Production of 10-oxo-19-nor-25-hydroxyvitamin D3 by solubilized kidney mitochondria from chick and rat

Cholate-solubilized chick kidney mitochondria that 1-hydroxylated 25-hydroxyvitamin-D3 (25-OH-D3) upon reconstitution also produced 10-oxo-19-nor-25-OH-D3, which co-eluted with 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) on normal phase high performance liquid chromatography (HPLC) with hexane:propanol-2 (9:1), the traditional chromatographic system for isolating 1,25-(OH)2-D3. The 10-oxo derivative was separated from 1,25-(OH)2-D3 by normal phase HPLC with dichloromethane:propanol-2 (19:1) or by reverse phase HPLC with methanol:water (4:1). Unlike 1,25-(OH)2-D3 production, formation of 10-oxo-19-nor-25-OH-D3 did not require a source of reducing equivalents and was blocked by the antioxidants, diphenyl-rho-phenylenediamine, and butylated hydroxytoluene, implicating a free radical or peroxidative synthetic mechanism. Rat kidney mitochondria solubilized with cholate or with cholate and Emulgen 911 produced 10-oxo-19-nor-25-OH-D3 but no detectable 1 alpha,25-(OH)2-D3. These results stress the importance of careful identification of vitamin D metabolites produced in vitro and suggest the use of alternate chromatographic conditions for isolating 1,25-(OH)2-D3 or inclusion of antioxidants in the assay of solubilized 1 alpha-hydroxylase to eliminate contamination of 1,25-dihydroxyvitamin D3 with 10-oxo-19-nor-25-OH-D3.     

Characterization of 19-nor-10-oxo-25-hydroxyvitamin D3 production by solubilized chick kidney mitochondria and bovine serum albumin

The vitamin D3 metabolite obtained from the incubation of 3-[(cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO)-solubilized chick kidney mitochondria with 25-hydroxyvitamin D3 (25-OH-D3) was identified to be 5(E)-19-nor-10-oxo-25-hydroxyvitamin D3 (5(E)-19-nor). The production of 19-nor was dependent on time and on protein concentration, but was not dependent on the pH of the incubation. 19-Nor was not formed in the absence of protein or when protein had been heat-treated following detergent solubilization. 19-Nor was not further metabolized to any other product upon incubation with the CHAPSO-solubilized proteins. No 19-nor-10-oxo derivative of 1,25(OH)2D3 was formed when 1,25(OH)2D3 was used as substrate in the incubation. Kinetic analysis showed a substrate saturation with an apparent Vmax of about 4.1 pmol/min.mg and S0.5 of approximately 1.3 x 10(-6) M. The production of 19-nor was not restricted to the CHAPSO-soluble protein fraction of kidney mitochondria but was also found in both the CHAPSO-soluble and -insoluble fractions of chick liver mitochondria and CHAPSO-treated bovine serum albumin (BSA). 19-Nor production by detergent-treated BSA also showed saturation kinetics with a similar S0.5 and an apparent Vmax which was about 5-fold higher than that obtained with CHAPSO-solubilized mitochondria. The evidence suggests that the formation of 19-nor is not mediated by a traditional enzyme, but does require protein. A mechanism for the conversion of 25-OH-E3 to 19-nor is proposed, in which the naturally-occurring 5(Z)-25-OH-D3 substrate binds to protein, isomerizes to 5(E)-25-OH-D3 and is oxidized by hydrogen peroxide to 5(E)-19-nor via a dioxetane intermediate.     

C-6 functionalized analogs of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3: synthesis and binding analysis with vitamin D-binding protein and vitamin D receptor.

