Repressed Expression of the Human Xanthine Oxidoreductase Gene
2000; Elsevier BV; Volume: 275; Issue: 8 Linguagem: Inglês
10.1074/jbc.275.8.5918
ISSN1083-351X
AutoresPing Xu, Patricia LaVallee, John R. Hoidal,
Tópico(s)Folate and B Vitamins Research
ResumoStudies were initiated to address the basis for the low xanthine oxidoreductase (XOR) activity in humans relative to nonprimate mammalian species. The expression of the XOR in humans is strikingly lower than in mice, and both transcription rates and core promoter activity of the gene are repressed. Analysis of humanXOR promoter activity in hepatocytes and vascular endothelial cells showed that the region from −258 to −1 contains both repressor and activator binding regions regulating core promoter activity. The region between −138 and −1 is necessary and sufficient for initiating, and the region between −258 and −228 is critical for restricting core promoter activity. Within the latter region, site-directed mutations identified a consensus sequence "acacaggtgtgg" (−242 to −230) that contains an E-box that binds a repressor. In addition, the TATA-like element is also required to restrict promoter activity and TFIID binds to this site. The results demonstrate that both an E-box and TATA-like element are required to restrict gene activity. A model is proposed to account for humanXOR regulation. Studies were initiated to address the basis for the low xanthine oxidoreductase (XOR) activity in humans relative to nonprimate mammalian species. The expression of the XOR in humans is strikingly lower than in mice, and both transcription rates and core promoter activity of the gene are repressed. Analysis of humanXOR promoter activity in hepatocytes and vascular endothelial cells showed that the region from −258 to −1 contains both repressor and activator binding regions regulating core promoter activity. The region between −138 and −1 is necessary and sufficient for initiating, and the region between −258 and −228 is critical for restricting core promoter activity. Within the latter region, site-directed mutations identified a consensus sequence "acacaggtgtgg" (−242 to −230) that contains an E-box that binds a repressor. In addition, the TATA-like element is also required to restrict promoter activity and TFIID binds to this site. The results demonstrate that both an E-box and TATA-like element are required to restrict gene activity. A model is proposed to account for humanXOR regulation. xanthine oxidoreductase reverse transcriptase-polymerase chain reaction human umbilical vein endothelial cells electrophoretic mobility shift assay base pair(s) Xanthine oxidoreductase (XOR1; EC 1.1.3.22) is a molybdoflavoprotein hydroxylase that is widely distributed in nature. It is a homodimer with each subunit being about 150,000 Da, and containing four redox active centers: two iron-sulfur, one FAD, and one molybdopterin (1.Massey V. Brumby P.E. Komai H. J. Biol. Chem. 1969; 244: 1682-1691Abstract Full Text PDF PubMed Google Scholar, 2.Waud W.R. Rajagopalan K.V. Arch. Biochem. Biophys. 1976; 172: 365-379Crossref PubMed Scopus (174) Google Scholar, 3.Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Abstract Full Text PDF PubMed Google Scholar). In mammals XOR exists in two interconvertible forms, xanthine dehydrogenase and xanthine oxidase. Xanthine dehydrogenase transfers the reducing equivalents generated by the oxidation of substrates to NAD+ whereas xanthine oxidase transfers them to oxygen with resultant production of superoxide anion and hydrogen peroxide. As the rate-limiting enzyme in nucleic acid degradation, XOR catalyzes the final two reactions of purine catabolism with resultant production of urate. Urate, which scavenges hydroxyl radical, singlet oxygen, hypochlorous