 domain promotes blood coagulation through suppressing the activity of tissue factor pathway inhibitor-1.

    Zheng PS, Reis M, Sparling C, Lee DY, La Pierre DP, Wong CK, Deng Z, Kahai S, Wen J and Yang BB

    Sunnybrook & Women's College Health Sciences Centre, University of Toronto, Ontario, Canada.

    We have detected versican, a member of the large chondroitin sulfate proteoglycans, and its degraded C-terminal G3 fragments in human plasma and observed that the versican G3 domain promoted blood coagulation. Silencing G3 expression with small interfering RNA reduced the effect of G3 on coagulation. Plasma coagulation assays suggest that G3 enhances coagulation irrespective of its actions on platelets and white blood cells. To examine how versican affected blood coagulation, we used normal human plasma and different types of coagulation factor-deficient plasmas. The experiments indicated that versican enhanced coagulation through the extrinsic pathway, and that Factor VII was the target molecule. FVII activity assays showed that G3 activated FVII in the presence of plasma but not with purified FVII directly. Yeast two-hybrid, immunoprecipitation, and gel co-migration assays showed that G3 interacted with the tissue factor pathway inhibitor-1 (TFPI-1). TFPI-1 activity assays suggested that G3 inhibited TFPI-1 activity, allowing FVIIa and FXa to facilitate the coagulation process. G3-induced blood coagulation was further confirmed with a mouse model in a real-time manner. Taken together, these results indicate that versican may represent a new target for the development of therapies against atherosclerosis.

    The Journal of biological chemistry 2006;281;12;8175-82

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Versican is induced in infiltrating monocytes in myocardial infarction.

    Toeda K, Nakamura K, Hirohata S, Hatipoglu OF, Demircan K, Yamawaki H, Ogawa H, Kusachi S, Shiratori Y and Ninomiya Y

    Department of Medicine and Medical Science, Okayama University Graduate School of Medicine and Dentistry, Japan.

    Versican, a large chondroitin sulfate proteoglycan, plays a role in conditions such as wound healing and tissue remodelling. To test the hypothesis that versican expression is transiently upregulated and plays a role in the infarcted heart, we examined its expression in a rat model of myocardial infarction. Northern blot analysis demonstrated increased expression of versican mRNA. Quantitative real-time RT-PCR analysis revealed that versican mRNA began to increase as early as 6 h and reached its maximal level 2 days after coronary artery ligation. Versican mRNA then gradually decreased, while the mRNA of decorin, another small proteoglycan, increased thereafter. Versican mRNA was localized in monocytes, as indicated by CD68-positive staining, around the infarct tissue. The induction of versican mRNA was accelerated by ischemia/reperfusion (I/R), which was characterized by massive cell infiltration and enhanced inflammatory response. To examine the alteration of versican expression in monocytes/macrophages, we isolated human peripheral blood mononuclear cells and stimulated them with granulocyte/macrophage colony-stimulating factor (GM-CSF). Stimulation of mononuclear cells with GM-CSF increased the expression of versican mRNA as well as cytokine induction. The production of versican by monocytes in the infarct area represents a novel finding of the expression of an extracellular matrix gene by monocytes in the infarcted heart. We suggest that upregulation of versican in the infarcted myocardium may have a role in the inflammatory reaction, which mediates subsequent chemotaxis in the infarcted heart.

    Molecular and cellular biochemistry 2005;280;1-2;47-56

  • Hair cycle-specific expression of versican in human hair follicles.

    Soma T, Tajima M and Kishimoto J

    Shiseido Life Science Research Center, 2-12-1 Fukuura, Kanazawa-ku, Yokohama 236-8643, Japan. tsutomu.souma@to.shiseido.co.jp

    Background: Versican, a large chondroitin sulfate proteoglycan molecule, is implicated in the induction of hair morphogenesis, the initiation of hair regeneration, and the maintenance of hair growth in mouse species. In contrast, in human hair follicles, the distribution and the roles of versican remains obscure.

    Objectives: To elucidate the implication of versican in normal human hair growth.

    Methods: Versican expression was examined by in situ hybridization (mRNA) and immunohistochemistry (protein).

    Results: The results clearly showed specific versican gene expression in the dermal papilla of anagen, which apparently decreased in the dermal papilla of catagen hair follicles. No specific signal was detectable in telogen hair follicles. Consistent with ISH results, versican immunoreactivity was extended over the dermal papilla of anagen hair follicles, and again, this staining diminished in the catagen phase of human hair follicles. Interestingly, versican proteins were deposited outside K15-positive epithelial cells in the bulge throughout the hair cycle. Versican immunoreactivity in the dermal papilla was almost lost in vellus-like hair follicles affected by male pattern baldness.

    Conclusion: Specific expression of versican in the anagen hair follicles suggests its importance to maintain the normal growing phase of human as well as mouse.

    Journal of dermatological science 2005;39;3;147-54

  • Versican splice variants in human trabecular meshwork and ciliary muscle.

    Zhao X and Russell P

    Section on Aging and Ocular Disease, National Eye Institute, National Institutes of Health, Bethesda, MD, USA. xiujun.zhao@mssm.edu

    Purpose: Versican, chondroitin sulfate glycoprotein 2, is thought to play a role in regulating aqueous humor outflow and intraocular pressure via the human trabecular meshwork (HTM) of the eye. This protein was upregulated in HTM cells when they were treated with TGF-beta. There are four splice variant forms of versican (V0-V3) with different numbers of glycoaminoglycan (GAG) attachment domains. In this study, we investigated the various isoforms of versican from ocular tissues and cultured cells.

    Methods: HTM and human ciliary muscle (HCM) tissues were dissected from three pairs of donors eyes with no histories of eye disease. Cultures of HTM and HCM cells were established from five donor eyes, and HTM cells were treated for 72 h with 1 ng/ml of either with hrTGF-beta1 or hrTGF-beta2. Total RNA was isolated from cells and tissues from each of the samples. Relative quantitation of gene expression of each variant was detected by real-time PCR with SYBR Green dye.

    Results: All four variants of versican existed in each sample. In cultured HTM cells, the two variants with the largest number of GAG domains predominate. The V1 form is about 20% greater than the V0 form. There is an upregulation, particularly in the V0 form, when the cells are cultured. In tissue, the V1 form is about five fold greater than the other three. In the ocular ciliary muscle, the V1 form is the most prominent, but with this tissue, the relative amount of the V0 form did not change when cells were cultured. There was an upregulation of all splice variants in HTM cells when they were cultured with either TGF-beta1 or TGF-beta2. The increase in expression in the HTM with TGF-beta treatments were greatest with the V0 and V2 forms.

    Conclusions: This is the first report about the presence of various forms of versican in the anterior segment of the eye and the alterations in the mRNA patterns of these forms when cells are placed in culture. The results indicate the variants with the largest numbers of GAG attachment domains are the most prominent in the HTM and HCM. The increases in these forms that have the most GAG domains would be consistent with the increases in chondroitin sulfate reported in glaucoma.

    Molecular vision 2005;11;603-8

  • Identification of a novel splice site mutation of the CSPG2 gene in a Japanese family with Wagner syndrome.

    Miyamoto T, Inoue H, Sakamoto Y, Kudo E, Naito T, Mikawa T, Mikawa Y, Isashiki Y, Osabe D, Shinohara S, Shiota H and Itakura M

    Division of Genetic Information, Institute for Genome Research, The University of Tokushima, Tokushima, Japan.

    Purpose: To investigate the genetic basis and clinical variability of Wagner syndrome, a rare, dominantly inherited vitreoretinopathy.

    Methods: Clinical examination, linkage analysis, and mutational screening were performed in a large, three-generation, consanguineous Japanese family with Wagner syndrome. The effect of splice site mutation was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis with lymphoblastoid cell total RNAs generated from affected individuals.

    Results: Ocular phenotypes of affected members included an empty vitreous with fibrillary condensations, avascular membrane, perivascular sheathing, and progressive chorioretinal dystrophy and were similar to those of the original Wagner syndrome family. All affected eyes examined exhibited pseudoexotropia with ectopic fovea. No systemic manifestations were observed. Genetic linkage confirmed disease segregation with the previously identified WGN1 locus on 5q13-q14. A heterozygous A-->G transversion at the second base of the 3'-acceptor splice site of intron 7 (c.4004-2 A-->G) of the chondroitin sulfate proteoglycan 2 (CSPG2) gene that cosegregated with the disease was identified. Results of RT-PCR analysis indicated that the c.4004-2 A-->G mutation activates a cryptic splice site, located 39 bp downstream from the authentic 3' splice acceptor site.

    Conclusions: This linkage study confirmed the genetic homogeneity of the Wagner syndrome. CSPG2 encodes versican, a large chondroitin sulfate proteoglycan, which, in vitreous, binds to hyaluronan and link protein and forms large aggregates that are important for maintaining structural integrity. Although the CSPG2 gene has been excluded as a candidate for causing Wagner syndrome, these data emphasize the necessity of further mutational screening in new families and careful functional characterization.

    Investigative ophthalmology & visual science 2005;46;8;2726-35

  • The expression and regulation of ADAMTS-1, -4, -5, -9, and -15, and TIMP-3 by TGFbeta1 in prostate cells: relevance to the accumulation of versican.

    Cross NA, Chandrasekharan S, Jokonya N, Fowles A, Hamdy FC, Buttle DJ and Eaton CL

    Academic Urology Unit, Division of Clinical Sciences (South), University of Sheffield, Royal Hallamshire Hospital, Sheffield, United Kingdom. n.a.cross@shef.ac.uk

    Background: Benign prostatic hyperplasia (BPH) is characterized by a proportional increase in the size of the stromal compartment of the gland, involving alterations to extracellular matrix (ECM) components. Some of these changes have been associated with the activity and expression of transforming growth factor beta1 (TGFbeta1). Versican (chondroitin sulphate proteoglycan-2) is overexpressed in BPH and prostate cancer and potentially contributes to disease pathology. A sub-group of the ADAMTS lineage of metalloproteases possess versican-degrading properties and are potential regulators of proteoglycan accumulation associated with BPH. These enzymes have one major inhibitor in the ECM, tissue inhibitor of metalloproteinases (TIMP)-3.