In this article, we describe the development of a general synthetic strategy to functionalize the C-6 position of vitamin D3 and its biologically important metabolites, i.e. 25-hydroxyvitamin D3 (25-OH-D3) and 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We employed Mazur's cyclovitamin D method to synthesize vitamin D3 analogs with several functionalities at the C-6 position. In addition, we synthesized 6-(3-hydroxypropyl) and 6-[(2-bromoacetoxy)propyl] derivatives of 25-OH-D3 15 and 16, respectively, and 6-(3-hydroxypropyl) derivative of 1,25(OH)2D3 17. Competitive binding assays of 15-17 with human serum vitamin D-binding protein showed that all these analogs specifically bound to this protein, although with significantly lower affinity than the 25-OH-D3, the strongest natural binder, but with comparable affinity with 1,25(OH)2D3, the hormone. On the other hand, 6-[3-hydroxypropyl], 1alpha,25-dihydroxyvitamin D3 17 did not show any specific binding for recombinant nuclear vitamin D receptor. These results indicated that the region containing the C-6 position of the parent seco-steroid [1,25(OH)2D3] may be an important recognition marker towards vitamin D receptor binding. Information, delineated in this article, will be important for evaluating structure-activity relationship in synthetic analogs of vitamin D and its metabolites.     

Cultured human keratinocytes cannot metabolize vitamin D3 to 25-hydroxyvitamin D3.

The metabolism of [3H]vitamin D3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [3H]vitamin D3 and the lipid soluble fractions from the media and cells were extracted by high-performance liquid chromatography (HPLC). Vitamin D3 and its metabolites, 25-OH-D3, 24,25(OH)2D3 and 1,25(OH)2D3 were added to the extracts, as markers, prior to HPLC. HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [3H]25-OH-D3 showed a 10 +/- 4% conversion to [3H]1,25(OH)2D3 whereas no conversion to [3H]1,25(OH)2D3 was observed in control CHKs that were boiled prior to incubation with [3H]25-OH-D3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D3 to 25-OH-D3.     

1alpha,25-Dihydroxyvitamin D hydroxylase in adipocytes

High vitamin D intake is associated with reduced insulin resistance. Expression of extra-renal 1alpha,25-dihydroxyvitamin D hydroxylase (1alpha-hydroxylase) has been reported in several tissues and contributes to local synthesis of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D) from the substrate 25-hydroxyvitamin D (25OHD). Expression and dietary regulation of 1alpha-hydroxylase in tissues associated with energy metabolism, including adipose tissue, has not been assessed. Male Wistar rats were fed a high calcium (1.5%) and high vitamin D (10,000IU/kg) or a low calcium (0.25%), low vitamin D (400IU/kg) with either a high fat (40% energy) or high sucrose (66% energy) dietary background for 14 weeks. Expression of 1alpha-hydroxylase, assessed by real time PCR, was detected in adipose tissue and did not differ with dietary level of calcium and vitamin D. 1alpha-Hydroxylase mRNA was also detected in 3T3-L1 preadipocytes and 25OHD treatment at 10nM levels induced 1,25(OH)(2)D responsive gene, CYP24, and this response was reduced in the presence of the p450 inhibitor, ketoconazole. In addition, (3)H 25OHD was converted to (3)H 1,25(OH)(2)D in intact 3T3-L1 preadipocytes. Cumulatively, these results demonstrate that 1alpha-hydroxylase is expressed in adipose tissue and is functional in cultured adipocytes. Thus, the capacity for local production may play a role in regulating adipocyte growth and metabolism.     

Kinetics of kidney mitochondrial 25-hydroxycholecalciferol-1 alpha-hydroxylase in vitamin D-repleted weanling guinea pigs

Kinetics of vitamin D-repleted guinea pig kidney mitochondrial 25-hydroxycholecalciferol-1 alpha-hydroxylase were studied. Omission of malate, source of mitochondrial reducing equivalents, abolished the 1 alpha-hydroxylase activity as well as the degradation of 1 alpha, 25-dihydroxycholecalciferol [1,25(OH)2D3], indicating that both functions shared elements of a common pathway. Preincubation of the mitochondrial preparation in presence of 10 nM 1,25(OH)2D3 for 15 min protected the labeled 1,25(OH)2D3 from degradation. Under these conditions an apparent Km of 605 nM and a Vmax of 40 pmol/30 min/mg mitochondrial protein were observed. These data show that this particular mammalian model may be used to study the modulation of mammalian 1 alpha-hydroxylase activity.     

