G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
heme oxygenase (decycling) 2
G00001109 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000073116 (Vega human gene)
ENSG00000103415 (Ensembl human gene)
3163 (Entrez Gene)
260 (G2Cdb plasticity & disease)
HMOX2 (GeneCards)
141251 (OMIM)
Marker Symbol
HGNC:5014 (HGNC)
Protein Sequence
P30519 (UniProt)

Synonyms (1)

  • HO-2

Literature (38)

Pubmed - other

  • Association of an intronic variant of the heme oxygenase-1 gene with hypertension in northern Chinese Han population.

    Yun L, Xiaoli L, Qi Z, Laiyuan W, Xiangfeng L, Chong S, Jianfeng H, Shufeng C, Hongfan L and Gu D

    Department of Evidence-Based Medicine and Division of Population Genetics, Cardiovascular Institute and Fuwai Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, PR China.

    Heme oxygenase was regarded as a regulator of oxidative stress, which was believed to underlie the etiology of hypertension. To assess the effect of its encoded genes (HMOX1 and HMOX2) on hypertension, we designed a case-control study in 503 cases and 490 controls. The results indicated that the rs9607267 of the HMOX1 gene was significantly associated with essential hypertension (EH) and the Hap3(T-C-G) of the HMOX1 gene was also significantly associated with the risk of EH. No association was observed between the HMOX2 gene and EH. The multifactor-dimensionality reduction analyses results did not show any interaction between the HMOX1 and HMOX2 genes underlying the development of hypertension.

    Clinical and experimental hypertension (New York, N.Y. : 1993) 2009;31;7;534-43

  • Heme regulatory motifs in heme oxygenase-2 form a thiol/disulfide redox switch that responds to the cellular redox state.

    Yi L, Jenkins PM, Leichert LI, Jakob U, Martens JR and Ragsdale SW

    Departments of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

    Heme oxygenase (HO) catalyzes the rate-limiting step in heme catabolism to generate CO, biliverdin, and free iron. Two isoforms of HO have been identified in mammals: inducible HO-1 and constitutively expressed HO-2. HO-1 and HO-2 share similar physical and kinetic properties but have different physiological roles and tissue distributions. Unlike HO-1, which lacks cysteine residues, HO-2 contains three Cys-Pro signatures, known as heme regulatory motifs (HRMs), which are known to control processes related to iron and oxidative metabolism in organisms from bacteria to humans. In HO-2, the C-terminal HRMs constitute a thiol/disulfide redox switch that regulates affinity of the enzyme for heme (Yi, L., and Ragsdale, S. W. (2007) J. Biol. Chem. 282, 20156-21067). Here, we demonstrate that the thiol/disulfide switch in human HO-2 is physiologically relevant. Its redox potential was measured to be -200 mV, which is near the ambient intracellular redox potential. We expressed HO-2 in bacterial and human cells and measured the redox state of the C-terminal HRMs in growing cells by thiol-trapping experiments using the isotope-coded affinity tag technique. Under normal growth conditions, the HRMs are 60-70% reduced, whereas oxidative stress conditions convert most (86-89%) of the HRMs to the disulfide state. Treatment with reductants converts the HRMs largely (81-87%) to the reduced dithiol state. Thus, the thiol/disulfide switch in HO-2 responds to cellular oxidative stress and reductive conditions, representing a paradigm for how HRMs can integrate heme homeostasis with CO signaling and redox regulation of cellular metabolism.

    Funded by: NHLBI NIH HHS: R21 HL089837, R21HL089837; NIA NIH HHS: AG027349, R01 AG027349; NIDCD NIH HHS: T32 DC000011; NIGMS NIH HHS: GM065318, R01 GM065318

    The Journal of biological chemistry 2009;284;31;20556-61

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Cellular mRNA expressions of anti-oxidant factors in the blood of preeclamptic women.

    Nakamura M, Sekizawa A, Purwosunu Y, Okazaki S, Farina A, Wibowo N, Shimizu H and Okai T

    Department of Obstetrics and Gynecology, Showa University School of Medicine, Tokyo, Japan.

    Objective: To assess the alterations of mRNA expressions associated with oxidative stress in the cellular component of blood from pregnant women with pre-eclampsia.

    Methods: Peripheral blood samples were obtained from pregnant women with and without pre-eclampsia. Cellular RNA was subjected to a reverse transcription polymerase chain reaction (PCR) assay in order to examine the mRNA distribution among women with pre-eclampsia (n = 24) and control subjects (n = 24) during 35-41 weeks of gestation. The data were analyzed by non-parametric statistics.

    Results: Significant differences between the pre-eclampsia subjects and the controls were observed in the gene expressions associated with oxidative stress. Lower values in the pre-eclampsia group were found for heme oxygenase (HO)-1, HO-2, catalase and superoxide dismutase (SOD). The HO-1, HO-2 and the catalase levels significantly correlated with proteinuria, and the HO-2 level with systolic blood pressure.

    Conclusion: Significantly lower concentrations of HO-1, HO-2, SOD and catalase are found in the cellular component of blood from pre-eclamptic patients. The values correlate with the severity of pre-eclampsia. These findings indicate that enhanced oxidative stress and a decrease in the number of anti-oxidant enzymes may be associated with pre-eclampsia.

