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Kjell Wikvall - One of the best experts on this subject based on the ideXlab platform.
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androgen receptor mediated regulation of the anti atherogenic enzyme CYP27A1 involves the jnk c jun pathway
Archives of Biochemistry and Biophysics, 2011Co-Authors: Maria Norlin, Wanjin Tang, H Pettersson, Kjell WikvallAbstract:CYP27A1, an enzyme with several important roles in cholesterol homeostasis and vitamin D3 metabolism, has been ascribed anti-atherogenic properties. This study addresses an important problem regarding how this enzyme, involved in cholesterol metabolism in the liver and peripheral tissues, is regulated. Our results identify the human CYP27A1 gene as a new target for the JNK/c-jun pathway. Initial experiments showed that an inhibitor of c-Jun N-terminal kinase (JNK) downregulated basal CYP27A1 promoter activity whereas overexpression of JNK slightly enhanced promoter activity. Androgen receptor (AR)-mediated upregulation of mRNA levels and endogenous enzyme activity was recently reported. In the present study, the AR antagonist nilutamide blocked the androgen induction of CYP27A1. The present data revealed that inhibition of the JNK/c-jun pathway abolishes the AR-mediated effect on CYP27A1 transcription and enzyme activity, whereas overexpression of JNK markedly increased androgenic upregulation of CYP27A1. In conclusion, the current results indicate involvement of the JNK/c-jun pathway in AR-mediated upregulation of human CYP27A1. The link to JNK signaling is interesting since inflammatory processes may upregulate CYP27A1 to clear cholesterol from peripheral tissues.
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Glucocorticoid receptor-mediated upregulation of human CYP27A1, a potential anti-atherogenic enzyme.
Biochimica et Biophysica Acta, 2008Co-Authors: Wanjin Tang, Maria Norlin, Kjell WikvallAbstract:Sterol 27-hydroxylase (CYP27A1) is required for the hepatic conversion of cholesterol into bile acids and for production of 27-hydroxycholesterol which affects cholesterol homeostasis in several ways. Dexamethasone increases hepatic bile acid biosynthesis and CYP27A1-mediated enzyme activity in HepG2 cells. This study examines the mechanism of the dexamethasone-induced effect on the human CYP27A1 promoter. Dexamethasone treatment of HepG2 cells overexpressed with glucocorticoid receptor alpha (GRalpha) increased the CYP27A1 promoter activity more than four-fold as compared with untreated cells. The GR-antagonist mifepristone almost completely abolished the dexamethasone-induced effect on the promoter activity. Progressive deletion analysis of the CYP27A1 promoter indicated that sequences involved in GR-mediated induction by dexamethasone are present in a region between -1094 and -792. Several putative GRE sites could be found in this region and EMSA experiments revealed that two of these could bind GR. Site-directed mutagenesis of GR-binding sequences in the CYP27A1 promoter identified a GRE at -824/-819 important for GR-mediated regulation of the transcriptional activity. Endogenous and pharmacological glucocorticoids may have a strong impact on several aspects of cholesterol homeostasis and other processes related to CYP27A1-mediated metabolism. The glucocorticoid-mediated induction of human CYP27A1 transcription is of particular interest due to the anti-atherogenic properties ascribed to this enzyme.
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regulation of human CYP27A1 by estrogens and androgens in hepg2 and prostate cells
Archives of Biochemistry and Biophysics, 2007Co-Authors: Wanjin Tang, Maria Norlin, Kjell WikvallAbstract:The regulation of the human CYP27A1 gene by estrogens and androgens was studied in human liver-derived HepG2 and prostate cells. Our results show that the promoter activity, enzymatic activity and mRNA levels of CYP27A1 in HepG2 cells are downregulated by estrogen in presence of ERalpha or ERbeta. Similar effects by estrogen were found in RWPE-1 prostate cells. In contrast, estrogen markedly upregulated the transcriptional activity of CYP27A1 in LNCaP prostate cancer cells. 5alpha-Dihydrotestosterone and androgen receptor upregulated the transcriptional activity of CYP27A1 in HepG2 cells. Progressive deletion experiments indicate that the ERbeta-mediated effects in HepG2 and LNCaP cells are conferred to the same region (-451/+42) whereas ERalpha-mediated effects on this promoter are more complex. The results indicate that the stimulating effect of androgen in HepG2 cells is conferred to a region upstream from -792 in the CYP27A1 promoter. In summary, we have identified the human CYP27A1 gene as a target for estrogens and androgens. The results imply that expression of CYP27A1 may be affected by endogenous sex hormones and pharmacological compounds with estrogenic or androgenic effects.
