Evidence that TRPC3 is a molecular component of the 1alpha,25(OH)2D3-activated capacitative calcium entry (CCE) in muscle and osteoblast cells

In chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8), 1alpha,25-dihydroxy-Vitamin-D(3) [1alpha,25(OH)(2)D(3)] stimulates release of Ca(2+) from inner stores and extracellular cation influx through both voltage-dependent and capacitative Ca(2+) entry (CCE) channels. We investigated the involvement of TRPC proteins in CCE induced by 1alpha,25(OH)(2)D(3). Two fragments were amplified by RT-PCR, exhibiting >85% sequence homology with human TRPC3. Northern and Western blots employing TRPC3-probes and anti-TRPC3 antibodies, respectively, confirmed endogenous expression of a TRPC3-like protein. Both cell types transfected with anti-TRPC3 antisense oligodeoxynucleotides showed reduced CCE and Mn(2+) entry induced by either thapsigargin or 1alpha,25(OH)(2)D(3). In muscle cells, anti-VDR antisense inhibited steroid-induced Ca(2+) and Mn(2+) influx and co-immunoprecipitation of TRPC3 and VDR was observed, suggesting an association between both proteins and a functional role of the receptor in 1alpha,25(OH)(2)D(3) activation of CCE. In osteoblasts, two PCR fragments showing high homology with human INAD-like sequences were obtained. Northern blot and antisense functional assays suggested the involvement of the INAD-like protein in CCE regulation by the hormone. Therefore, we propose that an endogenous TRPC3 protein mediates 1alpha,25(OH)(2)D(3) modulation of CCE in muscle and osteoblastic cells, which seems to implicate VDR-TRPC3 association and the participation of a INAD-like scaffold protein.     

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.     

1alpha,25(OH)2D3 induces capacitative calcium entry involving a TRPC3 protein in skeletal muscle and osteoblastic cells

This work describes the involvement of TRPC proteins in capacitative calcium entry (CCE) induced by 1alpha,25-dihydroxy-vitamin-D3 [1alpha,25(OH)2D3] in chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8) and the role of the vitamin D receptor (VDR) in this non-genomic rapid response mediated by the hormone. We propose that an endogenous TRPC3 protein mediates 1alpha,25(OH)2D3 modulation of CCE in these cells, which seems to implicate VDR-TRPC3 association and the participation of an INAD-like scaffold protein.     

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.     

1,25 dihydroxyvitamin D(3) activates sphingomyelin turnover in ROS17/2.8 osteosarcoma cells without sphingolipid-induced changes in cytosolic Ca(2+)

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] initiates the hydrolysis of sphingomyelin in ROS 17/2.8 osteosarcoma cells with the resultant generation of cell-associated ceramide. Increases in ceramide levels were detectable at 15 min and maximal one hour after exposure of cells to 1,25(OH)(2)D(3). Neither 1,25(OH)(2)D(3) nor exogenous ceramide elicited a change in cytosolic free Ca(2+) ([Ca(2+)](i)). Transient elevations in [Ca(2+)](i) were observed when cells were exposed to exogenous sphingosine, but there was no detectable conversion of ceramide to sphingosine in 1, 25(OH)(2)D(3)-treated cells. Ceramide also did not stimulate Ca(2+) uptake across ROS 17/2.8 cell plasma membranes. Collectively, these results suggest that 1,25(OH)(2)D(3) activates sphingomyelin turnover in ROS 17/2.8 osteosarcoma cells but that the sphingolipid metabolite ceramide is not responsible for 1,25(OH)(2)D(3)-induced activation of plasma membrane Ca(2+) channels.     

Characterization of a 1,25(OH)2-vitamin D3-responsive capacitative Ca2+ entry pathway in rat osteoblast-like cells

