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1α,25-Dihydroxyvitamin D3-24-Hydroxylase (CYP24) Hydroxylates the Carbon at the End of the Side Chain (C-26) of the C-24-fluorinated Analog of 1α,25-Dihydroxyvitamin D3

1997; Elsevier BV; Volume: 272; Issue: 22 Linguagem: Inglês

10.1074/jbc.272.22.14115

1083-351X

Yoichi Miyamoto, Toshimasa Shinki, Keiko Yamamoto, Yoshihiko Ohyama, Hiroshi Iwasaki, Ryuzo Hosotani, Toshio Kasama, Hiroaki Takayama, Sachiko Yamada, Tatsuo Suda,

Per- and polyfluoroalkyl substances research

The sequential oxidation and cleavage of the side chain of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1α,25(OH)2D3, 24,24-difluoro-1α,25-dihydroxyvitamin D3(F2-1α,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1α,25(OH)2D3-supplemented rats were incubated with F2-1α,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1α,25,26-trihydroxyvitamin D3(F2-1α,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1α,25,26(OH)3D3. Metabolism of F2-1α,25(OH)2D3into F2-1α,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1α,25(OH)2D3 into rats. The C-24 oxidation of 1α,25(OH)2D3 in renal homogenates was inhibited by F2-1α,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1α,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1α,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1α,25(OH)2D3 but also at C-26 of F2-1α,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered. The sequential oxidation and cleavage of the side chain of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1α,25(OH)2D3, 24,24-difluoro-1α,25-dihydroxyvitamin D3(F2-1α,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1α,25(OH)2D3-supplemented rats were incubated with F2-1α,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1α,25,26-trihydroxyvitamin D3(F2-1α,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1α,25,26(OH)3D3. Metabolism of F2-1α,25(OH)2D3into F2-1α,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1α,25(OH)2D3 into rats. The C-24 oxidation of 1α,25(OH)2D3 in renal homogenates was inhibited by F2-1α,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1α,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1α,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1α,25(OH)2D3 but also at C-26 of F2-1α,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered. Metabolic inactivation of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3), 1The abbreviations used are:1α25(OH)2D31α25-dihydroxyvitamin D325(OH)D325-hydroxyvitamin D323S25(OH)2D323S25-dihydroxyvitamin D31α(OH)D31α-hydroxyvitamin D31α24R25(OH)3D31α24R25-trihydroxyvitamin D324-oxo-1α25(OH)2D324-oxo-1α25-dihydroxyvitamin D324-oxo-1α23S25(OH)3D324-oxo-1α23S25trihydroxyvitamin D3F2-1α25(OH)2-D324,24-difluoro-1α25-dihydroxy-vitamin D3F2-1α25,26(OH)3D324,24-difluoro-1α25,26-trihydroxy-vitamin D31α25(OH)2[1β-3H]D31α25-dihydroxy[1β-3H]vitamin D3F2-1α25-(OH)2[1β-3H]D3F2-1α25-dihydroxy[1β-3H]vitamin D3HPLChigh pressure liquid chromatographyFABfast atom bombardmentLCliquid chromatographyMSmass spectroscopyVDRvitamin D receptorVDREvitamin D-responsive element the biologically active metabolite of vitamin D3, in its target cells is initiated by side chain hydroxylation at C-23, C-24, and C-26 (1Ishizuka S. Kiyoki M. Orimo H. Norman A.W. Norman A.W. Scheafer K. Grigoleit H.-G. Harrath D.v. Vitamin D: Chemical, Biochemical and Clinical Update. Walter de Gruyter & Co., Berlin, New York1985: 402-403Google Scholar, 2Mayer E. Bishop J.E. Chandraratna R.A.S. Okamura W.H. Kruse J.R. Popjak G. Ohmura N. Norman A.W. J. Biol. Chem. 1983; 258: 13458-13465Abstract Full Text PDF PubMed Google Scholar, 3Reddy G.S. Tserng K.-Y. Thoma B.R. Dayal R. Norman A.W. Biochemistry. 1987; 26: 324-331Crossref PubMed Scopus (57) Google Scholar, 4Tanaka Y. Schnoes H.K. Smith C.M. DeLuca H.F. Arch. Biochem. Biophys. 1981; 210: 104-109Crossref PubMed Scopus (28) Google Scholar). Of these hydroxylation sites, it is now accepted that the sequential oxidation and cleavage of the side chain initiated by the hydroxylation at C-24 catalyzed by mitochondrial 1α,25(OH)2D3-24-hydroxylase (CYP24) is the major pathway by which the hormone is inactivated (5Makin G. Lohnes D. Byford V. Ray R. Jones G. Biochem. J. 1989; 262: 173-180Crossref PubMed Scopus (211) Google Scholar). Because transcription of the CYP24 gene is highly up-regulated by 1α,25(OH)2D3 in its target cells (6Armbrecht H.J. Boltz M.A. FEBS Lett. 1991; 292: 17-20Crossref PubMed Scopus (48) Google Scholar, 7Shinki T. Jin C.H. Nishimura A. Nagai Y. Ohyama Y. Noshiro M. Okuda K. Suda T. J. Biol. Chem. 1992; 267: 13757-13762Abstract Full Text PDF PubMed Google Scholar, 8Nishimura A. Shinki T. Jin C.H. Ohyama Y. Noshiro M. Okuda K. Suda T. Endocrinology. 1994; 134: 1794-1799Crossref PubMed Google Scholar), CYP24 is regarded as the key enzyme for the breakdown of the hormone (9Suda T. Shinki T. Kurokawa K. Curr. Opin. Nephrol. Hypertens. 1994; 3: 59-64Crossref PubMed Scopus (14) Google Scholar). Metabolism of 1α,25(OH)2D3 initiated by C-23 hydroxylation is induced by 1α,25(OH)2D3itself (10Siu-Caldera M.-L. Zou L. Ehrlich M.G. Schwartz E.R. Ishizuka S. Reddy G.S. Endocrinology. 1995; 136: 4195-4203Crossref PubMed Google Scholar), and recombinant human CYP24 also catalyzes C-23 hydroxylation of 25-hydroxyvitamin D3(25(OH)D3) to yield 23S,25-dihydroxyvitamin D3 (23S,25(OH)2D3) (11Beckman M.J. Tadikonda P. Werner E. Prahl J. Yamada S. DeLuca H.F. Biochemistry. 