1,25-dihydroxyvitamin D3 receptor is upregulated in aortic smooth muscle cells during hypervitaminosis D

Several studies have demonstrated that excess of vitamin D3 is toxic particularly to vascular tissues. A notable pathological feature is arterial calcification. The nature of the toxic metabolite in hypervitaminosis D and the pathogenesis of arterial calcification are not clearly understood. The present study was undertaken to explore whether arterial calcification is a sequel of increased calcium uptake by arterial smooth muscle mediated by up regulation of vitamin D receptor in the cells in response to elevated circulating levels of vitamin D3 in serum. The experimental study was performed in 20 New Zealand white female rabbits aged 6 months. Animals in the test group were injected 10,000 IU of cholecalciferol intramuscularly twice a week for one month. Six control animals were given intra-muscular injections of plain cottonseed oil. Animals were sacrificed and aortas were examined for pathological lesions, 1,25-dihyroxyvitamin D3 (1,25(OH)2 D3) receptor levels and 45Ca uptake in smooth muscle cells. Serum samples collected at intervals were assayed for levels of 25-OH-D3 and calcium. The results showed that in animals given injections of cholecalciferol, serum levels of 25-OH-D3 were elevated. In four of these animals calcification and aneurysmal changes were seen in the aorta. Histological lesions comprised of fragmentation of elastic fibers as well as extensive loss of elastic layers. 1,25(OH)2 D3 receptor levels were up regulated and 45Ca uptake enhanced in aortas of animals which were given excessive vitamin D3. The evidences gathered suggest that excess vitamin D is arteriotoxic and that the vitamin induces arterial calcification through up regulation of 1,25(OH)2D3 receptor and increased calcium uptake in smooth muscle cells of the arteries.     

Loss of CYP3A7 gene induction by 1,25-dihydroxyvitamin D3 is caused by less binding of VDR to the proximal ER6 in CYP3A7 gene

Cytochrome P450 3A4 and 3A7 (CYP3A4 and CYP3A7, respectively) are predominant forms in the human adult and fetal liver, respectively. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is known to be a potent inducer of CYP3A4 in human colon carcinoma Caco-2 via vitamin D receptor (VDR). However, whether CYP3A7 is inducible by 1,25(OH)(2)D(3) has not yet been elucidated. In the present study, we examined the effect of 1,25(OH)(2)D(3) on CYP3A7 gene expression in Caco-2 cells, which express CYP3A4 and CYP3A7 mRNAs. 1,25(OH)(2)D(3) hardly induced the expression of CYP3A7 mRNA in contrast to the marked induction of CYP3A4 mRNA. Reporter assay using 5'-franking region CYP3A4 and CYP3A7 genes also revealed that 1,25(OH)(2)D(3) activates CYP3A4 promoter, but not CYP3A7 promoter, which has two mutations in the proximal ER6 site compared with CYP3A4 promoter. In addition, we found that the binding of VDR to the proximal ER6 in CYP3A7 gene was markedly less than that to the proximal ER6 in CYP3A4 gene using gel shift assay. Taken together, the decrease of VDR binding to the proximal ER6 caused by the mutation results in the loss of CYP3A7 gene activation by 1,25(OH)(2)D(3).     

The vitamin D receptor is necessary for 1alpha,25-dihydroxyvitamin D(3) to suppress experimental autoimmune encephalomyelitis in mice

The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3), suppresses autoimmune disease in several animal models including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The molecular mechanism of this immunosuppression is at present unknown. While 1alpha,25-dihydroxyvitamin D(3) is believed to function through a single vitamin D receptor, there are reports of other vitamin D receptors as well as a "nongenomic" mode of action. We have prepared the EAE model possessing the vitamin D receptor null mutation and determined if 1alpha,25-dihydroxyvitamin D(3) can suppress this disease in the absence of a functional vitamin D receptor. Vitamin D receptor null mice develop EAE although the incidence rate is one-half that of wild-type controls. The administration of 1alpha,25-dihydroxyvitamin D(3) had no significant effect on the incidence of EAE in the vitamin D receptor null mice, while it completely blocked EAE in the wild-type mice. We conclude that 1alpha,25-dihydroxyvitamin D(3) functions to suppress EAE through the well-known VDR and not through an undiscovered receptor or through a "nongenomic" mechanism.     

