Development of vitamin D3 25-hydroxylase activity in rat liver microsomes

The ontogeny of vitamin D3 25-hydroxylase activity has been determined in liver microsomes of rat fetuses and neonates. Production of 25-hydroxyvitamin D3 was low (0.11 pmol/g liver/h) 3 days prior to birth. Production rates were 1.2, 2.2, 1.8, and 2.8 pmol/g liver/h on Day 0, Day 2, Day 7, and Day 15, respectively. 25-Hydroxyvitamin D3 production in neonates increased sixfold from Day 15 to Day 22 to a value twice that of the mothers (17.6 pmol/g liver/h compared with 7.3 pmol/g liver/h). Activity in the maternal microsomes was constant (0.22 to 0.30 pmol/mg protein/h) except for the day of parturition (0.54 pmol/mg protein/h) and Day 22 postpartum (0.44 pmol/mg protein/h). A cytosolic factor, present as early as 3 days prior to birth, was required for vitamin D3 25-hydroxylase activity in the fetuses and stimulated the 25-hydroxylase reaction (up to 2.5-fold) in neonates and mothers. The ability of cytosol to prevent degradation of vitamin D3 was also present in the fetal stage. These data suggest that microsomal vitamin D3 25-hydroxylase activity in rat liver microsomes develops slowly and reaches full activity near the weaning stage. Since the cytosolic factor(s) is/are present in the fetal stage, the limiting component in the maturation of vitamin D3 25-hydroxylase activity in liver microsomes is the development of the cytochrome P-450 vitamin D3 25-hydroxylase.     

The rapid effects of 1,25-dihydroxyvitamin D3 require the vitamin D receptor and influence 24-hydroxylase activity: studies in human skin fibroblasts bearing vitamin D receptor mutations

If both rapid and genomic pathways may co-exist in the same cell, the involvement of the nuclear vitamin D receptor (VDR) in the rapid effects of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) remains unclear. We therefore studied rapid and long term effects of 1,25-(OH)(2)D(3) in cultured skin fibroblasts from three patients with severe vitamin D-resistant rickets and one age-matched control. Patients bear homozygous missense VDR mutations that abolished either VDR binding to DNA (patient 1, mutation K45E) or its stable ligand binding (patients 2 and 3, mutation W286R). In patient 1 cells, 1,25-(OH)(2)D(3) (1 pm-10 nm) had no effect on either intracellular calcium or 24-hydroxylase (enzyme activity and mRNA expression). In contrast, cells bearing the W286R mutation had calcium responses to 1,25-(OH)(2)D(3) (profile and magnitude) and 24-hydroxylase responses to low (1 pm-100 pm) 1,25-(OH)(2)D(3) concentrations (activity, CYP24, and ferredoxin mRNAs) similar to those of controls. The blocker of Ca(2+) channels, verapamil, impeded both rapid (calcium) and long term (24-hydroxylase activity, CYP24, and ferredoxin mRNAs) responses in patient and control fibroblasts. The MEK 1/2 kinase inhibitor PD98059 also blocked the CYP24 mRNA response. Taken together, these results suggest that 1,25-(OH)(2)D(3) rapid effects require the presence of VDR and control, in part, the first step of 1,25-(OH)(2)D(3) catabolism via increased mRNA expression of the CYP24 and ferredoxin genes in the 24-hydroxylase complex.     

Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3

Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3.     

Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-hydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25- and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D2, vitamin D3, 1alpha-hydroxyvitamin D2 (1alphaOHD2), and 1alpha-hydroxyvitamin D3 (1alphaOHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1alphaOHD3 > 1alphaOHD2 > vitamin D2 > vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1alphaOHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1alphaOHD3 was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24- and 25-hydroxylation of 1alphaOHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24- and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1alphaOHD2, and 1alphaOHD3. CYP2C11 is also a vitamin D 24-hydroxylase.     

Regulation of the procine 1,25-dihydroxyvitamin D3-24-hydroxylase (CYP24) by 1,25-dihydroxyvitamin D3 and parathyroid hormone in AOK-B50 cells

The 24-hydroxylase is the enzyme responsible for the first step in the catabolism of 1,25-dihydroxyvitamin D3, the active form of vitamin D. This enzyme was shown to be upregulated by 1,25-dihydroxyvitamin D3 itself and downregulated by parathyroid hormone (PTH). Upregulation of 24-hydroxylase by 1,25-dihydroxyvitamin D3 has been characterized; however, the mechanism by which PTH acts to downregulate 24-hydroxylase expression remains unknown. Here we report the cloning of the porcine 24-hydroxylase, and show that 1,25-dihydroxyvitamin D3-stimulated 24-hydroxylase mRNA and activity are repressed by PTH in AOK-B50 cells, a porcine kidney proximal tubule cell line with stably transfected opossum PTH receptors. Forskolin mimicked the effects of PTH consistent with in vivo data, and suppression by PTH was not due to changes in VDR levels. The first 1400 bp of the 24-hydroxylase promoter were not able to mediate the effects of PTH on a reporter gene. In view of the above findings we concluded that AOK-B50 cells are a suitable model for further studying the mechanism of action of PTH on 24-hydroxylase mRNA.     

