Immunopurified 25-hydroxyvitamin D 1 alpha-hydroxylase and 1,25-dihydroxyvitamin D 24-hydroxylase are closely related but distinct enzymes.

The chick kidney mitochondrial cytochrome P-450 1,25-dihydroxyvitamin D3 24-hydroxylase was partially purified by sequential polyethylene glycol precipitation, aminohexyl-Sepharose 4B, and hydroxylapatite chromatography. The specific activity of the final preparation, when reconstituted with NADPH, adrenodoxin, and adrenodoxin reductase, was 245 pmol/min/mg of protein or 0.56 pmol/min/pmol of P-450. The specific cytochrome P-450 content was 0.45-0.73 nmol/mg of protein. BALB/c mice immunized with this preparation developed serum polyclonal antibodies to the 24-hydroxylase, as demonstrated by immunoprecipitation. Splenic lymphocytes from an immunized mouse were fused with myeloma NSI/1-Ag-4-1 cells, and hybridomas secreting monoclonal antibodies to the 24-hydroxylase were detected by immunoprecipitation. The hybridoma lines were cloned by limiting dilution and further characterized as IgG1, IgG3, and IgM subclasses. In one-dimensional immunoblots of soluble 24-hydroxylase preparations, the monoclonal antibodies revealed a single band with an apparent molecular weight of 59,000. The monoclonal antibodies did not cross-react with cytochrome P-450s from other species but immunoprecipitated and immunoblotted a soluble chick renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase preparation, demonstrating the close similarity of these two hydroxylases. These antibodies were coupled to Sepharose CL-4B and used to isolate to homogeneity the two enzymes from chick kidney mitochondria. Amino-terminal sequences and amino acid composition data demonstrate that these enzymes are different but homologous.     

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.     

Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase,of 25-hydroxyvitamin D(3)-24-hydroxylase,and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3)

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

25-hydroxyvitamin D3 1alpha-hydroxylase and vitamin D receptor expression in papillary thyroid carcinoma.

Vitamin D receptor (VDR) and 25-hydroxyvitamin D3 1-alpha-hydroxylase expression have recently been shown to be upregulated in several tumors and thought to represent an important endogenous response to tumor progression. Little is known about the expression of these proteins in thyroid carcinoma, although previous reports have documented evidence of the biological effect of vitamin D in thyroid cells. Using paraffin-embedded and frozen sections of papillary thyroid carcinoma, we utilized real-time quantitative RT-PCR and immunohistochemistry to characterize the expression of VDR and 1-alpha-hydroxylase in thyroid follicular cells, with special emphasis on papillary thyroid carcinoma (PTC). VDR and 1-alpha-hydroxylase expression were increased in PTC compared with normal thyroid tissue and especially high in areas of lymphocyte infiltration. Expression of VDR and 1-alpha-hydroxylase in PTC may be compatible with an overall favorable prognosis for this tumor type and may constitute important prerequisites for using vitamin D and/or vitamin D analogs in the treatment of PTC.     

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.     

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.     

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.     

Effects of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on cytokine production by human decidual cells

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) is a potent immunomodulatory seco-steroid. We have demonstrated that several components of vitamin D metabolism and signaling are strongly expressed in human uterine decidua from first trimester pregnancies, suggesting that locally produced 1,25(OH)(2)D(3) may exert immunosuppressive effects during early stages of gestation. To investigate this further, we used primary cultures of human decidual cells from first and third trimester pregnancies to demonstrate expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)(2)D(3), 1alpha-hydroxylase (CYP27B1). Synthesis of 1,25(OH)(2)D(3) was higher in first trimester decidual cells (41 +/- 11.8 fmoles/h/mg protein) than in third trimester cells (8 +/- 4.4 fmoles/h/mg protein; P < 0.05). Purification of decidual cells followed by quantitative RT-PCR analysis showed that CYP27B1 was expressed by both CD10(+VE) stromal-enriched and CD10(-VE) stromal-depleted cells, with higher levels of mRNA in first trimester pregnancies. Expression of CYP27B1 correlated with TLR4 and IDO. Functional responses to 1,25(OH)(2)D(3) were studied using CD56(+VE) natural killer (NK) cells isolated from first trimester decidua. Decidual NK cells treated with 1,25(OH)(2)D(3) or precursor 25-hydroxyvitamin D(3) (25OHD(3)) for 28 h showed decreased synthesis of cytokines, such as granulocyte-macrophage colony stimulating factor 2 (CSF2), tumor necrosis factor, and interleukin 6, but increased expression of mRNA for the antimicrobial peptide cathelicidin antimicrobial peptide. These data indicate that human decidual cells are able to synthesize active 1,25(OH)(2)D(3), particularly in early gestation, and this may act in an autocrine/paracrine fashion to regulate both acquired and innate immune responses at the fetal-maternal interface.     