acid, oxoheme oxidants, and hydroperoxyl radicals, and is a potent iron chelator (4.Ames B.N. Cathcart R. Schwiers E. Hochstein P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6858-6862Crossref PubMed Scopus (2262) Google Scholar, 5.Howell R.R. Wyngaarden J.B. J. Biol. Chem. 1960; 235: 3544-3551Abstract Full Text PDF PubMed Google Scholar, 6.Kellogg III, E.W. Fridovich I. J. Biol. Chem. 1977; 252: 6721-6729Abstract Full Text PDF PubMed Google Scholar, 7.Davies K.J. Sevanian A. Muakkassah-Kelly S.F. Hochstein P. Biochem. J. 1986; 235: 747-754Crossref PubMed Scopus (455) Google Scholar), has been proposed as a major antioxidant in plasma (4.Ames B.N. Cathcart R. Schwiers E. Hochstein P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6858-6862Crossref PubMed Scopus (2262) Google Scholar, 5.Howell R.R. Wyngaarden J.B. J. Biol. Chem. 1960; 235: 3544-3551Abstract Full Text PDF PubMed Google Scholar, 8.Becker B.F. Reinhoklz N. Ozcelik T. Leipert B. Gerlach E. Pflugers Arch. 1989; 415: 127-138Crossref PubMed Scopus (115) Google Scholar) and epithelial secretions (9.Peden D.B. Hohman R. Brown M.E. Mason R.T. Berkebile C. Fales H.M. Kaliner M.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 97: 7638-7646Crossref Scopus (98) Google Scholar). Blood urate concentrations are approximately 10-fold higher in humans and most primates than in other mammals due to the evolutionary loss of uricase. These higher concentrations present a paradox. That is, urate functioning as an antioxidant has been cited as a possible basis for the increase in life span and decrease in cancer rates of humans compared with other species (4.Ames B.N. Cathcart R. Schwiers E. Hochstein P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6858-6862Crossref PubMed Scopus (2262) Google Scholar). The high concentrations, however, predispose to crystalline deposition, the histopathologic hallmark of gout, the "king of diseases." In Western industrialized countries approximately 5% of the population has hyperuricaemia (10.Slot O. Ugeskr-Laeger. 1994; 156: 2396-2401PubMed Google Scholar).Juxtaposed to the role of urate as a protective antioxidant is that XOR-derived superoxide anion and hydrogen peroxide lead to oxidative tissue injury in a variety of animal models simulating several clinical disorders including renal failure, endotoxin-induced mucosal injury, viral pneumonia, ischemia-reperfusion injury, and cutaneous photosensitivity to hematoporphyrins (11.Diamond J.R. Bonventre J.V. Karnovsky M.J. Kidney Int. 1986; 29: 478-483Abstract Full Text PDF PubMed Scopus (245) Google Scholar, 12.Kawamura T. Yoshika T. Bills T. Fogo A. Ichikawa I. Kidney Int. 1991; 40: 291-301Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 13.Linas S.L. Wittenburg D. Repine J.E. Am. J. Physiol. 1990; 258: F711-F716Crossref PubMed Google Scholar, 14.Paller M.S. Neumann T.V. Kidney Int. 1991; 40: 1041-1049Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 15.Rodell T.C. Cheronis J.C. Ohnemus C.L. Piermattie D.J. Repine J.E. J. Appl. Physiol. 1987; 63: 2159-2163Crossref PubMed Scopus (67) Google Scholar, 16.Akaike T. Ando M.M. Oda T. Doi T. Ijiri S. Araki S. Maeda H. J. Clin. Invest. 1990; 85: 739-745Crossref PubMed Scopus (293) Google Scholar, 17.Granger D.N. Am. J. Physiol. 1988; 255: H1269-H1275PubMed Google Scholar, 18.Athar M. Elmet C.A. Bickers D.R. Mukhtar H. J. Clin. Invest. 1989; 83: 1137-1143Crossref PubMed Scopus (88) Google Scholar). Recent studies demonstrate that XOR is a highly regulated enzyme. XOR activity and gene expression are increased by cytokines in a profile consistent with an acute phase response and during ischemia/reperfusion (19.Dupont G.P. Huecksteadt T.P. Marshall B.C. Ryan U.S. Michael J.R. Hoidal J.R. J. Clin. Invest. 