A specific binding protein for 1 alpha,25-dihydroxyvitamin D in the chick embryo chorioallantoic membrane

A 3.7 S binding protein for the steroid hormone and vitamin D metabolite 1 alpha-25-dihydroxyvitamin D (1,25-(OH)2-D) was observed in high salt cytosol extracts of chick embryo chorioallantoic membrane. The binding protein was characterized after partial purification of cytosol extracts by ammonium sulfate fractionation. The binding of 1,25-(OH)2-D was saturable, had a high affinity (Kd = 0.16 nM), and was specific for hormonally active vitamin D metabolites. Analysis of the displacement of [3H]1,25-(OH)2-D by unlabeled analogues showed the affinities of vitamin D metabolites to be in the order of 1,25-(OH)2-D = 1,24R,25-(OH)3-D much greater than 25-OH-D = 1-OH-D greater than 24R,25-(OH)2-D. Hormone binding was sensitive to pretreatment with sulfhydryl-blocking reagents. The chorioallantoic membrane 1,25-(OH)2-D-binding protein associated with the chromatin fraction after homogenization of membranes in low salt buffer, and bound to DNA-cellulose columns, eluting as a single peak at 0.215 M KCl. These findings support identification of this 1,25-(OH)2-D-binding protein as a steroid hormone receptor, with properties indistinguishable from 1,25-(OH)2-D receptors in other chick tissues. The chorioallantoic membrane functions in the last third of embryonic development to reabsorb calcium from the eff shell for deposition in embryonic bone. 1,25-(OH)2-D binding activity in the chorioallantoic membrane increased 4- to 5-fold from day 12 to day 16 of incubation, immediately preceding the onset of shell reabsorption. This finding suggests that 1,25-(OH)2-D may act to regulate shell mobilization and transepithelial calcium transport by the chorioallantoic membrane. Finally, the similarity of shell mobilization to bone resorption, which is also stimulated by 1,25-(OH)2-D, suggests that the chorioallantoic membrane is a useful alternate model for the study of 1,25-(OH)2-D action on bone mineral metabolism.     

Targeting 1alpha,25-dihydroxyvitamin D3 antiproliferative insensitivity in breast cancer cells by co-treatment with histone deacetylation inhibitors

Proliferation of the non-malignant breast epithelial cell line, MCF-12A, is sensitively and completely inhibited by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) (ED90 = 70 nM), We used real time RT-PCR to demonstrate that the relative resistance to 1alpha,25(OH)(2)D(3) of MDA-MB-231 cells (ED50 > 100 nM) correlated with significantly reduced Vitamin D receptor (VDR) and increased NCoR1 nuclear receptor co-repressor mRNA (0.1-fold reduction in VDR and 1.7-fold increase in NCoR1 relative to MCF-12A (P < 0.05)). This molecular lesion can be targeted by co-treating cells with 1alpha,25(OH)(2)D(3) or potent analogs and the histone deacetylation inhibitor trichostatin A (TSA). For example, the co-treatment of 1,25-dihydroxy-16,23,Z-diene-26,27-hexafluoro-19-nor Vitamin D(3) (RO-26-2198) (100 nM) plus TSA results in strong additive antiproliferative effects in MDA-MB-231 cells. This may represent novel chemotherapeutic regime for hormone insensitive breast cancer.     

Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60

Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.     

A-ring hydroxymethyl 19-nor analogs of the natural hormone 1alpha,25-dihydroxyvitamin D(3): synthesis and preliminary biological evaluation

A series 5-8 of 1- and 3-CH(2)OH 19-nor analogs of the hormone calcitriol (1) has been prepared. Surprisingly, 19-nor 1alpha-CH(2)OH analog 5a is more antiproliferative at 100 nM concentration than the corresponding regioisomeric analog 6a with the natural 1alpha-OH group, and 1alpha-CH(2)OH hybrid analog 7a is similar in antiproliferative potency to calcitriol (1) even at low nanomolar concentrations.     

Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney

Until recently measurement of 25-OH-D3-1 alpha-hydroxylase activity in mammalian kidney has not been possible due to the presence of a protein which inhibits the enzyme by reducing available substrate. However, utilization of sufficient unlabeled 25-OH-D3 (80 nmol/ml renal homogenate) to overcome the effect of the inhibitor while maintaining optimal concentration for 1-hydroxylation has made quantitation of enzyme activity possible. We have modified this existing technique in order to increase the sensitivity and to permit detailed study of 1 alpha-hydroxylate regulation in mouse kidney. The modifications that we have incorporated include (i) simplifying the purification scheme for obtaining measurable 1,25-(OH)2D3 by reducing to one the necessary number of high-performance liquid chromatography steps and (ii) quantifying 1,25-(OH)2D3 by radioligand assay. The sensitivity of the assay is 10 pg, which, corrected for fractionation and recovery (50-60%), allows the measurement of 0.5 fmol 1,25-(OH)2D3 produced per milligram kidney per minute. Moreover, reliability and precision of the assay have been confirmed by demonstrating that samples from carefully matched, identically treated mice have reproducible enzyme activity (interassay coefficient of variation = 9.1%, n = 5) and show appropriate dilution characteristics. We have also demonstrated appropriate modulation of enzyme activity by known effectors of 1-hydroxylation. Kidneys from D-deficient mice exhibit significantly higher enzyme activity (15.28 +/- 1.17, n = 21) than do normal mouse kidneys (5.14 +/- 0.26, n = 33). In contrast, enzyme activity is suppressed significantly in kidneys obtained from calcium-loaded (1.20 +/- 0.04, n = 5) and parathyroidectomized animals (2.94 +/- 0.29, n = 5). Our assay now permits the indepth study of 1 alpha-hydroxylase regulation in mammalian (mouse) kidneys.     

Side chain metabolism of vitamin D3 in osteosarcoma cell line UMR-106. Characterization of products

Previous work has shown that 25-hydroxyvitamin D3 (25-OH-D3) and 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) may be metabolized in the mammalian kidney through a side chain oxidation pathway resulting in C23-C24 cleavage, yielding 24,25,26,27-tetranor-23-OH-D3. In the present study, we have used UMR-106 clonal osteoblast cells to demonstrate that products of the side chain oxidation pathway are produced by an osteoblast-like cell. Cells cultured on microcarrier beads and incubated in the presence of pharmacological levels of substrate (1.4 microM, either 25-OH-D3 or 1,25-(OH)2D3) produced sufficient quantities of metabolite to allow identification through mass spectrometry. In addition, putative metabolites were identified through comigration with authentic standards on three high pressure liquid chromatography systems, chemical modification by NaBH4 and periodate, and UV spectral characterization. The pathway was undetectable unless the cells had been exposed to 1,25-(OH)2D3 prior to incubation with substrate. We have shown that 1,25-(OH)2D3 induces the 24-hydroxylase and perhaps also the other enzymes of this pathway in the bone cell. Although we used pharmacological concentrations of substrate to demonstrate the existence of the side chain oxidation pathway in bone cells, physiological levels of 25-OH-D3 or 1,25-(OH)2D3 were also metabolized through the pathway, at least as far as the penultimate product. We speculate that the side chain oxidation pathway may be ubiquitous among vitamin D target tissues.     

Effects of 1,25(OH)2D3, 25OHD3, and EB1089 on cell growth and Vitamin D receptor mRNA and 1alpha-hydroxylase mRNA expression in primary cultures of the canine prostate

The aim of this study was to investigate effects of 1,25(OH)(2)D(3) (calcitriol), 25OHD(3), and EB1089 on cell growth and on Vitamin D receptor (VDR) mRNA and 1alpha-hydroxylase (1alpha-OHase) mRNA expression in normal canine prostatic primary cultures. Canine prostatic epithelial cells were isolated, cultured, and treated with vehicle (ethanol), calcitriol, 25OHD(3), and EB1089 at 10(-9) and 10(-7)M. The VDR was present in epithelial and stromal cells of the canine prostate gland. 1,25(OH)(2)D(3), 25OHD(3), and EB1089 inhibited epithelial cell growth at 10(-7)M compared to vehicle-treated controls [calcitriol (P < 0.01), EB1089 (P < 0.01), and 25OHD(3) (P < 0.05)]. Epithelial cells treated with calcitriol and EB1089 at 10(-7)M had slightly increased VDR mRNA expression (0.2-0.3-fold) at 6 and 12h compared to controls. There was no difference in 1alpha-OHase mRNA expression in epithelial cells treated with these three compounds. 1,25(OH)(2)D(3) and its analogs may be effective antiproliferative agents of epithelial cells in certain types of prostate cancer.     