24R,25-dihydroxyvitamin D3 regulates 1,25-dihydroxyvitamin D3 binding to its chick intestinal receptor

We have studied the binding of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to its crude chromatin chick intestinal receptor in the absence or presence of a ten-fold excess of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] for each concentration of [3H]-1,25(OH)2D3 studied. We have found a significant shift to the right in the binding of 1,25(OH)2D3 to its receptor in the presence of this excess of 24R,25(OH)2D3. As a result, the affinity was found to be significantly reduced, the apparent dissociation constants varied from 0.97 +/- 0.09 (n = 5) to 1.36 +/- 0.04 nM (p less than 0.01). This reduction was related to a significant decrease in the positive cooperativity for the apparent Hill coefficient from nH = 1.49 +/- 0.06 to nH = 1.26 +/- 0.06 (p less than 0.03) in the binding of 1,25(OH)2D3 to its receptor. There was no significant change in the capacity of the receptor (189 +/- 11 compared to 200 +/- 9 fmoles/mg protein). These results suggest that the intestinal 1,25(OH)2D3 receptor must also have a binding recognition site for 24R,25(OH)2D3 which is postulated to play a regulatory role in the 1,25(OH)2D3 receptor's ligand binding properties.     

Regulation of human pulmonary surfactant protein gene expression by 1alpha,25-dihydroxyvitamin D3

1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has been reported to stimulate lung maturity, alveolar type II cell differentiation, and pulmonary surfactant synthesis in rat lung. We hypothesized that 1,25(OH)(2)D(3) stimulates expression of surfactant protein-A (SP-A), SP-B, and SP-C in human fetal lung and type II cells. We found that immunoreactive vitamin D receptor was detectable in fetal lung tissue and type II cells only when incubated with 1,25(OH)(2)D(3). 1,25(OH)(2)D(3) significantly decreased SP-A mRNA in human fetal lung tissue but did not significantly decrease SP-A protein in the tissue. In type II cells, 1,25(OH)(2)D(3) alone had no significant effect on SP-A mRNA or protein levels but reduced SP-A mRNA and protein in a dose-dependent manner when the cells were incubated with cAMP. SP-A mRNA levels in NCI-H441 cells, a nonciliated bronchiolar epithelial (Clara) cell line, were decreased in a dose-dependent manner in the absence or presence of cAMP. 1,25(OH)(2)D(3) had no significant effect on SP-B mRNA levels in lung tissue but increased SP-B mRNA and protein levels in type II cells incubated in the absence or presence of cAMP. Expression of SP-C mRNA was unaffected by 1,25(OH)(2)D(3) in lung tissue incubated +/- cAMP. These results suggest that regulation of surfactant protein gene expression in human lung and type II cells by 1,25(OH)(2)D(3) is not coordinated; 1,25(OH)(2)D(3) decreases SP-A mRNA and protein levels in both fetal lung tissue and type II cells, increases SP-B mRNA and protein levels only in type II cells, and has no effect on SP-C mRNA levels.     

Parathyroid hormone inhibits 25-hydroxyvitamin D3-24-hydroxylase mRNA expression stimulated by 1 alpha,25-dihydroxyvitamin D3 in rat kidney but not in intestine.

Using a cDNA probe for rat renal 24-hydroxylase, expression of its mRNA was compared in the rat kidney and intestine. Vitamin D-deficient rats received a single injection of 1 alpha,25-dihydroxyvitamin D3. Expression of 24-hydroxylase mRNA was first detected in the kidney at 3-h post-injection and increased thereafter. Similarly, 24-hydroxylase mRNA was expressed in the intestine after 1 alpha,25-dihydroxyvitamin D3 injection. However, the dose level of 1 alpha,25-dihydroxyvitamin D3 required to induce the intestinal 24-hydroxylase mRNA expression was only 1/100 the amount required to induce renal 24-hydroxylase mRNA. Induction of intestinal 24-hydroxylase mRNA expression by 1 alpha,25-dihydroxyvitamin D3 was far more rapid than that of renal 24-hydroxylase mRNA. Thyroparathyroidectomy shortened the time required to induce expression of renal, but not intestinal, 24-hydroxylase mRNA. Administration of either parathyroid hormone or cAMP to vitamin D-deficient rats greatly reduced the expression of 24-hydroxylase mRNA in the kidney but not in the intestine. When rats were fed a vitamin D-repleted diet containing 0.7% (adequate) or 0.03% (low) calcium for 2 weeks, intestinal expression of 24-hydroxylase mRNA could be induced only in the low calcium group. In contrast, renal mRNA expression was preferentially stimulated in the adequate calcium group. These results clearly demonstrate that the expression of 24-hydroxylase mRNA is down-regulated by parathyroid hormone in the kidney but not in the intestine.     

Calvarial cells synthesize 1 alpha,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3

A metabolite of vitamin D has been isolated in pure form from incubation of 25-hydroxyvitamin D3 with embryonic chick calvarial cells that had been grown on Cytodex 1 microcarrier beads. The isolation involved dichloromethane extraction of the cells and incubation medium, followed by Sephadex LH-20 column chromatography and high-performance liquid chromatography of the extract. The metabolite was identified as 1 alpha,25-dihydroxyvitamin D3 by means of ultraviolet absorption spectroscopy, mass spectrometry, and sensitivity to oxidation by periodate. This metabolite was not produced by cell-free medium or by cells from embryonic chick liver, skin, or heart. In conclusion, (1) kidney cells are not unique in having 25-hydroxyvitamin D3:1 alpha-hydroxylase activity as previously believed and (2) vitamin D target tissues such as the skeleton may play a direct role in mediating the metabolism of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3, a vitamin D metabolite active at those sites.     

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.