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glucocorticoid receptor mediated regulation of human CYP27A1
2007Co-Authors: Wanjin Tang, Maria Norlin, Kjell WikvallAbstract:CYP27A1 and CYP7B1 are widely expressed in various human tissues and are two key enzymes involved in the pathways for conversion of cholesterol to bile acids. Also, CYP27A1 is involved in bioactivation of vitamin D3 and CYP7B1 plays a role in 7alpha-hydroxylation of dehydroepiandrosterone and other steroids. Both enzymes have been reported to be relevant to prostate cell proliferation. The current study examines the hormonal regulation of CYP27A1 and CYP7B1.CYP7B1 was shown to be regulated by estrogens and androgens in human embryonic kidney HEK293 and prostate cancer LNCaP cells. Quantitation of CYP7B1 mRNA in adult and fetal human tissues showed markedly higher CYP7B1 mRNA levels in fetal tissues compared with the corresponding adult ones, except in the liver. This indicates a tissue-specific, developmental regulation of CYP7B1 and suggests an important function for this enzyme in fetal life. CYP7B1 regulation by estrogens may be of importance in fetal development and in other processes where CYP7B1 is involved, including cholesterol homeostasis, cellular proliferation, and CNS function. The regulation of CYP7B1 by sex hormones also suggests an important role for CYP7B1 in balancing prostate hormone levels in human cells. Results show that CYP27A1 can be regulated by dexamethasone, growth hormone, IGF-1, PMA, estrogens and androgens in liver-derived HepG2 cells. Dexamethasone, growth hormone and IGF-1 stimulated the promoter and endogenous activity of CYP27A1, whereas thyroid hormones and PMA inhibited CYP27A1. The regulatory effects of estrogens and androgens are different depending on the cell types. Thus, the results imply that human CYP27A1 gene is a target for estrogens and androgens, and the expression of CYP27A1 may be affected by endogenous sex hormones and pharmacological compounds with estrogenic or androgenic effects. The mechanism for the dexamethasone-induced effect on the human CYP27A1 promoter was examined. A GRE was identified important for GR-mediated regulation of CYP27A1 transcriptional activity.
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25 hydroxylation of vitamin d3 in primary cultures of pig hepatocytes evidence for a role of both cyp2d25 and CYP27A1
Biochemical and Biophysical Research Communications, 2003Co-Authors: Fardin Hosseinpour, Wanjin Tang, Ines Ibranovic, Kjell WikvallAbstract:There has been some controversy over whether the 25-hydroxylation of vitamin D(3) is carried out by one enzyme or two and whether this cytochrome P450 enzyme is found in the mitochondrial or microsomal fractions of liver. The pig is currently the only species in which both the microsomal 25-hydroxylase (CYP2D25) and the mitochondrial 25-hydroxylase (CYP27A1) have been cloned and characterized. In this paper, the roles of the two enzymes in 25-hydroxylation of vitamin D(3) are examined in primary cultures of hepatocytes. Inhibition experiments indicated that tolterodine and 7 alpha-hydroxy-4-cholesten-3-one were selective inhibitors of the CYP2D25- and CYP27A-mediated 25-hydroxylation of vitamin D(3), respectively. Addition of each inhibitor to primary hepatocytes decreased the total 25-hydroxylation of vitamin D(3) to about the same extent. No inhibition of other hydroxylase activities tested was found. Phorbol 12-myristate 13-acetate down-regulated the expression of both CYP2D25 and CYP27A1 as well as the 25-hydroxylase activity of the hepatocytes. The results implicate that both CYP2D25 and CYP27A1 contribute to the 25-hydroxylation in hepatocytes and are important in the bioactivation of vitamin D(3).
Klaus Badenhoop - One of the best experts on this subject based on the ideXlab platform.