    Prenatal diagnosis 2009;29;7;691-6

  • Inhibitors of heme oxygenase reduce invasion of human primary cytotrophoblast cells in vitro.

    McCaig D and Lyall F

    Institute of Medical Genetics, Yorkhill Hospital, Glasgow G3 8SJ, UK.

    Having previously demonstrated that heme oxygenase (HO) is expressed on invasive trophoblast within the human placental bed, we have now further hypothesised that HO may play a role in trophoblast invasion. To begin to test this hypothesis we have used a well characterised in vitro model of trophoblast invasion to determine whether antibodies raised against HO-1 and HO-2, or selective inhibition of HO with the HO inhibitor zinc protoporhyrin-9 (Zn PP-9), would affect the invasive ability of trophoblast cells. Cytotrophoblast cells were purified from term human placenta then cultured on Matrigel-coated chambers in the presence or absence of HO antibodies or Zn PP-9. The HO-1 antibody had no effect on invasion whereas the presence of the HO-2 antibody significantly inhibition invasion (p<0.05). The presence of Zn PP-9 resulted in a significant reduction in invasion (p<0.05) whereas the vehicle alone had no effect. Taken together these results suggest, that at least in vitro, HO-2 may be important in controlling trophoblast invasion.

    Funded by: British Heart Foundation

    Placenta 2009;30;6;536-8

  • No genetic association between polymorphisms of heme oxygenase 1 and 2 and Alzheimer's disease in a Japanese population.

    Shibata N, Ohnuma T, Baba H and Arai H

    Department of Psychiatry, Juntendo University School of Medicine, Tokyo, Japan. nobuto.shibata@nifty.ne.jp

    Recently, it has been shown that the heme oxygenases HMOX1 and HMOX2 might play a role in the pathogenesis of Alzheimer's disease (AD).

    Methods: To investigate whether there is any association between the HMOX1 and HMOX2 genes and AD, five single nucleotide polymorphisms (SNPs) in each gene were genotyped in 180 AD patients and 132 age-matched controls using TaqMan technology.

    Results: Our study failed to detect any association between the SNPs of the HMOX1 and HMOX2 genes and AD. In addition, we did not observe any synergetic association between the SNPs studied and apolipoprotein E in our AD patients.

    Conclusion: Further genetic studies are needed to clarify the relationship between the two genes and AD.

    Dementia and geriatric cognitive disorders 2009;27;3;273-7

  • Oxidative stress, telomere length and biomarkers of physical aging in a cohort aged 79 years from the 1932 Scottish Mental Survey.

    Starr JM, Shiels PG, Harris SE, Pattie A, Pearce MS, Relton CL and Deary IJ

    MRC Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Royal Victoria Hospital, Edinburgh EH4 2DN, UK. jstarr@staffmail.ed.ac.uk

    Telomere shortening is a biomarker of cellular senescence and is associated with a wide range of age-related disease. Oxidative stress is also associated with physiological aging and several age-related diseases. Non-human studies suggest that variants in oxidative stress genes may contribute to both telomere shortening and biological aging. We sought to test whether oxidative stress-related gene polymorphisms contribute to variance in both telomere length and physical biomarkers of aging in humans. Telomere lengths were calculated for 190 (82 men, 108 women) participants aged 79 years and associations with 384 SNPs, from 141 oxidative stress genes, identified 9 significant SNPS, of which those from 5 genes (GSTZ1, MSRA, NDUFA3, NDUFA8, VIM) had robust associations with physical aging biomarkers, respiratory function or grip strength. Replication of associations in a sample of 318 (120 males, 198 females) participants aged 50 years confirmed significant associations for two of the five SNPs (MSRA rs4841322, p=0.008; NDUFA8 rs6822, p=0.048) on telomere length. These data indicate that oxidative stress genes may be involved in pathways that lead to both telomere shortening and physiological aging in humans. Oxidative stress may explain, at least in part, associations between telomere shortening and physiological aging.

    Funded by: Biotechnology and Biological Sciences Research Council: S18386; Chief Scientist Office: CZB/4/505, ETM/55; Medical Research Council; Wellcome Trust

    Mechanisms of ageing and development 2008;129;12;745-51

  • Comparison of apo- and heme-bound crystal structures of a truncated human heme oxygenase-2.

    Bianchetti CM, Yi L, Ragsdale SW and Phillips GN

    Graduate Program in Biophysics, Center for Eukaryotic Structural Genomics, University of Wisconsin, Madison, WI 53706, USA.

    Heme oxygenase (HO) catalyzes the first step in the heme degradation pathway. The crystal structures of apo- and heme-bound truncated human HO-2 reveal a primarily alpha-helical architecture similar to that of human HO-1 and other known HOs. Proper orientation of heme in HO-2 is required for the regioselective oxidation of the alpha-mesocarbon. This is accomplished by interactions within the heme binding pocket, which is made up of two helices. The iron coordinating residue, His(45), resides on the proximal helix. The distal helix contains highly conserved glycine residues that allow the helix to flex and interact with the bound heme. Tyr(154), Lys(199), and Arg(203) orient the heme through direct interactions with the heme propionates. The rearrangements of side chains in heme-bound HO-2 compared with apoHO-2 further elucidate HO-2 heme interactions.

