Neurosteroid Hydroxylase CYP7B
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m011564200
ISSN1083-351X
AutoresKen A. Rose, Adrian K. Allan, Stephan D. Gauldie, Genevieve Stapleton, Lorraine Dobbie, Karin Dott, Cécile Martin, Ling Wang, Eva Hedlund, Jonathan R. Seckl, Jan-Ακε Gustafsson, Richard Lathe,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoThe major adrenal steroid dehydroepiandrosterone (DHEA) enhances memory and immune function but has no known dedicated receptor; local metabolism may govern its activity. We described a cytochrome P450 expressed in brain and other tissues, CYP7B, that catalyzes the 7α-hydroxylation of oxysterols and 3β-hydroxysteroids including DHEA. We report here that CYP7B mRNA and 7α-hydroxylation activity are widespread in rat tissues. However, steroids related to DHEA are reported to be modified at positions other than 7α, exemplified by prominent 6α-hydroxylation of 5α-androstane-3β,17β-diol (A/anediol) in some rodent tissues including brain. To determine whether CYP7B is responsible for these and other activities we disrupted the mouse Cyp7b gene by targeted insertion of an IRES-lacZ reporter cassette, placing reporter enzyme activity (β-galactosidase) underCyp7b promoter control. In heterozygous mouse brain, chromogenic detection of reporter activity was strikingly restricted to the dentate gyrus. Staining did not exactly reproduce the in situ hybridization expression pattern; post-transcriptional control is inferred. Lower level staining was detected in cerebellum, liver, and kidney, and which largely paralleled mRNA distribution. Liver and kidney expression was sexually dimorphic. Mice homozygous for the insertion are viable and superficially normal, but ex vivo metabolism of DHEA to 7α-hydroxy-DHEA was abolished in brain, spleen, thymus, heart, lung, prostate, uterus, and mammary gland; lower abundance metabolites were also eliminated. 7α-Hydroxylation of 25-hydroxycholesterol and related substrates was also abolished, as was presumed 6α-hydroxylation of A/anediol. These different enzyme activities therefore derive from the Cyp7bgene. CYP7B is thus a major extrahepatic steroid and oxysterol hydroxylase and provides the predominant route for local metabolism of DHEA and related molecules in brain and other tissues. The major adrenal steroid dehydroepiandrosterone (DHEA) enhances memory and immune function but has no known dedicated receptor; local metabolism may govern its activity. We described a cytochrome P450 expressed in brain and other tissues, CYP7B, that catalyzes the 7α-hydroxylation of oxysterols and 3β-hydroxysteroids including DHEA. We report here that CYP7B mRNA and 7α-hydroxylation activity are widespread in rat tissues. However, steroids related to DHEA are reported to be modified at positions other than 7α, exemplified by prominent 6α-hydroxylation of 5α-androstane-3β,17β-diol (A/anediol) in some rodent tissues including brain. To determine whether CYP7B is responsible for these and other activities we disrupted the mouse Cyp7b gene by targeted insertion of an IRES-lacZ reporter cassette, placing reporter enzyme activity (β-galactosidase) underCyp7b promoter control. In heterozygous mouse brain, chromogenic detection of reporter activity was strikingly restricted to the dentate gyrus. Staining did not exactly reproduce the in situ hybridization expression pattern; post-transcriptional control is inferred. Lower level staining was detected in cerebellum, liver, and kidney, and which largely paralleled mRNA distribution. Liver and kidney expression was sexually dimorphic. Mice homozygous for the insertion are viable and superficially normal, but ex vivo metabolism of DHEA to 7α-hydroxy-DHEA was abolished in brain, spleen, thymus, heart, lung, prostate, uterus, and mammary gland; lower abundance metabolites were also eliminated. 