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vitamin d hydroxylases cyp2r1 cyp27b1 and cyp24a1 in renal cell carcinoma
European Journal of Clinical Investigation, 2013Co-Authors: Anja Urbschat, Patrick Paulus, Quirine Freiin Von Quernheim, Patrick Bruck, Klaus Badenhoop, Stefan Zeuzem, Elizabeth RamoslopezAbstract:Background There is increasing evidence that vitamin D metabolites influence carcinogenesis. Besides its role in mineral homoeostasis, calcitriol, the active metabolite of vitamin D (1,25(OH)2D3), is known to possess antiproliferative, proapoptotic and immunomodulatory effects in cancer. Concerning the synthesis of vitamin D, the hydroxylases CYP2R1, CYP27B1 and CYP24A1 play a critical role, and the latter molecule determines the biological half-life of 1,25(OH)2D3, which is synthesized in the proximal renal tubules. Materials and methods The adjacency of these two biological processes prompted us to investigate the gene expression of CYP2R1, CYP27B1 and CYP24A1 in patients with ccRCC. Using RT-PCR, we retrospectively compared mRNA expression profiles from human ccRCC tumour samples with those derived from the corresponding adjacent healthy tissues (n = 30). Results We observed that all three genes (CYP2R1, CYP27B1 and CYP24A1) were upregulated in tumours compared with normal tissue (P < 0·0001). Moreover, CYP24A1 displayed a significantly higher expression in tumours than CYP27B1 (P < 0·05) and CYP2R1 (P < 0·0001), whereas no differences in the expression of these genes were found in healthy renal tissue. Gene expression of CYP2R1, CYP27B1 and CYP24A did not differ between pathological classifications (TNM, grading, presence of metastasis). Conclusion We thus conclude that upregulated gene expression of the catabolizing CYP24A1 as well as the synthesizing CYP2R1 and CYP27B1 may lead to a misbalance of vitamin D metabolites in ccRCC and thus contributing to its pathogenesis.
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association of the vitamin d metabolism gene cyp27b1 with type 1 diabetes
Diabetes, 2007Co-Authors: Rebecca Bailey, Elina Hyppönen, Elizabeth Ramoslopez, Jason D Cooper, Lauren R Zeitels, Deborah J Smyth, Jennie H M Yang, Neil Walker, David B Dunger, Klaus BadenhoopAbstract:OBJECTIVE: Epidemiological studies have linked vitamin D deficiency with the susceptibility to type 1 diabetes. Higher levels of the active metabolite 1 alpha,25-dihydroxyvitamin D (1 alpha,25(OH)(2)D) could protect from immune destruction of the pancreatic beta-cells. 1 alpha,25(OH)(2)D is derived from its precursor 25-hydroxyvitamin D by the enzyme 1 alpha-hydroxylase encoded by the CYP27B1 gene and is inactivated by 24-hydroxylase encoded by the CYP24A1 gene. Our aim was to study the association between the CYP27B1 and CYP24A1 gene polymorphisms and type 1 diabetes. RESEARCH DESIGN AND METHODS: We studied 7,854 patients with type 1 diabetes, 8,758 control subjects from the U.K., and 2,774 affected families. We studied four CYP27B1 variants, including common polymorphisms -1260C>A (rs10877012) and +2838T>C (rs4646536) and 16 tag polymorphisms in the CYP24A1 gene. RESULTS: We found evidence of association with type 1 diabetes for CYP27B1 -1260 and +2838 polymorphisms, which are in perfect linkage disequilibrium. The common C allele of CYP27B1 -1260 was associated with an increased disease risk in the case-control analysis (odds ratio for the C/C genotype 1.22, P = 9.6 x 10(-4)) and in the fully independent collection of families (relative risk for the C/C genotype 1.33, P = 3.9 x 10(-3)). The combined P value for an association with type 1 diabetes was 3.8 x 10(-6). For the CYP24A1 gene, we found no evidence of association with type 1 diabetes (multilocus test, P = 0.23). CONCLUSIONS: The present data provide evidence that common inherited variation in the vitamin D metabolism affects susceptibility to type 1 diabetes.
John Y L Chiang - One of the best experts on this subject based on the ideXlab platform.
