Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 (PTH1-34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [3H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylase activity appeared to follow Michaelis-Menten kinetics, and 1,25-dihydroxyvitamin D-3 treatment increased the Vmax of 24-hydroxylase from 33 to 95 pmol/h per 10(6) cells without affecting the apparent Km value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 X 10(-10) M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 microM cycloheximide. Treatment of the cells with PTH1-34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 X 10(-9) M PTH1-34. When 2.4 X 10(-9) M PTH1-34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to 70.7 +/- 2.9% of control. Higher concentrations of PTH1-34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH1-34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the Vmax of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH1-34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

Characteristics of the 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3-24-hydroxylase(s) from HL-60 cells.

1,25-Dihydroxyvitamin D3 induces the human promyelocyte leukemia cell line, HL-60, to differentiate into macrophages/monocytes via a steroid-receptor mechanism. This system is a relevant one for an investigation of the molecular mechanism of 1,25-dihydroxyvitamin D3. We have now examined the effect of 1,25-dihydroxyvitamin D3 on the induction of 1,25-dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities in HL-60 cells. The hydroxylase activities were measured by a periodate-based assay, which was validated by comparison with well-established HPLC analysis. HPLC analysis also suggested that 1,25-dihydroxyvitamin D3 induces a 23-hydroxylase in addition to the 24-hydroxylase. 1,25-Dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities were stimulated as early as 4 h after the addition of 10(-7) M 1,25-dihydroxyvitamin D3 and became maximal by 24 h. 1,25-Dihydroxyvitamin D3 stimulated both activities in a dose-dependent manner up to 10(-6) M. The Km of 24-hydroxylase for 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were 2 x 10(-8) M and 5.2 x 10(-7) M, respectively. Cycloheximide (5 microM) inhibited 1,25-dihydroxyvitamin D3-mediated stimulation of 24-hydroxylase activity. Other differentiation inducers, such as retinoic acid and phorbol ester, did not induce either activity. 1,25-Dihydroxyvitamin D3-24-hydroxylase in HL-60 mitochondria was solubilized with 0.6% cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for 24-hydroxylase activity. These results strongly suggest that the 24-hydroxylase in HL-60 cells is a three-component cytochrome P450-dependent mixed-function oxidase.     

The mechanism of 1,25-dihydroxyvitamin D(3) autoregulation in keratinocytes

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) from its precursor, 25-dihydroxyvitamin D(3) (25(OH)D(3)), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase). It has been generally assumed that 1,25(OH)(2)D(3) inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)(2)D(3) reduced the apparent conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) while stimulating the conversion of 1,25(OH)(2)D(3) and 25(OH)D(3) to 1,24,25(OH)(3)D(3) and 24,25(OH)(2)D(3), respectively. However, 1,25(OH)(2)D(3) failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)(2)D(3) was blocked with a specific inhibitor of the 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1alpha-hydroxylase activity by 1,25(OH)(2)D(3) was reversed. Thus, the apparent reduction in 1alpha-hydroxylase activity induced by 1,25(OH)(2)D(3) is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.     

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.     

Kinetic properties of 25-hydroxyvitamin D- and 1,25-dihydroxyvitamin D-24-hydroxylase from chick kidney

1,25-Dihydroxyvitamin D3 induces both 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3- 24-hydroxylase activities. However, whether 24-hydroxylation of these substrates is catalyzed by a single enzyme is unknown. We have examined the substrate specificity of the enzyme using the solubilized and reconstituted chick renal mitochondrial 24-hydroxylase enzyme system. The soluble enzyme catalyzes 24-hydroxylation of both substrates. The apparent Km of the 24-hydroxylase for 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 were 1.47 and 0.14 microM, respectively. Kinetic studies demonstrated that 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 act as competitive inhibitors with respect to each other. 1,25-Dihydroxyvitamin D3 inhibited the production of 24,25-dihydroxyvitamin D3 with an apparent Ki of 0.09 microM and 25-hydroxyvitamin D3 inhibited the production of 1,24,25-trihydroxyvitamin D3 with an apparent Ki of 3.9 microM. These results indicate that chick 24-hydroxylase preferentially hydroxylates 1,25-dihydroxyvitamin D3 and support the idea that the 24-hydroxylation of these substrates is catalyzed by a single enzyme.     

Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of porcine preadipocyte in vitro

1,25-Dihydroxyvitamin D3, the physiologically active form of vitamin D3, exerts its functions through a receptor-mediated mechanism and plays an important role in the cell differentiation. This study investigated the effects of 1,25-dihydroxyvitamin D3 on the proliferation and differentiation of porcine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from dorsal subcutaneous adipose tissue of approximately 3-day-old Chinese male crossbred pigs. After confluence, the differentiation was induced by transferrin, dexamethasone and insulin for 2 days, and then subsequently cultured for 6 days. The cells were treated with 1,25-dihydroxyvitamin D3 during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. The terminal differentiation markers, such as glycerol-3-phosphate dehydrogenase activity and lipid accumulation were measured during the process of cultures. The treatment with 1,25-dihydroxyvitamin D3 severely affected the induction of all differentiation markers throughout the differentiation period. 1,25-Dihydroxyvitamin D3 suppressed the expression of peroxisome proliferator-activated receptor gamma mRNA and interfered with the induction of retinoid X receptor alpha mRNA. The mRNAs of the adipogenesis-related genes, lipoprotein lipase, stearoyl-CoA desaturase, phosphoenolpyruvate carboxykinase, glycerol-3-phosphate dehydrogenase and glucose transporter 4 were reduced when 1,25-dihydroxyvitamin D3 was added into differentiation medium. Also, 1,25-dihydroxyvitamin D3 inhibited preadipocyte differentiation in dose-dependent manner. These results suggested that 1,25-dihydroxyvitamin D3 inhibited porcine preadipocyte differentiation through suppressing PPAR gamma and RXR alpha mRNA expressions and then down regulating the expression of adipogenesis-related genes.     

C(24)- and C(23)-oxidation, converging pathways of intestinal 1,25-dihydroxyvitamin D3 metabolism: identification of 24-keto-1,23,25-trihydroxyvitamin D3

24-Keto-1,23,25-trihydroxyvitamin D3 has been identified as a major 1,25-dihydroxyvitamin D3 metabolite, produced by intestinal mucosa cells isolated from rats dosed chronically with 1,25-dihydroxyvitamin D3. The identification was based on ultraviolet absorbance spectroscopy, mass spectroscopy, and chemical derivatization. The pathway of biosynthesis proceeded through 1,24,25-trihydroxyvitamin D3 and 24-keto-1,25-dihydroxyvitamin D3, which are physiological metabolites of 1,25-dihydroxyvitamin D3. Previous work [Napoli, J. L., Pramanik, B. C., Royal, P. M., Reinhardt, T. A., & Horst, R. L. (1983) J. Biol. Chem. 258, 9100-9107] had shown that the amount of 24-keto-1,23,25-trihydroxyvitamin D3 in intestine in vivo, relative to its C(24)-oxidized precursors, is enhanced by chronically dosing rats with 1,25-dihydroxyvitamin D3. These results establish the C(24)-oxidation pathway as a predominant route of intestinal 1,25-dihydroxyvitamin D3 metabolism under physiological conditions and indicate that treatment of the rat with exogenous 1,25-dihydroxyvitamin D3 causes expression of C(23)-hydroxylase activity, which uses C(24)-oxidized 1,25-dihydroxyvitamin D3 metabolites as substrates.     

The role of 1,25-dihydroxyvitamin D3 and parathyroid hormone in the regulation of chick renal 25-hydroxyvitamin D3-24-hydroxylase.