1996; 35: 8465-8472Crossref PubMed Scopus (181) Google Scholar). Therefore, it is likely that CYP24 initiates both C-24 and C-23 hydroxylation pathways of 1α,25(OH)2D3. In contrast, mitochondrial vitamin d-25-hydroxylase (CYP27) catalyzes the hydroxylation at C-25 and C-26 of vitamin D3and 1α-hydroxyvitamin D3 (1α(OH)D3) (12Guo Y.-D. Strugnell S. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (138) Google Scholar), but it is not clear whether or not this enzyme hydroxylates C-26 of 1α,25(OH)2D3. Recently, a model for the mechanism of the hydroxylation site selection by CYP24 and CYP27 was proposed. This model postulates that CYP24 directs its hydroxylation site(s) by the distance of C-24 and C-23 from the vitamin D ring structure and that CYP27 does so by the distance between the hydroxylation sites and the end of the side chain (13Dilworth F.J. Scott I. Green A. Strugnell S. Guo Y.-D. Roberts E.A. Kremer R. Calverley M.J. Makin H.L.J. Jones G. J. Biol. Chem. 1995; 270: 16766-16774Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).Of numerous synthetic analogs of 1α,25(OH)2D3, 24,24-difluoro-1α,25-dihydroxyvitamin D3(F2-1α,25(OH)2D3) (14Yamada S. Ohmori M. Takayama H. Chem. & Pharm. Bull. ( Tokyo ). 1979; 27: 3196-3198Crossref Scopus (19) Google Scholar) was the first that had a higher biological activity than the parental 1α,25(OH)2D3 (15Kobakoff B.D. Kendrick N.C. Faber D. DeLuca H.F. Arch. Biochem. Biophys. 1982; 215: 582-588Crossref PubMed Scopus (27) Google Scholar, 16Shiina Y. Abe E. Miyaura C. Tanaka H. Yamada S. Ohmori M. Nakayama K. Takayama H. Matsunaga I. Nishii Y. DeLuca H.F. Suda T. Arch. Biochem. Biophys. 1983; 220: 90-94Crossref PubMed Scopus (45) Google Scholar, 17Miyamoto Y. Shinki T. Ohyama Y. Kasama T. Iwasaki H. Hosotani R. Sato T. Suda T. J. Biochem. ( Tokyo ). 1995; 118: 1068-1076Crossref PubMed Scopus (7) Google Scholar). Although the biological activity of F2-1α,25(OH)2D3 was higher, the binding affinity of this analog to VDR was almost identical to that of 1α,25(OH)2D3 (16Shiina Y. Abe E. Miyaura C. Tanaka H. Yamada S. Ohmori M. Nakayama K. Takayama H. Matsunaga I. Nishii Y. DeLuca H.F. Suda T. Arch. Biochem. Biophys. 1983; 220: 90-94Crossref PubMed Scopus (45) Google Scholar, 17Miyamoto Y. Shinki T. Ohyama Y. Kasama T. Iwasaki H. Hosotani R. Sato T. Suda T. J. Biochem. ( Tokyo ). 1995; 118: 1068-1076Crossref PubMed Scopus (7) Google Scholar). It is accepted that the resistance of the C-F bond at C-24 of this analog to metabolic inactivation contributes to its higher biological activity. The metabolic fate of F2-1α,25(OH)2D3, however, has not yet been clarified. We recently reported that F2-1α,25(OH)2D3 is metabolized into a more polar compound(s) in rat osteoblastic ROB-C26 cells (17Miyamoto Y. Shinki T. Ohyama Y. Kasama T. Iwasaki H. Hosotani R. Sato T. Suda T. J. Biochem. ( Tokyo ). 1995; 118: 1068-1076Crossref PubMed Scopus (7) Google Scholar). The metabolism was initiated after transcription of the CYP24 gene, which was induced by the substrate, F2-1α,25(OH)2D3 itself (17Miyamoto Y. Shinki T. Ohyama Y. Kasama T. Iwasaki H. Hosotani R. Sato T. Suda T. J. Biochem. ( Tokyo ). 1995; 118: 1068-1076Crossref PubMed Scopus (7) Google Scholar).In this study, we examined whether CYP24 can metabolize vitamin D analogs, the C-24 of which is resistant to hydroxylation. We identified 24,24-difluoro-1α,25,26-trihydroxyvitamin D3(F2-1α,25,26(OH)3D3) as a major metabolite of F2-1α,25(OH)2D3. Moreover, the enzyme catalyzing the conversion of F2-1α,25(OH)2D3 into F2-1α,25,26(OH)3D3 was CYP24.DISCUSSIONCYP24 was discovered as the enzyme responsible for the hydroxylation at C-24 in the metabolism of 1α,25(OH)2D3 and 25(OH)D3 (25Akiyoshi-Shibata M. Sasaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (141) Google Scholar). Recently, it was found that human recombinant CYP24 also catalyzes the C-23 hydroxylation of 25(OH)D3 (11Beckman M.J. Tadikonda P. Werner E. Prahl J. Yamada S. DeLuca H.F. Biochemistry. 1996; 35: 8465-8472Crossref PubMed Scopus (181) Google Scholar), indicating that this enzyme has multicatalytic functions. However, there is no evidence that CYP24 hydroxylates any other carbons than C-24 or C-23 of vitamin D compounds. In this study, we showed that F2-1α,25(OH)2D3, C-24 of which is protected from the hydroxylation by fluorination, is metabolized into F2-1α,25,26(OH)3D3 by a 1α,25(OH)2D3-induced enzyme in the rat kidney (Figs. 1, 2, 3). The enzyme involved in this hydroxylation was CYP24 (Figs. 4 and 5A). This is the first report to describe that CYP24 hydroxylates a carbon other than C-24 and C-23 of vitamin D compounds. It is generally accepted that the fluorine atom mimics the hydrogen atom. A computer analysis confirmed that 24,24-F2-1α,25(OH)2D3 was very similar to that of 1α,25(OH)2D3, though the electronegativity and hydrophobicity of the fluorine atom were stronger than those of the hydrogen atom (data not shown). Thus the possibility cannot be ruled out at present that the fluorine atoms at C-24 influence the susceptibility of the neighboring carbons to CYP24. Hydroxylation at C-26 of F2-1α,25(OH)2D3 made C-25 asymmetric. The stereochemical configuration at C-25 of this metabolite has yet to be determined.According to the model proposed by Dilworth et al., CYP24 selects its hydroxylation site(s) by the distance from the vitamin D ring structure (13Dilworth F.J. Scott I. Green A. Strugnell S. Guo Y.