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pxr induces CYP27A1 and regulates cholesterol metabolism in the intestine
Journal of Lipid Research, 2007Co-Authors: Wenling Chen, John Y L ChiangAbstract:Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyzes oxidative cleavage of the sterol side chain in the bile acid biosynthetic pathway in the liver and 27-hydroxylation of cholesterol in most tissues. Recent studies suggest that 27-hydroxycholesterol (27-HOC) activates liver orphan receptor alpha (LXRalpha) and induces the cholesterol efflux transporters ABCA1 and ABCG1 in macrophages. The steroid- and bile acid-activated pregnane X receptor (PXR) plays critical roles in the detoxification of bile acids, cholesterol metabolites, and xenobiotics. The role of CYP27A1 in the intestine is not known. This study investigated PXR and CYP27A1 regulation of cholesterol metabolism in the human intestinal cell lines Caco2 and Ls174T. A human PXR ligand, rifampicin, induced CYP27A1 mRNA expression in intestine cells but not in liver cells. Rifampicin induced CYP27A1 gene transcription, increased intracellular 27-HOC levels, and induced ABCA1 and ABCG1 mRNA expression only in intestine cells. A functional PXR binding site was identified in the human CYP27A1 gene. Chromatin immunoprecipitation assays revealed that rifampicin induced the PXR recruitment of steroid receptor coactivator 1 to CYP27A1 chromatin. Cholesterol loading markedly increased intracellular 27-HOC levels in intestine cells. Rifampicin, 27-HOC, and a potent LXRalpha agonist, T0901317, induced ABCA1 and ABCG1 protein expression and stimulated cholesterol efflux from intestine cells to apolipoprotein A-I and HDL. This study suggests an intestine-specific PXR/CYP27A1/LXRalpha pathway that regulates intestine cholesterol efflux and HDL assembly.
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regulation of bile acid synthesis pathways nuclear receptors and mechanisms
Journal of Hepatology, 2004Co-Authors: John Y L ChiangAbstract:Cholesterol degradation to bile acids in the liver can be initiated by either cholesterol 7a-hydroxylase (CYP7A1) of the classic (neutral) pathway, or by mitochondrial sterol 27-hydroxylase (CYP27A1) of the alternative (or acidic) pathway. In the classic pathway, modification of the sterol nucleus including saturation of the double bond, epimerization of the 3b-hydroxyl group, and hydroxylation at the 7a and 12a-positions precedes oxidative cleavage of the side chain. In the alternative pathway, side-chain oxidation precedes steroid ring modification.
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regulation of human sterol 27 hydroxylase gene CYP27A1 by bile acids and hepatocyte nuclear factor 4α hnf4α
Gene, 2003Co-Authors: Wenling Chen, John Y L ChiangAbstract:Abstract Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyses sterol side-chain oxidation of bile acid synthesis from cholesterol, and the first reaction of the acidic bile acid biosynthetic pathway. Hydrophobic bile acids suppress human CYP27A1 gene reporter activity when assayed in human hepatocellular blastoma HepG2 cells. Bile acids also inhibit CYP27A1 reporter activity in human embryonic kidney 293 cells. A putative bile acid response element (BARE) was mapped to a region downstream of nt −147 of the human CYP27A1 gene, within which a binding site for a liver-specific nuclear receptor, HNF4α, is identified. HNF4α strongly stimulates CYP27A1 gene transcription and mutation of its binding site markedly reduced promoter activity. Results suggest that human CYP27A1 gene transcription is suppressed by bile acids and HNF4α plays a pivotal role in transcriptional regulation of this gene.
Toshiyuki Sakaki - One of the best experts on this subject based on the ideXlab platform.
