1 alpha,25(OH)2 vitamin D3: a steroid hormone capable of producing pleiotropic receptor-mediated biological responses by both genomic and nongenomic mechanisms

Vitamin D3 is now considered a pro-hormone and its biological activities are in a great majority due to its hormonally active derivative 1 alpha,25-dihydroxyvitamin D3. The actions of vitamin D3 have been extensively studied both in vivo and in vitro using different animal models as well as a diversity of cell types including intestinal epithelial cells, kidney cells, lymphocytes, macrophages and a number of malignant cell lines. Results thus obtained have considerably extended the number of target tissues for this vitamin and hormone far beyond its classical target organs, parathyroid gland, kidney, intestine and bone. Studies performed at the cellular, subcellular and molecular levels have led to the establishment of new concepts on the mode of action of vitamin D3 which is not believed to act through receptor-mediated genomic and nongenomic events leading to the expression of the full biological response.     

1alpha,25(OH)2 vitamin D3 actions on ion channels in osteoblasts

Membrane-initiated cellular responses to steroids include modulation of ion channel activities via signal transduction pathways. However, the molecular mechanisms involved in nongenomic actions remain only partially understood. Our research has focused on the rapid effects of 1alpha,25(OH)(2) Vitamin D(3) [1,25D] on L-type Ca(2+) [L-Ca] and DIDS-sensitive Cl(-) channels in osteoblasts. Physiological nanomolar concentrations of hormonally active 1,25D promote rapid (1-5 min) potentiation of outward Cl(-) currents in osteosarcoma ROS 17/2.8 cells and mouse primary osteoblasts. In addition, 1,25D increases inward barium currents through L-Ca channels at low depolarizing potentials within seconds in a fashion similar to the 1,4-dihydropyridine [DHP] agonist Bay K8644. We found that second messenger cAMP is involved in 1,25D potentiation of Cl(-) and Ca(2+) channels. Nongenomic 1,25D effects on ion channel activities in osteoblasts appear to involve different mechanisms that include a possible direct interaction with the L-Ca channel molecule, on one hand, and signaling through the cAMP pathway, on the other. Rapid 1,25D actions on Cl(-) and Ca(2+) currents seem to couple to secretory activities in osteoblasts, thus contributing to bone mass formation.     

Mechanisms regulating differential activation of membrane-mediated signaling by 1alpha,25(OH)2D3 and 24R,25(OH)2D3

Vitamin D metabolites 1alpha,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) regulate endochondral ossification in a cell maturation-dependent manner via membrane-mediated mechanisms. 24R,25(OH)(2)D(3) stimulates PKC activity in chondrocytes from the growth plate resting zone, whereas 1alpha,25(OH)(2)D(3) stimulates PKC in growth zone chondrocytes. We used the rat costochondral growth plate cartilage cell model to study how these responses are differentially regulated. 1alpha,25(OH)(2)D(3) acts on PKC, MAP kinase, and downstream physiological responses via phosphatidylinositol-specific PLC-beta; 24R,25(OH)(2)D(3) acts via PLD. In both cases, diacylglycerol (DAG) is increased, activating PKC. Both cell types possess membrane and nuclear receptors for 1alpha,25(OH)(2)D(3), but the mechanisms that render the 1alpha,25(OH)(2)D(3) pathway silent in resting zone cells or the 24R,25(OH)(2)D(3) pathway silent in growth zone cells are unclear. PLA(2) is pivotal in this process. 1alpha,25(OH)(2)D(3) stimulates PLA(2) activity in growth zone cells and 24R,25(OH)(2)D(3) inhibits PLA(2) activity in resting zone cells. Both processes result in PKC activation. To understand how negative regulation of PLA(2) results in increased PKC activity in resting zone cells, we used PLA(2) activating peptide to stimulate PLA(2) activity and examined cell response. PLAP is not expressed in resting zone cells in vivo, supporting the hypothesis that PLA(2) activation is inhibitory to 24R,25(OH)(2)D(3) action in these cells.     