    Methods: The effect of TGFbeta on mRNA expression in prostatic stromal cells was determined by real-time qRT-PCR using primers to ADAMTS-1, -4, -5, -9, -15, versican, and TIMP-3. MMP-inhibitory potential (TIMP activity) of conditioned medium was measured using a fluorometric peptide substrate.

    Results: Prostatic stromal cell cultures consistently expressed ADAMTS-1, -4, -5, -9, -15 and TIMP-3, in contrast to PC3, DU145, and LNCaP cells which failed to express at least two ADAMTS transcripts. In stromal cells, TGFbeta1 decreased ADAMTS-1, -5, -9, and -15 transcripts and increased ADAMTS-4, versican, and TIMP-3. TGFbeta also increased TIMP activity in conditioned medium.

    Conclusions: The induction of versican expression by TGFbeta in BPH stromal cells is in agreement with histological studies. The negative effect of TGFbeta1 on ADAMTS-1, -5, -9, and -15 coupled with increases in their inhibitor, TIMP-3 may aid the accumulation of versican in the stromal compartment of the prostate in BPH and prostate cancer.

    The Prostate 2005;63;3;269-75

  • Expression and purification of functionally active hyaluronan-binding domains from human cartilage link protein, aggrecan and versican: formation of ternary complexes with defined hyaluronan oligosaccharides.

    Seyfried NT, McVey GF, Almond A, Mahoney DJ, Dudhia J and Day AJ

    Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

    The chondroitin sulfate proteoglycan aggrecan forms link protein-stabilized complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other chondroitin sulfate proteoglycans (i.e. versican, brevican, and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this study, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells. The recombinant human proteins were found to have properties similar to those described for the native molecules (e.g. cLP was able to form oligomers, and HA decasaccharides were the minimum size that could compete effectively for their binding to polymeric HA). Gel filtration and protein cross-linking/matrix-assisted laser desorption ionization time-of-flight peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other in solution yet formed ternary complexes with HA24. N-linked glycosylation of AG1 and VG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Surprisingly, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences in the conformation of HA stabilized on binding these proteins.

    Funded by: Medical Research Council: MC_U138274352

    The Journal of biological chemistry 2005;280;7;5435-48

  • Versican protects cells from oxidative stress-induced apoptosis.

    Wu Y, Wu J, Lee DY, Yee A, Cao L, Zhang Y, Kiani C and Yang BB

    Sunnybrook and Women's College Health Sciences Centre, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5.

    Oxidant injury plays a critical role in the degenerative changes that are characterized by a decline in parenchymal cell numbers and viability, and occur with aging and in the etiology of many diseases. The extracellular proteoglycan versican is widely distributed in the extracellular matrix surrounding the cells. This study examines whether versican plays a role in protecting cells from free radical-induced apoptosis. Stable expression of versican or its C-terminal domain significantly decreased H(2)O(2)-induced cellular apoptosis. Cells in adherent monolayer were more resistant to H(2)O(2)-induced apoptosis than cells cultured in suspension. While vigorous trypsinization caused integrin cleavage and rendered the cells more susceptible to H(2)O(2)-induced damages, expression of versican or its C-terminal domain enhanced cell attachment and expression of beta1 integrin and fibronectin. Enhanced cell-matrix interaction by addition of manganese (MnCl(2)) to cultures also significantly diminished H(2)O(2)-induced apoptosis. The results suggest that versican plays an important role in reducing oxidant injury through an enhancement of cell-matrix interaction.

    Matrix biology : journal of the International Society for Matrix Biology 2005;24;1;3-13

  • Versican in nonsmall cell lung cancer: relation to hyaluronan, clinicopathologic factors, and prognosis.

    Pirinen R, Leinonen T, Böhm J, Johansson R, Ropponen K, Kumpulainen E and Kosma VM

    Department of Pathology and Forensic Medicine, University of Kuopio and Kuopio Univeristy Hospital, Finland.

    The aim of the study was to investigate the expression and prognostic role of versican in 212 patients with resected nonsmall cell lung cancer. Tumor samples were stained immunohistochemically, and the versican staining was evaluated both in tumor stroma and cancer cells. The staining results were compared to the clinical data of the patients, the tumor cell proliferation, and the expression of hyaluronan. In the whole material, low and high area percentages of stromal versican staining were observed in 135 and 77 carcinomas, respectively. Tumor cell-associated staining signal for versican was observed in 33 cases. In the whole material, the significant relationship between high stromal staining of versican and that of hyaluronan was noticed (P = .001). The expression of stromal versican was related to tumor type (P = .008) and high stromal staining was inversely correlated with poor tumor differentiation (P = .045), but not with tumor cell proliferation. Among adenocarcinomas, the high stromal staining of versican was associated with tumor recurrence (P = .024), higher tumor stage (P = .022), and lymph node metastases (P = .042). Versican expression was not related to patient outcome in the whole material, but among adenocarcinomas, the high stromal staining was related to poor disease-free survival (P = .0056). However, in Cox multivariate analysis with tumor stage, versican expression did not retain its prognostic significance. The results indicate that increased stromal versican is related to higher tumor recurrence rate and more advanced disease. Despite the important role of versican in nonsmall cell lung cancer, traditional clinicopathologic factors remained most significant in the current study.

    Human pathology 2005;36;1;44-50

  • PG-M/versican binds to P-selectin glycoprotein ligand-1 and mediates leukocyte aggregation.

    Zheng PS, Vais D, Lapierre D, Liang YY, Lee V, Yang BL and Yang BB

    Sunnybrook and Women's College Health Sciences Centre, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada.

    P-selectin glycoprotein ligand-1 (PSGL-1), a glycoprotein expressed on the cell surface of leukocytes, binds to selectins and mediates leukocyte rolling on the vascular endothelium. Here we report that PSGL-1 binds to the C-terminal (G3 domain) of the extracellular proteoglycan PG-M/versican. Cells transfected with PSGL-1 or a shorter form containing the binding site, or cells expressing endogenous PSGL-1 aggregate in the presence of versican or G3 product. The aggregation appears to be induced by G3 multimers that bind to PSGL-1 and form a network. Endogenous versican and/or G3-containing fragments also bind to PSGL-1 in human plasma. Removal of the endogenous G3-containing fragments reduces the effect of plasma on leukocyte aggregation. Finally, the roles of G3-containing fragments in leukocyte aggregation were confirmed in a mouse model. Taken together, our results strongly support a physiologically relevant role for PSGL-1/versican binding and may have implications in the immunoresponse.

    Journal of cell science 2004;117;Pt 24;5887-95

  • Pancreatic tumor cells influence the composition of the extracellular matrix.

    Köninger J, Giese T, di Mola FF, Wente MN, Esposito I, Bachem MG, Giese NA, Büchler MW and Friess H

    Department of General Surgery, University of Heidelberg, Germany.

    The malignant behavior of cancers depends on the microenvironmental context. We investigated compositional alterations of the extracellular matrix (ECM) in pancreatic cancer, with special emphasis on the proteoglycans decorin, lumican, and versican. Compared with normal controls (n=18), marked overexpression of these proteoglycans was observed in pancreatic cancer tissues (n=30) by quantitative RT-PCR (p<0.0001). Immunohistochemistry revealed abundance of proteoglycans in the ECM of pancreatic cancer specimens, whereas tumor cells themselves were devoid of either decorin, lumican or versican. RT-PCR confirmed pancreatic stellate cells (PSCs) as the major source of these proteins. Interestingly, TGFbeta1 and conditioned medium derived from pancreatic cancer cell lines synergistically suppressed the expression of known anti-tumor factors decorin and lumican, but stimulated the expression of pro-metastatic factor versican in cultured PSCs. These findings indicate that malignant cells can actively influence the composition of the ECM through TGFbeta1 and other soluble factors, altering their microenvironment in a tumor-favorable way.

    Biochemical and biophysical research communications 2004;322;3;943-9

  • 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

  • Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation.

    Brandenberger R, Wei H, Zhang S, Lei S, Murage J, Fisk GJ, Li Y, Xu C, Fang R, Guegler K, Rao MS, Mandalam R, Lebkowski J and Stanton LW

    Geron Corporation, Menlo Park, California 94025, USA. rbrandenberger@geron.com

    Human embryonic stem (hES) cells hold promise for generating an unlimited supply of cells for replacement therapies. To characterize hES cells at the molecular level, we obtained 148,453 expressed sequence tags (ESTs) from undifferentiated hES cells and three differentiated derivative subpopulations. Over 32,000 different transcripts expressed in hES cells were identified, of which more than 16,000 do not match closely any gene in the UniGene public database. Queries to this EST database revealed 532 significantly upregulated and 140 significantly downregulated genes in undifferentiated hES cells. These data highlight changes in the transcriptional network that occur when hES cells differentiate. Among the differentially regulated genes are several components of signaling pathways and transcriptional regulators that likely play key roles in hES cell growth and differentiation. The genomic data presented here may facilitate the derivation of clinically useful cell types from hES cells.

    Nature biotechnology 2004;22;6;707-16

  • Proteoglycans in atherosclerosis and restenosis: key roles for versican.

    Wight TN and Merrilees MJ

    Department of Vascular Biology, The Hope Heart Institute, 1124 Columbia St, No. 783, Seattle, Wash 98104-2046, USA. twight@hopeheart.org

    The proteoglycan versican is one of several extracellular matrix (ECM) molecules that accumulate in lesions of atherosclerosis and restenosis. Its unique structural features create a highly interactive molecule that binds growth factors, enzymes, lipoproteins, and a variety of other ECM components to influence fundamental events involved in vascular disease. Versican is one of the principal genes that is upregulated after vascular injury and is a prominent component in stented and nonstented restenotic lesions. The synthesis of versican is highly regulated by specific growth factors and cytokines and the principal source of versican is the smooth muscle cell. Versican interacts with hyaluronan, a long chain glycosaminoglycan, to create expanded viscoelastic pericellular matrices that are required for arterial smooth muscle cell (ASMC) proliferation and migration. Versican is also prominent in advanced lesions of atherosclerosis, at the borders of lipid-filled necrotic cores as well as at the plaque-thrombus interface, suggesting roles in lipid accumulation, inflammation, and thrombosis. Versican influences the assembly of ECM and controls elastic fiber fibrillogenesis, which is of fundamental importance in ECM remodeling during vascular disease. Collectively, these studies highlight the critical importance of this specific ECM component in atherosclerosis and restenosis.