Subcellular localization of vitamin D3 25-hydroxylase in human liver

Vitamin D3 25-hydroxylase activity was measured in subcellular and submitochondrial fractions of human liver. Quantitation of 25-hydroxyvitamin D3 was based on high performance liquid chromatography. Vitamin D3 25-hydroxylase activity was detected in the mitochondrial fraction only. The mitochondrial 25-hydroxylase activity was linear with time up to 60 min and with mitochondrial protein up to 1 mg/ml. An apparent Km value of about 10(-5) M was found. Substrate satuation level was not reached. In the presence of 2.4 X 10(-4) M vitamin D3, the rate of 25-hydroxyvitamin D3 formation was 0.19 nmol X mg of protein-1 X h-1 After fractionation of the mitochondria, 86% of the 25-hydroxylase activity was recovered in the mitoplast fraction. The outer membrane fraction was devoid of activity. It is concluded that human liver contains only one detectable vitamin D3 25-hydroxylase enzyme localized to the mitochondrial inner membrane.     

Saposin C下调LNCaP细胞雄激素受体的多泛素化降解（英）

本研究旨在证实鞘脂活化蛋白C(saposin C)对雄激素受体（AR）多泛素化降解的影响及其机制. 通过将真核表达载体saposin C转染LNCaP细胞,发现saposin C上调AR的蛋白水平和转录激活活性. 进一步将野生型和突变型泛素质粒Ubwt和UbK48R分别与saposin C 共转染LNCaP细胞发现,saposin C能够促进AR蛋白的单泛素化形式的稳定性,抑制AR的多泛素化修饰及其在蛋白酶体中的降解. 其分子机制是saposin C、Ub和AR三者形成复合体,抑制了AR的进一步多泛素化过程. 同时还发现,在这一机制中,细胞内低浓度的雄激素（0.1 nmol/L）与saposin C具有协同作用.     

25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria

Assay conditions for the measurement of 25-hydroxyvitamin D3-24-hydroxylase activity in rat kidney mitochondria have been worked out. The product, 24,25-dihydroxyvitamin D3 was quantitated either by high pressure liquid chromatography or by isotope dilution-mass spectrometry. By these procedures, the enzyme activity could be measured with saturating concentration (greater than 2.5 X 10(-6) M) of substrate. Pretreatment of the animals by aminophylline (Kulkowski, J. A., Chow, T., Martinez, J., and Ghazarian, J. G. (1979) Biochem. Biophys. Res. Commun. 90, 50-57) stimulated the 24-hydroxylase activity in vitro at least 2 to 3-fold. The identity of the product was verified by gas chromatography-mass spectrometry. The rates of the reaction varied between 1.5 and 5 pmol/mg of mitochondrial protein.min (at 25 degrees C), and the K'm was determined to be 4.2 X 10(-7) M. Malate, succinate, and isocitrate were all able to support the reaction. Low O2 tension, CO, KCN, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited the reaction, while the respiratory inhibitor rotenone had no effect. Metyrapone inhibited the reaction with 50% inhibition at a concentration of 2.5 mumol/ml. The enzyme was found to be localized inside the inner mitochondrial membrane. The results indicate that in the rat the renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.     

Prostaglandin E2 regulates vitamin D receptor expression, vitamin D-24-hydroxylase activity and cell proliferation in an adherent human myeloid leukemia cell line (Ad-HL60).