7α-Hydroxylation of 25-hydroxycholesterol and related substrates was also abolished, as was presumed 6α-hydroxylation of A/anediol. These different enzyme activities therefore derive from the Cyp7bgene. CYP7B is thus a major extrahepatic steroid and oxysterol hydroxylase and provides the predominant route for local metabolism of DHEA and related molecules in brain and other tissues. dehydroepiandrosterone 5α-androstane-3β,17β-diol (androstanediol) 5-androstene-3β,17β-diol (androstenediol) 7α-hydroxy-DHEA internal ribosomal entry signal hydroxysteroid dehydrogenase thin layer chromatography embryonic stem cells 2-aminoproply triethoxyslilane Brain function is subject to hormonal control, notably by steroids synthesized from the adrenal glands and gonads. Accumulating evidence also points to local steroid synthesis and metabolism in brain; a growing field of investigation focuses on the biological role of brain-active steroids, or "neurosteroids" (1Baulieu E.E. Psychoneuroendocrinology. 1998; 23: 963-987Crossref PubMed Scopus (502) Google Scholar, 2Baulieu E.E. Robel P. J. Steroid. Biochem. Mol. Biol. 1990; 37: 395-403Crossref PubMed Scopus (495) Google Scholar, 3De Kloet E.R. 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Neuroendocrinol. 1997; 9: 923-928Crossref PubMed Scopus (38) Google Scholar, 34Doostzadeh J. Cotillon A.C. Morfin R. Steroids. 1998; 63: 383-392Crossref PubMed Scopus (13) Google Scholar, 35Doostzadeh J. Cotillon A.C. Benalycherif A. Morfin R. Steroids. 1998; 63: 608-614Crossref PubMed Scopus (32) Google Scholar) and, in brain and other tissues, for oxysterols/bile acids (25- and 27-hydroxycholesterol, 3β-hydroxy-5-cholestenoic acid, 3β-hydroxy-5-cholenoic acid; Ref. 29Zhang J. Larsson O. Björkhem I. Biochem. Biophys. Acta. 1995; 1256: 353-359Crossref PubMed Scopus (40) Google Scholar), although 7β- and 6α-hydroxylation have also been recorded. Nevertheless, the identities of the enzymes responsible for these diverse activities have not been fully elucidated.We reported the molecular cloning, from rat and mouse hippocampus, of a new cytochrome P450 with steroid-modifying potential (36Stapleton G. Steel M. Richardson M. Mason J.O. Rose K.A. Morris R.G. Lathe R. J. Biol. Chem. 1995; 270: 29739-29745Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The enzyme is most homologous (39%) to hepatic cholesterol 7α-hydroxylase, CYP7A (37Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Abstract Full Text PDF PubMed Google Scholar), and more distantly related to CYP8A (prostacyclin synthase; Ref. 38Pereira B. Wu K.K. Wang L.H. Biochem. Biophys. Res. Commun. 1994; 203: 59-66Crossref PubMed Scopus (37) Google Scholar) and CYP8B (sterol 12α-hydroxylase; Ref. 39Eggertsen G. Olin M. Andersson U. Ishida H. Kubota S. Hellman U. Okuda K.I. Björkhem I. J. Biol. Chem. 1996; 271: 32269-32275Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Expressed from recombinant vaccinia virus, the mouse enzyme metabolized DHEA to 7α-hydroxy-DHEA (7HD) (Ref. 40Rose K.A. Stapleton G. Dott K. Kieny M.P. Best R. Schwarz M. Russell D.W. Björkhem I. Seckl J. Lathe R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4925-4930Crossref PubMed Scopus (204) Google Scholar); pregnenolone, estradiol (E2), and oxysterols, including 25-hydroxycholesterol, are also converted by the recombinant enzyme (40Rose K.A. Stapleton G. Dott K. Kieny M.P. Best R. Schwarz M. Russell D.W. Björkhem I. Seckl J. Lathe R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4925-4930Crossref PubMed Scopus (204) Google Scholar, 41Schwarz M. Lund E.G. Lathe R. Björkhem I. Russell D.W. J. Biol. Chem. 1997; 272: 23995-24001Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) (Fig. 1 A). However, the exact relationship of CYP7B enzyme to the observed ex vivosteroid hydroxylation activities is not known.Multiple enzymes may be present. First, CYP7B emerged from a screen for hippocampus-specific genes (36Stapleton G. Steel M. Richardson M. Mason J.O. Rose K.A. Morris R.G. Lathe R. J. Biol. Chem. 1995; 270: 29739-29745Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar); steroid hydroxylation in different brain regions may be mediated by other enzyme(s). Second, inhibitor studies argue that separate enzymes catalyze the 7α- and 7β-hydroxylation of DHEA and pregnenolone in brain (31Doostzadeh J. Morfin R. Steroids. 