Targeted disruption of the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in ras-transformed keratinocytes demonstrates that locally produced 1alpha,25-dihydroxyvitamin D3 suppresses growth and induces differentiation in an autocrine fashion

It has been previously shown that keratinocytes express a high level of 25-hydroxyvitamin D(3) (25-OHD(3)) 1alpha-hydroxylase (1alpha-hydroxylase). 1alpha-Hydroxylase catalyzes the conversion of 25-OHD(3) to 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. 1,25(OH)(2)D(3) is both antiproliferative (i.e., suppresses cell growth) and prodifferentiative (i.e., induces cell differentiation) in many cell types. We hypothesized that local production of 1,25(OH)(2)D(3) by keratinocytes may suppress their growth and induce their differentiation in an autocrine fashion. To test this hypothesis, we inactivated both 1alpha-hydroxylase alleles in a ras-transformed keratinocyte cell line, HPK1Aras, which typically produces squamous carcinoma in nude mice. To inactivate 1alpha-hydroxylase expression by HPK1Aras cells, we disrupted both alleles of the 1alpha-hydroxylase gene by homologous recombination. Lack of expression and activity of 1alpha-hydroxylase was confirmed by Northern blot analysis and detected conversion of 25-OHD(3) to 1,25(OH)(2)D(3). We then examined the effect of substrate 25-OHD(3) on parameters of growth and differentiation in the double knockout cell line as compared to wild-type HPK1Aras cells in vitro. It was found that 1alpha-hydroxylase inactivation blocked the antiproliferative and prodifferentiative effect of 25-OHD(3). These in vitro effects were further analyzed in vivo by injecting knockout or control cells subcutaneously in severely compromised immunodeficient mice. Tumor growth was accelerated and differentiation was inhibited in mice given injections of knockout cells as compared to control cells in the presence of substrate 25-OHD(3). Our results demonstrate, for the first time, that 1alpha-hydroxylase expression by keratinocytes plays an important role in autocrine growth and differentiation of these cells, and suggest that expression of this enzyme may modulate tumor growth in squamous carcinomas.     

The photobiogenesis and metabolism of vitamin D.

Provitamin D3 (7-dehydrocholesterol) is converted to previtamin D3 by the action of ultraviolet radiation on the skin. Previtamin D3 thermally isomerizes to vitamin D3 in the skin and the vitamin is then transported to the liver on the vitamin D-binding protein. Although there are extrahepatic vitamin D-25-hydroxylases, the liver is the major site for the 25-hydroxylation of vitamin D. In response to hypocalcemia and hypophosphatemia, 25-OH-D is metabolized by a renal-cytochrome. P450-dependent mixed function oxidase system is 1alpha,25(OH)2D. When calcium and phosphate homeostasis prevails the renal 25-OH-D-1alpha-hydroxylase activity is limited and instead a non-cytochrome P450 mixed function oxidase metabolizes 25-OH-D to 24R,25(OH)2D. Parathyroid hormone has clearly been shown to be a trophin for the renal synthesis of 1,25(OH)2D; however, the role and significance of the adrenal steroids, or gonadal and pituitary hormones, on the renal 25-OH-D-1alpha-hydroxylase is not well defined. The regulation of the photometabolism of provitamin D3 to vitamin D3, the role and significance of the side-chain metabolism of 1,25(OH)2D by the small intestine, and the metabolism of 25-OH-D to 24R,25(OH)2D by chondrocytes and its stimulation of protein synthesis in these cells are just a few issues that will require further investigation.     