The effects of prostaglandin E2, forskolin, and phorbol 12-myristate 13-acetate on cell proliferation, cell surface antigen expression, vitamin D-24-hydroxylase activity and vitamin D receptor (VDR) expression have been studied in an adherent variant (Ad-HL60) of the human HL60 promyelomonocytic leukemia cell line. Ad-HL60 cells have a more differentiated phenotype than the nonadherent HL60 cells from which they were derived and, unlike the parent cell line, constitutively express vitamin D-24-hydroxylase activity. Treatment of Ad-HL60 cells with 1 microM PGE2 resulted in a decrease in the rate of cell proliferation (cell numbers were approximately 23% of control values after 72 h treatment), a change in expression of leukocyte surface antigens (decreased CD13 and CD14, increased CD11b and CD49d expression), an increase in the synthesis of 24,25-dihydroxyvitamin D3 from substrate 25-hydroxyvitamin D3 (control 5.76 +/- 0.17, 72 h PGE2-treated cells 12.10 +/- 1.90 pmol/h/10(6) cells), and an increase in receptors for the active metabolite of vitamin D, 1 alpha,25-dihydroxyvitamin D3, from 3910 to 11285 receptors per cell in control and 7-day treated cells, respectively. Prostaglandin E2 may be acting via a mechanism involving cyclic AMP in these cells, as we have also demonstrated that 10 microM forskolin, an adenylate cyclase activator, has similar effects. Phorbol 12-myristate 13-acetate had little effect on any of the parameters measured in this cell line.     

24-Dehydrovitamin D is potent inhibitor of rat liver microsomal vitamin D-25-hydroxylase

A pathway has been described in the skin for the synthesis of 24-dehydrovitamin D3 (delta 24D3) from 24-dehydroprovitamin D3. The physiologic function of delta 24D3 is unknown, but has been proposed as a potential inhibitor of hepatic vitamin D-25-hydroxylase. We validated an assay for vitamin D-25-hydroxylase in rat hepatic microsomes, using nanomolar amounts of [3H]D3 as substrate, and found that delta 24D3 competitively inhibits vitamin D-25-hydroxylase activity. The apparent Ki was approximately 17 nM, indistinguishable from the Km of approximately 15 nM, suggesting that both delta 24D3 and cholecalciferol have similar affinity for the enzyme. We found no [3H]delta 24D3 in serum or liver extracts after repletion of vitamin D-depleted rats with [3H]vitamin D3 for 4 h or 6 days. A dose of 1 microgram delta 24D3 to vitamin D- and calcium-depleted rats was unable to promote any elevation in the 45Ca transport by everted duodenal sacs or to increase levels of plasma calcium: thus no evidence for biological conversion of delta 24D3 to vitamin D3 was observed. Further studies are needed to determine whether delta 24D3 is released from the skin to the circulation and is taken up by the liver, before physiological relevance can be attributed to this inhibitor.     

Sex-related difference in vitamin D3 25-hydroxylase of rat liver microsomes

Cholecalciferol 25-hydroxylase was partially purified by polyethylene glycol fractionation and chromatographies on octylamino-Sepharose and hydroxylapatite columns starting from the liver microsomes of female rats, and compared with P-450cc25 purified from the liver microsomes of male rats (Hayashi, et al. (1986) J. Biochem. 99, 1753-1763). On octylamino-Sepharose 4B column chromatography, most of the activity was recovered in the fraction eluted with 0.08% Emulgen 913 in the case of the male enzyme, whereas the female enzyme was recovered in the fraction eluted with 0.2% Emulgen. Anti-cc25 antibodies against purified male P-450cc25 inhibited the 25-hydroxylation activity of male polyethylene glycol (PEG) fraction and partially purified male enzyme, but did not inhibit the activities of the corresponding female fractions. The antibodies formed a single precipitation line with male P-450cc25, but did not form a precipitation line with partially purified female 25-hydroxylase on immuno-diffusion. These observations indicated that the vitamin D3 25-hydroxylase in female rat liver microsomes is a different entity from that of male rats.     

25-Hydroxyvitamin D-24-hydroxylase (CYP24A1): its important role in the degradation of vitamin D

CYP24A1 is the cytochrome P450 component of the 25-hydroxyvitamin D(3)-24-hydroxylase enzyme that catalyzes the conversion of 25-hydroxyvitamin D(3) (25-OH-D(3)) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) into 24-hydroxylated products, which constitute the degradation of the vitamin D molecule. This review focuses on recent data in the CYP24A1 field, including biochemical, physiological and clinical developments. Notable among these are: the first crystal structure for rat CYP24A1; mutagenesis studies which change the regioselectivity of the enzyme; and the finding that natural inactivating mutations of CYP24A1 cause the genetic disease idiopathic infantile hypercalcemia (IIH). The review also discusses the emerging correlation between rising serum phosphate/FGF-23 levels and increased CYP24A1 expression in chronic kidney disease, which in turn underlies accelerated degradation of both serum 25-OH-D(3) and 1,25-(OH)(2)D(3) in this condition. This review concludes by evaluating the potential clinical utility of blocking this enzyme with CYP24A1 inhibitors in various disease states.     

Identification of a novel rat microsomal vitamin D3 25-hydroxylase

Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1alpha-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50-60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1alpha-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver.