MicroRNA-31 Negatively Regulates Interleukin-34 Expression In Vitro.

Interleukin-34 (IL-34) is a recently discovered cytokine that promotes tissue macrophage maturation and differentiation. We previously found that 1α,25-Dihydroxyvitamin D3 up-regulated IL-34 expression in SH-SY5Y neural cells. However, whether microRNA regulates IL-34 expression is not completely clear. By using on-line TargetScan and MiRanda software, we found that there was only one conserved microRNA-31 (miR-31) binding site in the 3' untranslated region (3'UTR) of IL-34 mRNA. Intriguingly, using qPCR we demonstrated that miR-31 levels were negatively correlated to IL-34 mRNA levels in different cell lines. By examining the effect of miR-31 on IL-34 3' UTR reporter luciferase activity and on IL-34 mRNA and argonaute RISC catalytic component 2 (AGO2) binding, it was found that miR-31 bound directly to IL-34 3'UTR and regulated the post-transcriptional expression of IL-34 in MGC-803 cells. Moreover, a miR-31 mimic significantly reduced IL-34 expression levels while a miR-31 inhibitor up-regulated IL-34 expression in KYSE-45 and HT-29 cells. Taken together, these results show that miR-31 negatively regulates IL-34 expression by directly binding to the IL-34 3' UTR in vitro.

Vitamin D3 Is Transformed into 1,25(OH)2D3 by Triggering CYP3A11(CYP3A4) Activity and Hydrolyzing Midazolam.

BACKGROUND Vitamin D3 (VD3) is a commonly used supplement in clinical practice. Cytochrome P450 3A11 (CYP3A11) is the most important monomeric enzyme involved in metabolism of drugs. This study aimed to investigate effects of vitamin D3 (VD3) on CYP3A11 activity. MATERIAL AND METHODS Forty male Sprague-Dawley (SD) rats were randomly divided a Control group (peanut oil 0.1 ml/kg/d), a Low-VD3 group (100 IU/kg/d), a Medium-VD3 group (400 IU/kg/d), and a High-VD3 (1600 IU/kg/d) group. Blood samples were collected from the jugular vein after midazolam (MDZ) administration. CYP3A11 expressions in liver and colon were detected by Western blotting and immunohistochemistry (IHC) assay. The concentration of serum 25(OH)D3 and serum 1,25(OH)2D3 were evaluated using ELISA. Effects of different dosages of vitamin D3 on metabolism of MDZ were evaluated using high-performance liquid chromatography (HPLC). RESULTS Vitamin D3 significantly enhanced serum 25(OH)D3 and 1,25(OH)2D3 levels in rats compared to Control rats (p<0.05). Expressions of hepatic CYP3A11 were more than 10-fold higher in rats treated with vitamin D3 compared to Control rats (p<0.05). Expressions of colon CYP3A11 were 5-fold higher than in Control rats (p<0.05). CYP3A11 expressions in vitamin D3-treated groups were significantly higher compared to the Control group (p<0.05). MDZ levels were significantly higher in Vitamin D3-treated rats compared to that in Control rats (p<0.05). Concentrations of serum MDZ at every sampling point were remarkably lower in the vitamin D3-treated rats than in Control rats (p<0.05). CONCLUSIONS Vitamin D3 was transformed into 1,25(OH)2D3 by triggering CYP3A11 and CYP3A11 activity and by hydrolyzing MDZ.

Lithocholic Acid Is a Vitamin D Receptor Ligand That Acts Preferentially in the Ileum.

The vitamin D receptor (VDR) is a nuclear receptor that mediates the biological action of the active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], and regulates calcium and bone metabolism. Lithocholic acid (LCA), which is a secondary bile acid produced by intestinal bacteria, acts as an additional physiological VDR ligand. Despite recent progress, however, the physiological function of the LCA−VDR axis remains unclear. In this study, in order to elucidate the differences in VDR action induced by 1,25(OH)2D3 and LCA, we compared their effect on the VDR target gene induction in the intestine of mice. While the oral administration of 1,25(OH)2D3 induced the Cyp24a1 expression effectively in the duodenum and jejunum, the LCA increased target gene expression in the ileum as effectively as 1,25(OH)2D3. 1,25(OH)2D3, but not LCA, increased the expression of the calcium transporter gene Trpv6 in the upper intestine, and increased the plasma calcium levels. Although LCA could induce an ileal Cyp24a1 expression as well as 1,25(OH)2D3, the oral LCA administration was not effective in the VDR target gene induction in the kidney. No effect of LCA on the ileal Cyp24a1 expression was observed in the VDR-null mice. Thus, the results indicate that LCA is a selective VDR ligand acting in the lower intestine, particularly the ileum. LCA may be a signaling molecule, which links intestinal bacteria and host VDR function.