-D. Roberts E.A. Kremer R. Calverley M.J. Makin H.L.J. Jones G. J. Biol. Chem. 1995; 270: 16766-16774Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The results of the present study, however, suggest that the hydroxylation site selected by the enzyme is not necessarily strict and that CYP24 can hydroxylate a carbon other than C-24 and C-23 when C-24 is protected from metabolic hydroxylation. Therefore, it is highly likely that CYP24 is also responsible for the C-26 hydroxylation of the vitamin D3 metabolites in vivo. At present, the possibility cannot be excluded that enzymes other than CYP24, such as CYP27, hydroxylate C-26 of 1α,25(OH)2D3in vivo. In fact, CYP27 reportedly hydroxylates C-25 and C-26 of vitamin D3and 1α(OH)D3 (12Guo Y.-D. Strugnell S. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (138) Google Scholar). Under our conditions, however, kidney homogenates obtained from rats given either 1α,25(OH)2D3 or vehicle did not metabolize vitamin D3 into 25(OH)D3 or other metabolites (data not shown), indicating that no CYP27 is present in the kidney. In addition, CYP27-transfected ROS17/2.8 cells did not metabolize F2-1α,25(OH)2D3 into F2-1α,25,26(OH)3D3 (Fig.5B). Therefore, the C-26 hydroxylation of F2-1α,25(OH)2D3 in kidney homogenates does not appear to be mediated by CYP27.Two methyl groups at C-25 of 1α,25(OH)2D3 (or 25(OH)D3) are heterotopic. Hydroxylation of one of the methyls yields a new chiral center at C-25. Hydroxylation of the pro-S-methyl group produces 25R configuration and pro-R-methyl group 25S configuration. Two types of 26-oxygenated vitamin D3 metabolites have been found; one is the metabolites with 25S configuration such as 1α,25S,26-trihydroxyvitamin D3 (26Partridge J.J. Shiuey S.-J. Chandha N.K. Baggiolini E.G. Hennessy B.M. Uskokovic M.R. Napoli J.L. Reinhardt T.A. Horst R.L. Helv. Chim. Acta. 1981; 64: 2138-2141Crossref Scopus (20) Google Scholar) and 25S,26-dihydroxyvitamin D3 (27Partridge J.J. Shiuey S.-J. Chandha N.K. Baggiolini E.G. Blount J.F. Uskokovic M.R. J. Am. Chem. Soc. 1981; 103: 1253-1255Crossref Scopus (69) Google Scholar), and the other is those with 25R configuration such as 25R-hydroxyvitamin D3-26,23S-lactone (28Yamada S. Nakayama K. Takayama H. Shinki T. Takasaki Y. Suda T. J. Biol. Chem. 1984; 259: 884-889Abstract Full Text PDF PubMed Google Scholar), 1α,25R-dihydroxyvitamin D3-26,23S-lactone (29Ishizuka S. Oshida J. Tsuruta H. Norman A.W. Arch. Biochem. Biophys. 1985; 242: 82-89Crossref PubMed Scopus (31) Google Scholar), and their precursors. It has also been reported that natural 25,26-dihydroxyvitamin D3 is a mixture of 25R- and 25S-isomers (30Ikekawa N. Noizumi N. Ohshima E. Ishizuka S. Takeshita T. Tanaka Y DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5286-5288Crossref PubMed Scopus (25) Google Scholar). These results suggest that there are two C-26 hydroxylation enzymes; one catalyzes the hydroxylation of the pro-S-methyl and the other catalyzes the pro-R-methyl. It may be likely that these two types of hydroxylation at C-26 are catalyzed by CYP24 and CYP27, respectively. The stereochemical configuration at C-25 of the metabolite X, F2-1α,25,26(OH)3D3, is now under investigation.CYP24 has been found in all the target tissues of vitamin D that possess VDR (31Pike J.W. Annu. Rev. Nutr. 1991; 11: 189-216Crossref PubMed Scopus (283) Google Scholar). Cloning the cDNA and characterizing the CYP24 gene (20Ohyama Y. Noshiro M. Okuda K. FEBS Lett. 1990; 278: 195-198Crossref Scopus (216) Google Scholar, 32Ohyama Y. Noshiro M. Eggertsen G. Gotoh O. Kato Y. Björkhem I. Okuda K. Biochemistry. 1993; 32: 76-82Crossref PubMed Scopus (77) Google Scholar) has allowed the mechanism of regulation of its gene expression to be studied. Northern blotting has revealed that the expression of this enzyme is induced exclusively by 1α,25(OH)2D3 at the transcriptional level (6Armbrecht H.J. Boltz M.A. FEBS Lett. 1991; 292: 17-20Crossref PubMed Scopus (48) Google Scholar, 7Shinki T. Jin C.H. Nishimura A. Nagai Y. Ohyama Y. Noshiro M. Okuda K. Suda T. J. Biol. Chem. 1992; 267: 13757-13762Abstract Full Text PDF PubMed Google Scholar, 8Nishimura A. Shinki T. Jin C.H. Ohyama Y. Noshiro M. Okuda K. Suda T. Endocrinology. 1994; 134: 1794-1799Crossref PubMed Google Scholar). Three groups independently identified functional but different vitamin D-responsive elements (VDRE-1 and VDRE-2) in the antisense strand in rat CYP24 gene promoter at −151 to −137 (VDRE-1) (33Ohyama Y. Ozono K. Uchida M. Shinki T. Kato S. Suda T. Yamamoto O. Noshiro M. Kato Y. J. Biol. Chem. 1994; 269: 10545-10550Abstract Full Text PDF PubMed Google Scholar, 34Hahn C.N. Kerry D.M. Omdahl J.L. May B.K. Nucleic Acids Res. 1994; 22: 2410-2416Crossref PubMed Scopus (64) Google Scholar) and at −259 to −245 (VDRE-2) (35Zierold C. Darwish H.M. DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 900-902Crossref PubMed Scopus (145) Google Scholar) in rats. The presence of the two VDREs in the CYP24 gene promoter may be important for regulating intracellular concentration as well as the half-life of 1α,25(OH)2D3. Makin et