    Funded by: NCI NIH HHS: Y1 CO1020; NCRR NIH HHS: 1 P20 RR17675, P20 RR017675; NHLBI NIH HHS: R21 HL089837, R21 HL089837-02; NIGMS NIH HHS: 1 U54 GM074901-01, P50 GM064598, U54 GM074901, Y1 GM1104

    The Journal of biological chemistry 2007;282;52;37624-31

  • Membrane potential gradient is carbon monoxide-dependent in mouse and human small intestine.

    Sha L, Farrugia G, Harmsen WS and Szurszewski JH

    Enteric NeuroScience Program, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.

    The aims of this study were to quantify the change in resting membrane potential (RMP) across the thickness of the circular muscle layer in the mouse and human small intestine and to determine whether the gradient in RMP is dependent on the endogenous production of carbon monoxide (CO). Conventional sharp glass microelectrodes were used to record the RMPs of circular smooth muscle cells at different depths in the human small intestine and in wild-type, HO2-KO, and W/W(V) mutant mouse small intestine. In the wild-type mouse and human intestine, the RMP of circular smooth muscle cells near the myenteric plexus was -65.3 +/- 2 mV and -58.4 +/- 2 mV, respectively, and -60.1 +/- 2 mV and -49.1 +/- 1 mV, respectively, in circular smooth muscle cells at the submucosal border. Oxyhemoglobin (20 microM), a trapping agent for CO, and chromium mesoporphyrin IX, an inhibitor of heme oxygenase, abolished the transwall gradient. The RMP gradients in mouse and human small intestine were not altered by N(G)-nitro-l-arginine (200 microM). No transwall RMP gradient was found in HO2-KO mice and W/W(V) mutant mice. TTX (1 microM) and 1H-[1,2,4-]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM) had no effect on the RMP gradient. These data suggest that the gradient in RMP across the thickness of the circular muscle layer of mouse and human small intestine is CO dependent.

    Funded by: NIDDK NIH HHS: DK-17238, DK-52766

    American journal of physiology. Gastrointestinal and liver physiology 2007;293;2;G438-45

  • Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding.

    Yi L and Ragsdale SW

    Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.

    Heme oxygenase (HO) catalyzes the O(2)- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568-12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys(265) of HRM1 and Cys(282) of HRM2, binds heme tightly. Reduction of the disulfide bond increases the K(d) for ferric heme from 0.03 to 0.3 microm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the K(d) for heme, they do not alter the k(cat) for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca(2+)-activated potassium channel.

    Funded by: NCRR NIH HHS: 1P20RR17675, P20 RR017675; NHLBI NIH HHS: R21 HL089837

    The Journal of biological chemistry 2007;282;29;21056-67

  • A genetic association analysis of cognitive ability and cognitive ageing using 325 markers for 109 genes associated with oxidative stress or cognition.

    Harris SE, Fox H, Wright AF, Hayward C, Starr JM, Whalley LJ and Deary IJ

    Department of Psychology, University of Edinburgh, Edinburgh, UK. Sarah.Harris@hgu.mrc.ac.uk <Sarah.Harris@hgu.mrc.ac.uk&gt;

    Background: Non-pathological cognitive ageing is a distressing condition affecting an increasing number of people in our 'ageing society'. Oxidative stress is hypothesised to have a major role in cellular ageing, including brain ageing.

    Results: Associations between cognitive ageing and 325 single nucleotide polymorphisms (SNPs), located in 109 genes implicated in oxidative stress and/or cognition, were examined in a unique cohort of relatively healthy older people, on whom we have cognitive ability scores at ages 11 and 79 years (LBC1921). SNPs showing a significant positive association were then genotyped in a second cohort for whom we have cognitive ability scores at the ages of 11 and 64 years (ABC1936). An intronic SNP in the APP gene (rs2830102) was significantly associated with cognitive ageing in both LBC1921 and a combined LBC1921/ABC1936 analysis (p < 0.01), but not in ABC1936 alone.

    Conclusion: This study suggests a possible role for APP in normal cognitive ageing, in addition to its role in Alzheimer's disease.

    Funded by: Medical Research Council: MC_U127561128

    BMC genetics 2007;8;43

  • Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell lines.

    Ding Y, Zhang YZ, Furuyama K, Ogawa K, Igarashi K and Shibahara S

    Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Japan.

    Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase. Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2. Recent studies have identified HO-2 as a potential oxygen sensor. To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells. Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma. Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells. In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression. HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells. Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner. Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin. HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis. These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2.

    The FEBS journal 2006;273;23;5333-46

  • 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

  • Hemeoxygenase-2 as an O2 sensor in K+ channel-dependent chemotransduction.