    Funded by: NHLBI NIH HHS: HL-18645

    Circulation research 2004;94;9;1158-67

  • 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

  • Link protein has greater affinity for versican than aggrecan.

    Shi S, Grothe S, Zhang Y, O'Connor-McCourt MD, Poole AR, Roughley PJ and Mort JS

    Joint Diseases Laboratory, Shriners Hospitals for Children, 1529 Cedar Avenue, Montreal, Quebec, Canada H3G 1A6.

    The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues.

    The Journal of biological chemistry 2004;279;13;12060-6

  • Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation.

    Russell DL, Doyle KM, Ochsner SA, Sandy JD and Richards JS

    Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

    ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs-1) is a member of the ADAMTS family of metalloproteases which, together with ADAMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans. ADAMTS-1 mRNA is induced in granulosa cells of periovulatory follicles by the luteinizing hormone surge through a progesterone receptor-dependent mechanism. Female progesterone receptor knockout (PRKO) mice are infertile primarily due to ovulatory failure and lack the normal periovulatory induction of ADAMTS-1 mRNA. We therefore investigated the protein localization and function of ADAMTS-1 in ovulating ovaries. Antibodies against two specific peptide regions, the pro-domain and the metalloprotease domain of ADAMTS-1, were generated. Pro-ADAMTS-1 of 110 kDa was identified in mural granulosa cells and appears localized to cytoplasmic secretory vesicles. The mature (85-kDa pro-domain truncated) form accumulated in the extracellular matrix of the cumulus oocyte complex (COC) during the process of matrix expansion. Each form of ADAMTS-1 protein increased >10-fold after the ovulatory luteinizing hormone surge in wild-type but not PRKO mice. Versican is also localized selectively to the ovulating COC matrix and was found to be cleaved yielding a 70-kDa N-terminal fragment immunopositive for the neoepitope DPEAAE generated by ADAMTS-1 and ADAMTS-4 protease activity. This extracellular processing of versican was reduced in ADAMTS-1-deficient PRKO mouse ovaries. These observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded COC matrix and that the anovulatory phenotype of PRKO mice is at least partially due to loss of this function.

    The Journal of biological chemistry 2003;278;43;42330-9

  • Distinct interaction of versican/PG-M with hyaluronan and link protein.

    Matsumoto K, Shionyu M, Go M, Shimizu K, Shinomura T, Kimata K and Watanabe H

    Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195.

    The proteoglycan aggregate is the major structural component of the cartilage matrix, comprising hyaluronan (HA), link protein (LP), and a large chondroitin sulfate (CS) proteoglycan, aggrecan. Here, we found that another member of aggrecan family, versican, biochemically binds to both HA and LP. Functional analyses of recombinant looped domains (subdomains) A, B, and B' of the N-terminal G1 domain revealed that the B-B' segment of versican is adequate for binding to HA and LP, whereas A and B-B' of aggrecan bound to LP and HA, respectively. BIAcore trade mark analyses showed that the A subdomain of versican G1 enhances HA binding but has a negligible effect on LP binding. Overlay sensorgrams demonstrated that versican G1 or its B-B' segment forms a complex with both HA and LP. We generated a molecular model of the B-B' segment, in which a deletion and an insertion of B' and B are critical for stable structure and HA binding. These results provide important insights into the mechanisms of formation of the proteoglycan aggregate and HA binding of molecules containing the link module.

    The Journal of biological chemistry 2003;278;42;41205-12

  • Experimental immunity to the G1 domain of the proteoglycan versican induces spondylitis and sacroiliitis, of a kind seen in human spondylarthropathies.

    Shi S, Ciurli C, Cartman A, Pidoux I, Poole AR and Zhang Y

    Shriners Hospitals for Children, Montreal, Quebec, Canada.

    Objective: Experimental immunity to the G1 domain of the cartilage proteoglycan (PG) aggrecan (AG1) leads to the development of spondylitis as well as polyarthritis in BALB/c mice. The PG versican contains a structurally similar G1 domain (VG1). This study was conducted to determine whether immunity to VG1 would elicit similar pathology in these mice.

    Methods: Recombinant natively folded VG1 and AG1 were prepared. BALB/c mice received either a series of 5 injections of human VG1 or AG1, or no protein. Polyarthritis was determined clinically, and spondylitis and sacroiliitis histologically. Immunohistochemistry of rat tissues was used to study the localization of versican. Enzyme-linked immunosorbent assays were employed to study humoral immunity to the recombinant proteins as well as to overlapping synthetic peptides covering all these human G1 domains and mouse homologs. Affinity-purified antibodies to human AG1 and VG1 were isolated from sera of hyperimmunized mice. T lymphocyte proliferation assays were performed using recombinant human proteins. T cell lines reactive with specific immunodominant T cell epitopes in human AG1 and VG1 were isolated. Synthetic peptides encoding sequences in these human proteins and in corresponding mouse proteins were used in these analyses. Guanidinium chloride extracts of mouse spines were also used in Western blots to study antibody cross-reactivity.

    Results: Immunity to recombinant VG1 did not result in clinical polyarthritis. There was, however, clear evidence that VG1, like AG1, could induce spondylitis in the lumbar spine and sacroiliitis. Accumulation of mononuclear cells was observed in spinal ligaments adjacent to the intervertebral disc, in the intervertebral disc, and in the sacroiliac joints, the same sites where versican is localized. In contrast to AG1-immunized mice, in which T cells reactive with human AG1 cross-reacted with mouse AG1, there was no evidence in VG1-immunized mice that T cell immunity to human VG1 was cross-reactive with a mouse synthetic peptide that contained the sequence corresponding to the single immunodominant T cell sequence recognized in human VG1. Antibodies to specific sequences in human VG1 did, however, cross-react with human AG1 and with corresponding peptide sequences in mouse versican and aggrecan and with mouse proteins containing VG1 and AG1, present in mouse spine extracts. Similarly, antibodies to human AG1 cross-reacted with human VG1 and with extracted mouse VG1 and AG1 and synthetic peptides containing mouse sequences that corresponded to the reactive human epitopes in AG1 and VG1.

    Conclusion: These observations suggest that humoral immunity to human VG1 is involved in the induction of experimental spondylitis and sacroiliitis in BALB/c mice. This humoral immunity is cross-reactive with mouse versican and aggrecan but is not associated with polyarthritis, probably because of the lack of cross-reactive T cell immunity and the absence of detectable versican in articular cartilage limbs. Induction of polyarthritis by bovine or human aggrecan requires the involvement of immunity mediated by T lymphocytes that are cross-reactive to a mouse aggrecan epitope. Together these observations suggest that humoral immunity to versican as well as immunity to aggrecan may be of importance in the development of the spinal pathology characteristic of spondylarthropathies.

    Arthritis and rheumatism 2003;48;10;2903-15

  • Differential expression of versican isoforms is a component of the human melanoma cell differentiation process.

    Domenzain C, Docampo MJ, Serra M, Miquel L and Bassols A

    Departament de Bioqui;mica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra 081893, Spain

    Versican is a large chondroitin sulfate proteoglycan produced by human melanoma cell lines and malignant melanocytic lesions. In the present work, we have analyzed the expression of versican spliced variants V0, V1, V2 and V3 in human melanoma cell lines at several differentiation degrees. The isoform expression pattern depends on the degree of cell differentiation. Differentiated cell lines do not produce any of the versican isoforms as analyzed by Western blot, Northern blot and RT-PCR. All cell lines with an early or intermediate degree of differentiation (AX3, SK-mel-37, Rider, SK-mel-1.36-1-5 and SK-mel-3.44) expressed V0 and V1 transcripts, whereas V2 and V3 expression was shown only by the undifferentiated cell lines SK-mel-1.36-1-5 and Rider. Furthermore, we have analyzed the expression of versican isoforms in SK-mel-3.44 and SK-mel-1.36-1-5 cells induced to differentiate by TPA treatment. The expression of the large V0, V1 and V2 isoforms practically disappears in differentiated cells, whereas V3 remains detectable by RT-PCR analysis.

    Biochimica et biophysica acta 2003;1642;1-2;107-14

  • Distribution of PG-M/versican variants in human tissues and de novo expression of isoform V3 upon endothelial cell activation, migration, and neoangiogenesis in vitro.

    Cattaruzza S, Schiappacassi M, Ljungberg-Rose A, Spessotto P, Perissinotto D, Mörgelin M, Mucignat MT, Colombatti A and Perris R

    Department of Evolutionary and Functional Biology, University of Parma, Viale delle Scienze 11/A, 43100 Parma, Italy.

    We have carried out a comprehensive molecular mapping of PG-M/versican isoforms V0-V3 in adult human tissues and have specifically investigated how the expression of these isoforms is regulated in endothelial cells in vitro. A survey of 21 representative tissues highlighted a prevalence of V1 mRNA; demonstrated that the relative frequency of expression was V1 > V2 > V3 >or= V2; and showed that <15% of the tissues transcribed significant levels of all four isoforms. By employing novel and previously described anti-versican antibodies we verified a ubiquitous versican deposition in normal and tumor-associated vascular structures and disclosed differences in the glycanation profiles of versicans produced in different vascular beds. Resting endothelial cells isolated from different tissue sources transcribed several of the versican isoforms but consistently failed to translate these mRNAs into detectable proteoglycans. However, if stimulated with tumor necrosis factor-alpha or vascular endothelial growth factor, they altered their versican expression by de novo transcribing the V3 isoform and by exhibiting a moderate V1/V2 production. Induced versican synthesis and de novo V3 expression was also observed in endothelial cells elicited to migrate in a wound-healing model in vitro and in angiogenic endothelial cells forming tubule-like structures in Matrigel or fibrin clots. The results suggest that, independent of the degree of vascularization, human adult tissues show a limited expression of versican isoforms V0, V2, and V3 and that endothelial cells may contribute to the deposition of versican in vascular structures, but only following proper stimulation.