A single 325-pmol dose of 1,25-dihydroxyvitamin D₃ given to chicks fed a vitamin D-deficient diet containing 3% calcium and 0.6% phosphorus suppresses renal mitochondrial 25-hydroxyvitamin D₃-1α-hydroxylase and stimulates the 25-hydroxyvitamin D₃-24-hydroxylase as measured by in vitro assay. This alteration in the enzymatic activity takes place over a period of hours. The administration of parathyroid hormone rapidly suppresses the 25-hydroxyvitamin D₃-24-hydroxylase. The alterations in the hydroxylases by parathyroid hormone or 1,25-dihydroxyvitamin D₃ are not related to changes in serum clacium or phosphate but could be related to changes in intracellular levels of these ions. Actinomycin D or cycloheximide given in vivo reduces the 25-hydroxyvitamin D₃-24-hydroxylase activity rapidly which suggests that the turnover of the enzyme and its messenger RNA is rapid (1- and 5-h half-life, respectively). The half-lives of the hydroxylases are sufficiently short to permit a consideration that the regulation by 1,25-dihydroxyvitamin D₃ and parathyroid hormone may involve enzyme synthesis and degradation.     

A rapid assay for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D 24-hydroxylase

A rapid method for the measurement of the 24-hydroxylated metabolites of 25-hydroxy[26,27-3H]vitamin D3 and 1,25-dihydroxy[26,27-3H]vitamin D3 has been developed. This measurement has, in turn, made possible a rapid assay for the 24-hydroxylases of the vitamin D system. The assay involves the use of 26,27-3H-labeled 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 as the substrate and treatment of the enzyme reaction mixture with sodium periodate, which specifically cleaves the 24-hydroxylated products between carbons 24 and 25, releasing tritiated acetone. The acetone is measured after its separation from the labeled substrate by using a reversed-phase cartridge. The results obtained with this assay were validated by comparison with the results obtained with a well-established high-performance liquid chromatography assay. The activity of the enzyme determined by both methods was equal. This assay has been successfully used for the rapid screening of column fractions during purification of the enzyme and in the screening for monoclonal antibodies to the 24-hydroxylase.     

Regulation of rat yolk sac 25-hydroxy- and 1,25-dihydroxyvitamin D3 24-hydroxylase activities

The yolk sac of the pregnant rat which functions as a true placenta is a target organ for vitamin D. This tissue can hydroxylate in position 24 both 25-hydroxy- and 1,25-dihydroxyvitamin D3 (25-OHD3 and 1,25-(OH)2D3). The present report describes an in vitro model for the study of 1,25-(OH)2D3 action on the further metabolism of 25-OH[3H]D3 and 1,25-(OH)2[3H]D3 by yolk sac. The tissue explants were preincubated with 1,25-(OH)2D3 for 18 h in a serum-free culture medium. Physiological concentrations of 1,25-(OH)2D3 were the most effective in stimulating (7.5-fold) the 1,25-(OH)2D3 24-hydroxylase, while the 25-OHD3 24-hydroxylase stimulation (4-fold) required a 1,25-(OH)2D3 concentration of 10(-7) M. The stimulating effect of 1,25-(OH)2D3 on the 1,25-(OH)2D3 24-hydroxylase was temperature-dependent, and, since its was inhibited by actinomycin D and cycloheximide, required de novo protein synthesis. 1,24,25-(OH)3D3, 25-OHD3, and 24,25-(OH)2D3 were 10- to 1000-fold less potent than 1,25-(OH)2D3 in inducing the 1,25-(OH)2D3 hydroxylase. Our results strongly suggest that 1,25-(OH)2D3 regulated the 1,25-(OH)2D3 24-hydroxylase by a receptor-mediated process. Furthermore, 1,25-(OH)2D3 at 10(-9) M induced within 4 h an increase of its own degradation and the formation of an as yet unidentified major 1,25-(OH)2[3H]D3 metabolite. We conclude that the yolk sac can participate in the regulation of 1,25-(OH)2D3 concentration in the fetoplacental unit.     