    Kemp PJ

    School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, UK. kemp@cf.ac.uk

    The physiological response of the carotid body is critically dependent upon oxygen-sensing by potassium channels expressed in glomus cells. One such channel is the large conductance, voltage- and calcium-dependent potassium channel, BK(Ca). Although it is well known that a decrease in oxygen evokes glomus cell depolarization, voltage-gated calcium entry, and transmitter release, the molecular identity of the upstream oxygen sensor has been the subject of some controversy for decades. Recently, we have demonstrated that hemeoxygenase-2 associates tightly with recombinant BK(Ca) and that activity of this enzyme confers oxygen sensitivity to the BK(Ca) channel complex. Similar observations were also made in native channels recorded from carotid body glomus cells, suggesting that hemoxygenase-2 functions as an oxygen sensor of native and recombinant BK(Ca) channels.

    Funded by: Wellcome Trust

    Biochemical and biophysical research communications 2005;338;1;648-52

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Catalytically inactive heme oxygenase-2 mutant is cytoprotective.

    Kim YS and Doré S

    Department of Anesthesiology/Critical Care Medicine, Johns Hopkins University School of Medicine, Neuro. Res. Div., 720 Rutland Ave., Ross Research Bldg. 365, Baltimore, MD 21205, USA.

    Heme oxygenase (HO) catalyzes the rate-limiting step in heme degradation, producing iron, carbon monoxide, and bilirubin/biliverdin. HO consists of two isozymes: HO-1, which is an oxidative stress-response protein, and HO-2, which is constitutively expressed. HO-2 accounts for most HO activity within the nervous system. Its posttranslational modifications and/or interactions with other proteins make HO-2 a unique regulator of cellular homeostasis. Our previous results revealed that brain infarct volume was enlarged in HO-2 knockout mice. A similar neuroprotective role of HO-2 was shown using primary cortical neurons. To better understand the neuroprotective mechanism of HO-2, we used a catalytically inactive mutant, HO-2H45A, and investigated its cellular effects in response to hemin and hydrogen peroxide-induced cytotoxicity. We observed that HO-2WT overexpression in the HEK293 cell lines became less sensitive to hemin, whereas the inactive mutant HO-2H45A was more sensitive to hemin as compared to control. Interestingly, HO-2WT- and HO-2H45A-overexpressing cells were both protected against H2O2-induced oxidative stress and had less oxidatively modified proteins as compared to control cells. These data indicate that when HO-2 cannot metabolize the prooxidant heme, more cytotoxicity is found, whereas, interestingly, the catalytically inactive HO-2H45A was also able to protect cells against oxidative stress injury. These results suggest the multiplicity of action of the HO-2 protein itself.

    Funded by: NCCIH NIH HHS: AT001836; NIA NIH HHS: AG022971; NIAAA NIH HHS: AA014911; NINDS NIH HHS: NS046400

    Free radical biology & medicine 2005;39;4;558-64

  • Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel.

    Williams SE, Wootton P, Mason HS, Bould J, Iles DE, Riccardi D, Peers C and Kemp PJ

    School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK.

    Modulation of calcium-sensitive potassium (BK) channels by oxygen is important in several mammalian tissues, and in the carotid body it is crucial to respiratory control. However, the identity of the oxygen sensor remains unknown. We demonstrate that hemoxygenase-2 (HO-2) is part of the BK channel complex and enhances channel activity in normoxia. Knockdown of HO-2 expression reduced channel activity, and carbon monoxide, a product of HO-2 activity, rescued this loss of function. Inhibition of BK channels by hypoxia was dependent on HO-2 expression and was augmented by HO-2 stimulation. Furthermore, carotid body cells demonstrated HO-2-dependent hypoxic BK channel inhibition, which indicates that HO-2 is an oxygen sensor that controls channel activity during oxygen deprivation.

    Science (New York, N.Y.) 2004;306;5704;2093-7

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • 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

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Effect of glucose and oxygen deprivation on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells.

    Appleton SD, Lash GE, Marks GS, Nakatsu K, Brien JF, Smith GN and Graham CH

    Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario, Canada.

    Although hypoxia induces heme oxygenase (HO)-1 mRNA and protein expression in many cell types, recent studies in our laboratory using human placental tissue have shown that a preexposure to hypoxia does not affect subsequent HO enzymatic activity for optimized assay conditions (20% O2; 0.5 mM NADPH; 25 microM methemalbumin) or HO-1 protein content. One of the consequences of impaired blood flow is glucose deprivation, which has been shown to be an inducer of HO-1 expression in HepG2 hepatoma cells. The objective of the present study was to test the effects of a 24-h preexposure to glucose-deprived medium, in 0.5 or 20% O2, on HO protein content and enzymatic activity in isolated chorionic villi and immortalized HTR-8/SVneo first-trimester trophoblast cells. HO protein content was determined by Western blot analysis, and microsomal HO enzymatic activity was measured by assessment of the rate of CO formation. HO enzymatic activity was increased (P < 0.05) in both placental models after 24-h preexposure to glucose-deficient medium in 0.5 or 20% O2. Preexposure (24 h) in a combination of low O2 and low glucose concentrations decreased the protein content of the HO-1 isoform by 59.6% (P < 0.05), whereas preexposure (24 h) to low glucose concentration alone increased HO-2 content by 28.2% in chorionic villi explants (P < 0.05). In this preparation, HO enzymatic activity correlated with HO-2 protein content (r = 0.825). However, there was no correlation between HO-2 protein content and HO enzymatic activity in HTR-8/SVneo trophoblast cells preexposed to 0.5% O2 and low glucose concentration for 24 h. These findings indicate that the regulation of HO expression in the human placenta is a complex process that depends, at least in part, on local glucose and oxygen concentrations.