Phosphorylation of human vitamin D receptor serine-182 by PKA suppresses 1,25(OH)2D3-dependent transactivation

The human vitamin D receptor (hVDR), which is a substrate for several protein kinases, mediates the actions of its 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ligand to regulate gene expression. To determine the site, and functional impact, of cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of hVDR, we generated a series of C-terminally truncated and point mutant receptors. Incubation of mutant hVDRs with PKA and [gamma-32P]ATP, in vitro, or overexpressing them in COS-7 kidney cells labeled with [32P]orthophosphate, revealed that serine-182 is the predominant residue in hVDR phosphorylated by PKA. An aspartate substituted mutant (S182D), incorporating a negative charge to mimic phosphorylation, displayed only 50% of the transactivation capacity in response to 1,25(OH)2D3 of either wild-type or an S182A-altered hVDR. When the catalytic subunit of PKA was overexpressed, a similar reduction in wild-type but not S182D hVDR transactivity was observed. In a mammalian two-hybrid system, S182D bound less avidly than wild-type or S182A hVDR to the retinoid X receptor (RXR) heterodimeric partner that co-mediates vitamin D responsive element recognition and transactivation. These data suggest that hVDR serine-182 is a primary site for PKA phosphorylation, an event that leads to an attenuation of both RXR heterodimerization and resultant transactivation of 1,25(OH)2D3 target genes.     

Synthesis of 2α-substituted-14-epi-previtamin D3 and its genomic activity

We synthesized and isolated 2α-substituted analogs of 14-epi-previtamin D₃ after thermal isomerization at 80 °C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the 2α-methyl-substituted analog was found to have greater genomic activity than 14-epi-previtamin D₃.     

Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein

Vitamin D-binding protein (DBP), a multi-functional serum glycoprotein, has a triple-domain modular structure. Mutation of Trp145 (in Domain I) to Ser decreased 25-OH-D(3)-binding by 80%. Furthermore, recombinant Domain I (1-203) and Domain I + II (1-330) showed specific and strong binding for 25-OH-D(3), but Domain III (375-427) did not, suggesting that only Domains I and II might be required for vitamin D sterol-binding. Past studies have suggested that Domain III is independently capable of binding G-actin. We exploited this apparently independent ligand-binding property of DBP to purify DBP-actin complex from human serum and rabbit muscle actin by 25-OH-D(3) affinity chromatography. Competitive (3)H-25-OH-D(3) binding curves for native DBP and DBP-actin complex were almost identical, further suggesting that vitamin D sterol- and actin-binding activities by DBP might be largely independent of each other. Trypsin treatment of DBP produced a prominent 25 kDa band (Domain I, minus 5 amino acids in N-terminus), while actin was completely fragmented by such treatment. In contrast, tryptic digestion of purified DBP-actin complex showed two prominent bands, 52 (DBP, minus 5 amino acids in the N-terminus) and 34 kDa (actin, starting with amino acid position 69) indicating that DBP, particularly its Domains II and III were protected from trypsin cleavage upon actin-binding. Similarly, actin, except its N-terminus, was also protected from tryptic digestion when complexed with DBP. These results provided the basis for our studies to crystallize DBP-actin complex, which produced a 2.5 A crystal, primitive orthorhombic with unit cell dimensions a=80.2A, b=87.3A, and c=159.6A, P2(1)2(1)2(1) space group, V(m)=2.9. Soaking of crystals of actin-DBP in crystallization buffer containing various concentrations of 25-OH-D(3) resulted in cracking of the crystal, which was probably a reflection of a ligand-induced conformational change in the complex, disrupting crystal contacts. In conclusion, we have provided data to suggest that although binding of 25-OH-D(3) to DBP might result in discrete conformational changes in the holo-protein to influence actin-binding, these binding processes are largely independent of each other in solution.     