Lysophosphatidic acid signaling promotes proliferation, differentiation, and cell survival in rat growth plate chondrocytes.

Growth plate cartilage is responsible for long bone growth in children and adolescents and is regulated by vitamin D metabolites in a cell zone-specific manner. Resting zone chondrocytes (RC cells) are regulated by 24,25-dihydroxyvitamin D3 via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH)2D3 stimulates rat costochondral RC chondrocytes to release lysophosphatidic acid (LPA) and, therefore sought to determine the role of LPA signaling in these cells. RC cells expressed the G-protein coupled receptors LPA1-5 and peroxisome proliferator-activated receptor gamma (PPAR-gamma). LPA and the LPA1/3 selective agonist OMPT increased proliferation and two maturation markers, alkaline phosphatase activity and [35S]-sulfate incorporation. LPA and 24R,25(OH)2D3's effects were inhibited by the LPA1/3 selective antagonist VPC32183(S). Furthermore, apoptosis induced by either inorganic phosphate or chelerythrine was attenuated by LPA, based on DNA fragmentation, TUNEL staining, caspase-3 activity, and Bcl-2:Bax protein ratio. LPA prevented apoptotic signaling by decreasing the abundance, nuclear localization, and transcriptional activity of the tumor-suppressor p53. LPA treatment also regulated the expression of the p53-target genes Bcl-2 and Bax to enhance cell survival. Collectively, these data suggest that LPA promotes differentiation and survival in RC chondrocytes, demonstrating a novel physiological function of LPA-signaling.

A kinetic overview of the receptors involved in 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 signaling: a systems biology approach.

The vitamin D endocrine system modulates an arsenal of important biological functions in more than 30 different tissues in short- and long-term perspectives. Two membrane receptors and one nuclear receptor are suggested to be involved in the vitamin D signaling system, but the function and physiological relevance of the receptors are debated. The complexity of the vitamin D endocrine system makes it necessary to combine experimental data with in silico simulations to get a holistic view of vitamin D-dependent regulation of tissue and cell physiology. This review focus on binding characteristics for the three putative vitamin D receptors and proposes a future systems biology approach including mathematical modeling that will be helpful together with experimental methods in depicting antitumoral and other biological effects promoted by the vitamin D endocrine system.

24R,25-dihydroxyvitamin D3 modulates tumorigenicity in breast cancer in an estrogen receptor-dependent manner.

Vitamin D has long been prescribed as a supplement to breast cancer patients. This is partially motivated by data indicating that low serum vitamin D, measured as 25-hydroxyvitamin D3 [25(OH)D3], is associated with worsened cancer prognosis and decreased survival rates in cancer patients. However, clinical studies investigating the role of vitamin D supplementation in breast cancer treatment are largely inconclusive. One reason for this may be that many of these studies ignore the complexity of the vitamin D metabolome and the effects of these metabolites at the cellular level. Once ingested, vitamin D is metabolized into 37 different metabolites, including 25(OH)D3, which is the metabolite actually measured clinically, as well as 1,25(OH)2D3 and 24,25(OH)2D3. Recent work by our lab and others has demonstrated a role for 24R,25(OH)2D3, in the modulation of breast cancer tumors via an estrogen receptor α-dependent mechanism. This review highlights the importance of considering estrogen receptor status in vitamin d-associated prognostic studies of breast cancer and proposes a potential mechanism for 24R,25(OH)2D3 signaling in breast cancer cells.

24R,25-Dihydroxyvitamin D3 regulates breast cancer cells in vitro and in vivo.