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eldecalcitol is more effective in promoting osteogenesis than alfacalcidol in cyp27b1 knockout mice
PLOS ONE, 2018Co-Authors: Yoshihisa Hirota, Keigo Isomoto, Noboru Kubodera, Maya Kamao, Yoshitomo Suhara, Kimie Nakagawa, Toshiyuki Sakaki, Naomi Osakabe, Toshio OkanoAbstract:: Calcium (Ca) absorption from the intestinal tract is promoted by active vitamin D (1α,25D3). Vitamin D not only promotes Ca homeostasis, but it also inhibits bone resorption and promotes osteogenesis, thus playing a role in the maintenance of normal bone metabolism. Because 1α,25D3 plays an important role in osteogenesis, vitamin D formulations, such as alfacalcidol (ALF) and eldecalcitol (ELD), are used for treating osteoporosis. While it is known that, in contrast to ALF, ELD is an active ligand that directly acts on bone, the reason for its superior osteogenesis effects is unknown. Cyp27b1-knockout mice (Cyp27b1-/-mice) are congenitally deficient in 1α,25D3 and exhibit marked hypocalcemia and high parathyroid hormone levels, resulting in osteodystrophy involving bone hypocalcification and growth plate cartilage hypertrophy. However, because the vitamin D receptor is expressed normally in Cyp27b1-/-mice, they respond normally to 1α,25D3. Accordingly, in Cyp27b1-/-mice, the pharmacological effects of exogenously administered active vitamin D derivatives can be analyzed without being affected by 1α,25D3. We used Cyp27b1-/-mice to characterize and clarify the superior osteogenic effects of ELD on the bone in comparison with ALF. The results indicated that compared to ALF, ELD strongly induces ECaC2, calbindin-D9k, and CYP24A1 in the duodenum, promoting Ca absorption and decreasing the plasma concentration of 1α,25D3, resulting in improved osteogenesis. Because bone morphological measurements demonstrated that ELD has stronger effects on bone calcification, trabecular formation, and cancellous bone density than ALF, ELD appears to be a more effective therapeutic agent for treating postmenopausal osteoporosis, in which cancellous bone density decreases markedly. By using Cyp27b1-/-mice, this study was the first to succeed in clarifying the osteogenic effect of ELD without any influence of endogenous 1α,25D3. Furthermore, ELD more strongly enhanced bone mineralization, trabecular proliferation, and cancellous bone density than did ALF. Thus, ELD is expected to show an effect on postmenopausal osteoporosis, in which cancellous bone mineral density decreases markedly. In the future, this study may enable the development of next-generation active vitamin D derivatives with higher affinity for bone than ELD.
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eldecalcitol is more effective for promoting osteogenesis than alfacalcidol in cyp27b1 knockout mice
bioRxiv, 2018Co-Authors: Yoshihisa Hirota, Keigo Isomoto, Noboru Kubodera, Maya Kamao, Yoshitomo Suhara, Kimie Nakagawa, Toshiyuki Sakaki, Naomi Osakabe, Toshio OkanoAbstract:?Calcium (Ca) absorption from the intestinal tract is promoted by active vitamin D (1α,25D3). Vitamin D not only promotes Ca homeostasis, but it also inhibits bone resorption and promotes osteogenesis, thus playing a role in the maintenance of normal bone metabolism. Because 1α,25D3 plays an important role in osteogenesis, vitamin D formulations, such as alfacalcidol (ALF) and eldecalcitol (ELD), are used for treating osteoporosis. While it is known that, in contrast to ALF, ELD is an active ligand that directly acts on bone, the reason for its superior osteogenesis effects is unknown. Cyp27b1-knockout mice (Cyp27b1-/- mice) are congenitally deficient in 1α,25D3 and exhibit marked hypocalcemia and high parathyroid hormone levels, resulting in osteodystrophy involving bone hypocalcification and growth plate cartilage hypertrophy. However, because the vitamin D receptor is expressed normally in Cyp27b1-/- mice, they respond normally to 1α,25D3. Accordingly, in Cyp27b1-/- mice, the pharmacological effects of exogenously administered active vitamin D derivatives can be analyzed without being affected by 1α,25D3. We used Cyp27b1-/- mice to characterize and clarify the superior osteogenic effects of ELD on the bone in comparison with ALF. The results indicated that compared to ALF, ELD strongly induces ECaC2, calbindin-D9k, and CYP24A1 in the duodenum, promoting Ca absorption and decreasing the blood concentration of 1α,25D3, resulting in improved osteogenesis. Because bone morphological measurements demonstrated that ELD has stronger effects on bone calcification, trabecular formation, and cancellous bone density than ALF, ELD appears to be a more effective therapeutic agent for treating postmenopausal osteoporosis, in which cancellous bone density decreases markedly. By using Cyp27b1-/- mice, this study was the first to succeed in clarifying the osteogenic effect of ELD without any influence of endogenous 1α,25D3. Furthermore, ELD more strongly enhanced bone mineralization, trabecular proliferation, and cancellous bone density than did ALF. Thus, ELD is expected to show an effect on postmenopausal osteoporosis, in which cancellous bone mineral density decreases markedly.