Crystal structure of the vitamin D nuclear receptor ligand binding domain in complex with a locked side chain analog of calcitriol

The crystal structures of vitamin D nuclear receptor (VDR) have revealed that all compounds are anchored by the same residues to the ligand binding pocket (LBP). Based on this observation, a synthetic analog with a locked side chain (21-nor-calcitriol-20(22),23-diyne) has been synthesized in order to gain in entropy energy with a predefined active side chain conformation. The crystal structure of VDR LBD bound to this locked side chain analogue while confirming the docking provides a structural basis for the activity of this compound.     

Measurement and characterization of C-3 epimerization activity toward vitamin D3

Recently, epimerization of the hydroxyl group at C-3 has been identified as a unique metabolic pathway of vitamin D compounds. We measured C-3 epimerization activity in subcellular fractions prepared from cultured cells and investigated the basic properties of the enzyme responsible for the epimerization. C-3 epimerization activity was detected using a NADPH-generating system containing glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and Mg(2+). The highest level of activity was observed in a microsomal fraction prepared from rat osteoblastic UMR-106 cells but activity was also observed in microsomal fractions prepared from MG-63, Caco-2, Hep G2, and HUH-7 cells. In terms of maximum velocity (V(max)) and the Michaelis constant (K(m)), 25-hydroxyvitamin D(3) [25(OH)D(3)] exhibited the highest specificity for the epimerization at C-3 among 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], 25(OH)D(3), 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], and 22-oxacalcitriol (OCT). The epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha-->beta)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D(3) at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.     

Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats

Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D₃ in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D₃ treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D₃ supplementation improved the behavioural deficit. In conclusion, vitamin D₃ shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D₃ modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D₃ supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D₃ and muscarinic M3 receptors in regulating insulin secretion from pancreas.     

Tissue-specific and embryonic expression of the retinoid X receptors in Sebastiscus marmoratus

Retinoid X receptors (RXRs) are highly conserved members of the nuclear receptor family and mediate various physiological processes in vertebrates. Most studies on RXRs have concentrated on their structure and function in mammals and their characterization and developmental expression in Danio rerio. However, there is little information concerning the distribution of RXRs in teleost tissues. In the present study, we cloned partial sequences of three RXR subtypes (RXRa, -b, -g) from Sebastiscus marmoratus by RACE PCR and analyzed the phylogeny of the teleost and the tetrapod RXR genes, and identified some inconsistencies with previous studies. The tissue-specific and embryonic expression profiles of each RXR gene were explored using real time quantitative PCR. This analysis demonstrated that these RXRs were expressed in all test tissues indicating their participation in many physiological processes. However, we found a great difference in the distribution of RXRg between teleosts and mammals. Furthermore, we followed expression of the three subtypes through various embryo developmental stages and found that the RXRa orthologues of teleosts might be involved in the development of the anterior hindbrain, tailbud and neural crest and in the formation of the pharynx and fin, that RXRb played ubiquitous roles in fish early development, and that RXRg probably played a role in brain and nervous system development and function.     

In vivo effects of chronic contamination with depleted uranium on vitamin D3 metabolism in rat

The extensive use of depleted uranium (DU) in today's society results in the increase of the number of human population exposed to this radionuclide. The aim of this work was to investigate in vivo the effects of a chronic exposure to DU on vitamin D₃ metabolism, a hormone essential in mineral and bone homeostasis. The experiments were carried out in rats after a chronic contamination for 9 months by DU through drinking water at 40 mg/L (1 mg/rat/day). This dose corresponds to the double of highest concentration found naturally in Finland. In DU-exposed rats, the active vitamin D (1,25(OH)₂D₃) plasma level was significantly decreased. In kidney, a decreased gene expression was observed for cyp24a1, as well as for vdr and rxrα, the principal regulators of CYP24A1. Similarly, mRNA levels of vitamin D target genes ecac1, cabp-d28k and ncx-1, involved in renal calcium transport were decreased in kidney. In the brain lower levels of messengers were observed for cyp27a1 as well as for lxrβ, involved in its regulation. In conclusion, this study showed for the first time that DU affects both the vitamin D active form (1,25(OH)₂D₃) level and the vitamin D receptor expression, and consequently could modulate the expression of cyp24a1 and vitamin D target genes involved in calcium homeostasis.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.