al. reported that the target cells of vitamin D metabolize 1α,25(OH)2D3 sequentially into calcitroic acid by the 24-oxidation pathway (5Makin G. Lohnes D. Byford V. Ray R. Jones G. Biochem. J. 1989; 262: 173-180Crossref PubMed Scopus (211) Google Scholar). Using a bacterially expressed enzyme, Akiyoshi-Shibata et al. showed that CYP24 alone can catalyze all of the following reactions; 1α,25(OH)2D3 → 1α,24R,25(OH)3D3 → 24-oxo-1α,25(OH)2D3 → 24-oxo-1α,23S,25(OH)3D3 (25Akiyoshi-Shibata M. Sasaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (141) Google Scholar). The ability of CYP24 to catalyze not only 24-hydroxylation but also its successive reactions implies that the role of this enzyme is to decrease the binding affinity of vitamin D compounds to VDR in the target cells, because 1α,24R,25(OH)3D3 still has about 40% of the affinity of 1α,25(OH)2D3 for VDR (36Bouillon R. Okamura W.H. Norman A.W. Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar). Although the bacterially expressed CYP24 also catalyzed the sequential metabolism of 25(OH)D3, namely 25(OH)D3 → 24R,25-dihydroxyvitamin D3 → 24-oxo-25-hydroxyvitamin D3 → 24-oxo-23S,25-dihydroxyvitamin D3, theKm value of the enzyme for 1α,25(OH)2D3 was one-tenth of that for 25(OH)D3 (25Akiyoshi-Shibata M. Sasaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (141) Google Scholar), suggesting that the former is the real substrate of CYP24.In conclusion, CYP24 appears to solely regulate the intracellular concentration of the VDR ligand and hence the VDR-mediated transactivation in the target cells of vitamin D. It is highly likely that CYP24 catalyzes all three known catabolic pathways of 1α,25(OH)2D3, namely the C-23, C-24, and C-26 hydroxylation pathways, further emphasizing the importance of this enzyme in regulating vitamin D metabolism and function. Metabolic inactivation of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3), 1The abbreviations used are:1α25(OH)2D31α25-dihydroxyvitamin D325(OH)D325-hydroxyvitamin D323S25(OH)2D323S25-dihydroxyvitamin D31α(OH)D31α-hydroxyvitamin D31α24R25(OH)3D31α24R25-trihydroxyvitamin D324-oxo-1α25(OH)2D324-oxo-1α25-dihydroxyvitamin D324-oxo-1α23S25(OH)3D324-oxo-1α23S25trihydroxyvitamin D3F2-1α25(OH)2-D324,24-difluoro-1α25-dihydroxy-vitamin D3F2-1α25,26(OH)3D324,24-difluoro-1α25,26-trihydroxy-vitamin D31α25(OH)2[1β-3H]D31α25-dihydroxy[1β-3H]vitamin D3F2-1α25-(OH)2[1β-3H]D3F2-1α25-dihydroxy[1β-3H]vitamin D3HPLChigh pressure liquid chromatographyFABfast atom bombardmentLCliquid chromatographyMSmass spectroscopyVDRvitamin D receptorVDREvitamin D-responsive element the biologically active metabolite of vitamin D3, in its target cells is initiated by side chain hydroxylation at C-23, C-24, and C-26 (1Ishizuka S. Kiyoki M. Orimo H. Norman A.W. Norman A.W. Scheafer K. Grigoleit H.-G. Harrath D.v. Vitamin D: Chemical, Biochemical and Clinical Update. Walter de Gruyter & Co., Berlin, New York1985: 402-403Google Scholar, 2Mayer E. Bishop J.E. Chandraratna R.A.S. Okamura W.H. Kruse J.R. Popjak G. Ohmura N. Norman A.W. J. Biol. Chem. 1983; 258: 13458-13465Abstract Full Text PDF PubMed Google Scholar, 3Reddy G.S. Tserng K.-Y. Thoma B.R. Dayal R. Norman A.W. Biochemistry. 1987; 26: 324-331Crossref PubMed Scopus (57) Google Scholar, 4Tanaka Y. Schnoes H.K. Smith C.M. DeLuca H.F. Arch. Biochem. Biophys. 1981; 210: 104-109Crossref PubMed Scopus (28) Google Scholar). Of these hydroxylation sites, it is now accepted that the sequential oxidation and cleavage of the side chain initiated by the hydroxylation at C-24 catalyzed by mitochondrial 1α,25(OH)2D3-24-hydroxylase (CYP24) is the major pathway by which the hormone is inactivated (5Makin G. Lohnes D. Byford V. Ray R. Jones G. Biochem. J. 1989; 262: 173-180Crossref PubMed Scopus (211) Google Scholar). Because transcription of the CYP24 gene is highly up-regulated by 1α,25(OH)2D3 in its target cells (6Armbrecht H.J. Boltz M.A. FEBS Lett. 1991; 292: 17-20Crossref PubMed Scopus (48) Google Scholar, 7Shinki T. Jin C.H. Nishimura A. Nagai Y. Ohyama Y. Noshiro M. Okuda K. Suda T. J. Biol. Chem. 1992; 267: 13757-13762Abstract Full Text PDF PubMed Google Scholar, 8Nishimura A. Shinki T. Jin C.H. Ohyama Y. Noshiro M. Okuda K. Suda T. Endocrinology. 1994; 134: 1794-1799Crossref PubMed Google Scholar), CYP24 is regarded as the key enzyme for the breakdown of the hormone (9Suda T. Shinki T. Kurokawa K. Curr. Opin. Nephrol. Hypertens. 1994; 3: 59-64Crossref PubMed Scopus (14) Google Scholar). Metabolism of 1α,25(OH)2D3 initiated by C-23 hydroxylation is induced by 1α,25(OH)2D3itself (10Siu-Caldera M.-L. Zou L. Ehrlich M.G. Schwartz E.R. Ishizuka S. Reddy G.S. Endocrinology. 1995; 136: 4195-4203Crossref PubMed Google Scholar), and recombinant human CYP24 also catalyzes C-23 hydroxylation of 25-hydroxyvitamin D3(25(OH)D3) to yield 23S,25-dihydroxyvitamin D3 (23S,25(OH)2D3) (11Beckman M.J. Tadikonda P. Werner E. Prahl J. Yamada S. DeLuca H.F. Biochemistry. 1996; 35: 8465-8472Crossref PubMed Scopus (181) Google Scholar). Therefore, it is likely that CYP24 initiates both C-24 and C-23 hydroxylation pathways of 1α,25(OH)2D3. In contrast, mitochondrial vitamin d-25-hydroxylase (CYP27) catalyzes the hydroxylation at C-25 and C-26 of vitamin D3and 1α-hydroxyvitamin D3 (1α(OH)D3) (12Guo Y.-D. Strugnell S. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (138) Google Scholar), but it is not clear whether or not this enzyme hydroxylates C-26 of 1α,25(OH)2D3. Recently, a model for the mechanism of the hydroxylation site selection by CYP24 and CYP27 was proposed. This model postulates that CYP24 directs its hydroxylation site(s) by the distance of C-24 and C-23 from the vitamin D ring structure and that CYP27 does so by the distance between the hydroxylation sites and the end of the side chain (13Dilworth F.J. Scott I. Green A. Strugnell S. Guo Y.-D. Roberts E.A. Kremer R. Calverley M.J. Makin H.L.J. Jones G. J. Biol. Chem. 1995; 270: 16766-16774Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 25(OH)2D3 25-dihydroxyvitamin D3 25-hydroxyvitamin D3 25(OH)2D3 25-dihydroxyvitamin D3 1α-hydroxyvitamin D3 24R 1α 25-trihydroxyvitamin D3 25(OH)2D3 25-dihydroxyvitamin D3 23S 24-oxo-1α 25trihydroxyvitamin D3 25(OH)2-D3 25-dihydroxy-vitamin D3 25,26(OH)3D3 25,26-trihydroxy-vitamin D3 25(OH)2[1β-3H]D3 25-dihydroxy[1β-3H]vitamin D3 25-(OH)2[1β-3H]D3 25-dihydroxy[1β-3H]vitamin D3 high pressure liquid chromatography fast atom bombardment liquid chromatography mass spectroscopy vitamin D receptor vitamin D-responsive element Of numerous synthetic analogs of 1α,25(OH)2D3, 24,24-difluoro-1α,25-dihydroxyvitamin D3(F2-1α,25(OH)2D3) (14Yamada S. Ohmori M. Takayama H. Chem. & Pharm. Bull. ( Tokyo ). 1979; 27: 3196-3198Crossref Scopus (19) Google Scholar) was the first that had a higher biological activity than the parental 1α,25(OH)2D3 (15Kobakoff B.D. Kendrick N.C. Faber D. DeLuca H.F. Arch. Biochem. Biophys. 1982; 215: 582-588Crossref PubMed Scopus (27) Google Scholar, 16Shiina Y. Abe E. Miyaura C. Tanaka H. Yamada S. Ohmori M. Nakayama K. Takayama H. Matsunaga I. Nishii Y. DeLuca H.F. Suda T. Arch. Biochem. Biophys. 1983; 220: 90-94Crossref PubMed Scopus (45) Google Scholar, 17Miyamoto Y. Shinki T. Ohyama Y. Kasama T. Iwasaki H. Hosotani R. Sato T. Suda T. J. Biochem. ( Tokyo ). 1995; 118: 1068-1076Crossref PubMed Scopus (7) Google Scholar). Although the biological activity of F2-1α,25(OH)2D3 was higher, the binding affinity of this analog to VDR was almost identical to that of 1α,25(OH)2D3 (16Shiina Y. Abe E. Miyaura C. Tanaka H. Yamada S. Ohmori M. Nakayama K. Takayama H. Matsunaga I. Nishii Y. DeLuca H.F. Suda T. Arch. Biochem. Biophys. 1983; 220: 90-94Crossref PubMed Scopus (45) Google Scholar, 17Miyamoto Y. Shinki T. Ohyama Y. Kasama T. Iwasaki H. Hosotani R. Sato T. Suda T. J. Biochem. ( Tokyo ). 1995; 118: 1068-1076Crossref PubMed Scopus (7) Google Scholar). It is accepted that the resistance of the C-F bond at C-24 of this analog to metabolic inactivation contributes to its higher biological activity. The metabolic fate of F2-1α,25(OH)2D3, however, has not yet been clarified. We recently reported that F2-1α,25(OH)2D3 is metabolized into a more polar compound(s) in rat osteoblastic ROB-C26 cells (17Miyamoto Y. Shinki T. Ohyama Y. Kasama T. Iwasaki H. Hosotani R. Sato T. Suda T. J. Biochem. ( Tokyo ). 1995; 118: 1068-1076Crossref PubMed Scopus (7) Google Scholar). The metabolism was initiated after transcription of the CYP24 gene, which was induced by the substrate, F2-1α,25(OH)2D3 itself (17Miyamoto Y. Shinki T. Ohyama Y. Kasama T. Iwasaki H. Hosotani R. Sato T. Suda T. J. Biochem. ( Tokyo ). 1995; 118: 1068-1076Crossref PubMed Scopus (7) Google Scholar). In this study, we examined whether CYP24 can metabolize vitamin D analogs, the C-24 of which is resistant to hydroxylation. We identified 24,24-difluoro-1α,25,26-trihydroxyvitamin D3(F2-1α,25,26(OH)3D3) as a major metabolite of F2-1α,25(OH)2D3. Moreover, the enzyme catalyzing the conversion of F2-1α,25(OH)2D3 into F2-1α,25,26(OH)3D3 was CYP24. DISCUSSIONCYP24 was discovered as the enzyme responsible for the hydroxylation at C-24 in the metabolism of 1α,25(OH)2D3 and 25(OH)D3 (25Akiyoshi-Shibata M. Sasaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (141) Google Scholar). Recently, it was found that human recombinant CYP24 also catalyzes the C-23 hydroxylation of 25(OH)D3 (11Beckman M.J. Tadikonda P. Werner E. Prahl J. Yamada S. DeLuca H.F. Biochemistry. 1996; 35: 8465-8472Crossref PubMed Scopus (181) Google Scholar), indicating that this enzyme has multicatalytic functions. However, there is no evidence that CYP24 hydroxylates any other carbons than C-24 or C-23 of vitamin D compounds. In this study, we showed that F2-1α,25(OH)2D3, C-24 of which is protected from the hydroxylation by fluorination, is metabolized into F2-1α,25,26(OH)3D3 by a 1α,25(OH)2D3-induced enzyme in the rat kidney (Figs. 1, 2, 3). The enzyme involved in this hydroxylation was CYP24 (Figs. 4 and 5A). This is the first report to describe that CYP24 hydroxylates a carbon other than C-24 and C-23 of vitamin D compounds. It is generally accepted that the fluorine atom mimics the hydrogen atom. A computer analysis confirmed that 24,24-F2-1α,25(OH)2D3 was very similar to that of 1α,25(OH)2D3, though the electronegativity and hydrophobicity of the fluorine atom were stronger than those of the hydrogen atom (data not shown). Thus the possibility cannot be ruled out at present that the fluorine atoms at C-24 influence the susceptibility of the neighboring carbons to CYP24. Hydroxylation at C-26 of F2-1α,25(OH)2D3 made C-25 asymmetric. The stereochemical configuration at C-25 of this metabolite has yet to be determined.According to the model proposed by Dilworth et al., CYP24 selects its hydroxylation site(s) by the distance from the vitamin D ring structure (13Dilworth F.J. Scott I. Green A. Strugnell S. Guo Y.