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metabolism of 2α propoxy 1α 25 dihydroxyvitamin d3 and 2α 3 hydroxypropoxy 1α 25 dihydroxyvitamin d3 by human CYP27A1 and cyp24a1
Drug Metabolism and Disposition, 2005Co-Authors: Daisuke Abe, Yoshitomo Suhara, Toshiyuki Sakaki, Tatsuya Kusudo, Atsushi Kittaka, Nozomi Saito, Toshie Fujishima, Hiroaki Takayama, Hiromi Hamamoto, Masaki KamakuraAbstract:Recently, we demonstrated that some A-ring-modified vitamin D3 analogs had unique biological activity. Of these analogs, 2alpha-propoxy-1alpha,25(OH)2D3 (C3O1) and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)2D3 (O2C3) were examined for metabolism by CYP27A1 and CYP24A1. Surprisingly, CYP27A1 catalyzed the conversion from C3O1 to O2C3, which has 3 times more affinity for vitamin D receptor than C3O1. Thus, the conversion from C3O1 to O2C3 by CYP27A1 is considered to be a metabolic activation process. Five metabolites were detected in the metabolism of C3O1 and O2C3 by human CYP24A1 including both C-23 and C-24 oxidation pathways. On the other hand, three metabolites of the C-24 oxidation pathway were detected in their metabolism by rat CYP24A1, indicating a species-based difference in the CYP24A1-dependent metabolism of C3O1 and O2C3 between humans and rats. Kinetic analysis revealed that the Km and kcat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1alpha,25(OH)2D3. Thus, the catalytic efficiency, kcat/Km, of human CYP24A1 for O2C3 is only 2% of 1alpha,25(OH)2D3. These results and a high calcium effect of C3O1 and O2C3 in animal experiments using rats suggest that C3O1 and O2C3 are promising for clinical treatment of osteoporosis.
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metabolism of vitamin d3 by cytochromes p450
Frontiers in Bioscience, 2005Co-Authors: Toshiyuki Sakaki, Norio Kagawa, Keiko Yamamoto, Kuniyo InouyeAbstract:The vitamin D3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) and 1alpha,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha,25(OH)2D3 in addition to the major metabolite 25(OH)D3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH)D3 and 24R,25(OH)2D3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 supports the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH)D3 and 1alpha,25(OH)2D3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha,25(OH)2D3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha,25(OH)2D3-26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.
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purification and characterization of mouse cyp27b1 overproduced by an escherichia coli system coexpressing molecular chaperonins groel es
Biochemical and Biophysical Research Communications, 2004Co-Authors: Eriko Uchida, Toshiyuki Sakaki, Norio Kagawa, Natsumi Sawada, Shigeaki Kato, Masaki Kamakura, Naoko Urushino, Miho Ohta, Kuniyo InouyeAbstract:Abstract The expression of mouse CYP27B1 in Escherichia coli has been dramatically enhanced by coexpression of GroEL/ES. To reveal the enzymatic properties of CYP27B1, we measured its hydroxylation activity toward vitamin D3 and 1α-hydroxyvitamin D3 (1α(OH)D3) in addition to the physiological substrate 25(OH)D3. Surprisingly, CYP27B1 converted vitamin D3 to 1α,25(OH)D3. Both 1α-hydroxylation activity toward vitamin D3, and 25-hydroxylation activity toward 1α(OH)D3 were observed. The Km and Vmax values for 25-hydroxylation activity toward 1α(OH)D3 were estimated to be 1.7 μM and 0.51 mol/min/mol P450, respectively, while those for 1α-hydroxylation activity toward 25(OH)D3 were 0.050 μM and 2.73 mol/min/mol P450, respectively. Note that the substrate must be fixed in the opposite direction in the substrate-binding pocket of CYP27B1 between 1α-hydroxylation and 25-hydroxylation. Based on these results and the fact that human CYP27A1 and Streptomyces CYP105A1 also convert vitamin D3 to 1α,25(OH)D3, 1α-hydroxylation, and 25-hydroxylation of vitamin D3 appear to be closely linked together.
Kuniyo Inouye - One of the best experts on this subject based on the ideXlab platform.