Novel vitamin D3 antipsoriatic antedrugs: 16-En-22-oxa-1alpha,25-(OH)2D3 analogs

A series of 16-en-22-oxa-derivatives of vitamin D3 based on the structure of maxacalcitol (2) were prepared. Maxacalcitol is currently used topically for the treatment of psoriasis and is recognized as the most successful antedrug of natural vitamin D(3) because it retains the original antiproliferative activity of calcitriol without increased calcemic activity. We introduced 16-olefinic functionality to accelerate the oxidative metabolism of the drug in liver, presumed to be essential for the reduction of calcemic activity, and modified the side-chain moiety by placing the 22-oxygen on the more labile allylic carbon center. Novel 22-oxa analogs (7a-i), carrying either the 24-alkynyl bond or 24-hydroxy functionality in addition to the 16-double bond were synthesized and their pharmacokinetics were evaluated.     

Chemical synthesis and biological activity of 3-deoxy-1alpha-hydroxyvitamin D3 an analog of 1alpha,25-(OH)2-D3, the active form of vitamin D3

The chemical synthesis of 3-deoxy-1α-hydroxyvitamin D₃ from cholesterol is described. This steroid is a highly important analog of the hormonally active form of vitamin D, 1α, 25-dihydroxyvitamin D₃; it is the only analog presently available for structure-function studies which lacks the 3β-hydroxyl but retains the key 1α-hydroxyl of 1α, 25-dihydroxyvitamin D₃. The new steroid is highly biologically active; it stimulated intestinal calcium absorption significantly more rapidly than vitamin D₃ and as rapidly as 1α, 25-dihydroxyvitamin D₃.     

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.     

1Alpha,25(OH)2D3-induced transrepression by vitamin D receptor through E-box-type elements in the human parathyroid hormone gene promoter

Although transactivation by the liganded vitamin D receptor (VDR) is well described at the molecular level, the precise molecular mechanism of negative regulation by the liganded VDR remains to be elucidated. We have previously reported a novel class of negative vitamin D response element (nVDRE) called 1alphanVDRE in the human 25(OH)D31alpha-hydroxylase [1alpha(OH)ase] gene by 1alpha,25(OH)2D3-bound VDR. This element was composed of two E-box-type motifs that bound to VDIR for transactivation, which was attenuated by liganded VDR. Here, we explore the possible functions of VDIR and E-box motifs in the human (h) PTH and hPTHrP gene promoters. Functional mapping of the hPTH and hPTHrP promoters identified E-box-type elements acting as nVDREs in both the hPTH promoter (hPTHnVDRE; -87 to -60 bp) and in the hPTHrP promoter (hPTHrPnVDRE; -850 to -600 bp; -463 to -104 bp) in a mouse renal tubule cell line. The hPTHnVDRE alone was enough to direct ligand-induced transrepression mediated through VDR/retinoid X receptor and VDIR. Direct DNA binding of hPTHnVDRE to VDIR, but not VDR/retinoid X receptor, was observed and ligand-induced transrepression was coupled with recruitment of VDR and histone deacetylase 2 (HDAC2) to the hPTH promoter. These results suggest that negative regulation of the hPTH gene by liganded VDR is mediated by VDIR directly binding to the E-box-type nVDRE at the promoter, together with recruitment of an HDAC corepressor for ligand-induced transrepression.     

TRPC3-like protein and vitamin D receptor mediate 1alpha,25(OH)2D3-induced SOC influx in muscle cells

1alpha,25-Dihydroxy-Vitamin-D3 (1alpha,25(OH)2-Vitamin D3) stimulates in skeletal muscle cells Ca2+ release from inner stores and influx through both voltage-dependent and store-operated Ca2+ (SOC, CCE) channels. We investigated the involvement of TRPC proteins and Vitamin D receptor (VDR) in CCE induced by 1alpha,25(OH)2D3 in chick muscle cells. Two fragments were amplified by RT-PCR, exhibiting approximately 80% sequence homology with mammalian TRPC3/6/7. Northern and Western blots employing a TRPC3-probe and anti-TRPC3 antibodies, respectively, confirmed endogenous expression of a TRPC3-like protein of 140 kDa. Spectrofluorimetric measurements in Fura-2 loaded cells showed reduced CCE and Mn2+ entry in response to either thapsigargin or 1alpha,25(OH)2D3 upon transfection with anti-TRPC3/6/7 antisense oligodeoxynucleotides (ODNs). Transfection with anti-VDR antisense ODNs diminished 1alpha,25(OH)2D3-dependent Ca2+ and Mn2+ influx. Co-immunoprecipitation of TRPC3-like protein and VDR under non-denaturating conditions was observed. We propose that endogenous TRPC3-like proteins and the VDR participate in the modulation of CCE by 1alpha,25(OH)2D3 in muscle cells, which could be mediated by an interaction between these proteins.     