-D. Roberts E.A. Kremer R. Calverley M.J. Makin H.L.J. Jones G. J. Biol. Chem. 1995; 270: 16766-16774Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The results of the present study, however, suggest that the hydroxylation site selected by the enzyme is not necessarily strict and that CYP24 can hydroxylate a carbon other than C-24 and C-23 when C-24 is protected from metabolic hydroxylation. Therefore, it is highly likely that CYP24 is also responsible for the C-26 hydroxylation of the vitamin D3 metabolites in vivo. At present, the possibility cannot be excluded that enzymes other than CYP24, such as CYP27, hydroxylate C-26 of 1α,25(OH)2D3in vivo. In fact, CYP27 reportedly hydroxylates C-25 and C-26 of vitamin D3and 1α(OH)D3 (12Guo Y.-D. Strugnell S. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (138) Google Scholar). Under our conditions, however, kidney homogenates obtained from rats given either 1α,25(OH)2D3 or vehicle did not metabolize vitamin D3 into 25(OH)D3 or other metabolites (data not shown), indicating that no CYP27 is present in the kidney. In addition, CYP27-transfected ROS17/2.8 cells did not metabolize F2-1α,25(OH)2D3 into F2-1α,25,26(OH)3D3 (Fig.5B). Therefore, the C-26 hydroxylation of F2-1α,25(OH)2D3 in kidney homogenates does not appear to be mediated by CYP27.Two methyl groups at C-25 of 1α,25(OH)2D3 (or 25(OH)D3) are heterotopic. Hydroxylation of one of the methyls yields a new chiral center at C-25. Hydroxylation of the pro-S-methyl group produces 25R configuration and pro-R-methyl group 25S configuration. Two types of 26-oxygenated vitamin D3 metabolites have been found; one is the metabolites with 25S configuration such as 1α,25S,26-trihydroxyvitamin D3 (26Partridge J.J. Shiuey S.-J. Chandha N.K. Baggiolini E.G. Hennessy B.M. Uskokovic M.R. Napoli J.L. Reinhardt T.A. Horst R.L. Helv. Chim. Acta. 1981; 64: 2138-2141Crossref Scopus (20) Google Scholar) and 25S,26-dihydroxyvitamin D3 (27Partridge J.J. Shiuey S.-J. Chandha N.K. Baggiolini E.G. Blount J.F. Uskokovic M.R. J. Am. Chem. Soc. 1981; 103: 1253-1255Crossref Scopus (69) Google Scholar), and the other is those with 25R configuration such as 25R-hydroxyvitamin D3-26,23S-lactone (28Yamada S. Nakayama K. Takayama H. Shinki T. Takasaki Y. Suda T. J. Biol. Chem. 1984; 259: 884-889Abstract Full Text PDF PubMed Google Scholar), 1α,25R-dihydroxyvitamin D3-26,23S-lactone (29Ishizuka S. Oshida J. Tsuruta H. Norman A.W. Arch. Biochem. Biophys. 1985; 242: 82-89Crossref PubMed Scopus (31) Google Scholar), and their precursors. It has also been reported that natural 25,26-dihydroxyvitamin D3 is a mixture of 25R- and 25S-isomers (30Ikekawa N. Noizumi N. Ohshima E. Ishizuka S. Takeshita T. Tanaka Y DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5286-5288Crossref PubMed Scopus (25) Google Scholar). These results suggest that there are two C-26 hydroxylation enzymes; one catalyzes the hydroxylation of the pro-S-methyl and the other catalyzes the pro-R-methyl. It may be likely that these two types of hydroxylation at C-26 are catalyzed by CYP24 and CYP27, respectively. The stereochemical configuration at C-25 of the metabolite X, F2-1α,25,26(OH)3D3, is now under investigation.CYP24 has been found in all the target tissues of vitamin D that possess VDR (31Pike J.W. Annu. Rev. Nutr. 1991; 11: 189-216Crossref PubMed Scopus (283) Google Scholar). Cloning the cDNA and characterizing the CYP24 gene (20Ohyama Y. Noshiro M. Okuda K. FEBS Lett. 1990; 278: 195-198Crossref Scopus (216) Google Scholar, 32Ohyama Y. Noshiro M. Eggertsen G. Gotoh O. Kato Y. Björkhem I. Okuda K. Biochemistry. 1993; 32: 76-82Crossref PubMed Scopus (77) Google Scholar) has allowed the mechanism of regulation of its gene expression to be studied. Northern blotting has revealed that the expression of this enzyme is induced exclusively by 1α,25(OH)2D3 at the transcriptional level (6Armbrecht H.J. Boltz M.A. FEBS Lett. 1991; 292: 17-20Crossref PubMed Scopus (48) Google Scholar, 7Shinki T. Jin C.H. Nishimura A. Nagai Y. Ohyama Y. Noshiro M. Okuda K. Suda T. J. Biol. Chem. 1992; 267: 13757-13762Abstract Full Text PDF PubMed Google Scholar, 8Nishimura A. Shinki T. Jin C.H. Ohyama Y. Noshiro M. Okuda K. Suda T. Endocrinology. 1994; 134: 1794-1799Crossref PubMed Google Scholar). Three groups independently identified functional but different vitamin D-responsive elements (VDRE-1 and VDRE-2) in the antisense strand in rat CYP24 gene promoter at −151 to −137 (VDRE-1) (33Ohyama Y. Ozono K. Uchida M. Shinki T. Kato S. Suda T. Yamamoto O. Noshiro M. Kato Y. J. Biol. Chem. 1994; 269: 10545-10550Abstract Full Text PDF PubMed Google Scholar, 34Hahn C.N. Kerry D.M. Omdahl J.L. May B.K. Nucleic Acids Res. 1994; 22: 2410-2416Crossref PubMed Scopus (64) Google Scholar) and at −259 to −245 (VDRE-2) (35Zierold C. Darwish H.M. DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 900-902Crossref PubMed Scopus (145) Google Scholar) in rats. The presence of the two VDREs in the CYP24 gene promoter may be important for regulating intracellular concentration as well as the half-life of 