BACKGROUND: Epidemiological studies indicate high serum 25(OH)D3 is associated with increased survival in breast cancer patients. Pre-clinical studies attributed this to anti-tumorigenic properties of its metabolite 1α,25(OH)2D3. However, 1α,25(OH)2D3 is highly calcemic and thus has a narrow therapeutic window. Here we propose another metabolite, 24R,25(OH)2D3, as an alternative non-calcemic vitamin D3 supplement. METHODS: NOD-SCID-IL2γR null female mice with MCF7 breast cancer xenografts in the mammary fat pad were treated with 24R,25(OH)2D3 and changes in tumor burden and metastases were assessed. ERα66+ MCF7 and T47D cells, and ERα66- HCC38 cells were treated with 24R,25(OH)2D3in vitro to assess effects on proliferation and apoptosis. Effects on migration and metastatic markers were assessed in MCF7. RESULTS: 24R,25(OH)2D3 reduced MCF7 tumor growth and metastasis in vivo. In vitro results indicate that this was not due to an anti-proliferative effect; 24R,25(OH)2D3 stimulated DNA synthesis in MCF7 and T47D. In contrast, markers of invasion and metastasis were decreased. 24R,25(OH)2D3 caused dose-dependent increases in apoptosis in MCF7 and T47D, but not HCC38 cells. Inhibitors to palmitoylation, caveolae integrity, phospholipase-D, and estrogen receptors (ER) demonstrate that 24R,25(OH)2D3 acts on MCF7 cells through caveolae-associated, phospholipase D-dependent mechanisms via cross-talk with ERs. CONCLUSION: These results indicate that 24R,25(OH)2D3 shows promise in treatment of breast cancer by stimulating tumor apoptosis and reducing metastasis. GENERAL SIGNIFICANCE: 24R,25(OH)2D3 regulates breast cancer cell survival through ER-associated mechanisms similar to 24R,25(OH)2D3 effects on chondrocytes. Thus, 24R,25(OH)2D3 may modulate cell survival in other estrogen-responsive cell types, and its therapeutic potential should be investigated in ER-associated pathologies.

Intake of ergosterol increases the vitamin D concentrations in serum and liver of mice.

Factors that can modify the bioavailability of orally administered vitamin D are not yet widely known. Ergosterol is a common fungal sterol found in food which has a chemical structure comparable to that of vitamin D. This study aimed to investigate the effect of ergosterol on vitamin D metabolism. Therefore, 36 male wild type-mice were randomly subdivided into three groups (n = 12) and received a diet containing 25 µg vitamin D3 and either 0 mg (control), 2 mg or 7 mg ergosterol per kg diet for 6 weeks. To elucidate the impact of ergosterol on hepatic hydroxylation of vitamin D, human hepatoma cells (HepG2) were treated with different concentrations of ergosterol. Concentrations of vitamin D3 and 25-hydroxyvitamin D3 (25(OH)D3) in cells, livers and kidneys of mice and additionally 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in serum were quantified by LC-MS/MS. The concentration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in serum was analyzed by commercially-available enzyme immuno assay. The concentrations of cholesterol and triglycerides were analyzed in livers of mice by photometric assays. Analyses revealed that mice receiving 7 mg/kg ergosterol with their diet had 1.3-, 1.7- and 1.5-times higher concentrations of vitamin D3 in serum, liver and kidney, respectively, than control mice (P < 0.05), whereas no significant effects were observed in mice fed 2 mg/kg ergosterol. The hydroxylation of vitamin D remained unaffected by dietary ergosterol, since the concentration of 25-hydroxyvitamin D3 in serum and tissues and the concentrations of 1,25(OH)2D3 and 24,25(OH)2D3 in serum were not different between the three groups of mice. The lipid concentrations in liver were also not affected by dietary ergosterol. Data from the cell culture studies showed that ergosterol did not influence the conversion of vitamin D3 to 25(OH)D3. To conclude, ergosterol appears to be a modulator of vitamin D3 concentrations in the body of mice, without modulating the hydroxylation of vitamin D3 in liver.

Regulation of vitamin D metabolism following disruption of the microbiota using broad spectrum antibiotics.