1996; 61: 613-620Crossref PubMed Scopus (64) Google Scholar, 33Doostzadeh J. Cotillon A.C. Morfin R. J. Neuroendocrinol. 1997; 9: 923-928Crossref PubMed Scopus (38) Google Scholar, 34Doostzadeh J. Cotillon A.C. Morfin R. Steroids. 1998; 63: 383-392Crossref PubMed Scopus (13) Google Scholar, 35Doostzadeh J. Cotillon A.C. Benalycherif A. Morfin R. Steroids. 1998; 63: 608-614Crossref PubMed Scopus (32) Google Scholar). Third, metabolism of A/anediol in rodent brain and also prostate is principally at the 6α position (24Warner M. Strömstedt M. Möller L. Gustafsson J.-Å. Endocrinology. 1989; 124: 2699-2706Crossref PubMed Scopus (59) Google Scholar, 25Akwa Y. Morfin R.F. Robel P. Baulieu E.E. Biochem. J. 1992; 288: 959-964Crossref PubMed Scopus (172) Google Scholar, 26Gemzik B. Green J. Parkinson A. Arch. Biochem. Biophys. 1992; 296: 355-365Crossref PubMed Scopus (9) Google Scholar, 28Strömstedt M. Warner M. Banner C.D. MacDonald P.C. Gustafsson J.-Å. Mol. Pharmacol. 1993; 44: 1077-1083PubMed Google Scholar), perhaps indicative of a separate A/anediol hydroxylase enzyme.To address these questions the identity of the brain enzyme, its relationship to the observed activities, and expression pattern, we prepared mice harboring a targeted insertion of a reporter gene cassette (IRES-lacZ) into Cyp7b. We describe reporter gene activity in brain and other tissues and explore alterations in ex vivo steroid hydroxylation in tissues ofCyp7b −/− mice. We argue that the Cyp7b locus encodes a major pathway of extrahepatic steroid and oxysterol hydroxylation.DISCUSSIONWe have used gene targeting to address the relationship of CYP7B enzyme to the steroid and sterol hydroxylation activities reported in rodent tissues. Li-Hawkins et al. (47Li-Hawkins J. Lund E.G. Turley S.D. Russell D.W. J. Biol. Chem. 2000; 275: 16536-16542Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) recently and independently reported mice in which the Cyp7b gene had been disrupted; their studies centered on cholesterol metabolism in liver. We now describe transgenic mice in which a reporter gene (lacZ) is inserted into the Cyp7b gene; we report expression studies and steroid/sterol metabolism in extrahepatic tissues of the mutant mouse.Our principal observations are as follows. First, steroid hydroxylation activity and CY7B mRNA are widely distributed in brain and other tissues. Second, targeting the mouse Cyp7b gene with anIRES-lacZ construct generates reporter enzyme activity in multiple tissues including brain, kidney, and liver; brain reporter expression was dramatically, if superficially, restricted to the dentate gyrus. Third, the pattern of reporter activity in brain failed to match exactly the distribution of CYP7B mRNA or LacZ mRNA, although the reporter activity/mRNA patterns in liver and kidney were largely coincident. Fourth, mice lacking CYP7B activity are viable and superficially normal. Fifth, ex vivoextracts of homozygous Cyp7b −/− animals fail to catalyze hydroxylation of the steroid (and sterol) substrates tested, including DHEA and 25-hydroxycholesterol. Sixth, the targeted mutation also abolished hydroxylation (presumed to be at 6α) of the 5α-reduced steroid, A/anediol. These aspects are discussed separately below.CYP7B expression, originally suspected to be most robustly expressed in hippocampus, is found more widely. Transcripts were readily detected in rat brain, but also at significant levels in spleen, heart, prostate, lung, and ovary, in addition to kidney and liver expression in both mouse and rat (this work and Ref. 36Stapleton G. Steel M. Richardson M. Mason J.O. Rose K.A. Morris R.G. Lathe R. J. Biol. Chem. 1995; 270: 29739-29745Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Relative levels of expression are difficult to assess precisely, but appeared highest in brain, and particularly in the dentate gyrus of the hippocampus; high levels are present in prostate, with significant levels in liver, kidney, heart, and spleen. The level of mRNA expression in ovary was lower. Three mRNAs are present in brain and some other tissues: a pair of transcripts at 2 kb (1.8 and 2.1) and a much larger RNA (∼5 kb) that is prominent in rat but not mouse brain; while the two smaller transcripts are thought to arise by alternative polyadenylation site utilization (36Stapleton G. Steel M. Richardson M. Mason J.O. Rose K.A. Morris R.G. Lathe R. J. Biol. Chem. 1995; 270: 29739-29745Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), the origin of the larger transcript is not known. 