    American journal of physiology. Regulatory, integrative and comparative physiology 2003;285;6;R1453-60

  • Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2.

    Boehning D, Moon C, Sharma S, Hurt KJ, Hester LD, Ronnett GV, Shugar D and Snyder SH

    Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

    Carbon monoxide (CO) is a putative gaseous neurotransmitter that lacks vesicular storage and must be synthesized rapidly following neuronal depolarization. We show that the biosynthetic enzyme for CO, heme oxygenase-2 (HO2), is activated during neuronal stimulation by phosphorylation by CK2 (formerly casein kinase 2). Phorbol ester treatment of hippocampal cultures results in the phosphorylation and activation of HO2 by CK2, implicating protein kinase C (PKC) in CK2 stimulation. Odorant treatment of olfactory receptor neurons augments HO2 phosphorylation and activity as well as cyclic guanosine monophosphate (cGMP) levels, with all of these effects selectively blocked by CK2 inhibitors. Likewise, CO-mediated nonadrenergic, noncholinergic (NANC) relaxation of the internal anal sphincter requires CK2 activity. Our findings provide a molecular mechanism for the rapid neuronal activation of CO biosynthesis, as required for a gaseous neurotransmitter.

    Funded by: NIDA NIH HHS: DA-000266, DA-00074; NIDCD NIH HHS: DC-02979; NINDS NIH HHS: NS-043850, NS-39657

    Neuron 2003;40;1;129-37

  • Heme oxygenases in pregnancy II: HO-2 is downregulated in human pathologic pregnancies.

    Zenclussen AC, Lim E, Knoeller S, Knackstedt M, Hertwig K, Hagen E, Klapp BF and Arck PC

    Charité, Biomedizinisches Forschungszentrum, Campus Virchow, Humboldt University of Berlin, Germany.

    Problem: We previously reported a diminished expression of the heme-degrading enzymes heme oxygenases (HO)-1 and HO-2 in decidua and placenta from mice undergoing Th1-mediated abortion, strongly indicating the protective effect of HO in murine pregnancy maintenance. Here we investigated whether the expression of HO-1 and HO-2 is also reduced at the feto-maternal interface of pathologic human pregnancies.

    Immunohistochemistry was used to detect HOs expression in placental and decidual first-trimester tissue from patients with: spontaneous abortion (n = 14), choriocarcinoma (n = 14), hydatidiform mole (H-mole) (n = 12), compared with normally progressing pregnancies (n = 15). Further, we investigated early third-trimester decidual and placental tissue from patients with pre-eclampsia (n = 13) compared with fetal growth retardation (n = 14) as age-matched controls.

    Results: In first trimester tissue, we observed a significant reduction of HO-2 expression in invasive trophoblast cells, endothelial cells, and syncytiotrophoblasts in samples from patients with spontaneous abortion compared with normal pregnancy. H-mole samples showed a diminished expression of HO-2 in invasive trophoblast cells and endothelial cells in comparison with NP, whereas choriocarcinoma samples showed no significant differences compared with the control. In third trimester tissue, HO-2 was also reduced in syncytiotrophoblasts and invasive trophoblast cells from pre-eclampsia compared with samples from fetal growth retardation. HO-1 expression was diminished in all pathologies investigated; however, the differences did not reach levels of significance.

    Conclusions: Our data indicate that HOs play a crucial role in pregnancy and low expression of HO-2, as observed in pathologic pregnancies, may lead to enhanced levels of free heme at the feto-maternal interface, with subsequent upregulation of adhesion molecules, allowing enhanced inflammatory cells migration to the feto-maternal interface.

    American journal of reproductive immunology (New York, N.Y. : 1989) 2003;50;1;66-76

  • Differential transcriptional regulation by human immunodeficiency virus type 1 and gp120 in human astrocytes.

    Galey D, Becker K, Haughey N, Kalehua A, Taub D, Woodward J, Mattson MP and Nath A

    Department of Neurology, Johns Hopkins School of Medicine, Baltimore, Maryland 21287, USA.

    Astrocytes may be infected with the human immunodeficiency virus type 1 (HIV-1) or exposed to the HIV protein gp120, yet their role in the pathogenesis of HIV dementia is largely unknown. To characterize the effects of HIV on astrocytic transcription, microarray analysis and ribonuclease protection assays (RPA) were performed. Infection of astrocytes by HIV or treatment with gp120 had differential and profound effects on gene transcription. Of the 1153 oligonucleotides on the immune-based array, the expression of 108 genes (53 up; 55 down) and 82 genes (32 up; 50 down) were significantly modulated by gp120 and HIV infection respectively. Of the 1153 oligonucleotides on the neuro-based array, 58 genes (25 up; 33 down) and 47 genes (17 up; 30 down) were significantly modulated by gp120 and HIV infection respectively. Chemokine and cytokine induction occurred predominantly by HIV infection, whereas gp120 had no significant effect. These results were confirmed by RPA. The authors conclude that profound alterations of astrocytic function occur in response to HIV infection or interaction with viral proteins, suggesting that astrocytes may play an important role in the pathogenesis of HIV dementia.