Vitamin D, 25hydroxyvitamin D (25D), and 24,25dihydroxyvitamin D (24,25D) were measured before and after broad spectrum antibiotic (Abx) treatment for 2 wks. Abx treatments increased 25D and 24,25D levels suggesting that the microbiota or Abx were altering vitamin D metabolism. Increased 25D, but not 24,25D, following Abx treatments were found to be dependent on toll like receptor signaling. Conversely, the effects of Abx on 24,25D levels required that the vitamin D receptor (VDR) be expressed in tissues outside of the hematopoietic system (kidney) and not the immune system. Fibroblast growth factor (FGF)23 increased following Abx treatment and the effect of Abx treatment on FGF23 (like the effect on 24,25D) was not present in VDR knockout (KO) mice. The Abx mediated increase in 24,25D was due to changes to the endocrine regulation of vitamin D metabolism. Conversely, 25D levels went up with Abx treatment of the VDR KO mice. Host sensing of microbial signals regulates the levels of 25D in the host.

Membrane receptors for vitamin D metabolites.

Membrane-initiated signaling by steroid hormones is now widely accepted. Current debate is centered upon which protein moieties act as membrane-associated receptors. In this review, we consider evidence for the classical vitamin D receptor (VDR) in this role, as well as the more recently identified 1,25D3-MARRS (membrane-associated, rapid response steroid binding) receptor, also known as ERp57/GRp58. The structure of the 1,25D3-MARRS receptor is discussed, with emphasis on two thioredoxin domains that promote dimerization and ligand binding. We then summarize recent studies on a 24,25(OH)2D3 binding protein--catalase--and how ligand-induced decreases in enzymatic activity produce increased reactive oxygen species that target both the 1,25D3-MARRS receptor--but not the VDR--and the protein kinase C signaling pathway. Finally, we briefly discuss the available literature suggesting that the metabolite 25(OH)D3 may also be biologically active.

Placental vitamin D metabolism and its associations with circulating vitamin D metabolites in pregnant women.

Background: Little is known about placental vitamin D metabolism and its impact on maternal circulating vitamin D concentrations in humans.Objective: This study sought to advance the current understanding of placental vitamin D metabolism and its role in modulating maternal circulating vitamin D metabolites during pregnancy.Design: Nested within a feeding study, 24 healthy pregnant women (26-29 wk of gestation) consumed a single amount of vitamin D (511 IU/d from diet and a cholecalciferol supplement) for 10 wk. Concentrations of placental and blood vitamin D metabolites and placental messenger RNA (mRNA) abundance of vitamin D metabolic pathway components were quantified. In addition, cultured human trophoblasts were incubated with 13C-cholecalciferol to examine the intracellular generation and secretion of vitamin D metabolites along with the regulation of target genes.Results: In placental tissue, 25-hydroxyvitamin D3 [25(OH)D3] was strongly correlated (r = 0.83, P < 0.001) with 24,25-dihydroxyvitamin D3 Moreover, these placental metabolites were strongly correlated (r ≤ 0.85, P ≤ 0.04) with their respective metabolites in maternal circulation. Positive associations (P ≤ 0.045) were also observed between placental mRNA abundance of vitamin D metabolic components and circulating vitamin D metabolites [i.e., LDL-related protein 2 (LRP2, also known as megalin) with 25(OH)D3 and the C3 epimer of 25(OH)D3 [3-epi-25(OH)D3]; cubilin (CUBN) with 25(OH)D3; 25-hydroxylase (CYP2R1) with 3-epi-25(OH)D3; 24-hydroxylase (CYP24A1) with 25(OH)D3, 3-epi-25(OH)D3, and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]; and 1α-hydroxylase [(CYP27B1) with 3-epi-25(OH)D3 and 1,25(OH)2D3]. Notably, in vitro experiments with trophoblasts showed increased production and secretion of 25(OH)D3 and higher CYP24A1 gene transcript abundance in response to cholecalciferol treatment.Conclusions: The numerous associations of many of the placental biomarkers of vitamin D metabolism with circulating vitamin D metabolites among pregnant women [including a CYP27B1-associated increase in 1,25(OH)2D3] and the evidence of trophoblast production and secretion of vitamin D metabolites, especially 25(OH)D3, suggest that the placenta may play an active role in modulating the vitamin D metabolite profile in maternal circulation in human pregnancy. This trial was registered at clinicaltrials.gov as NCT03051867.

Role of dihydroxyvitamin D(3) and its nuclear receptor in novel directed therapies for cancer.

Dihydroxyvitamin D(3) is known to affect broad spectrum of various biochemical and molecular biological reactions in organisms. Research on the role and function of nuclear vitamin D(3) receptors (VDR) playing a role as dihydroxyvitamin D(3) inducible transcription factor belongs to dynamically developing branches of molecular endocrinology. In higher organisms, full functionality of VDR in the form of heterodimer with nuclear 9-cis retinoic acid receptor is essential for biological effects of dihydroxyvitamin D(3). This article summarizes selected effects of biologically active vitamin D(3) acting through their cognate nuclear receptors, and also its potential use in therapy and prevention of various types of cancer.

25-Hydroxyvitamin D assays: Potential interference from other circulating vitamin D metabolites.

The vitamin D External Quality Assessment Scheme (DEQAS) for 25-hydroxyvitamin D (25-OHD) has approximately 1100 participants in 53 countries using 26 different methods or variants of methods (October 2014). In April 2015, the scheme was extended to cover 24,25-dihydroxyvitamin D (24,25(OH)2D). Since 2013, the 25-OHD scheme has been accuracy-based with values assigned by the NIST reference measurement procedure (RMP). DEQAS is uniquely placed to assess the accuracy (bias) and specificity of 25-OHD methods in a routine laboratory setting. Other vitamin D metabolites are known to interfere in 25-OHD assays and DEQAS has distributed samples spiked with 3-epi-25-OHD3 (52.4nmol/L), 24R,25(OH)2D3 (14.4nmol/L) and 24S,25(OH)2D3 (57.9nmol/L). The 3-epimer showed a cross reactivity of 56% in a competitive protein binding assay but was not detected in any antibody-based methods. Not all HPLC/UV or LC-MS/MS methods were able to resolve 3-epi-25-OHD3 from 25-OHD3 and thus overestimated total 25-OHD. The cross reactivity of 24R,25(OH)2D3 (24S,25(OH)2D3) ranged from <5% (<5%) to 548% (643%) in ligand binding assays. Both 24-hydroxylated metabolites were resolved by HPLC/UV and LC-MS/MS methods and thus caused no complications in the measurement of 25-OHD. Most antibodies to 25-OHD are known to cross-react with dihydroxylated metabolites but interference in some assays was far greater than expected. This may be related to the anomalous behaviour of exogenously added metabolites in these 25-OHD methods.

Pro-inflammatory signaling by 24,25-dihydroxyvitamin D3 in HepG2 cells.

The vitamin D metabolite 24,25-dihydroxyvitamin D3 (24, 25[OH]2D3) was shown to induce nongenomic signaling pathways in resting zone chondrocytes and other cells involved in bone remodeling. Recently, our laboratory demonstrated that 24,25-[OH]2D3 but not 25-hydroxyvitamin D3, suppresses apolipoprotein A-I (apo A-I) gene expression and high-density lipoprotein (HDL) secretion in hepatocytes. Since 24,25-[OH]2D3 has low affinity for the vitamin D receptor (VDR) and little is known with regard to how 24,25-[OH]2D3 modulates nongenomic signaling in hepatocytes, we investigated the capacity of 24,25-[OH]2D3 to activate various signaling pathways relevant to apo A-I synthesis in HepG2 cells. Treatment with 24,25-[OH]2D3 resulted in decreased peroxisome proliferator-activated receptor alpha (PPARα) expression and retinoid-X-receptor alpha (RXRα) expression. Similarly, treatment of hepatocytes with 50 nM 24,25-[OH]2D3 for 1-3 h induced PKCα activation as well as c-jun-N-terminal kinase 1 (JNK1) activity and extracellular-regulated kinase 1/2 (ERK1/2) activity. These changes in kinase activity correlated with changes in c-jun phosphorylation, an increase in AP-1-dependent transcriptional activity, as well as repression of apo A-I promoter activity. Furthermore, treatment with 24,25-[OH]2D3 increased IL-1ß, IL-6, and IL-8 expression by HepG2 cells. These observations suggest that 24,25-[OH]2D3 elicits several novel rapid nongenomic-mediated pro-inflammatory protein kinases targeting AP1 activity, increasing pro-inflammatory cytokine expression, potentially impacting lipid metabolism and hepatic function.

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.

In vivo metabolism of 24R,25-dihydroxyvitamin D3: structure of its major bile metabolite.