1992; 89: 197-202Crossref PubMed Scopus (119) Google Scholar, 20.Pfeffer K.D. Huecksteadt T.P. Hoidal J.R. J. Immunol. 1994; 153: 1789-1797PubMed Google Scholar, 21.Falciani F. Ghezzi P. Terao M. Cazzaniga G. Garattini E. Biochem. J. 1992; 285: 1001-1008Crossref PubMed Scopus (49) Google Scholar, 22.Hassoun P.M., Yu, F.S. Shedd A.L. Zulueta J.J. Thannickal V.J. Lanzillo J.J. Fanburg B.L. Am. J. Physiol. 1994; 266: L163-L171PubMed Google Scholar).Somewhat surprising in view of the high urate concentrations, XOR enzyme activity in humans is 100 times lower than that in nonprimate species including rats and mice (23.Wajner M. Harkness R.A. Biochim. Biophys. Acta. 1989; 991: 79-84Crossref PubMed Scopus (93) Google Scholar, 24.Muxfeldt M. Schaper W. Basic Res. Cardiol. 1987; 82: 486-492Crossref PubMed Scopus (120) Google Scholar, 25.Abadeh S. Killacky J. Benboubetra M. Harrison R. Biochim. Biophys. Acta. 1992; 1117: 25-32Crossref PubMed Scopus (76) Google Scholar). Post-translational mechanisms appear to only partially explain the low activity (26.Harrison R. Biochem. Soc. Trans. 1997; 25: 786-791Crossref PubMed Scopus (41) Google Scholar, 27.Sanders S. Eisenthal R.S. Harrison R. Eur. J. Biochem. 1997; 245: 541-548Crossref PubMed Scopus (143) Google Scholar). We hypothesized that the low activity in humans represents an adaptive mechanism to control the potentially deleterious formation of urate crystals and that repression of XOR expression is partially responsible. To begin to pursue this hypothesis, we cloned the human XOR (hXOR) cDNA and characterized the chromosomal location and genomic organization of the gene (28.Xu P. Huechsteadt T.P. Harrison R. Hoidal J.R. Biochem. Biophys. Res. Commun. 1994; 199: 998-1004Crossref PubMed Scopus (56) Google Scholar, 29.Xu P. Zhu X.L. Huecksteadt T.P. Brothman A. Hoidal J.R. Genomics. 1994; 23: 289-291Crossref PubMed Scopus (30) Google Scholar, 30.Xu P. Huecksteadt T.P. Hoidal J.R. Genomics. 1994; 34: 173-180Crossref Scopus (85) Google Scholar). In the present study, we determined that expression of XOR in human tissues is strikingly restricted relative to that in mouse. Analysis ofXOR transcription rates and core promoter function of the 5′-untranslated region of the human and mouse genes indicates that transcription and core promoter activity of hXOR are repressed. An E-box with a consensus GTTTC and a TATA-like element present in the human, but not in the mouse 5′-untranslated region are required for the repression. A model is proposed to account for humanXOR regulation.DISCUSSIONXOR is a key enzyme in the catabolism of purines. Over the past century the enzymology of XOR has been intensively investigated. Despite a welter of information about its mechanisms of action, very little is known about factors that control its expression. Of note, relative to most other species, low XOR activity has been shown in a variety of human tissues. Post-translational mechanisms only partially explain the low activity (26.Harrison R. Biochem. Soc. Trans. 1997; 25: 786-791Crossref PubMed Scopus (41) Google Scholar, 27.Sanders S. Eisenthal R.S. Harrison R. Eur. J. Biochem. 1997; 245: 541-548Crossref PubMed Scopus (143) Google Scholar). We hypothesized that repression of XOR expression is another mechanism contributing to the low activity in humans.The Expression of hXORGene Is Transcriptionally RepressedIn support of the hypothesis, in the present investigation we demonstrated that XOR transcript levels and transcription rates were substantially lower in human than mouse tissues. Moreover, the lower activity of the human promoter compared with that of the mouse in hepatocytes and vascular endothelial cells support the contention that a generalized repression ofh XOR gene expression may contribute to the