Natural metabolites of 1alpha,25-dihydroxyvitamin D(3) retain biologic activity mediated through the vitamin D receptor

1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, mediates many of its effects through the intranuclear vitamin D receptor (VDR, NR1I1), that belongs to the large superfamily of nuclear receptors. Vitamin D receptor can directly regulate gene expression by binding to vitamin D response elements (VDREs) located in promoter or enhancer regions of various genes. Although numerous synthetic analogs of 1alpha,25(OH)(2)D(3) have been analysed for VDR binding and transactivation of VDRE-driven gene expression, the biologic activity of many naturally occurring metabolites has not yet been analyzed in detail. We therefore studied the transactivation properties of 1alpha,24R, 25-trihydroxyvitamin D(3) (1alpha,24R,25(OH)(3)D(3)), 1alpha, 25-dihydroxy-3-epi-vitamin D(3) (1alpha,25(OH)(2)-3-epi-D(3)), 1alpha,23S,25-trihydroxyvitamin D(3) (1alpha,23S,25(OH)(3)D(3)), and 1alpha-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D(3) (1alpha(OH)-24,25,26,27-tetranor-23-COOH-D(3); calcitroic acid) using the human G-361 melanoma cell line. Cells were cotransfected with a VDR expression plasmid and luciferase reporter gene constructs driven by two copies of the VDRE of either the mouse osteopontin promoter or the 1alpha,25(OH)(2)D(3) 24-hydroxylase (CYP24) promoter. Treatment with 1alpha,25(OH)(2)D(3) or the metabolites 1alpha,24R,25(OH)(3)D(3), 1alpha,25(OH)(2)-3-epi-D(3), and 1alpha,23S,25(OH)(3)D(3) resulted in transactivation of both constructs in a time- and dose-dependent manner, and a postitive regulatory effect was observed even for calcitroic acid in the presence of overexpressed VDR. The metabolites that were active in the reporter gene assay also induced expression of CYP24 mRNA in the human keratinocyte cell line HaCaT, although with less potency than the parent hormone. A ligand-binding assay based on nuclear extracts from COS-1 cells overexpressing human VDR demonstrated that the metabolites, although active in the reporter gene assay, were much less effective in displacing [(3)H]-labeled 1alpha,25(OH)(2)D(3) from VDR than the parent hormone. Thus, we report that several natural metabolites of 1alpha,25(OH)(2)D(3) retain significant biologic activity mediated through VDR despite their apparent low affinity for VDR.     

Chronic 1alpha,25-(OH)2 vitamin D3 treatment reduces Ca2+ -mediated hippocampal biomarkers of aging

Aging in the hippocampus of several species is characterized by alterations in multiple Ca(2+)-mediated processes, including an increase in L-type voltage-gated Ca(2+) channel (L-VGCC) current, an enhanced Ca(2+)-dependent slow afterhyperpolarization (AHP), impaired synaptic plasticity and elevated Ca(2+) transients. Previously, we found that 1alpha,25-dihydoxyvitamin D(3) (1,25VitD), a major Ca(2+) regulating hormone, down-regulates L-VGCC expression in cultured hippocampal neurons. Here, we tested whether in vivo treatment of aged F344 rats with 1,25VitD would reverse some of the Ca(2+) -mediated biomarkers of aging seen in hippocampal CA1 neurons. As previously reported, L-VGCC currents and the AHP were larger in aged than in young neurons. Treatment with 1,25VitD over 7 days decreased L-VGCC activity in aged rats, as well as the age-related increase in AHP amplitude and duration. In addition, reduced L-VGCC activity was correlated with reduced AHPs in the same animals. These data provide direct evidence that 1,25VitD can regulate multiple Ca(2+)-dependent processes in neurons, with particular impact on reducing age-related changes associated with Ca(2+) dysregulation. Thus, these results may have therapeutic implications and suggest that 1,25VitD, often taken to maintain bone health, may also retard some consequences of brain aging.     