In vivo metabolism of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) in female dogs has been studied thoroughly, and its major bile metabolite identified. After single oral administration of 24,25-(OH)2 [6,19,19-3H]D3 the plasma concentrations of radioactive metabolites were monitored for 504 h, and the metabolites in the bile collected and analyzed. The concentration of 24,25-(OH)2D3 in plasma reached a maximum after 6 h and decayed in two distinct phases; a fast-phase with a half-life of 17 h, followed by a slow-phase with a 17-day half-life. The area under the concentration/time curve (AUC) was 78-84% (0-504 h). The only detectable metabolite in the plasma was 25-hydroxy-24-oxovitamin D3 whose AUC was less than 5%. At 504 h, about 50% of administered radioactivity has been excreted, of which about 90% was found in the feces, indicating most of the administered 24,25-(OH)2D3 to be excreted in bile. A major metabolite, which constituted 23% of the total bile radioactivity at 504 h, was found in the bile. This metabolite was efficiently deconjugated by beta-glucuronidase to afford an aglycone which was identified as 23S,25-dihydroxy-24-oxovitamin D3 (23S,25-(OH)2-24-oxo-D3), by co-chromatography on HPLC with synthetic standards. The glucuronide was isolated from the bile of dogs given large doses of 24,25-(OH)2D3, and the structure determined being 23-(beta-glucuronide) of 23S,25-(OH)2-24-oxo-D3, by analyzing its negative ion mass spectrum and the positive ion mass spectrum of its derivatives. Thus it was concluded that, in dogs, 24,25-(OH)2D3 is a long lasting vitamin D metabolite, is mainly excreted in bile when metabolized to 23S,25-(OH)2-24-oxo-D3 and is conjugated at 23-OH as glucuronide.

Regulation of arachidonic acid turnover by 1,25-(OH)2D3 and 24,25-(OH)2D3 in growth zone and resting zone chondrocyte cultures.

Previous studies have shown that phospholipase A2 activity in rat costochondral chondrocyte cultures is differentially regulated by 1,25-(OH)2D3 and 24,25-(OH)2D3. 1,25-(OH)2D3 stimulates enzyme activity in growth zone chondrocytes but has no effect on the resting zone chondrocyte enzyme activity. 24,25-(OH)2D3 inhibits the resting zone enzyme but has no effect on the growth zone chondrocyte phospholipase A2. This study examined whether the metabolites affect arachidonic acid turnover in their target cell populations. Incorporation and release of [14C]arachidonate was measured at various times following addition of hormone to the cultures. Acylation and reacylation were measured independently by incubating half of the [14C]arachidonate-labeled cultures with p-chloromercuribenzoate. The results demonstrated that the distribution of [14C]arachidonate in membrane phospholipids differed between growth zone and resting zone chondrocytes and between the plasma membranes and matrix vesicles isolated from the growth zone chondrocyte cultures. Plasma membrane phospholipids were more susceptible to the release of [14C]arachidonic acid by exogenous phospholipases than were matrix vesicle phospholipids. The effect of 1,25-(OH)2D3 on growth zone chondrocytes was observed within 5 min. Incorporation was greatest after 60 min; release was greatest after 30 min. 24,25-(OH)2D3 stimulated consistently elevated incorporation throughout the incubation period, peaking at 15 min. Peak release was at 60 min. The results confirm that resting zone chondrocytes and growth zone chondrocytes retain a differential phenotype in culture and demonstrate that matrix vesicles are distinct from the plasma membrane in terms of lipid composition and arachidonic acid incorporation. 1,25-(OH)2D3 and 24,25-(OH)2D3 appear to stimulate arachidonic acid turnover in their target cells by different mechanisms. Changes in fatty acid acylation and reacylation may be one mode of vitamin D-3 action in cartilage.

Vitamin D receptor expression, 24-hydroxylase activity, and inhibition of growth by 1alpha,25-dihydroxyvitamin D3 in seven human prostatic carcinoma cell lines.