7α-Hydroxylation activity is also widespread, and we report conversion of DHEA to a molecule comigrating with 7α-hydroxy-DHEA in several rat and mouse tissues including brain, spleen, thymus, heart, lung, testis, prostate, uterus, ovary, and mammary gland, although the efficiency of conversion varied between tissues. Liver and kidney metabolism was complex, precluding specific analysis of B-ring hydroxylation. Most of these tissues hydroxylate A/anediol and 25-hydroxycholesterol (this work and data not presented) in addition to DHEA (this work). In contrast, the potent anesthetic steroid 5α-androstane-3α,17β-diol was very poorly metabolized by the majority of these tissues, including brain (not presented). In addition to 7α-hydroxylation of DHEA, we also observed a slightly more slowly migrating species, molecule 1, that is inferred, on the basis of other experiments 4E. DeGryse, P. Vico, and R. Lathe, unpublished data. , to be the 17β-HSD product of DHEA, androstenediol, also a substrate for CYP7B (40Rose K.A. Stapleton G. Dott K. Kieny M.P. Best R. Schwarz M. Russell D.W. Björkhem I. Seckl J. Lathe R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4925-4930Crossref PubMed Scopus (204) Google Scholar). This emphasizes previous reports that 17β-HSD activity is also widespread in brain and other tissues (48Martel C. Rheaume E. Takahashi M. Trudel C. Couet J. Luu-The V. Simard J. Labrie F. J. Steroid Biochem. Mol. Biol. 1992; 41: 597-603Crossref PubMed Scopus (234) Google Scholar).We generated mice harboring a targeted insertion of anIRES-lacZ reporter cassette. Chromogenic staining was observed in both liver and kidney, organs in which CYP7B mRNA is present (Ref. 36Stapleton G. Steel M. Richardson M. Mason J.O. Rose K.A. Morris R.G. Lathe R. J. Biol. Chem. 1995; 270: 29739-29745Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar and this work); the staining pattern was similar to the patterns of CYP7B mRNA or LacZ sequences detected by in situ hybridization. Staining in kidney was associated with the S3 segment of the outer stripe of the medulla, a region of acid-base and electrolyte exchange, fuel resorption, and metabolic activity. Liver staining appeared largely in the perivenous zone of the lobules, where glycolysis predominates. The function of CYP7B in these regions is not known. No staining was observed in other tissues analyzed (not presented), probably because of the insensitivity of this technique, with the exception of peri-follicular staining in ovary and intense staining in seminiferous tubules.3In the brain, strikingly vivid reporter staining was seen in the dentate gyrus of the hippocampus, with only lower levels in cerebellum. Overt coloration was absent from other brain regions including cortex and olfactory bulb. Punctate staining in the dentate gyrus is reminiscent of the pattern seen previously with the kin gene trap insertion (49Steel M. Moss J. Clark K.A. Kearns I.R. Davies C.H. Morris R.G. Skarnes W.C. Lathe R. Hippocampus. 1998; 8: 444-457Crossref PubMed Scopus (11) Google Scholar) and suggestive of association of the reporter enzyme with discrete membrane components; these could be a subclass of synapses or other unidentified structures. Here, in contrast to liver and kidney, reporter expression failed to parallel either CYP7B or LacZ mRNA. Staining was restricted to dentate gyrus and, at lower levels, cerebellum; hippocampal regions CA1–3 were essentially negative. In contrast, CYP7B mRNA and transgene-encoded LacZ mRNA were through much of cortex, hippocampus, olfactory bulb, and cerebellum. Nevertheless, localized LacZ activity reiterates the