    The Journal of biological chemistry 2002;277;49;47626-35

  • Gene expression profiling of isogenic cells with different TP53 gene dosage reveals numerous genes that are affected by TP53 dosage and identifies CSPG2 as a direct target of p53.

    Yoon H, Liyanarachchi S, Wright FA, Davuluri R, Lockman JC, de la Chapelle A and Pellegata NS

    Human Cancer Genetics Program, Comprehensive Cancer Center, Ohio State University, 420 West 12th Avenue, Columbus 43210, USA.

    TP53 does not fully comply with the Knudson model [Knudson, A. G., Jr. (1971) Proc. Natl. Acad. Sci. USA 68, 820-823] in that a reduction of constitutional expression of p53 may be sufficient for tumor predisposition. This finding suggests a gene-dosage effect for p53 function. To determine whether TP53 gene dosage affects the transcriptional regulation of target genes, we performed oligonucleotide-array gene expression analysis by using human cells with wild-type p53 (p53 +/+), or with one (p53 +/-), or both (p53 -/-) TP53 alleles disrupted by homologous recombination. We identified 35 genes whose expression is significantly correlated to the dosage of TP53. These genes are involved in a variety of cellular processes including signal transduction, cell adhesion, and transcription regulation. Several of them are involved in neurogenesis and neural crest migration, developmental processes in which p53 is known to play a role. Motif search analysis revealed that of the genes highly expressed in p53 +/+ and +/- cells, several contain a putative p53 consensus binding site (bs), suggesting that they could be directly regulated by p53. Among those genes, we chose CSPG2 (which encodes versican) for further study because it contains a bona fide p53 bs in its first intron and its expression highly correlates with TP53 dosage. By using in vitro and in vivo assays, we showed CSPG2 to be directly transactivated by p53. In conclusion, we developed a strategy to demonstrate that many genes are affected by TP53 gene dosage for their expression. We report several candidate genes as potential downstream targets of p53 in nonstressed cells. Among them, CSPG2 is validated as being directly transactivated by p53. Our method provides a useful tool to elucidate additional mechanisms by which p53 exerts its functions.

    Funded by: NCI NIH HHS: P01 CA16058, P30 CA016058

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;24;15632-7

  • Synthesis and expression of mRNA encoding for different versican splice variants is related to the aggregation of human epithelial mesothelioma cells.

    Syrokou A, Dobra K, Tzanakakis GN, Hjerpe A and Karamanos NK

    Department of Chemistry, Section of Organic Chemistry, Biochemistry and Natural Products, Laboratory of Biochemistry, University of Patras, 26110 Patras, Greece.

    Malignant mesothelioma often has a biphasic growth pattern of epithelial and/or sarcomatous morphology. In culture, epithelial cells form aggregates, whereas fibroblast-like cells do not. Two human mesothelioma cell sub-lines, one with epithelial differentiation and the other with fibroblast-like phenotype were studied. We have previously shown (Dobra et al, 2000) that distinct types of the cell-associated syndecans are involved in the regulation of mesothelioma cell differentiation, whereas the role of matrix proteoglycans (PGs) remains unknown. This study was undertaken to examine whether cell aggregation of the epithelial mesothelioma cells correlates to the differential expression of the matrix PGs versican and perlecan at different degrees of confluence. PGs were isolated from the culture medium using ion-exchange chromatography and identified by high-performance liquid chromatography, capillary electrophoresis and Western blotting. Fibroblast-like cells express substantially more versican than epithelial cells. RT-PCR showed that both cell lines express mRNA coding for versican splice variants V0 and V1, but not for V2. The dominating splice variant in both cell lines is the V0. Screening of versican splice variants in various degrees of culture confluence showed that the expression of mRNA conding for the versican splice variants V0 and V1 is different only in confluent cultures. No significant differences in the expression of perlecan between the two cell lines were recorded. These results suggest that the aggregation of epithelial cells is related to a significant decrease (p < or = 0.001) of the splice variant V1. This variant seems to be a biologically active constituent that affects tumor biology.

    Anticancer research 2002;22;6C;4157-62

  • Cleavage of the carboxyl tail from the G3 domain of aggrecan but not versican and identification of the amino acids involved in the degradation.

    Lee V, Chen L, Paiwand F, Cao L, Wu Y, Inman R, Adams ME and Yang BB

    Sunnybrook & Women's College Health Sciences Centre, the Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario M4N 3M5, Canada.

    Aggrecan, a major structural proteoglycan in cartilage, contains three globular domains, G1, G2, and G3, as well as sequences for glycosaminoglycan modification. A large number of proteases are implicated in aggrecan cleavage in normal metabolism, aging, and arthritis. These proteases are known to cleave at the IGD, KS, and CS domains. Here we report for the first time evidence of cleavage at a novel site, the carboxyl tail of aggrecan. Results from deletion mutants of the tail indicated that the likely cleavage sites were two consensus sequences, RRLXK and RSPR, present in the aggrecan analogs of many species. This was confirmed by site-directed mutagenesis. A construct containing two G3 domains (G3G3) was also found to cleave between the G3 duplicates. When G3 tail was linked to a glycosaminoglycan-modifying sequence, it was protected from cleavage. Furin inhibitor also reduced the levels of tail cleavage. The carboxyl tails of chicken and human versican were not cleaved, despite the presence of the consensus sequence. Our studies indicate that the basic amino acids present in the tail play an important role in cleavage, and this mechanism is specific to aggrecan.

    The Journal of biological chemistry 2002;277;25;22279-88

  • beta 1-Integrin-mediated glioma cell adhesion and free radical-induced apoptosis are regulated by binding to a C-terminal domain of PG-M/versican.

    Wu Y, Chen L, Zheng PS and Yang BB

    Sunnybrook and Women's College Health Sciences Centre and the Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M4N 3M5, Canada.

    Integrins are cell-surface glycoproteins that mediate cell activities, including tissue morphogenesis, development, immune response, and cancer, through interaction with extracellular proteins. Here we report a novel means by which integrin signaling and functions are regulated. In pull-down assays and immunoprecipitation, beta(1)-integrin bound to the C-terminal domain of PG-M/versican, an extracellular chondroitin sulfate proteoglycan. This was confirmed by cell-surface binding assays. Binding was calcium- and manganese-dependent. Upon native gel electrophoresis, beta(1)-integrin comigrated with the C-terminal domain of PG-M/versican. The interaction of beta(1)-integrin with the C-terminal domain of PG-M/versican activated focal adhesion kinase, enhanced integrin expression, and promoted cell adhesion. As a result, cells expressing the C-terminal domain of PG-M/versican were resistant to free radical-induced apoptosis. As the PG-M/versican peptide used in this study does not contain the RGD consensus-binding motif for integrins, the mechanism of the observed binding represents an entirely new function.

    The Journal of biological chemistry 2002;277;14;12294-301

  • Versican interacts with fibrillin-1 and links extracellular microfibrils to other connective tissue networks.

    Isogai Z, Aspberg A, Keene DR, Ono RN, Reinhardt DP and Sakai LY

    Shriners Hospital for Children and the Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA.

    Fibrillin-containing microfibrils are polymeric structures that are difficult to extract from connective tissues. Proteolytic digestion of tissues has been utilized to release microfibrils for study. Few of the molecules that connect microfibrils to other elements in the matrix have been identified. In this study, electron microscopic immunolocalization of anti-versican antibodies in tissues and in extracted microfibrils demonstrated that the C-terminal region of versican is found associated with fibrillin microfibrils. Extraction of microfibrils followed by treatment of microfibrils under dissociating conditions suggested that the versican C terminus is covalently bound to microfibrils. Binding assays using recombinant fibrillin-1 polypeptides and recombinant lectican lectin domains indicated that the versican lectin domain binds to specific fibrillin-1 polypeptides. The versican lectin domain also bound to molecules comigrating with authentic fibrillin-1 monomers in an assay using cell culture medium. In assays using microfibrils, the versican lectin domain demonstrated preferential binding compared with other lecticans. Binding was calcium-dependent. The binding site for versican in microfibrils is most likely within a region of fibrillin-1 between calcium-binding epidermal growth factor-like domains 11 and 21. Human mutations in this region can result in severe forms of the Marfan syndrome ("neonatal" Marfan syndrome). The connection between versican and fibrillin microfibrils may be functionally significant, particularly in cardiovascular tissues.

    The Journal of biological chemistry 2002;277;6;4565-72

  • Versican is differentially expressed in human melanoma and may play a role in tumor development.

    Touab M, Villena J, Barranco C, Arumí-Uría M and Bassols A

    Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 01893 Bellaterra, Barcelona, Spain.

    Undifferentiated human melanoma cell lines produce a large chondroitin sulfate proteoglycan, different from the well-known melanoma-specific proteoglycan mel-PG (Heredia and colleagues, Arch Biochem Biophys, 333: 198-206, 1996). We have identified this proteoglycan as versican and analyzed the expression of versican in several human melanoma cell lines. Versican isoforms are expressed in undifferentiated cell lines but not in differentiated cells, and the isoform expression pattern depends on the degree of cell differentiation. The V0 and V1 isoforms are found on cells with an early degree of differentiation, whereas the V1 isoform is present in cells with an intermediate degree of differentiation. We have also characterized some functional properties of versican on human melanoma cells: the purified proteoglycan stimulates cell growth and inhibits cell adhesion when cells are grown on fibronectin or collagen type I as substrates, and thus may facilitate tumor cell detachment and proliferation. Furthermore, we have analyzed the expression of versican in human melanocytic nevi and melanoma: 10 benign melanocytic nevi, 10 dysplastic nevi, 11 primary malignant melanomas, and 8 metastatic melanomas were tested. Immunoreactivity for versican was negative in benign melanocytic nevi, weakly to strongly positive in dysplastic nevi, and intensely positive in primary malignant melanomas and metastatic melanomas. Our results indicate that versican is involved in the progression of melanomas and may be a reliable marker for clinical diagnosis.