Although prostatic cancer is often viewed as an androgen-dependent malignancy, a number of other hormones including 1alpha, 25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] are now recognized to modulate its growth and differentiated phenotype. Seven different continuous human prostatic carcinoma cell lines were examined for the presence of biologically active receptors for 1alpha,25(OH)2D3. All seven lines were found to contain mRNA for the vitamin D receptor using an RNase protection assay. Six of the seven cell lines were found to have high-affinity saturable binding sites for 1alpha,25(OH)2D3. The seventh line was found to contain vitamin D receptors by sucrose gradient analysis. All seven lines were found to express 24-hydroxylase activity by a HPLC assay that measures the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3. 24-Hydroxylase activity was up-regulated in all seven cell lines by preincubation with 1alpha,25(OH)2D3. In the presence of fetal bovine serum, the growth of four of the seven cell lines was inhibited. In the majority of cell lines growth inhibition was related not only to the number of receptors per cell, but also in inverse proportion to the 24-hydroxylase activity of each cell line. The ubiquitous presence of vitamin D receptor and 24-hydroxylase activity in human prostatic carcinoma cells suggests new alternatives for the pharmacological treatment of advanced prostatic cancer and implies that chemoprevention strategies could also make use of this endocrine axis.

Regulation of 25-hydroxyvitamin D3 metabolism in cultures of osteoblastic cells.

This study was designed to investigate the mechanisms involved in the regulation of the conversion of 25-hydroxyvitamin D3 (25-OHD3) to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] in primary cultures of osteoblastlike cells from neonatal mouse calvariae. These cells, when incubated with tritiated 25-OHD3 ([3H]25-OHD3), spontaneously synthesized [3H]24,25-(OH)2D3 20-50 times more efficiently than [3H]1,25-(OH)2D3 at a rate of conversion that was substrate dependent and linear from 1 to 36 h. Gas chromatography-mass spectrometry verified the identity of the dihydroxylated metabolites. The calcium ionophore A23187 (5 microM) consistently stimulated the synthesis of 1,25-(OH)2D3 while suppressing the production of 24,25-(OH)2D3. This effect was sustained for 36 h and was dose dependent for concentrations from 0.05 to 10 microM. Furthermore, A23187 stimulated cAMP production and indomethacin (50 ng/ml) blocked the A23187-induced production of cAMP and 1,25-(OH)2D3 but had no effect on the suppression of 24,25-(OH)2D3 by A23187. This led to other experiments to find out whether the stimulative effect of A23187 on 1,25-(OH)2D3 synthesis is mediated by prostaglandins or cAMP, or both. PGE2 (10(-8)-10(-6) M) increased the production of 1,25-(OH)2D3 and of 24,25-(OH)2D3. Forskolin (0.01-10 microM) and dibutyryl cAMP (0.1-10 mM) increased the production of both metabolites but to a lesser degree than PGE2. These data suggest that osteoblastlike cells are stimulated by A23187 to increase the synthesis of 1,25-(OH)2D3 through mechanisms involving prostaglandins and cAMP. The synthesis of 24,25-(OH)2D3 is suppressed by A23187 through different mechanisms.

Novel mutations in the 1alpha-hydroxylase (P450c1) gene in three families with pseudovitamin D-deficiency rickets resulting in loss of functional enzyme activity in blood-derived macrophages.

Pseudovitamin D-defiency rickets (PDDR) is an autosomal recessive disorder characterized by hypocalcemia, rickets (which are resistant to treatment with vitamin D), and low or undetectable serum levels of 1,25-dihydroxyvitamin D (1,25(OH)2D). The symptoms are corrected with 1,25(OH)2D treatment, and the disease is now believed to result from a defect in the cytochrome P450 component (P450c1; CYP27B1) of the renal 25-hydroxyvitamin D-1alpha-hydroxylase (1-OHase). We have studied genomic DNA from three families with PDDR and have identified the same homozygous mutation in the P450c1 gene in two of the index cases, causing a frameshift in exon 8, resulting in a premature stop codon in the heme-binding domain. The two cases in the third kindred were compound heterozygotes with missense mutations in exons 6 and 9. We have also identified a C/T polymorphism in intron 6 of the P450c1 genomic DNA. Interferon gamma-inducible 1-OHase activity in blood-derived macrophages was shown by 1,25(OH)2D synthesis in all control cells tested (37-184 fmol/h/106 cells) and those from the PDDR family parents (34-116 fmol/h/106 cells) but was totally absent from the patients' cells, indicating a defect in their macrophage 1-OHase, similar to the presumed renal defect. The assumption of similarity between the renal and macrophage P450c1 was supported by our ability to clone a 514 bp sequence, including the heme-binding region of the macrophage P450c1 cDNA from controls, which was identical to that published for both the renal and keratinocyte P450c1 cDNAs.