low activity in humans.An E-box Regulates the Promoter Activity of the hXORGeneStudies to characterize the basis for repression ofh XOR transcription demonstrated that the core region from −138 to −1 is required for basal promoter activity and the sequence from −258 to −228 is required for repressive regulation of the basal activity. The requirement for both the core region and repressive sequence is not specific for a given cell type.We determined that the sequence acacaggtgtgg (−242 to −230) is critical for restricting promoter activity of the hXOR gene. This "core" sequence contains a consensus E box element (ACAGGTGT) located at −240. Site-directed mutations of the E-box element eliminated the repressive activity. This observation is consistent with previous studies demonstrating that the E-box may play a critical role as a regulatory site (43.Atchley W.R. Fitch W.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5172-5176Crossref PubMed Scopus (489) Google Scholar). Several transcription factors containing helix-loop-helix motifs have been identified that bind to the E-box, and augment or repress gene transcription. These include AREB6, USF, and TFII-I. AREB6, a zinc finger homeodomain protein, inhibits transcription by interacting with the repressor NC2. AREB6 is expressed in HepG2 and HUVECs (data not shown). In neuroblastoma cells, the repressive effect of AREB6 on the human 70-kDa heat shock gene promoter requires the sequence GTTTC in conjunction with the ACAGGTGT (38.Ikeda K. Kawakami K. Eur. J. Biochem. 1995; 233: 73-82Crossref PubMed Scopus (78) Google Scholar). AREB6 is the only known factor whose regulation of promoter activity requires the presence of this conjuncted sequence (38.Ikeda K. Kawakami K. Eur. J. Biochem. 1995; 233: 73-82Crossref PubMed Scopus (78) Google Scholar, 39.Ikeda K. Halle J.P. Stelzer G. Meisterernst M. Kawakami K. Mol. Cell. Biol. 1998; 18: 10-18Crossref PubMed Scopus (44) Google Scholar). This sequence is present 21 bp upstream (−260) of the E-box inh XOR gene and preliminary studies have demonstrated binding of nuclear proteins to the E-box site and a requirement for the GTTTC for the binding (data not shown). Further studies will be necessary to determine whether AREB6 is indeed involved in repressing promoter activity of h XOR.The TATA-like Element Also Regulates the Repressed Promoter Activity of the hXORGeneRecently, a novel transcriptional activity was reported (42.Manley J.L. Um M. Li C. Ashali H. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1996; 351: 517-526Crossref PubMed Scopus (15) Google Scholar) that represses transcription from promoters containing the TATA-like element, but activates that lacking the TATA-like element. Compared with the TATA-less rat or mouseXOR, the region between −138 and −1 ofh XOR contains a TATA-like element (30.Xu P. Huecksteadt T.P. Hoidal J.R. Genomics. 1994; 34: 173-180Crossref Scopus (85) Google Scholar, 44.Cazzaniga G. Terao M. Schiavo P.L. Galbiati F. Segalla F. Seldin M.F. Garattini E. Genomics. 1992; 23: 390-402Crossref Scopus (50) Google Scholar, 45.Chow C-W. Clark M. Rinaldo J. Chalkley R. Nucleic Acids Res. 1994; 22: 1846-1854Crossref PubMed Scopus (25) Google Scholar, 46.Chow C-W. Clark M. Rinaldo J. Chalkley R. Nucleic Acids Res. 1995; 23: 3132-3140Crossref PubMed Scopus (23) Google Scholar). Site-directed mutagenesis demonstrated that conversion of the TATA to a non-TATA site relieved repression. These results indicate a functional requirement of the TATA-like element in repressive regulation ofh XOR.It was proposed that the mechanism of this repressor activity of the TATA-like element involved the interaction between TFIID and repressors (42.Manley J.L. Um M. Li C. Ashali H. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1996; 351: 517-526Crossref PubMed Scopus (15) Google Scholar). The requirement of a TATA-like element in repressive regulation of h XOR promoter may involve a similar mechanism of interaction of TFIID and repressors. Analysis of DNA-nuclear protein interactions showed three specific band shifts within the region from −138 to −51, indicating multiple transcription factors binding or interacting within the region. Further studies with a subset of specific competitors demonstrated that the shifts required a common or core region (from −125 to −89) that contained a centered TATA-like element. To examine TFIID binding to the region, we studied interactions between TFIID and the core region from −138 to −51. The results demonstrated that antibodies to TFIID blocked a specific band shift and TFIID protected the TATA-like element from the DNase I digestion by binding to the region. In addition, competition studies demonstrated that the sequences flanking the TATA-like element ofh XOR are required for the binding of nuclear proteins to the TATA-centered region. Taken together, the results suggest that there is a DNA-nuclear protein complex formed via interactions between several transcription factors and regulatory sites within the core region (−125 to −89) that produce a repressive activity of the TATA-like element. In the complex, TFIID may play a central role via binding to the element.Models for composite core promoters containing both TATA elements and initiators, such as the adenovirus major late promoter and human β-globin promoter (47.Roy A.L. Du H. Gregor P. Novina C. Martinez E. Roeder R.G. EMBO J. 1997; 16: 7091-7104Crossref PubMed Scopus (171) Google Scholar, 48.Du H. Roy A.L. Roeder R.G. EMBO J. 1993; 12: 501-511Crossref PubMed Scopus (189) Google Scholar, 49.Lewis B.A. Orkin S.H. J. Biol. Chem. 1995; 270: 28139-28144Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 50.Roy A.L. Malik S. Meisterernst M. Roeder R. Nature. 1993; 365: 355-358Crossref PubMed Scopus (135) Google Scholar), have been shown to provide alternative preinitiation complex assembly pathways. In the model, the stabilization of TFIID binding to the TATA element either by TFII-I binding to the initiator or by interacting with TFII-A provides differential mechanisms in gene regulation by specific activators or repressors (51.Roy A.L. Carruthers C. Gutjahr T. Roeder R.G. Nature. 1993; 365: 359-361Crossref PubMed Scopus (221) Google Scholar, 52.Hanna-Rose W. Hansen U. Trends Genet. 1997; 12: 229-234Abstract Full Text PDF Scopus (280) Google Scholar). In the TATA-less rat XOR, a series of four initiators have been reported that induce promoter activity by interacting with the initiator-binding proteins TFII-I or YY-1 (45.Chow C-W. Clark M. Rinaldo J. Chalkley R. Nucleic Acids Res. 1994; 22: 1846-1854Crossref PubMed Scopus (25) Google Scholar, 46.Chow C-W. Clark M. Rinaldo J. Chalkley R. Nucleic Acids Res. 1995; 23: 3132-3140Crossref PubMed Scopus (23) Google Scholar,53.Clark M.P. Chow C.W. Rinaldo J.E. Chalkley R. Nucleic Acids Res. 1998; 26: 1801-1806Crossref PubMed Scopus (24) Google Scholar, 54.Clark M.P. Chow C.W. Rinaldo J.E. Chalkley R. Nucleic Acids Res. 1998; 26: 2813-2820Crossref PubMed Scopus (14) Google Scholar). In the TATA-containing hXOR promoter, the fourth initiator of the rat XOR promoter is conserved and overlaps partially with the TATA element. Therefore, hXOR is an example of a composite core promoter. TFIID binding to the TATA-like element of the hXOR may be stabilized by TFII-I or YY-1 binding to the initiator, thereby providing the opportunity for differential regulation of hXOR. It has been reported that TFII-I is able to bind to initiators and the consensus E-box and YY-1 have repressive effects on gene transcription (47.Roy A.L. Du H. Gregor P. Novina C. Martinez E. Roeder R.G. EMBO J. 1997; 16: 7091-7104Crossref PubMed Scopus (171) Google Scholar, 52.Hanna-Rose W. Hansen U. Trends Genet. 