1α,25(OH)2D3. Makin et al. reported that the target cells of vitamin D metabolize 1α,25(OH)2D3 sequentially into calcitroic acid by the 24-oxidation pathway (5Makin G. Lohnes D. Byford V. Ray R. Jones G. Biochem. J. 1989; 262: 173-180Crossref PubMed Scopus (211) Google Scholar). Using a bacterially expressed enzyme, Akiyoshi-Shibata et al. showed that CYP24 alone can catalyze all of the following reactions; 1α,25(OH)2D3 → 1α,24R,25(OH)3D3 → 24-oxo-1α,25(OH)2D3 → 24-oxo-1α,23S,25(OH)3D3 (25Akiyoshi-Shibata M. Sasaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (141) Google Scholar). The ability of CYP24 to catalyze not only 24-hydroxylation but also its successive reactions implies that the role of this enzyme is to decrease the binding affinity of vitamin D compounds to VDR in the target cells, because 1α,24R,25(OH)3D3 still has about 40% of the affinity of 1α,25(OH)2D3 for VDR (36Bouillon R. Okamura W.H. Norman A.W. Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar). Although the bacterially expressed CYP24 also catalyzed the sequential metabolism of 25(OH)D3, namely 25(OH)D3 → 24R,25-dihydroxyvitamin D3 → 24-oxo-25-hydroxyvitamin D3 → 24-oxo-23S,25-dihydroxyvitamin D3, theKm value of the enzyme for 1α,25(OH)2D3 was one-tenth of that for 25(OH)D3 (25Akiyoshi-Shibata M. Sasaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (141) Google Scholar), suggesting that the former is the real substrate of CYP24.In conclusion, CYP24 appears to solely regulate the intracellular concentration of the VDR ligand and hence the VDR-mediated transactivation in the target cells of vitamin D. It is highly likely that CYP24 catalyzes all three known catabolic pathways of 1α,25(OH)2D3, namely the C-23, C-24, and C-26 hydroxylation pathways, further emphasizing the importance of this enzyme in regulating vitamin D metabolism and function. CYP24 was discovered as the enzyme responsible for the hydroxylation at C-24 in the metabolism of 1α,25(OH)2D3 and 25(OH)D3 (25Akiyoshi-Shibata M. Sasaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (141) Google Scholar). Recently, it was found that human recombinant CYP24 also catalyzes the C-23 hydroxylation of 25(OH)D3 (11Beckman M.J. Tadikonda P. Werner E. Prahl J. Yamada S. DeLuca H.F. Biochemistry. 1996; 35: 8465-8472Crossref PubMed Scopus (181) Google Scholar), indicating that this enzyme has multicatalytic functions. However, there is no evidence that CYP24 hydroxylates any other carbons than C-24 or C-23 of vitamin D compounds. In this study, we showed that F2-1α,25(OH)2D3, C-24 of which is protected from the hydroxylation by fluorination, is metabolized into F2-1α,25,26(OH)3D3 by a 1α,25(OH)2D3-induced enzyme in the rat kidney (Figs. 1, 2, 3). The enzyme involved in this hydroxylation was CYP24 (Figs. 4 and 5A). This is the first report to describe that CYP24 hydroxylates a carbon other than C-24 and C-23 of vitamin D compounds. It is generally accepted that the fluorine atom mimics the hydrogen atom. A computer analysis confirmed that 24,24-F2-1α,25(OH)2D3 was very similar to that of 1α,25(OH)2D3, though the electronegativity and hydrophobicity of the fluorine atom were stronger than those of the hydrogen atom (data not shown). Thus the possibility cannot be ruled out at present that the fluorine atoms at C-24 influence the susceptibility of the neighboring carbons to CYP24. Hydroxylation at C-26 of F2-1α,25(OH)2D3 made C-25 asymmetric. The stereochemical configuration at C-25 of this metabolite has yet to be determined. According to the model proposed by Dilworth et al., CYP24 selects its hydroxylation site(s) by the distance from the vitamin D ring structure (13Dilworth F.J. Scott I. Green A. Strugnell S. Guo Y.-D. Roberts E.A. Kremer R. Calverley M.J. Makin H.L.J. Jones G. J. Biol. Chem. 1995; 270: 16766-16774Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The results of the present study, however, suggest that the hydroxylation site selected by the enzyme is not necessarily strict and that CYP24 can hydroxylate a carbon other than C-24 and C-23 when C-24 is protected from metabolic hydroxylation. Therefore, it is highly likely that CYP24 is also responsible for the C-26 hydroxylation of the vitamin D3 metabolites in vivo. At present, the possibility cannot be excluded that enzymes other than CYP24, such as CYP27, hydroxylate C-26 of 1α,25(OH)2D3in vivo. In fact, CYP27 reportedly hydroxylates C-25 and C-26 of vitamin D3and 1α(OH)D3 (12Guo Y.-D. Strugnell S. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (138) Google Scholar). Under our conditions, however, kidney homogenates obtained from rats given either 1α,25(OH)2D3 or vehicle did not metabolize vitamin D3 into 25(OH)D3 or other metabolites (data not shown), indicating that no CYP27 is present in the kidney. In addition, CYP27-transfected ROS17/2.8 cells did not metabolize F2-1α,25(OH)2D3 into F2-1α,25,26(OH)3D3 (Fig.5B). Therefore, the C-26 hydroxylation of F2-1α,25(OH)2D3 in kidney homogenates does not appear to be mediated by CYP27. Two methyl groups at C-25 of 1α,25(OH)2D3 (or 25(OH)D3) are heterotopic. Hydroxylation of one of the methyls yields a new chiral center at C-25. Hydroxylation of the pro-S-methyl group produces 25R configuration and pro-R-methyl group 25S configuration. Two types of 26-oxygenated vitamin D3 metabolites have been found; one is the metabolites with 25S configuration such as 1α,25S,26-trihydroxyvitamin D3 (26Partridge J.J. Shiuey S.-J. Chandha N.K. Baggiolini E.G. Hennessy B.M. Uskokovic M.R. Napoli J.L. Reinhardt T.A. Horst R.L. Helv. Chim. Acta. 1981; 64: 2138-2141Crossref Scopus (20) Google Scholar) and 25S,26-dihydroxyvitamin D3 (27Partridge J.J. Shiuey S.-J. Chandha N.K. Baggiolini E.G. Blount J.F. Uskokovic M.R. J. Am. Chem. Soc. 1981; 103: 1253-1255Crossref Scopus (69) Google Scholar), and the other is those with 25R configuration such as 25R-hydroxyvitamin D3-26,23S-lactone (28Yamada S. Nakayama K. Takayama H. Shinki T. Takasaki Y. Suda T. J. Biol. Chem. 1984; 259: 884-889Abstract Full Text PDF PubMed Google Scholar), 1α,25R-dihydroxyvitamin D3-26,23S-lactone (29Ishizuka S. Oshida J. Tsuruta H. Norman A.W. Arch. Biochem. Biophys. 1985; 242: 82-89Crossref PubMed Scopus (31) Google Scholar), and their precursors. It has also been reported that natural 25,26-dihydroxyvitamin D3 is a mixture of 25R- and 25S-isomers (30Ikekawa N. Noizumi N. Ohshima E. Ishizuka S. Takeshita T. Tanaka Y DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5286-5288Crossref PubMed Scopus (25) Google Scholar). These results suggest that there are two C-26 hydroxylation enzymes; one catalyzes the hydroxylation of the pro-S-methyl and the other catalyzes the pro-R-methyl. It may be likely that these two types of hydroxylation at C-26 are catalyzed by CYP24 and CYP27, respectively. The stereochemical configuration at C-25 of the metabolite X, F2-1α,25,26(OH)3D3, is now under investigation. CYP24 has been found in all the target tissues of vitamin D that possess VDR (31Pike J.W. Annu. Rev. Nutr. 1991; 11: 189-216Crossref PubMed Scopus (283) Google Scholar). Cloning the cDNA and characterizing the CYP24 gene (20Ohyama Y. Noshiro M. Okuda K. FEBS Lett. 1990; 278: 195-198Crossref Scopus (216) Google Scholar, 32Ohyama Y. Noshiro M. Eggertsen G. Gotoh O. Kato Y. Björkhem I. Okuda K. Biochemistry. 1993; 32: 76-82Crossref PubMed Scopus (77) Google Scholar) has allowed the mechanism of regulation of its gene expression to be studied. Northern blotting has revealed that the expression of this enzyme is induced exclusively by 1α,25(OH)2D3 at the transcriptional level (6Armbrecht H.J. Boltz M.A. FEBS Lett. 1991; 292: 17-20Crossref PubMed Scopus (48) Google Scholar, 7Shinki T. Jin C.H. Nishimura A. Nagai Y. Ohyama Y. Noshiro M. Okuda K. Suda T. J. Biol. Chem. 1992; 267: 13757-13762Abstract Full Text PDF PubMed Google Scholar, 8Nishimura A. Shinki T. Jin C.H. Ohyama Y. Noshiro M. Okuda K. Suda T. Endocrinology. 1994; 134: 1794-1799Crossref PubMed Google Scholar). Three groups independently identified functional but different vitamin D-responsive elements (VDRE-1 and VDRE-2) in the antisense strand in rat CYP24 gene promoter at −151 to −137 (VDRE-1) (33Ohyama Y. Ozono K. Uchida M. Shinki T. Kato S. Suda T. Yamamoto O. Noshiro M. Kato Y. J. Biol. Chem. 1994; 269: 10545-10550Abstract Full Text PDF PubMed Google Scholar, 34Hahn C.N. Kerry D.M. Omdahl J.L. May B.K. Nucleic Acids Res. 1994; 22: 2410-2416Crossref PubMed Scopus (64) Google Scholar) and at −259 to −245 (VDRE-2) (35Zierold C. Darwish H.M. DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 900-902Crossref PubMed Scopus (145) Google Scholar) in rats. The presence of the two VDREs in the CYP24 gene promoter may be important for regulating intracellular concentration as well as the half-life of 1α,25(OH)2D3. Makin et al. reported that the target cells of vitamin D metabolize 1α,25(OH)2D3 sequentially into calcitroic acid by the 24-oxidation pathway (5Makin G. Lohnes D. Byford V. Ray R. Jones G. Biochem. J. 1989; 262: 173-180Crossref PubMed Scopus (211) Google Scholar). Using a bacterially expressed enzyme, Akiyoshi-Shibata et al. showed that CYP24 alone can catalyze all of the following reactions; 1α,25(OH)2D3 → 1α,24R,25(OH)3D3 → 24-oxo-1α,25(OH)2D3 → 24-oxo-1α,23S,25(OH)3D3 (25Akiyoshi-Shibata M. Sasaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (141) Google Scholar). The ability of CYP24 to catalyze not only 24-hydroxylation but also its successive reactions implies that the role of this enzyme is to decrease the binding affinity of vitamin D compounds to VDR in the target cells, because 1α,24R,25(OH)3D3 still has about 40% of the affinity of 1α,25(OH)2D3 for VDR (36Bouillon R. Okamura W.H. Norman A.W. Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar). Although the bacterially expressed CYP24 also catalyzed the sequential metabolism of 25(OH)D3, namely 25(OH)D3 → 24R,25-dihydroxyvitamin D3 → 24-oxo-25-hydroxyvitamin D3 → 24-oxo-23S,25-dihydroxyvitamin D3, theKm value of the enzyme for 1α,25(OH)2D3 was one-tenth of that for 25(OH)D3 (25Akiyoshi-Shibata M. Sasaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (141) Google Scholar), suggesting that the former is the real substrate of CYP24. In conclusion, CYP24 appears to solely regulate the intracellular concentration of the VDR ligand and hence the VDR-mediated transactivation in the target cells of vitamin D. It is highly likely that CYP24 catalyzes all three known catabolic pathways of 1α,25(OH)2D3, namely the C-23, C-24, and C-26 hydroxylation pathways, further emphasizing the importance of this enzyme in regulating vitamin D metabolism and function.