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metabolism of vitamin d3 by cytochromes p450
Frontiers in Bioscience, 2005Co-Authors: Toshiyuki Sakaki, Norio Kagawa, Keiko Yamamoto, Kuniyo InouyeAbstract:The vitamin D3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) and 1alpha,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha,25(OH)2D3 in addition to the major metabolite 25(OH)D3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH)D3 and 24R,25(OH)2D3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 supports the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH)D3 and 1alpha,25(OH)2D3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha,25(OH)2D3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha,25(OH)2D3-26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.
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purification and characterization of mouse cyp27b1 overproduced by an escherichia coli system coexpressing molecular chaperonins groel es
Biochemical and Biophysical Research Communications, 2004Co-Authors: Eriko Uchida, Toshiyuki Sakaki, Norio Kagawa, Natsumi Sawada, Shigeaki Kato, Masaki Kamakura, Naoko Urushino, Miho Ohta, Kuniyo InouyeAbstract:Abstract The expression of mouse CYP27B1 in Escherichia coli has been dramatically enhanced by coexpression of GroEL/ES. To reveal the enzymatic properties of CYP27B1, we measured its hydroxylation activity toward vitamin D3 and 1α-hydroxyvitamin D3 (1α(OH)D3) in addition to the physiological substrate 25(OH)D3. Surprisingly, CYP27B1 converted vitamin D3 to 1α,25(OH)D3. Both 1α-hydroxylation activity toward vitamin D3, and 25-hydroxylation activity toward 1α(OH)D3 were observed. The Km and Vmax values for 25-hydroxylation activity toward 1α(OH)D3 were estimated to be 1.7 μM and 0.51 mol/min/mol P450, respectively, while those for 1α-hydroxylation activity toward 25(OH)D3 were 0.050 μM and 2.73 mol/min/mol P450, respectively. Note that the substrate must be fixed in the opposite direction in the substrate-binding pocket of CYP27B1 between 1α-hydroxylation and 25-hydroxylation. Based on these results and the fact that human CYP27A1 and Streptomyces CYP105A1 also convert vitamin D3 to 1α,25(OH)D3, 1α-hydroxylation, and 25-hydroxylation of vitamin D3 appear to be closely linked together.
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structure function analysis of cyp27b1 and CYP27A1 studies on mutants from patients with vitamin d dependent rickets type i vddr i and cerebrotendinous xanthomatosis ctx
FEBS Journal, 2001Co-Authors: Natsumi Sawada, Toshiyuki Sakaki, Sachiko Kitanaka, Shigeaki Kato, Kuniyo InouyeAbstract:We have determined eight types of missense mutants of CYP27B1 from Japanese vitamin D-dependent rickets type I (VDDR-I) patients [Kitanaka, S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi, H., Yanagisawa, J., Tanaka, T. & Kato, S. (1998) New England J. Med., 338, 653-661 and Kitanaka, S., Murayama, A., Sakaki, T., Inouye, K., Seino, Y., Fukumoto, S., Shima, M., Yukizane, S., Takayanagi, M., Niimi, H., Takeyama, K. & Kato, S. (1999) J. Clin. Endocrine Metab., 84, 4111-4117]. None of the CYP27B1 mutants showed 1alpha-hydroxylase activity towards 25-hydroxyvitamin D3. Thus, it was assumed that the mutated amino-acid residues play important roles in the 1alpha-hydroxylase activity, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the cytochrome P450 structure. To examine our hypothesis, we generated various mutants of CYP27B1 and studied their enzymatic properties. In addition, the corresponding mutations were introduced to CYP27A1, which belongs to the same family as CYP27B1. As CYP27A1 showed much higher expression level than CYP27B1 in Escherichia coli, further analysis including heme-binding and substrate-binding was performed with CYP27A1 in place of CYP27B1. Western blot analysis, spectral analysis including reduced CO-difference spectra and substrate-induced difference spectra, and enzymatic analysis of the mutant CYP27A1 gave information on the structure-function relationships of both CYP27A1 and CYP27B1. Although the sequence alignment suggested that Arg107, Gly125, and Pro497 of CYP27B1 might be involved in substrate binding, the experimental data strongly suggested that mutations of these amino-acid residues destroyed the tertiary structure of the substrate-heme pocket. It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme-propionate binding, and Asp164 stabilized the four-helix bundle consisting of D, E, I and J helices, possibly by forming a salt bridge. Thr321 was found to be responsible for the activation of molecular oxygen.