    Funded by: NCRR NIH HHS: RR15592; NINDS NIH HHS: NS38428, NS39253

    Journal of neurovirology 2003;9;3;358-71

  • Effects of hypoxia on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells.

    Appleton SD, Marks GS, Nakatsu K, Brien JF, Smith GN, Graham CH and Lash GE

    Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario, Canada K7L 3N6.

    Although hypoxia induces heme oxygenase (HO)-1 protein and mRNA expression in many cell types, hypoxia has also been shown to decrease HO-1 mRNA and protein expression. We tested the hypothesis that 24-h preexposure to hypoxia in human placental preparations suppresses HO protein expression and enzymatic function. Immortalized HTR-8/SVneo first-trimester trophoblast cells and explants of normal human chorionic villi (CV) from term placentas were cultured for 24 h in 1%, 5%, or 20% O(2). HO protein levels were determined by Western blot analysis, and microsomal HO activity was measured. HO-2 protein content was decreased by 17% and 5% in human trophoblast cells after 24-h exposure to 1% and 5% O(2), respectively, versus 20% O(2). In contrast, HO-2 protein content in CV explants was unaffected by changes in oxygenation. HO-1 protein content, which was barely detectable in both biological systems, was not affected by changes in oxygenation. Similarly, HO enzymatic activity was unchanged in both preparations after 24-h exposure to 1%, 5%, or 20% O(2). The above data do not support the hypothesis that hypoxia in the human placenta suppresses both HO protein content and HO protein function. The present observations reinforce the necessity to determine both HO protein expression and function.

    American journal of physiology. Heart and circulatory physiology 2003;284;3;H853-8

  • Decreased activity of the antioxidant heme oxygenase enzyme: implications in ischemia and in Alzheimer's disease.

    Doré S

    Department of Anesthesiology and Critical Care Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD 21287, USA. sd@jhmi.edu

    Heme oxygenase (HO) is the rate-limiting enzyme for the degradation of heme, a prooxidant, coming from a multitude of heme-containing proteins/enzymes. With the action of cytochrome P(450) reductase, HO cleaves the heme ring into biliverdin which is converted into bilirubin, both have been shown to have intrinsic radical scavenger activities. Iron is also released from the heme core and in its free form can act as a catalyst for oxidative stress damage or can be sequested by several iron-binding proteins. Under physiological conditions, the newly generated iron can be neutralized within the cell. The third product of the opening of the porphyrin ring is carbon monoxide, which role has been puzzling. It has been reported as a potential neuromodulator, it modulates guanylate cyclase activity and has vasodilation, anti-inflammatory and antiapoptotic effects. In the brain, HO2 accounts for the vast majority of HO activity. By decreasing HO2 activity, one would expect more neuronal damage after oxidative stress injury with possible direct implications to acute and chronic neurodegenerative disorders. Pharmacological ways to increase neuronal HO activity is likely to have therapeutic applications.

    Free radical biology & medicine 2002;32;12;1276-82

  • Neural roles for heme oxygenase: contrasts to nitric oxide synthase.

    Barañano DE and Snyder SH

    Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    The heme oxygenase (HO) and nitric oxide (NO) synthase (NOS) systems display notable similarities as well as differences. HO and NOS are both oxidative enzymes using NADPH as an electron donor. The constitutive forms of the enzyme are differentially activated, with calcium entry stimulating NOS by binding to calmodulin, whereas calcium entry activates protein kinase C to phosphorylate and activate HO2. Although both NO and carbon monoxide (CO) stimulate soluble guanylyl cyclase to form cGMP, NO also S-nitrosylates selected protein targets. Both involve constitutive and inducible biosynthetic enzymes. However, functions of the inducible forms are virtual opposites. Macrophage-inducible NOS generates NO to kill other cells, whereas HO1 generates bilirubin to exert antioxidant cytoprotective effects and also provides cytoprotection by facilitating iron extrusion from cells. The neuronal form of HO, HO2, is also cytoprotective. Normally, neural NO in the brain seems to exert some sort of behavioral inhibition. However, excess release of NO in response to glutamate's N-methyl-d-aspartate receptor activation leads to stroke damage. On the other hand, massive neuronal firing during a stroke presumably activates HO2, leading to neuroprotective actions of bilirubin. Loss of this neuroprotection after HO inhibition by mutant forms of amyloid precursor protein may mediate neurotoxicity in Familial Alzheimer's Disease. NO and CO both appear to be neurotransmitters in the brain and peripheral autonomic nervous system. They also are physiologic endothelial-derived relaxing factors for blood vessels. In the gastrointestinal pathway, NO and CO appear to function as coneurotransmitters, both stimulating soluble guanylyl cyclase to cause smooth muscle relaxation.

    Funded by: NIDA NIH HHS: DA-000266, DA-00074, DA-05900, F30 DA005900, K05 DA000074, P50 DA000266

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;20;10996-1002

  • Haem oxygenase-1: a novel player in cutaneous wound repair and psoriasis?

    Hanselmann C, Mauch C and Werner S

    Institute of Cell Biology, Swiss Federal Institute of Technology, Hönggerberg, CH-8093 Zürich, Switzerland.

    Haem oxygenase (HO) is the rate-limiting enzyme in the degradation of haem. In addition to its obvious role in iron metabolism, a series of findings indicate an important role for HO in cellular protection against oxidative stress. This effect might be of particular importance during wound healing and also in inflammatory disease. Therefore we determined the expression of the two HO isoenzymes, HO-1 and HO-2, during the healing process of full-thickness excisional wounds in mice. We show a remarkable induction of HO-1 mRNA and protein expression within three days after skin injury. After completion of wound healing, HO-1 expression declined to basal levels. By contrast, expression of HO-2 was not significantly modulated by skin injury. In situ hybridization and immunohistochemistry revealed high HO-1 expression in inflammatory cells of the granulation tissue and in keratinocytes of the hyperproliferative epithelium. A strong overexpression of HO-1 was also observed in the skin of patients suffering from the inflammatory skin disease psoriasis. In addition, HO-2 mRNA levels were increased in the skin of psoriatic patients. Similar to wounded skin, inflammatory cells and keratinocytes of the hyperthickened epidermis were the major producers of HO-1 in psoriatic skin. In vitro studies with cultured keratinocytes revealed a potential role for reactive oxygen species (ROS), but not for growth factors and pro-inflammatory cytokines, as inducers of HO-1 expression in inflamed skin. Our findings suggest a novel role for HO in wound healing and inflammatory skin disease, where it might be involved in haem degradation and in the protection of cells from the toxic effects of ROS.

    The Biochemical journal 2001;353;Pt 3;459-66

  • Expression and localization of heme oxygenase in human placental villi.

    Yoshiki N, Kubota T and Aso T

    Comprehensive Reproductive Medicine, Regulation of Internal Environment and Reproduction, Systemic Organ Regulation, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. n.yoshiki.gyne@med.tmd.ac.jp

    The aim of the present study was to investigate the expression and distribution patterns of heme oxygenase (HO)-1 and HO-2 in human placental villi at term and in the first trimester of pregnancy using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. RT-PCR detected mRNA for HO-1 and HO-2 in human placental villi during gestation. Western blotting also revealed the expression of the two distinct HO proteins throughout gestation. HO-1 was constitutively expressed, while HO-2 expression was apparently greater at term than in early pregnancy. Immunohistochemistry showed that distribution of the two HO isoforms had distinct topographic patterns: HO-1 was observed in villous trophoblastic cells, while HO-2 was found in endothelial cells and smooth muscle cells of blood vessels of placental villi. These results may provide a microtopographic basis for elucidating the mechanism of carbon monoxide (CO)-mediated vasodilatation, and it is suggested that the CO/HO system may be involved in the control of placental vascular function and may protect the syncytiotrophoblast and endothelium against oxidative injury.

    Biochemical and biophysical research communications 2000;276;3;1136-42

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • Assignment of the mouse heme oxygenase genes: heme oxygenase-1 (Hmox1) to chromosome 10 band C1 and heme oxygenase-2 (Hmox2) to chromosome 16 band B1.

    Saito-Ohara F, Ikeuchi T, Matsumoto M and Kurata S

    Department of Cytogenetics, Tokyo Medical and Dental University, Japan.

    Cytogenetics and cell genetics 1997;77;3-4;180-1

  • Expression of heme oxygenase isozyme mRNAs in the human brain and induction of heme oxygenase-1 by nitric oxide donors.

    Takahashi K, Hara E, Suzuki H, Sasano H and Shibahara S

    Department of Applied Physiology and Molecular Biology, Tohoku Univesity School of Medicine, Miyagi, Japan.

    Heme oxygenase is an essential enzyme in the heme catabolism that produces carbon monoxide (CO). This study was designed to examine the expression of two heme oxygenase isozyme mRNAs in the human brain and to explore the involvement of nitric oxide (NO) and various neuropeptides in the regulation of their expression. Northern blot analysis showed the expression of heme oxygenase-1 and heme oxygenase-2 mRNAs in every region of the brain examined, with the highest levels found in the frontal cortex, temporal cortex, occipital cortex, and hypothalamus. In a human glioblastoma cell line, T98G, treatment with any of three types of NO donors--sodium nitroprusside, 3-morpholinosydnonimine, and S-nitroso-L-glutathione--caused a significant increase in the levels of heme oxygenase-1 mRNA but not in the levels of heme oxygenase-2 and heat-shock protein 70 mRNAs. Sodium nitroprusside increased the levels of heme oxygenase-1 protein but not the levels of heat-shock protein 70 in T98G cells. The increase in content of heme oxygenase-1 mRNA caused by sodium nitro-prusside was completely abolished by the treatment with actinomycin D. On the other hand, the levels of heme oxygenase isozyme mRNAs were not noticeably changed in T98G cells following the treatment with 8-bromo cyclic, GMP sodium nitrite, or various neuropeptides, such as calcitonin gene-related peptide, endothelin-1, and corticotropin-releasing hormone. The present study has shown the expression profiles of heme oxygenase-1 and -2 mRNAs in the human brain and the induction of heme oxygenase-1 mRNA caused by NO donors in T98G cells. These findings raise a possibility that the CO/heme oxygenase system may function in concert with the NO/NO synthase system in the brain.