1997; 12: 229-234Abstract Full Text PDF Scopus (280) Google Scholar). In future studies of mechanisms of repressive regulation of the hXORpromoter, it will be important to determine the role of the initiator and its binding proteins TFII-I and YY-1, including their possible interactions with the E-box.To summarize potential mechanisms for the repressed expression ofhXOR, a model is proposed and illustrated in Fig.11. In the model, the region from −138 to −1 provides a core for formation of a complex containing TFIID that is required for basal promoter activity. In a speculated bent DNA structure, the repressive transcription factors that bind to the E-box, possibly AREB6, may inhibit the gene transcription via indirect (through a protein-protein interaction with repressor NC2) or direct interactions with the TFIID-containing complex. NC2 has been shown to negatively regulate gene transcription by interacting with TFIID or by binding to AREB6 (38.Ikeda K. Kawakami K. Eur. J. Biochem. 1995; 233: 73-82Crossref PubMed Scopus (78) Google Scholar, 39.Ikeda K. Halle J.P. Stelzer G. Meisterernst M. Kawakami K. Mol. Cell. Biol. 1998; 18: 10-18Crossref PubMed Scopus (44) Google Scholar, 55.Meisterernst M. Roy A. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Abstract Full Text PDF PubMed Scopus (225) Google Scholar, 56.Goppelt A. Stelzer G. Lottspeich F. Meisterernst M. EMBO J. 1996; 15: 3105-3116Crossref PubMed Scopus (129) Google Scholar, 57.Meisterernst M. Roeder R.G. Cell. 1991; 67: 557-567Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 58.Inostroza J.A. Mermelstein F.H. Ha I. Lane W.S. Reinberg D. Cell. 1992; 70: 477-489Abstract Full Text PDF PubMed Scopus (292) Google Scholar). Based on the results of EMSA, other potential bindings are labeled as "A-E", as illustrated in Fig. 11. The transcription factors "C" and "D" within the core region, possibly representing TFII-I, YY-1, or TFII-A, may alter the formation or stability of TFIID complex, thereby playing repressive roles in regulation of the gene transcription. The model suggests that alternative pathways may provide differential mechanisms of regulation of hXOR transcription in various pathological or physiological conditions. Xanthine oxidoreductase (XOR1; EC 1.1.3.22) is a molybdoflavoprotein hydroxylase that is widely distributed in nature. It is a homodimer with each subunit being about 150,000 Da, and containing four redox active centers: two iron-sulfur, one FAD, and one molybdopterin (1.Massey V. Brumby P.E. Komai H. J. Biol. Chem. 1969; 244: 1682-1691Abstract Full Text PDF PubMed Google Scholar, 2.Waud W.R. Rajagopalan K.V. Arch. Biochem. Biophys. 1976; 172: 365-379Crossref PubMed Scopus (174) Google Scholar, 3.Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Abstract Full Text PDF PubMed Google Scholar). In mammals XOR exists in two interconvertible forms, xanthine dehydrogenase and xanthine oxidase. Xanthine dehydrogenase transfers the reducing equivalents generated by the oxidation of substrates to NAD+ whereas xanthine oxidase transfers them to oxygen with resultant production of superoxide anion and hydrogen peroxide. As the rate-limiting enzyme in nucleic acid degradation, XOR catalyzes the final two reactions of purine catabolism with resultant production of urate. Urate, which scavenges hydroxyl radical, singlet oxygen, hypochlorous acid, oxoheme oxidants, and hydroperoxyl radicals, and is a potent iron chelator (4.Ames B.N. Cathcart R. Schwiers E. Hochstein P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6858-6862Crossref PubMed Scopus (2262) Google Scholar, 5.Howell R.R. Wyngaarden J.B. J. Biol. Chem. 1960; 235: 3544-3551Abstract Full Text PDF PubMed Google Scholar, 6.Kellogg III, E.W. Fridovich I. J. Biol. Chem. 1977; 252: 6721-6729Abstract Full Text PDF PubMed Google Scholar, 7.Davies K.J. Sevanian A. Muakkassah-Kelly S.F. Hochstein P. Biochem. J. 1986; 235: 747-754Crossref PubMed Scopus (455) Google Scholar), has been proposed as a major antioxidant in plasma (4.Ames B.N. Cathcart R. Schwiers E. Hochstein P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6858-6862Crossref PubMed Scopus (2262) Google Scholar, 5.Howell R.R. Wyngaarden J.B. J. Biol. Chem. 1960; 235: 3544-3551Abstract Full Text PDF PubMed Google Scholar, 8.Becker B.F. Reinhoklz N. Ozcelik T. Leipert B. Gerlach E. Pflugers Arch. 1989; 415: 127-138Crossref PubMed Scopus (115) Google Scholar) and epithelial secretions (9.Peden D.B. Hohman R. Brown M.E. Mason R.T. Berkebile C. Fales H.M. Kaliner M.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 97: 7638-7646Crossref Scopus (98) Google Scholar). Blood urate concentrations are approximately 10-fold higher in humans and most primates than in other mammals due to the evolutionary loss of uricase. These higher concentrations present a paradox. That is, urate functioning as an antioxidant has been cited as a possible basis for the increase in life span and decrease in cancer rates of humans compared with other species (4.Ames B.N. Cathcart R. Schwiers E. Hochstein P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6858-6862Crossref PubMed Scopus (2262) Google Scholar). The high concentrations, however, predispose to crystalline deposition, the histopathologic hallmark of gout, the "king of diseases." In Western industrialized countries approximately 5% of the population has hyperuricaemia (10.Slot O. Ugeskr-Laeger. 1994; 156: 2396-2401PubMed Google Scholar). Juxtaposed to the role of urate as a protective antioxidant is that XOR-derived superoxide anion and hydrogen peroxide lead to oxidative tissue injury in a variety of animal models simulating several clinical disorders including renal failure, endotoxin-induced mucosal injury, viral pneumonia, ischemia-reperfusion injury, and cutaneous photosensitivity to hematoporphyrins (11.Diamond J.R. Bonventre J.V. Karnovsky M.J. Kidney Int. 1986; 29: 478-483Abstract Full Text PDF PubMed Scopus (245) Google Scholar, 12.Kawamura T. Yoshika T. Bills T. Fogo A. Ichikawa I. Kidney Int. 1991; 40: 291-301Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 13.Linas S.L. Wittenburg D. Repine J.E. Am. J. Physiol. 1990; 258: F711-F716Crossref PubMed Google Scholar, 14.Paller M.S. Neumann T.V. Kidney Int. 1991; 40: 1041-1049Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 15.Rodell T.C. Cheronis J.C. Ohnemus C.L. Piermattie D.J. Repine J.E. J. Appl. Physiol. 1987; 63: 2159-2163Crossref PubMed Scopus (67) Google Scholar, 16.Akaike T. Ando M.M. Oda T. Doi T. Ijiri S. Araki S. Maeda H. J. Clin. Invest. 1990; 85: 739-745Crossref PubMed Scopus (293) Google Scholar, 17.Granger D.N. Am. J. Physiol. 1988; 255: H1269-H1275PubMed Google Scholar, 18.Athar M. Elmet C.A. Bickers D.R. Mukhtar H. J. Clin. Invest. 1989; 83: 1137-1143Crossref PubMed Scopus (88) Google Scholar). Recent studies demonstrate that XOR is a highly regulated enzyme. XOR activity and gene expression are increased by cytokines in a profile consistent with an acute phase response and during ischemia/reperfusion (19.Dupont G.P. Huecksteadt T.P. Marshall B.C. Ryan U.S. Michael J.R. Hoidal J.R. J. Clin. Invest. 1992; 89: 197-202Crossref PubMed Scopus (119) Google Scholar, 20.Pfeffer K.D. Huecksteadt T.P. Hoidal J.R. J. Immunol. 