Alternative splicing of vitamin D-24-hydroxylase: a novel mechanism for the regulation of extrarenal 1,25-dihydroxyvitamin D synthesis

Synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-(OH)(2)D), by renal epithelial cells is tightly controlled during normal calcium homeostasis. By contrast, macrophage production of 1,25-(OH)(2)D is often dysregulated with potential hypercalcemic complications. We have postulated that this is due to abnormal catabolism of 1,25-(OH)(2)D by the feedback control enzyme, vitamin D-24-hydroxylase (CYP24). Using chick HD-11 and human THP-1 myelomonocytic cell lines, we have shown that macrophage-like cells express a splice variant of the CYP24 gene (CYP24-SV), which encodes a truncated protein. Compared with the holo-CYP24 gene product in chick and human cells (508 and 513 amino acids, respectively), the truncated CYP24-SV versions consisted of 351 and 372 amino acids. These CYP24-SV proteins retained intact substrate-binding domains but lacked mitochondrial targeting sequences and were therefore catalytically inactive. In common with CYP24, expression of the CYP24 variants was induced by 1,25-(OH)(2)D but without a concomitant rise in 24-hydroxylase activity. However, overexpression of CYP24-SV in HD-11 and THP-1 cells reduced synthesis of 1,25-(OH)(2) D (40-50%), whereas antisense CYP24-SV expression increased 1,25-(OH)(2)D production by 2-7-fold. These data suggest that alternative splicing of CYP24 leads to the generation of a dominant negative-acting protein that is catalytically dysfunctional. We theorize that expression of the CYP24-SV may contribute to the extracellular accumulation of 1,25(OH)(2)D in human health and disease.     

UVB-induced production of 1,25-dihydroxyvitamin D3 and vitamin D activity in human keratinocytes pretreated with a sterol delta7-reductase inhibitor

The skin fulfills an important role in the vitamin D photo-endocrine system. Epidermis is not only the site of vitamin D3 photoproduction. In addition, epidermal keratinocytes contain the vitamin D receptor (VDR) and possess 25-hydroxylase and 1alpha-hydroxylase activity indicating that all components of the vitamin D system are present. We investigated whether these components cooperate in inducing vitamin D activity upon treatment with physiological UVB doses. Upon irradiation, 24-hydroxylase mRNA was induced in keratinocytes pretreated with a sterol Delta7-reductase inhibitor (BM15766) whereby the 7-dehydrocholesterol content increased by 300-fold. Transfection experiments with a vitamin D response element containing construct confirmed VDR-dependent gene activation. Furthermore, the UVB-dependent induction of 24-hydroxylase was blocked by the cytochrome-P450 inhibitor ketoconazole. The 24-hydroxylase inducing photoproduct was transferable to unirradiated keratinocytes by medium and cellular homogenates of UVB-irradiated, BM15766-pretreated cells and was identified as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by high-performance liquid chromatography with tandem mass spectrometric detection. Addition of vitamin D binding protein blunted UVB-induced 24-hydroxylase suggesting the possibility of a paracrine or autocrine role for 1,25(OH)2D3. In conclusion, epidermal keratinocytes can produce vitamin D3, convert it to 1,25(OH)2D3 and respond to it upon UVB irradiation in the absence of exogenous 7-dehydrocholesterol and therefore contain a unique and complete photo-endocrine vitamin D system.     

Induction of calbindin-D 9k mRNA but not calcium transport in rat intestine by 1,25-dihydroxyvitamin D3 24-homologs

The function and precise mechanism of regulation of calbindin-D 9k in intestine is largely unknown. It is suggested that this calcium binding protein is involved in active intestinal calcium transport and that its expression is mainly mediated by 1,25-dihydroxyvitamin D3. We examined the effect of two side chain modified analogs of 1,25-dihydroxyvitamin D3 as compared to 1,25-dihydroxyvitamin D3 itself on the regulation of the calbindin-D 9k at the mRNA level and on intestinal calcium transport in the rat. delta 22-24,24-dihomo-1,25-dihydroxyvitamin D3 at a single dose of 500, 1,000, and 2,000 pmol caused greater than 7.0-fold increase in calbindin-D 9k mRNA without stimulating intestinal calcium transport. A 10,000-pmol dose of delta 22-24,24,24-trihomo-1,25-dihydroxyvitamin D3 caused a 7.6-fold increase in calbindin-D 9k mRNA without significantly increasing intestinal absorption of calcium. In contrast, 1,25-dihydroxyvitamin D3 caused a parallel increase in calbindin-D 9k mRNA and intestinal absorption of calcium. Thus, calbindin 9k is not by itself responsible for 1,25-dihydroxyvitamin D3-mediated increase in intestinal absorption of calcium.