    The American journal of pathology 2002;160;2;549-57

  • Platelet-derived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells.

    Evanko SP, Johnson PY, Braun KR, Underhill CB, Dudhia J and Wight TN

    The Hope Heart Institute, Seattle, Washington 98104, USA.

    Hyaluronan and versican-rich pericellular matrices form around arterial smooth muscle cells (ASMC) preferentially during the detachment phase of proliferation and migration. PDGF is a potent mitogen and chemotactic agent for ASMC and also stimulates the production of extracellular matrix molecules which may regulate the proliferative and migratory capacity of the cells. We have examined the effect of PDGF on the formation of hyaluronan-dependent pericellular matrices, and on the synthesis and interaction of several major pericellular coat constituents. As demonstrated using a particle exclusion assay, PDGF stimulated the formation of pericellular matrices and was seen both in an increased proportion of cells with a coat and a greater coat size. This increase was accompanied by a transient increase in hyaluronan synthase 2 (HAS2) expression and an increase in hyaluronan synthesis and polymer length. PDGF also increased the synthesis of versican and link protein as measured at the mRNA and protein levels. The amount of native versican-hyaluronan aggregates and link-stabilized aggregate was also increased following PDGF treatment. Time lapse imaging showed that pericellular matrix formation occurred around trailing cell processes prior to their detachment. These data suggest that PDGF modulates the synthesis and organization of ASMC pericellular coat-forming molecules such as versican, hyaluronan, and link protein, which leads to extracellular matrix expansion and alterations in ASMC phenotype.

    Funded by: NHLBI NIH HHS: HL-18645

    Archives of biochemistry and biophysics 2001;394;1;29-38

  • Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4.

    Sandy JD, Westling J, Kenagy RD, Iruela-Arispe ML, Verscharen C, Rodriguez-Mazaneque JC, Zimmermann DR, Lemire JM, Fischer JW, Wight TN and Clowes AW

    Shriners Hospital for Children, Tampa, Florida 33612, USA. jsandy@shctampa.usf.edu

    Mature human aorta contains a 70-kDa versican fragment, which reacts with a neoepitope antiserum to the C-terminal peptide sequence DPEAAE. This protein therefore appears to represent the G1 domain of versican V1 (G1-DPEAAE(441)), which has been generated in vivo by proteolytic cleavage at the Glu(441)-Ala(442) bond, within the sequence DPEAAE(441)-A(442)RRGQ. Because the equivalent aggrecan product (G1-NITEGE(341)) and brevican product (G1-EAVESE(395)) are generated by ADAMTS-mediated cleavage of the respective proteoglycans, we tested the capacity of recombinant ADAMTS-1 and ADAMTS-4 to cleave versican at Glu(441)-Ala(442). Both enzymes cleaved a recombinant versican substrate and native human versican at the Glu(441)-Ala(442) bond and the mature form of ADAMTS-4 was detected by Western analysis of extracts of aortic intima. We conclude that versican V1 proteolysis in vivo can be catalyzed by one or more members of the ADAMTS family of metalloproteinases.

    Funded by: NCI NIH HHS: R01CA 77420; NCRR NIH HHS: RR00166; NHLBI NIH HHS: HL07828, HL30946

    The Journal of biological chemistry 2001;276;16;13372-8

  • Versican interacts with chemokines and modulates cellular responses.

    Hirose J, Kawashima H, Yoshie O, Tashiro K and Miyasaka M

    Department of Bioregulation, Biomedical Research Center, Osaka University Graduate School of Medicine, 2-2, Yamada-Oka, Suita 565-0871, Japan.

    We previously reported that versican, a large chondroitin sulfate proteoglycan, isolated from a renal adenocarcinoma cell line, ACHN, binds L-selectin. Here we report that versican also binds certain chemokines and regulates chemokine function. This binding was strongly inhibited by the chondroitinase digestion of versican or by the addition of soluble chondroitin sulfate (CS) B, CS E, or heparan sulfate. Furthermore, these glycosaminoglycans (GAGs) could bind directly to the chemokines that bind versican. Thus, versican appears to interact with chemokines via its GAGs. We next examined if versican or GAGs affect secondary lymphoid tissue chemokine (SLC)-induced integrin activation and Ca(2+) mobilization in lymphoid cells expressing a receptor for SLC, CC chemokine receptor 7. Interestingly, whereas heparan sulfate supported both alpha(4)beta(7) integrin-dependent binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-IgG and Ca(2+) mobilization induced by SLC, versican or CS B inhibited these cellular responses, and the extent of inhibition was dependent on the dose of versican or CS B added. These findings suggest that different proteoglycans have different functions in the regulation of chemokine activities and that versican may negatively regulate the function of SLC via its GAG chains.

    The Journal of biological chemistry 2001;276;7;5228-34

  • The proteoglycans aggrecan and Versican form networks with fibulin-2 through their lectin domain binding.

    Olin AI, Mörgelin M, Sasaki T, Timpl R, Heinegård D and Aspberg A

    Department of Cell and Molecular Biology, Section for Connective Tissue Biology, Lund University, BMC Plan C12, SE-221 84 Lund, Sweden.

    Aggrecan, versican, neurocan, and brevican are important components of the extracellular matrix in various tissues. Their amino-terminal globular domains bind to hyaluronan, but the function of their carboxyl-terminal globular domains has long remained elusive. A picture is now emerging where the C-type lectin motif of this domain mediates binding to other extracellular matrix proteins. We here demonstrate that aggrecan, versican, and brevican lectin domains bind fibulin-2, whereas neurocan does not. As expected for a C-type lectin, the interactions are calcium-dependent, with K(D) values in the nanomolar range as measured by surface plasmon resonance. Solid phase competition assays with previously identified ligands demonstrated that fibulin-2 and tenascin-R bind the same site on the proteoglycan lectin domains. Fibulin-1 has affinity for the common site on versican but may bind to a different site on the aggrecan lectin domain. By using deletion mutants, the interaction sites for aggrecan and versican lectin domains were mapped to epidermal growth factor-like repeats in domain II of fibulin-2. Affinity chromatography and solid phase assays confirmed that also native full-length aggrecan and versican bind the lectin domain ligands. Electron microscopy confirmed the mapping and demonstrated that hyaluronan-aggrecan complexes can be cross-linked by the fibulins.

    The Journal of biological chemistry 2001;276;2;1253-61

  • Binding of a large chondroitin sulfate/dermatan sulfate proteoglycan, versican, to L-selectin, P-selectin, and CD44.

    Kawashima H, Hirose M, Hirose J, Nagakubo D, Plaas AH and Miyasaka M

    Department of Bioregulation, Biomedical Research Center, Osaka University Graduate School of Medicine 2-2, Yamada-oka, Suita 565-0871, Japan.

    Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L- and P-selectin and CD44. The binding of versican to L- and P-selectin was inhibited by CS B, CS E, and heparan sulfate (HS) but not by any other glycosaminoglycans tested. On the other hand, the binding to CD44 was inhibited by hyaluronic acid, chondroitin (CH), CS A, CS B, CS C, CS D, and CS E but not by HS or keratan sulfate. A cross-blocking study indicated that L- and P-selectin recognize close or overlapping sites on versican, whereas CD44 recognizes separate sites. We also show that soluble L- and P-selectin directly bind to immobilized CS B, CS E, and HS and that soluble CD44 directly binds to immobilized hyaluronic acid, CH, and all the CS chains examined. Consistent with these results, structural analysis showed that versican is modified with at least CS B and CS C. Thus, proteoglycans sufficiently modified with the appropriate glycosaminoglycans should be able to bind L-selectin, P-selectin, and/or CD44.

    The Journal of biological chemistry 2000;275;45;35448-56

  • A novel hereditary developmental vitreoretinopathy with multiple ocular abnormalities localizing to a 5-cM region of chromosome 5q13-q14.

    Black GC, Perveen R, Wiszniewski W, Dodd CL, Donnai D and McLeod D

    University Department of Medical Genetics and Regional Genetic Service, St. Mary's Hospital, Manchester, England. gblack@fs1.cmht.nwest.nhs.uk

    Background: To undertake a clinical and molecular analysis of a previously unpublished kindred with a phenotypically distinct vitreoretinopathy characterized by associated ocular developmental abnormalities.

    Design: Family genetic study.

    Participants: A total of 23 members, both affected and unaffected, of 1 kindred with vitreoretinopathy.

    Method: Individuals within the kindred were examined clinically and blood samples taken for DNA analysis. Genetic analysis was performed for the proximal region of chromosome 5q by means of polymerase chain reaction (PCR).

    Detection of vitreoretinopathy and associated abnormalities.

    Results: This novel, hereditary vitreoretinopathy, showing the classic features of vitreous pathology and early-onset retinal detachments, was associated with a variety of ocular developmental abnormalities, including posterior embryotoxon, congenital glaucoma, iris hypoplasia, congenital cataract, ectopia lentis, microphthalmia, and persistent hyperplastic primary vitreous. There were no associated systemic features. Genetic mapping with markers from the proximal region of 5q13-q14 showed linkage to a 5-cM region between the markers D5S626 and D5S2103.

    Conclusions: The 5-cM region is within that implicated in the etiology of both Wagner and erosive vitreoretinopathies. This suggests that this novel condition may be allelic, refines the genetic mapping for vitreoretinopathies that map to 5q13-q14, and implicates a gene important not only in vitreous production but also in early ocular development.

    Funded by: Wellcome Trust

    Ophthalmology 1999;106;11;2074-81

  • Fibulin-1 is a ligand for the C-type lectin domains of aggrecan and versican.

    Aspberg A, Adam S, Kostka G, Timpl R and Heinegård D

    Department of Cell and Molecular Biology, Section for Connective Tissue Biology, Lund University, P. O. Box 94, SE-221 00 Lund, Sweden. anders.aspberg@medlem.lu.se

    The aggregating proteoglycans (aggrecan, versican, neurocan, and brevican) are important components of many extracellular matrices. Their N-terminal globular domain binds to hyaluronan, but the function of their C-terminal region containing a C-type lectin domain is less clear. We now report that a 90-kDa protein copurifies with recombinant lectin domains from aggrecan and versican, but not from the brain-specific neurocan and brevican. Amino acid sequencing of tryptic peptides from this protein identified it as fibulin-1. This extracellular matrix glycoprotein is strongly expressed in tissues where versican is expressed (blood vessels, skin, and developing heart), and also expressed in developing cartilage and bone. It is thus likely to interact with these proteoglycans in vivo. Surface plasmon resonance measurements confirmed that aggrecan and versican lectin domains bind fibulin-1, whereas brevican and neurocan do not. As expected for a C-type lectin, the interactions with fibulin-1 are Ca2+-dependent, with KD values in the low nanomolar range. Using various deletion mutants, the binding site for aggrecan and versican lectin domains was mapped to the epidermal growth factor-like repeats in domain II of fibulin-1. No difference in affinity was found for deglycosylated fibulin-1, indicating that the proteoglycan C-type lectin domains bind to the protein part of fibulin-1.

    The Journal of biological chemistry 1999;274;29;20444-9

  • Versican/PG-M isoforms in vascular smooth muscle cells.

    Lemire JM, Braun KR, Maurel P, Kaplan ED, Schwartz SM and Wight TN

    Department of Pathology, University of Washington, Seattle, WA, USA. joanlemi@u.washington.edu

    The expression of increased amounts of proteoglycans in the extracellular matrix may play a role in vascular stenosis and lipid retention. The large chondroitin sulfate proteoglycan versican is synthesized by vascular smooth muscle cells (SMCs), accumulates during human atherosclerosis and restenosis, and has been shown to bind LDLs. We recently demonstrated that adult rat aortic SMCs express several versican mRNAs. Four versican splice variants, V0, V1, V2, and V3, have recently been described, which differ dramatically in length. These variants differ in the extent of modification by glycosaminoglycan chains, and V3 may lack glycosaminoglycan chains. In this study, we characterized versican RNAs from rat SMCs by cloning, sequencing, and hybridization with domain-specific probes. DNA sequence was obtained for the V3 isoform, and for a truncated V0 isoform. By hybridization of polyadenylated RNA with domain-specific probes, we determined that the V0, V1, and V3 isoforms are present in vascular SMCs. We confirmed the presence of the V3 isoform in polyadenylated RNA and in RT-PCR products by hybridization with an oligonucleotide that spans the splice junction between the hyaluronan-binding domain and the epidermal growth factor-like domain. In addition, a novel splice variant was cloned by PCR amplification from both rat and human SMC RNA. This appears to be an incompletely spliced variant, retaining the final intron. PCR analysis shows that this intron can be retained in both V1 and V3 isoforms. The predicted translation product of this variant would have a different carboxy-terminus than previously described versican isoforms.

    Funded by: NHLBI NIH HHS: HL03174, HL18645, P01 HL018645

    Arteriosclerosis, thrombosis, and vascular biology 1999;19;7;1630-9

  • Refined genetic and physical localization of the Wagner disease (WGN1) locus and the genes CRTL1 and CSPG2 to a 2- to 2.5-cM region of chromosome 5q14.3.

    Perveen R, Hart-Holden N, Dixon MJ, Wiszniewski W, Fryer AE, Brunner HG, Pinkners AJ, van Beersum SE and Black GC

    University Department of Medical Genetics and Regional Genetic Service, St. Mary's Hospital, Hathersage Road, Manchester, M13 OJH, United Kingdom.

    Wagner syndrome (WGN1; MIM 143200), an autosomal dominant vitreoretinopathy characterized by chorioretinal atrophy, cataract, and retinal detachment, is linked to 5q14.3. Other vitreoretinopathies without systemic stigmata, including erosive vitreoretinopathy, are also linked to this region and are likely to be allelic. Within the critical region lie genes encoding two extracellular macromolecules, link protein (CRTL1) and versican (CSPG2), which are important in binding hyaluronan, a significant component of the mammalian vitreous gel, and which therefore represent excellent candidates for Wagner syndrome. Genetic mapping presented here in two further families reduces the critical region to approximately 2 cM. Subsequent refinement of the physical map allows ordering of known polymorphic microsatellites and excludes CRTL1 as a likely candidate for the disorder. CSPG2 is shown to lie within the critical region; however, analysis of the complete coding region of the mature peptide reveals no clear evidence that it is the gene underlying WGN1.

    Funded by: Wellcome Trust

    Genomics 1999;57;2;219-26

  • Phospholipase A2 type II binds to extracellular matrix biglycan: modulation of its activity on LDL by colocalization in glycosaminoglycan matrixes.

    Sartipy P, Bondjers G and Hurt-Camejo E

    Wallenberg Laboratory for Cardiovascular Research, Department of Heart and Lung Disease, Göteborg University, Gothenburg, Sweden. Peter.Sartipy@wlab.wall.gu.se

    We recently reported the presence of secretory, nonpancreatic phospholipase A2 type II (snpPLA2; EC in human atherosclerotic arteries (Hurt-Camejo et al, Arterioscler Thromb Vasc Biol. 1997;17:300-309). SnpPLA2 may generate the proinflammatory products lysophospholipids and free fatty acids, thus contributing to atherogenesis when acting on low density lipoproteins (LDLs) retained in the arterial wall. Immunohistochemical studies showed that smooth muscle cells (SMCs) in human arterial tissue are the main sources of snpPLA2. In cultures of human arterial SMCs, snpPLA2 interacts with versican and smaller heparan/chondroitin sulfate proteoglycans (PGs) secreted as soluble components into the medium. In the present study, we investigated the binding of snpPLA2 to extracellular matrix (ECM) PGs produced by SMCs. The results show that snpPLA2 can bind to the ECM at physiological salt concentrations. ECM-bound snpPLA2 was active, hydrolyzing phosphatidylcholine-containing micelles. Soluble chondroitin-6-sulfate at concentrations >1 micromol/L, but not heparin or heparan sulfate, was able to release ECM-bound snpPLA2. The PG mainly involved in the binding of snpPLA2 was identified as biglycan. Perlecan was also present in the ECM synthesized by SMCs, but it contributed less to the binding of snpPLA2. Experiments with immobilized glycosaminoglycans indicated that snpPLA2 hydrolyzed 7-fold more LDL phospholipids when the lipoprotein and the enzyme were colocalized in a matrix with chondroitin-6-sulfate compared with one with heparin. These data suggest that retention of snpPLA2 in ECMs of different composition may modulate the enzymatic activity of snpPLA2 toward LDL. The results presented in this work support the hypothesis of the potential contribution of snpPLA2 to atherosclerosis.

    Arteriosclerosis, thrombosis, and vascular biology 1998;18;12;1934-41

  • The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein-protein interactions independent of carbohydrate moiety.

    Aspberg A, Miura R, Bourdoulous S, Shimonaka M, Heinegârd D, Schachner M, Ruoslahti E and Yamaguchi Y

    The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

    The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate-protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein-protein interaction through the fibronectin type III domains 3-5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein-protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein-protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.

    Funded by: NCI NIH HHS: CA28896, CA30199, P30 CA030199; NINDS NIH HHS: NS32717

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;19;10116-21

  • Binding of human phospholipase A2 type II to proteoglycans. Differential effect of glycosaminoglycans on enzyme activity.

    Sartipy P, Johansen B, Camejo G, Rosengren B, Bondjers G and Hurt-Camejo E

    Wallenberg Laboratory for Cardiovascular Research, Department of Heart and Lung Disease, Göteborg University, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden.

    Phospholipase A2 acting on low density lipoproteins in the extracellular arterial intima may form proinflammatory lipid mediators. Human nonpancreatic secretory phospholipase A2 has three regions that may associate with sulfated glycosaminoglycans. The apoB-100 molecule in low density lipoproteins also has glycosaminoglycan binding regions that could mediate its retention in the arterial intima. Here we report that human nonpancreatic phospholipase A2 isolated from a transfected cell line binds to glycosaminoglycans secreted by cultured human arterial smooth muscle cells. A gel mobility shift assay showed that the affinity of phospholipase A2 for glycosaminoglycans from a heparan sulfate/chondroitin sulfate proteoglycan was higher than for chondroitin sulfate glycosaminoglycans from a larger versican-like proteoglycan. Affinity chromatography confirmed these results. All glycosaminoglycans tested, at concentrations up to 100 microM, increased the activity of phospholipase A2 toward phosphatidylcholine liposomes. Above this concentration, heparan sulfate and heparin inhibited the enzyme. Heparin and chondroitin 6-sulfate increased phospholipase A2 activity on low density lipoproteins up to 4-fold at 100 microM, whereas heparan sulfate had no effect. The results indicate that human nonpancreatic secretory phospholipase A2 interacts with proteoglycans via their glycosaminoglycan moiety and that the enzyme activity may be modulated by the association of the enzyme and its substrate to the sulfated polysaccharides.

    The Journal of biological chemistry 1996;271;42;26307-14

  • Differential expression of versican isoforms in brain tumors.

    Paulus W, Baur I, Dours-Zimmermann MT and Zimmermann DR

    Institute of Neuropathology, University Hospital, Zurich, Switzerland.

    Versican is a member of the family of large aggregating chondroitin sulfate proteoglycans which are one of the major constituents of brain extracellular matrix (ECM). We examined the expression of versican splice variants at mRNA and protein levels in normal human brain and in gliomas, medulloblastomas, schwannomas, neurofibromas, and meningiomas. RT-PCR revealed transcripts for V0 and V1 in all tissues. V2 mRNA was restricted to gliomas and normal brain, while V3 mRNA was detected in all tissues except for medulloblastomas. Immunohistochemistry using antibodies to the glycosaminoglycan (GAG)-alpha attachment domain of versican (present in V0 and V2) revealed decreased staining of most glioma ECMs compared to normal neuropil, while some abnormal tumor vessels, but not normal cerebral vessels, were GAG-alpha-positive. Expression of the GAG-beta attachment domain (present in V0 and V1) was faint in normal neuropil and cerebral vessels, but increased in tumor vessels and was absent in most glioma ECMs. Both GAG-alpha and GAG-beta were expressed in connective tissue of all nonglial tumors. Our data suggest that V2 is the major versican isoform of normal neuropil and glioma ECM. Furthermore, increased expression in tumor vessels and decreased expression in glioma ECM of the anti-adhesive versican may be related to marked local invasivity and rarity of extracranial metastasis of gliomas.

    Journal of neuropathology and experimental neurology 1996;55;5;528-33

  • Distribution of the large aggregating proteoglycan versican in adult human tissues.

    Bode-Lesniewska B, Dours-Zimmermann MT, Odermatt BF, Briner J, Heitz PU and Zimmermann DR

    Institute of Clinical Pathology, Department of Pathology, University of Zürich, Switzerland.

    We studied the distribution of the large hyaluronan-binding proteoglycan versican (also known as PG-M) in human adult tissues using affinity-purified polyclonal antibodies that recognize the core protein of the prominent versican splice variants VO and V1. Versican was present in the loose connective tissues of various organs and was often associated with the elastic fiber network. Furthermore, it was localized in most smooth muscle tissues and in fibrous and elastic cartilage. Versican staining was also noted in the central and peripheral nervous system, in the basal layer of the epidermis, and on the luminal surface of some glandular epithelia. In blood vessels, versican was present in all three wall layers of veins and elastic arteries. In muscular arteries the immunoreactivity was normally restricted to the tunica adventitia. However, it appeared in the media and the split elastica interna of atherosclerotically transformed vessel walls. Our survey of the distribution of versican in normal human tissues now forms the basis for extended studies of potentially aberrant versican expression during pathogenic processes.

    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 1996;44;4;303-12

  • Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence.

    Adams MD, Kerlavage AR, Fleischmann RD, Fuldner RA, Bult CJ, Lee NH, Kirkness EF, Weinstock KG, Gocayne JD, White O et al.

    Institute for Genomic Research, Rockville, Maryland 20850, USA.

    In an effort to identify new genes and analyse their expression patterns, 174,472 partial complementary DNA sequences (expressed sequence tags (ESTs)), totalling more than 52 million nucleotides of human DNA sequence, have been generated from 300 cDNA libraries constructed from 37 distinct organs and tissues. These ESTs have been combined with an additional 118,406 ESTs from the database dbEST, for a total of 83 million nucleotides, and treated as a shotgun sequence assembly project. The assembly process yielded 29,599 distinct tentative human consensus (THC) sequences and 58,384 non-overlapping ESTs. Of these 87,983 distinct sequences, 10,214 further characterize previously known genes based on statistically significant similarity to sequences in the available databases; the remainder identify previously unknown genes. Thirty tissues were sampled by over 1,000 ESTs each; only eight genes were matched by ESTs from all 30 tissues, and 227 genes were represented in 20 or more of the tissues sampled with more than 1,000 ESTs. Approximately 40% of identified human genes appear to be associated with basic energy metabolism, cell structure, homeostasis and cell division, 22% with RNA and protein synthesis and processing, and 12% with cell signalling and communication.

    Nature 1995;377;6547 Suppl;3-174

  • Genetic linkage of Wagner disease and erosive vitreoretinopathy to chromosome 5q13-14.

    Brown DM, Graemiger RA, Hergersberg M, Schinzel A, Messmer EP, Niemeyer G, Schneeberger SA, Streb LM, Taylor CM, Kimura AE et al.

    Department of Ophthalmology, University of Iowa College of Medicine, Iowa City, USA.

    Background: Wagner disease and erosive vitreoretinopathy are potentially blinding autosomal dominant diseases that share some similarities with Stickler syndrome. However, both disorders have associated retinal pigment epithelial changes, poor night vision, visual field defects, and abnormal electroretinographic findings, which are not found in families with COL2A1-associated Stickler syndrome. In addition, rhegmatogenous retinal detachments are uncommon in Wagner disease but occur in approximately 50% of patients with either Stickler syndrome or erosive vitreoretinopathy.

    Objectives: To identify the chromosomal location of the genes involved in Wagner disease and erosive vitreoretinopathy and to distinguish these conditions genetically from Stickler syndrome.

    Methods: Fifteen affected members of a family affected with erosive vitreoretinopathy and 24 affected descendants of the pedigree described by Wagner were genotyped with a set of short tandem repeat polymorphisms distributed across the genome.

    Results: Significant linkage was observed in each family between the disease phenotype and markers that map to chromosome 5q13-14. The highest lod score for the family affected with erosive vitreoretinopathy was 4.2 and was obtained with marker GATA3H06 (theta = 0). The highest lod score for the family affected with Wagner disease was 5.8 and was obtained with marker D5S815 (theta = 0). A candidate gene (cartilage link protein) that is known to lie near the linked interval was screened for mutations, but none was found in either family.

    Conclusions: These data suggest that erosive vitreoretinopathy and Wagner disease are allelic disorders and demonstrate that they are genetically distinct from COL2A1-associated Stickler syndrome.

    Funded by: NEI NIH HHS: EY10539, EY10564

    Archives of ophthalmology (Chicago, Ill. : 1960) 1995;113;5;671-5

  • Expression of PG-M(V3), an alternatively spliced form of PG-M without a chondroitin sulfate attachment in region in mouse and human tissues.

    Zako M, Shinomura T, Ujita M, Ito K and Kimata K

    Institute for Molecular Science of Medicine, Aichi Medical University, Japan.

    We showed previously that the alternative splicing of chondroitin sulfate attachment domains (CS alpha and CS beta) yielded multiforms of the PG-M core protein in mouse. A transcript encoding a new short form of the core protein PG-M(V3) was found in various mouse tissues using polymerase chain reaction. DNA sequences of the polymerase chain reaction products suggested that PG-M(V3) had no chondroitin sulfate attachment domain. PG-M(V3) was also detected in various human tissues. The presence of a transcript for PG-M(V3) was further supported by Northern blot analysis. Southern blot analysis confirmed that multiforms of the PG-M core protein, including PG-M(V3), were derived from a single genomic locus by an alternative splicing mechanism. Because PG-M(V3) has no chondroitin sulfate attachment region, which is the most distinctive portion of a proteoglycan molecule, this form may have a unique function.

    The Journal of biological chemistry 1995;270;8;3914-8

  • A novel glycosaminoglycan attachment domain identified in two alternative splice variants of human versican.

    Dours-Zimmermann MT and Zimmermann DR

    Department of Pathology, University of Zürich, Switzerland.

    We have cloned an alternatively spliced glycosaminoglycan attachment domain (GAG-alpha) of human versican from cDNA libraries derived from U251MG glioma cells. Inserted carboxyl-terminal of the hyaluronan-binding region, this domain adds another 987 amino acids to the original versican (V1) core protein giving rise to the large V0 isoform with 3396 amino acids and 17-23 putative glycosaminoglycan attachment sites. The GAG-alpha domain is encoded by exon 7 of the human versican gene (Naso et al., J. Biol. Chem., 32999-33008). Sequence comparisons revealed a slight similarity to the alternative splice domain of PG-M, further supporting the notion that PG-M is the chicken homologue of versican. On immunoblots of a proteoglycan preparation from U251MG culture medium, anti-GAG-alpha antibodies reacted exclusively with the larger of two versican core proteins recognized by antibodies against the original GAG-beta domain. Using reverse transcription-polymerase chain reaction, we detected both the V0 and V1 isoforms in the cerebral cortex, aorta, intervertebral disc, liver, myometrium, and prostate, whereas keratinocytes exclusively expressed versican V1. In brain tissue, we identified a short versican variant (V2) including only the GAG-alpha domain. By expressing particular splice forms of versican, cells may control the hydration properties of their pericellular hyaluronan coat and thus could modulate interactions with the extracellular matrix or neighboring cells.

    The Journal of biological chemistry 1994;269;52;32992-8

  • Characterization of the complete genomic structure of the human versican gene and functional analysis of its promoter.

    Naso MF, Zimmermann DR and Iozzo RV

    Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

    Versican is a modular proteoglycan involved in the control of cellular growth and differentiation. To understand versican gene regulation and transcriptional control, we have isolated genomic clones spanning the entire gene locus including 5'- and 3'-flanking sequences. Versican was encoded by 15 exons encompassing over 90 kilobase pairs of continuous DNA. The exon organization corresponded to the protein subdomains encoded by homologous proteins, with a remarkable conservation of exon size and intron phase. We discovered an additional exon just proximal to the glycosaminoglycan-binding region that was identical to a recently identified splice variant of versican (Dours-Zimmermann, M.T., and Zimmermann, D.R. (1994) J. Biol. Chem. 269, 32992-32998). The versican promoter harbored a typical TATA box located approximately 16 base pairs upstream of the transcription start site and binding sites for a number of transcription factors involved in regulated gene expression. This promoter was shown to be highly functional in transiently transfected cells of both mesenchymal and epithelial origin. Stepwise 5' deletions identified a strong enhancer element between -209 and -445 base pairs and a strong negative element between -445 and -632 base pairs. This study provides the molecular basis for discerning the transcriptional control of the versican gene and offers the opportunity to investigate genetic disorders linked to this important human gene.

    Funded by: NCI NIH HHS: CA-39481, CA-47282

    The Journal of biological chemistry 1994;269;52;32999-3008

  • Identification of the proteoglycan versican in aorta and smooth muscle cells by DNA sequence analysis, in situ hybridization and immunohistochemistry.

    Yao LY, Moody C, Schönherr E, Wight TN and Sandell LJ

    Department of Orthopaedics, University of Washington, Seattle.

    Versican is a large chondroitin sulfate proteoglycan (CSPG) initially identified in cultured human fibroblasts. Previous studies have shown that there is a versican-like molecule in cultured monkey smooth muscle cells. In this study, we have cloned and sequenced the large CSPG from cultured monkey smooth muscle cells, fetal and juvenile monkey aorta, and human fetal aorta. The cDNA sequence from human fetal aorta is completely homologous to the human fibroblast versican. We obtained 2.5 kb of cDNA sequence from monkey aortic RNA and cultured monkey smooth muscle cell RNA. This sequence covers three distinct domains of versican (hyaluronic acid binding domain, glycosaminoglycan attachment domain and protein binding domain) and demonstrates over 90% homology to the human versican sequence. In situ hybridization histochemistry indicates that the versican RNA transcript is located in the epithelium throughout the tunica media of the aorta. Western blot analysis and immunohistochemistry also confirm the presence of versican in human and monkey aorta.

    Funded by: NHLBI NIH HHS: HL 18645; NIAMS NIH HHS: R01 AR36994

    Matrix biology : journal of the International Society for Matrix Biology 1994;14;3;213-25

  • Mapping of the versican proteoglycan gene (CSPG2) to the long arm of human chromosome 5 (5q12-5q14).

    Iozzo RV, Naso MF, Cannizzaro LA, Wasmuth JJ and McPherson JD

    Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

    Versican is a major chondroitin sulfate proteoglycan of vascularized connective tissues whose eponym reflects its functional versatility in macromolecular affinity and interactions. In this report we have localized the versican gene (CSPG2) to the long arm of human chromosome 5 by utilizing a combination of somatic cell hybrids, Southern blotting, polymerase chain reaction, and chromosomal in situ hybridization. The proteoglycan gene segregated concordantly with hybrid cell lines containing the long arm of chromosome 5, comprising the 5q12-q14 band regions. To refine this locus further, we screened a chromosome 5-specific library and isolated several genomic clones encoding a portion of the 5' end of versican. One of these genomic clones was used as a probe for in situ hybridization of human chromosome metaphases. The results corroborated the data obtained using somatic cell hybrids and further refined the assignment of the versican gene to the narrow band region of 5q12-5q14, with the primary site likely to be 5q13.2. The availability of novel genomic clones and the mapping data presented here will make possible the identification of any defect genetically linked to this proteoglycan gene.

    Funded by: NCI NIH HHS: CA-39481, CA-47282; NHGRI NIH HHS: HG-00320

    Genomics 1992;14;4;845-51

  • Isolation of a large aggregating proteoglycan from human brain.

    Perides G, Rahemtulla F, Lane WS, Asher RA and Bignami A

    Department of Pathology, Harvard Medical School, Boston, Massachusetts.

    A large proteoglycan (365 kDa), identified with monoclonal antibodies raised against chondroitin sulfate, was isolated from human brain. The isolation required anion-exchange chromatography followed by gel filtration through a Sephacryl S-500 column. The proteoglycan bound specifically to [3H]hyaluronate (HA). The binding was not reduced by high salt concentrations (up to 4 M) and was inhibited at low pH (< 4.0). The binding was inhibited by the octamer and decamer (but not the hexamer) oligosaccharides of HA. Limited proteolysis of the proteoglycan gave rise to a relatively stable polypeptide (80 kDa). The amino-terminal sequence of the 80-kDa polypeptide was identical to the cDNA-derived amino-terminal sequence of versican, a large human fibroblast proteoglycan. A monoclonal antibody raised against bovine proteoglycans and recognizing the versican core protein reacted by immunoblotting with the proteoglycan isolated from human brain. The antibody was used to localize the proteoglycan in acetone-fixed cryostat sections of bovine spinal cord. The localization of the proteoglycan in the central nervous system was identical to that previously reported for glial hyaluronate-binding protein (GHAP), a 60-kDa glycoprotein of the brain extracellular matrix (ECM). However, a major difference was observed with respect to the sensitivity of the two antigens to hyaluronidase. As previously reported, GHAP was released from the tissue by hyaluronidase digestion, whereas the proteoglycan persisted under these conditions. We conclude that the protein-hyaluronate aggregates in brain ECM contain both GHAP and versican, that GHAP is only retained in the ECM by its interaction with hyaluronate, and that the proteoglycan is anchored in some other manner and probably connects cell surfaces with the ECM since it was not released by hyaluronidase digestion.

    Funded by: NIDCR NIH HHS: DE 08466; NINDS NIH HHS: NS 13034

    The Journal of biological chemistry 1992;267;33;23883-7

  • Multiple domains of the large fibroblast proteoglycan, versican.

    Zimmermann DR and Ruoslahti E

    La Jolla Cancer Research Foundation, CA 92037.

    The primary structure of a large chondroitin sulfate proteoglycan expressed by human fibroblasts has been determined. Overlapping cDNA clones code for the entire 2389 amino acid long core protein and the 20-residue signal peptide. The sequence predicts a potential hyaluronic acid-binding domain in the amino-terminal portion. This domain contains sequences virtually identical to partial peptide sequences from a glial hyaluronate-binding protein. Putative glycosaminoglycan attachment sites are located in the middle of the protein. The carboxy-terminal portion includes two epidermal growth factor (EGF)-like repeats, a lectin-like sequence and a complement regulatory protein-like domain. The same set of binding elements has also been identified in a new class of cell adhesion molecules. Amino- and carboxy-terminal portions of the fibroblast core protein are closely related to the core protein of a large chondroitin sulfate proteoglycan of chondrosarcoma cells. However, the glycosaminoglycan attachment regions in the middle of the core proteins are different and only the fibroblast core protein contains EGF-like repeats. Based on the similarities of its domains with various binding elements of other proteins, we suggest that the large fibroblast proteoglycan, herein referred to as versican, may function in cell recognition, possibly by connecting extracellular matrix components and cell surface glycoproteins.

    Funded by: NCI NIH HHS: CA 30199, CA 42507

    The EMBO journal 1989;8;10;2975-81

  • Isolation and partial characterization of a glial hyaluronate-binding protein.

    Perides G, Lane WS, Andrews D, Dahl D and Bignami A

    Department of Pathology, Harvard Medical School, Boston, Massachusetts.

    A glial hyaluronate-binding protein (GHAP) with an isoelectric point of 4.3-4.4 was isolated from human brain white matter. The 60-kDa glycoprotein appeared to be quite resistant to proteolysis, and comparison with GHAP from a viable glioma removed at surgery showed that the protein isolated from autopsy material was not a degradation product resulting from postmortem autolysis. The protein was localized immunohistochemically with mouse monoclonal and rabbit polyclonal antibodies in cerebral white matter. Only small amounts could be found in the gray matter. After enzymatic deglycosylation, an immunoreactive 47-kDa polypeptide was obtained. Two amino acid sequences of GHAP showed a striking similarity (up to 89%) with a highly conserved region of cartilage proteins (bovine nasal cartilage proteoglycan and rat and chicken link protein). However, the amino acid composition and other amino acid sequences suggested that there are also differences between brain-specific GHAP and cartilage proteins.

    Funded by: NINDS NIH HHS: NS13034

    The Journal of biological chemistry 1989;264;10;5981-7

  • Structural similarity of hyaluronate binding proteins in brain and cartilage.

    Bignami A, Lane WS, Andrews D and Dahl D

    Department of Pathology, Harvard Medical School, West Roxbury, MA 02132.

    A glial hyaluronate-binding protein (GHAP) was isolated from human brain white matter by affinity chromatography on immobilized hyaluronate. The 60 kDa protein appeared remarkably homogeneous by reversed-phase high pressure liquid chromatography analysis. Four cyanogen bromide peptides and 10 tryptic peptides were characterized by amino acid sequence, a total of 12 sequences since overlaps were found between 2 cyanogen bromide and 2 tryptic peptide sequences. Two sequences of brain GHAP had similarity with rat link protein, a hyaluronate binding protein in cartilage. The region of similarity was contained in the evolutionary conserved COOH-terminal half of link protein which is involved in the binding of hyaluronate. The remaining 10 amino acid sequences of brain GHAP had no similarity with link protein, nor with previously reported protein sequences. The findings suggest that the hyaluronate binding domains of such diverse proteins as brain GHAP and cartilage link protein are similar, probably due to the fact that hyaluronic acid is highly conserved in evolution.

    Funded by: NINDS NIH HHS: NS 13034

    Brain research bulletin 1989;22;1;67-70

  • A fibroblast chondroitin sulfate proteoglycan core protein contains lectin-like and growth factor-like sequences.

    Krusius T, Gehlsen KR and Ruoslahti E

    Cancer Research Center, La Jolla Cancer Research Foundation, California 92037.

    We have isolated cDNA clones that code for a proteoglycan-related polypeptide with unique properties. A lambda gt11 expression library made from human fibroblast mRNA was screened with an antiserum made against a proteoglycan fraction from human fetal membranes. One group of positive clones revealed an open reading frame coding for 685 amino acids from the COOH terminus of a polypeptide. This amino acid sequence contains a domain that is strongly homologous with the COOH-terminal core protein domain of the large aggregating cartilage proteoglycan. This domain also contains sequences that are homologous with vertebrate lectins that bind terminal galactosyl, N-acetyl-glucosaminyl or mannosyl residues. On the NH2-terminal side of the lectin-like domain the cDNA-derived amino acid sequence contains two epidermal growth factor-related segments. The cDNA clones were shown to belong to a chondroitin sulfate proteoglycan by using antisera made against two peptides predicted from the cDNA sequence. These antisera were reactive with a proteoglycan fraction from fibroblasts after chondroitinase treatment of the fraction but not after treatment with heparinase or no treatment. Among the several polypeptides reactive with the anti-peptide antibodies the largest one, corresponding to a molecular weight of about 400,000, is likely to be the intact core protein, whereas the smaller polypeptides may be processing products or products of artifactual proteolysis. These results show that the amino acid sequence belongs to a proteoglycan core protein, and the sequence, therefore, provides a molecular definition to this proteoglycan. The lectin-related and growth factor-like sequences in the core protein of this proteoglycan suggest that it may play a role in intercellular signaling.

    Funded by: NCI NIH HHS: CA 28896, CA 30199, CA 42507; ...

    The Journal of biological chemistry 1987;262;27;13120-5

Gene lists (6)

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
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© 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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