1994; 153: 1789-1797PubMed Google Scholar, 21.Falciani F. Ghezzi P. Terao M. Cazzaniga G. Garattini E. Biochem. J. 1992; 285: 1001-1008Crossref PubMed Scopus (49) Google Scholar, 22.Hassoun P.M., Yu, F.S. Shedd A.L. Zulueta J.J. Thannickal V.J. Lanzillo J.J. Fanburg B.L. Am. J. Physiol. 1994; 266: L163-L171PubMed Google Scholar). Somewhat surprising in view of the high urate concentrations, XOR enzyme activity in humans is 100 times lower than that in nonprimate species including rats and mice (23.Wajner M. Harkness R.A. Biochim. Biophys. Acta. 1989; 991: 79-84Crossref PubMed Scopus (93) Google Scholar, 24.Muxfeldt M. Schaper W. Basic Res. Cardiol. 1987; 82: 486-492Crossref PubMed Scopus (120) Google Scholar, 25.Abadeh S. Killacky J. Benboubetra M. Harrison R. Biochim. Biophys. Acta. 1992; 1117: 25-32Crossref PubMed Scopus (76) Google Scholar). Post-translational mechanisms appear to only partially explain the low activity (26.Harrison R. Biochem. Soc. Trans. 1997; 25: 786-791Crossref PubMed Scopus (41) Google Scholar, 27.Sanders S. Eisenthal R.S. Harrison R. Eur. J. Biochem. 1997; 245: 541-548Crossref PubMed Scopus (143) Google Scholar). We hypothesized that the low activity in humans represents an adaptive mechanism to control the potentially deleterious formation of urate crystals and that repression of XOR expression is partially responsible. To begin to pursue this hypothesis, we cloned the human XOR (hXOR) cDNA and characterized the chromosomal location and genomic organization of the gene (28.Xu P. Huechsteadt T.P. Harrison R. Hoidal J.R. Biochem. Biophys. Res. Commun. 1994; 199: 998-1004Crossref PubMed Scopus (56) Google Scholar, 29.Xu P. Zhu X.L. Huecksteadt T.P. Brothman A. Hoidal J.R. Genomics. 1994; 23: 289-291Crossref PubMed Scopus (30) Google Scholar, 30.Xu P. Huecksteadt T.P. Hoidal J.R. Genomics. 1994; 34: 173-180Crossref Scopus (85) Google Scholar). In the present study, we determined that expression of XOR in human tissues is strikingly restricted relative to that in mouse. Analysis ofXOR transcription rates and core promoter function of the 5′-untranslated region of the human and mouse genes indicates that transcription and core promoter activity of hXOR are repressed. An E-box with a consensus GTTTC and a TATA-like element present in the human, but not in the mouse 5′-untranslated region are required for the repression. A model is proposed to account for humanXOR regulation. DISCUSSIONXOR is a key enzyme in the catabolism of purines. Over the past century the enzymology of XOR has been intensively investigated. Despite a welter of information about its mechanisms of action, very little is known about factors that control its expression. Of note, relative to most other species, low XOR activity has been shown in a variety of human tissues. Post-translational mechanisms only partially explain the low activity (26.Harrison R. Biochem. Soc. Trans. 1997; 25: 786-791Crossref PubMed Scopus (41) Google Scholar, 27.Sanders S. Eisenthal R.S. Harrison R. Eur. J. Biochem. 1997; 245: 541-548Crossref PubMed Scopus (143) Google Scholar). We hypothesized that repression of XOR expression is another mechanism contributing to the low activity in humans.The Expression of hXORGene Is Transcriptionally RepressedIn support of the hypothesis, in the present investigation we demonstrated that XOR transcript levels and transcription rates were substantially lower in human than mouse tissues. Moreover, the lower activity of the human promoter compared with that of the mouse in hepatocytes and vascular endothelial cells support the contention that a generalized repression ofh XOR gene expression may contribute to the low activity in humans.An E-box Regulates the Promoter Activity of the hXORGeneStudies to characterize the basis for repression ofh XOR transcription demonstrated that the core region from −138 to −1
Referência(s)