restricted expression exploited for CYP7B cDNA isolation (36Stapleton G. Steel M. Richardson M. Mason J.O. Rose K.A. Morris R.G. Lathe R. J. Biol. Chem. 1995; 270: 29739-29745Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Because DHEA 7-hydroxylation activity is present in microdissected brain regions including olfactory bulb, cortex, cerebellum, brainstem, and is abolished by the mutation, 5K. Rose, S. Gauldie, and R. Lathe, unpublished data. our results would seem to exclude a second unidentified enzyme with significant ex vivo activity. Possible explanations for the reporter/mRNA discrepancy include: 1) tissue specificity of IRES elements (50Creancier L. Morello D. Mercier P. Prats A.C. J. Cell Biol. 2000; 150: 275-281Crossref PubMed Scopus (118) Google Scholar); 2) structure and/or processing of the hybrid RNA may differ between dentate and the CA regions; 3) β-galactosidase enzyme is multimeric: LacZ staining may show a threshold effect; 4) possible post-transcriptional control of CYP7B expression: 7-hydroxylation, presumably mediated by CYP7B, is modulated by cell density (51Akwa Y. Sananes N. Gouezou M. Robel P. Baulieu E.E. Le Goascogne C. J. Cell Biol. 1993; 121: 135-143Crossref PubMed Scopus (159) Google Scholar, 52Killinger D.W. Strutt B.J. Roncari D.A. Khalil M.W. J. Steroid Biochem. Mol. Biol. 1995; 52: 195-201Crossref PubMed Scopus (38) Google Scholar), although the mechanism has not been determined.Intercrossing heterozygous Cyp7b +/− animals generated homozygous −/− animals at or near the expected frequency. Although our data do not rule out some selective perinatal loss of male homozygotes, we conclude that Cyp7b gene function is not essential for viability in adult mice; a similar conclusion was reached by another group (47Li-Hawkins J. Lund E.G. Turley S.D. Russell D.W. J. Biol. Chem. 2000; 275: 16536-16542Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). This contrasts with the situation in human, where CY7B deficiency was associated with abormalities of hepatic cholesterol metabolism and was incompatible with survival (43Setchell K.D. Schwarz M. O'Connell N.C. Lund E.G. Davis D.L. Lathe R. Thompson H.R. Weslie Tyson R. Sokol R.J. Russell D.W. J. Clin. Invest. 1998; 102: 1690-1703Crossref PubMed Scopus (287) Google Scholar). Species differences in hepatic cholesterol metabolism were previously suggested by studies on CYP27 (cholesterol 27-hydroxylase): human mutations produce disordered lipid metabolism, atherosclerosis, and mental retardation (cerebrotendinous xanthomatosis; Ref. 53Björkhem I. Scand. J. Gastroenterol. 1994; 204S: 68-72Crossref Scopus (18) Google Scholar); mice lacking CYP27 display no CTX-related pathological abnormalities (54Rosen H. Reshef A. Maeda N. Lippoldt A. Shpizen S. Triger L. Eggertsen G. Björkhem I. Leitersdorf E. J. Biol. Chem. 1998; 273: 14805-14812Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar).We inspected tissues from Cyp7b −/− mice for their ability to catalyze steroid and oxysterol hydroxylation ex vivo. 7α-Hydroxylation of DHEA was abolished in brain, spleen, thymus, lung, heart, uterus, and mammary gland; gene disruption not only abolished 7α-hydroxylation of DHEA, but also of pregnenolone, A/enediol, and 25-hydroxycholesterol. The complexity of steroid and sterol conversions in transgenic and control liver precluded analysis of specific B-ring modification. In brain and other tissues, homozygous disruption of the Cyp7b gene also abolishes the production of two minor products, probably 7β-hydroxy-DHEA (ascertained by TLC comigration) and a second product with a TLC migration slightly faster than 7α-hydroxy-DHEA: this is inferred (but not proven), on the basis of this and other work (40Rose K.A. Stapleton G. Dott K. Kieny M.P. Best R. Schwarz M. Russell D.W. Björkhem I. Seckl J. Lathe R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4925-4930Crossref PubMed Scopus (204) Google Scholar, 31Doostzadeh J. Morfin R. Steroids. 1996; 61: 613-620Crossref PubMed Scopus (64) Google Scholar, 34Doostzadeh J. Cotillon A.C. Morfin R. Steroids. 