Mechanistic studies to evaluate the enhanced antiproliferation of human keratinocytes by 1alpha,25-dihydroxyvitamin D(3)-3-bromoacetate, a covalent modifier of vitamin D receptor, compared to 1alpha,25-dihydroxyvitamin D(3).

1alpha,25-Dihydroxyvitamin D(3)-3-bromoacetate (1, 25(OH)(2)D(3)-3-BE), an affinity labeling analog of 1alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), displayed stronger antiproliferative activities than 1,25(OH)(2)D(3) at 10(-10)-10(-6) M dose levels in cultured human keratinocytes (CHK). Additionally, preincubation of the cells with 10(-6) M 1,25(OH)(2)D(3), followed by treatment with various doses of 1,25(OH)(2)D(3)-3-BE, resulted in a significantly stronger antiproliferative activity by the mixture than individual reagents at every dose level. To search for a mechanism of this observation, we determined that [(14)C]1, 25(OH)(2)D(3)-3-BE covalently labeled human recombinant 1alpha, 25-dihydroxyvitamin D(3) receptor (reVDR) swiftly (<1 min) with a 1:1 stoichiometry and induced conformational changes (in VDR) that are different from 1,25(OH)(2)D(3), by limited tryptic digestion. Furthermore, a protein band, corresponding to reVDR, was specifically labeled by [(14)C]1,25(OH)(2)D(3)-3-BE in CHK extract, indicating that VDR is the main target of [(14)C]1, 25(OH)(2)D(3)-3-BE. The above-mentioned observations suggest that a rapid covalent labeling of VDR in CHK might alter the interaction between the holo-VDR and 1,25(OH)(2)D(3)-controlled genes. Furthermore, we observed that 1,25(OH)(2)D(3)-3-BE significantly decreased the binding of VDR to human osteocalcin vitamin D responsive element (hOCVDRE), as well as the dissociation rate of VDR from hOCVDRE, compared with 1,25(OH)(2)D(3) in COS-1 cells, transiently transfected with a VDR construct. Additionally, 1, 25(OH)(2)D(3)-3-BE was found to be more potent in inducing 1alpha, 25-dihydroxyvitamin D(3)-24-hydroxylase (24-OHase) promoter activity and mRNA expression in keratinocytes. The accumulation of 24-OHase message was also prolonged by the analog. Collectively these results indicated that rapid covalent labeling of VDR in keratinocytes (by 1, 25(OH)(2)D(3)-3-BE) might result in the conversion of apo-VDR to a holo-form, with a conformation that is different from that of the 1, 25(OH)(2)D(3)-VDR complex. This resulted in an enhanced stability of the 1,25(OH)(2)D(3)-3-BE/VDR-VDRE complex and contributed to the amplified antiproliferative effect of 1,25(OH)(2)D(3)-3-BE in keratinocytes.     

Extra-renal 25-hydroxyvitamin D3-1alpha-hydroxylase in human health and disease

Although ectopic expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase) has been recognized for many years, the precise function of this enzyme outside the kidney remains open to debate. Three specific aspects of extra-renal 1alpha-OHase have attracted most attention: (i) expression and regulation in non-classical tissues during normal physiology; (ii) effects on the immune system and inflammatory disease; (iii) expression and function in tumors. The most well-recognized manifestation of extra-renal 1alpha-OHase activity remains that found in some patients with granulomatous diseases where locally synthesized 1alpha,25(OH)(2)D(3) has the potential to spill-over into the general circulation. However, immunohistochemistry and mRNA analyses suggest that 1alpha-OHase is also expressed by a variety of normal human tissues including the gastrointestinal tract, skin, vasculature and placenta. This has promoted the idea that autocrine/paracrine synthesis of 1,25(OH)(2)D(3) contributes to normal physiology, particularly in mediating the potent effects of vitamin D on innate (macrophage) and acquired (dendritic cell) immunity. We have assessed the capacity for synthesis of 1,25(OH)(2)D(3) in these cells and the functional significance of autocrine responses to 1alpha-hydroxylase. Data suggest that local synthesis of 1,25(OH)(2)D(3) may be a preferred mode of response to antigenic challenge in many tissues.