Evidence for distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in osteoblasts

1 alpha,25-(OH)(2)D(3) exerts its effects on chondrocytes and enterocytes via nuclear receptors (1,25-nVDR) and a separate membrane receptor (1,25-mVDR) that activates protein kinase C (PKC). 24R,25-(OH)(2)D(3) also stimulates PKC in chondrocytes, but through other membrane mechanisms. This study examined the hypothesis that osteoblasts possess distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) that are involved in the activation of PKC and that receptor expression varies as a function of cell maturation state. 1 alpha,25-(OH)(2)D(3) stimulated PKC in well differentiated (UMR-106, MC-3T3-E1) and moderately differentiated (ROS 17/2.8) osteoblast-like cells, and in cultures of fetal rat calvarial (FRC) cells and 2T3 cells treated with rhBMP-2 to promote differentiation. 24R,25-(OH)(2)D(3) stimulated PKC in FRC and 2T3 cultures that had not been treated to induce differentiation, and in ROS 17/2.8 cells. MG63 cells, a relatively undifferentiated osteoblast-like cell line, had no response to either metabolite. Ab99, a polyclonal antibody generated to the chick enterocyte 1,25-mVDR, but not a specific antibody to the 1,25-nVDR, inhibited response to 1 alpha,25-(OH)(2)D(3). 1 alpha,25-(OH)(2)D(3) exhibited specific binding to plasma membrane preparations from cells demonstrating a PKC response to this metabolite that is typical of positive cooperativity. Western blots of these membrane proteins reacted with Ab99, and the Ab99-positive protein had an Mr of 64 kDa. There was no cross-reaction with antibodies to the C- or N-terminus of annexin II. The effect of 24,25-(OH)(2)D(3) on PKC was stereospecific; 24S,25-(OH)(2)D(3) had no effect. These results demonstrate that response to 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) depends on osteoblast maturation state and suggest that specific and distinct membrane receptors are involved.     

Autoradiographic studies with 3H 1,25 (OH)2 vitamin D3 and 3H 25 (OH) vitamin D3 in rat parathyroid glands

Summary After injection of radiolabeled 1,25 (OH)₂ vitamin D₃, nuclear concentration of radioactivity is observed in parenchymal cells of the parathyroid gland in pregnant, adult male, and 10-day male neonatal rats. In competition studies with unlabeled 1,25 (OH)₂ vitamin D₃, but not with 25 (OH) vitamin D₃, nuclear uptake is prevented. Experiments with ³H 25 (OH) vitamin D₃, in contrast to ³H 1,25 (OH)₂ vitamin D₃, do not show nuclear concentration in cells of the parathyroid. The results of the autoradiographic studies suggest the presence of receptors for a direct effect of 1,25 (OH)₂ vitamin D₃ on the parathyroid gland for modulation of parathyroid hormone secretion.     

1 beta, 25 (OH)2-vitamin D3 is an antagonist of 1 alpha,25 (OH)2-vitamin D3 stimulated transcaltachia (the rapid hormonal stimulation of intestinal calcium transport).

The steroid hormone 1 alpha,25-dihydroxyvitamin-D3 [1 alpha,25(OH)2D3] stimulates biological responses via both genomic mechanisms and nongenomic mechanisms (opening of voltage-gated Ca2+ channels). We report here that 1 beta, 25(OH)2-vitamin-D3 (a) is devoid of activity as an agonist for transcaltachia, (b) is a potent stereospecific antagonist of 1 alpha,25 (OH)2D3 stimulation of the nongenomic transcaltachia response and also (c) has less than 1% the ability of 1 alpha,25(OH)2D3 to bind to the chick intestinal nuclear 1 beta,25(OH)2D3 receptor. We conclude that the membrane response element(s) which generates the nongenomic response of transcaltachia has a different ligand specificity than the classic nuclear 1 alpha, 25(OH)2D3 receptor.