    Journal of neurochemistry 1996;67;2;482-9

  • Heme oxygenase-2. Properties of the heme complex of the purified tryptic fragment of recombinant human heme oxygenase-2.

    Ishikawa K, Takeuchi N, Takahashi S, Matera KM, Sato M, Shibahara S, Rousseau DL, Ikeda-Saito M and Yoshida T

    Department of Biochemistry, Yamagata University School of Medicine, Japan.

    Recombinant human microsomal heme oxygenase-2 was expressed in Escherichia coli. Tryptic digestion of the membrane fraction, in which the wild-type enzyme was localized, yielded a soluble tryptic peptide of 28 kDa, which retained the ability to accept electrons from NADPH-cytochrome P-450 reductase and the enzymatic activity for conversion of heme to biliverdin. The tryptic fragment, when purified to apparent homogeneity, bound one equivalent of heme to form a substrate-enzyme complex that had spectroscopic properties characteristic of heme proteins, such as myoglobin and hemoglobin. Optical absorption, Raman scattering, and EPR studies of the heme-tryptic fragment complex revealed that the ferric heme was six coordinate high spin at neutral pH and six coordinate low spin at alkaline pH, with a pK alpha value of 8.5. EPR and Raman scattering studies indicated that a neutral imidazole of a histidine residue served as the proximal ligand in the heme-heme oxygenase-2 fragment complex. The reaction with hydrogen peroxide converted the heme of the heme oxygenase-2 fragment complex into a verdoheme-like intermediate, while the reaction with m-chloroperbenzoic acid yielded a oxoferryl species. These spectroscopic properties are similar to those obtained for heme oxygenase-1, and thus the catalytic mechanism of heme oxygenase-2 appears to be similar to that of heme oxygenase-1.

    Funded by: NCRR NIH HHS: RR05659; NIGMS NIH HHS: GM39492, GM48714

    The Journal of biological chemistry 1995;270;11;6345-50

  • Chromosomal localization of the human heme oxygenase genes: heme oxygenase-1 (HMOX1) maps to chromosome 22q12 and heme oxygenase-2 (HMOX2) maps to chromosome 16p13.3.

    Kutty RK, Kutty G, Rodriguez IR, Chader GJ and Wiggert B

    Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.

    Heme oxygenase catalyzes the oxidation of heme to biliverdin, the precursor of the bile pigment bilirubin, and carbon monoxide, a putative neurotransmitter. We have employed polymerase chain reaction and fluorescence in situ hybridization to determine the chromosome localization of the genes coding for the two known heme oxygenase isozymes. Heme oxygenase-1 (HMOX1), the inducible form, was localized to human chromosome 22q12, while heme oxygenase-2 (HMOX2), the constitutive form, was localized to chromosome 16p13.3.

    Genomics 1994;20;3;513-6

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

  • Human heme oxygenase-2: characterization and expression of a full-length cDNA and evidence suggesting that the two HO-2 transcripts may differ by choice of polyadenylation signal.

    McCoubrey WK, Ewing JF and Maines MD

    Department of Biophysics, University of Rochester School of Medicine, New York 14642.

    We show by Northern blot analysis that human HO-2 is encoded by two transcripts (1.3 and 1.7 kb) and is a single-copy gene as judged by Southern blot analysis. We further provide evidence based on Northern blot and sequence analysis of a cDNA representing the larger transcript that the transcripts differ in the 3' untranslated region. A 274-base-pair DNA fragment from the rat heme oxygenase-2 gene (I. Cruse and M.D. Maines, 1988, J. Biol. Chem. 263, 3348-3353) was used to isolate a human HO-2 cDNA from a fetal kidney library in lambda gt11. The clone, designated hK-1, was sequenced and the cDNA insert was determined to be 1625 base pairs in length, encoding a protein of 313 amino acids. Two consensus polyadenylation signals separated by 440 nucleotides were identified in the 3' untranslated region. The size of the cDNA insert closely approximated the larger of two mRNAs. The nucleotide sequence was 88% identical to the rat HO-2 gene within the predicted coding region and the putative translation product was also estimated to be 88% identical to the rat gene product (M. O. Rotenberg and D. Maines, 1990, J. Biol. Chem. 265, 7501). The predicted size, 36 kDa, corresponded well with HO-2 detected in human testis microsomes by Western blot analysis. Further, the fusion protein expressed in Escherichia coli displayed significant heme oxygenase activity, which was inhibited by Zn- and Sn-protoporphyrins, known inhibitors of eukaryotic heme oxygenase, but not by sulfhydryl reagents.

    Funded by: NIEHS NIH HHS: ES01247, R37ES04391

    Archives of biochemistry and biophysics 1992;295;1;13-20

Gene lists (1)

Gene List Source Species Name Description Gene count
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
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