1998; 63: 383-392Crossref PubMed Scopus (13) Google Scholar, 35Doostzadeh J. Cotillon A.C. Benalycherif A. Morfin R. Steroids. 1998; 63: 608-614Crossref PubMed Scopus (32) Google Scholar), to correspond to the 6α-hydroxy derivative of DHEA. Both products are generated in vitro by recombinant CYP7B enzyme (40Rose K.A. Stapleton G. Dott K. Kieny M.P. Best R. Schwarz M. Russell D.W. Björkhem I. Seckl J. Lathe R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4925-4930Crossref PubMed Scopus (204) Google Scholar). We conclude that minor modification of DHEA at the 7β and probably 6α positions is an inherent property of CYP7B enzyme. This contrasts with the conclusion, on the basis of inhibitor studies, that different enzymes in brain and prostate are responsible for 7α- and 7β-hydroxylation of DHEA and pregnenolone (34Doostzadeh J. Cotillon A.C. Morfin R. Steroids. 1998; 63: 383-392Crossref PubMed Scopus (13) Google Scholar, 35Doostzadeh J. Cotillon A.C. Benalycherif A. Morfin R. Steroids. 1998; 63: 608-614Crossref PubMed Scopus (32) Google Scholar, 31Doostzadeh J. Morfin R. Steroids. 1996; 61: 613-620Crossref PubMed Scopus (64) Google Scholar).Incubation of A/anediol with extracts of mouse brain (this work) or recombinant CYP7B enzyme (this work and Ref. 40Rose K.A. Stapleton G. Dott K. Kieny M.P. Best R. Schwarz M. Russell D.W. Björkhem I. Seckl J. Lathe R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4925-4930Crossref PubMed Scopus (204) Google Scholar) yielded, like DHEA, one major and two minor polar metabolites. The major metabolite of A/anediol produced by brain ex vivo is 6α-hydroxy-A/anediol (5α-androstane-3β,6α,17β-triol) as reported previously (24Warner M. Strömstedt M. Möller L. Gustafsson J.-Å. Endocrinology. 1989; 124: 2699-2706Crossref PubMed Scopus (59) Google Scholar, 25Akwa Y. Morfin R.F. Robel P. Baulieu E.E. Biochem. J. 1992; 288: 959-964Crossref PubMed Scopus (172) Google Scholar, 26Gemzik B. Green J. Parkinson A. Arch. Biochem. Biophys. 1992; 296: 355-365Crossref PubMed Scopus (9) Google Scholar, 28Strömstedt M. Warner M. Banner C.D. MacDonald P.C. Gustafsson J.-Å. Mol. Pharmacol. 1993; 44: 1077-1083PubMed Google Scholar); this comigrated with the major CYP7B product. We infer, but have not formally proven, that CYP7B catalyzes 6α-hydroxylation of A/anediol and that the less abundant A/anediol metabolites correspond to 7α- and 7β-hydroxylated derivatives. Ex vivo production of all these molecules was abolished by Cyp7b gene disruption (<0.1% of the wild-type conversion level in Cyp7b −/− extracts).Disruption of the Cyp7b gene therefore abolishes hydroxyation of DHEA, pregnenolone, A/enediol, and 25-hydroxycholesterol, at both major (7α) and minor (7β, 6α?) positions. It also abolishes hydroxylation of A/anediol, both at the major (6α) position and at minor positions. We suggest that one gene product, CYP7B enzyme, is responsible for all these activities.This study does not rule out formally the possibility that different transcripts from the CYP7B gene might encode physically distinct enzymes with separate substrate specificities and hydroxylation stereochemistry. We think this unlikely. First, substrate specificity and stereochemistry for these conversions can vary according to reaction conditions (26Gemzik B. Green J. Parkinson A. Arch. Biochem. Biophys. 1992; 296: 355-365Crossref PubMed Scopus (9) Google Scholar, 31Doostzadeh J. Morfin R. Steroids. 1996; 61: 613-620Crossref PubMed Scopus (64) Google Scholar). Second, the hydroxylation profiles of DHEA and A/anediol by brain and recombinant CYP7B enzyme are indistinguishable.4CYP7B is thereby likely to furnish a major extrahepatic and broad-spectrum steroid/sterol B-ring hydroxylase, with predominant hydroxylation at the 7α position (exemplified by DHEA and oxysterols) complemented by 6α-hydroxylation of some atypical substrates (exemplified by A/anediol). CYP7B is not the only extrahepatic B-ring hydroxylase; a testosterone 7α-hydroxylase has been described in testis (CYP2A9/15: Refs. 55Sonderfan A.J. Arlotto M.P.
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