We investigated the existence of a capacitative Ca2+ entry (CCE) pathway in ROS 17/2.8 osteoblast-like cells and its responsiveness to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3]. Depletion of inner Ca2+ stores with thapsigargin or 1,25(OH)2D3 in the absence of extracellular Ca2+ transiently elevated cytosolic Ca2+ ([Ca2+]i); after recovery of basal values, Ca2+ re-addition to the medium markedly increased Ca2+ entry, reflecting pre-activation of a CCE pathway. Recovery of the Ca2+ overshoot that followed the induced CCE was mainly mediated by the plasma membrane Ca2+-ATPase. Addition of 1,25(OH)2D3 to the declining phase of the thapsigargin-induced CCE did not modify further [Ca2+]i, indicating that steroid activation of CCE was dependent on store depletion. Pre-treatment with 1 microM Gd3+ inhibited 30% both thapsigargin- and 1,25(OH)2D3-stimulated CCE, whereas 2.5 microM Gd3+ was required for maximal inhibition ( approximately 85%). The activated CCE was permeable to both Mn2+ and Sr2+. Mn2+ entry sensitivity to Gd3+ was the same as that of the CCE. However, 1-microM Gd3+ completely prevented capacitative Sr2+ influx, whereas subsequent Ca2+ re-addition was reduced only 30%. These results suggest that in ROS 17/2.8 cells CCE induced by thapsigargin or 1,25(OH)2D3 is contributed by at least two cation entry pathways: a Ca2+/Mn2+ permeable route insensitive to very low micromolar (1 microM) Gd3+ accounting for most of the CCE and a minor Ca2+/Sr2+/Mn2+ permeable route highly sensitive to 1 microM Gd3+. The Ca2+-mobilizing agonist ATP also stimulated CCE resembling the Ca2+/Sr2+/Mn2+ permeable entry activated by 1,25(OH)2D3. The data demonstrates for the first time, the presence of a hormone-responsive CCE pathway in an osteoblast cell model, raising the possibility that it could be an alternative Ca2+ influx route through which osteotropic agents influence osteoblast Ca2+ homeostasis. Copyright Wiley-Liss, Inc.     

1alpha,25(OH)2-vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival

1alpha,25(OH)(2)-vitamin D(3) (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca(2+) channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca(2+) concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca(2+)-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca(2+)-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.     

Plasma membrane requirements for 1alpha,25(OH)2D3 dependent PKC signaling in chondrocytes and osteoblasts

1,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] acts on chondrocytes and osteoblasts through traditional nuclear Vitamin D receptor (VDR) mechanisms as well as through rapid actions on plasma membranes that initiate intracellular signaling pathways. We have investigated the mechanisms involved in activation of protein kinase C (PKC) and downstream biological responses that depend on the latter pathway. These studies show that PKC activation depends on presence of a membrane receptor ERp60 and rapid increases in phospholipase A(2) (PLA(2)) activity. Cells that are responsive to 1alpha,25(OH)(2)D(3) express PLA(2) activating protein (PLAA), suggesting a link between ERp60 and PLA(2). Increased PLA(2) results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLCbeta), leading to rapid formation of inositol-trisphosphate (IP3) and diacylglycerol (DAG). PLA(2), PLC, and DAG are all associated with lipid rafts including caveolae in many cells, suggesting that the caveolar environment may be an important mediator of PKC activation by 1alpha,25(OH)(2)D(3). Here, we use the VDR(-/-) mouse costochondral cartilage growth plate to examine the expression of ERp60 and PLAA in vivo in 1alpha,25(OH)(2)D(3)-responsive hypertrophic chondrocytes (growth zone cells) and in resting zone cells that do not respond to this Vitamin D metabolite in vitro. In addition, we determined if intact lipid rafts are required for the response of rat costochondral cartilage growth zone cells to 1alpha,25(OH)(2)D(3). The results show that ERp60 and PLAA are localized to 1alpha,25(OH)(2)D(3)-responsive growth zone cells and metaphyseal osteoblasts, even in VDR(-/-) mice. Disruption of lipid rafts using beta-cyclodextrin blocks the activation of PKC by 1alpha,25(OH)(2)D(3) and reduces the ability of 1alpha,25(OH)(2)D(3) to regulate [(35)S]-sulfate incorporation.     

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.     

Isolation and characterization of the rat alpha 1(I) collagen promoter. Regulation by 1,25-dihydroxyvitamin D

Our previous work demonstrated that the inhibition of type I collagen synthesis by 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in fetal rat calvaria and cultured rat osteosarcoma cells is accompanied by equivalent reduction in steady state levels of alpha 1(I) and alpha 2(I) collagen mRNA. To pursue the mechanism for this effect, we isolated and sequenced a 3.6-kilobase DNA fragment that contained the promoter for the rat alpha 1(I) collagen gene. This promoter fragment was fused to the chloramphenicol acetyltransferase gene and was introduced into ROS 17/2.8 cells by calcium phosphate co-precipitation. Expression of this construct was diminished by 1,25-(OH)2D3 to the same degree as the endogenous collagen gene in both transient expression assays and in permanently selected bone cells. However, a fibroblast cell line did not show a similar reduction in the activity of the transgene or the endogenous collagen gene. These experiments indicate that the alpha 1(I) promoter contains cis-active elements which are regulated by the 1,25-(OH)2D3 receptor in ROS 17/2.8 cells.     

Evidence for the functional involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 in bone.

In the present study the involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast-like cells and in the stimulation of in vitro bone resorption by 1,25(OH)2D3 was examined. Incubation for 24 h with 1,25(OH)2D3 potently stimulated osteocalcin synthesis by ROS 17/2.8 cells. This stimulation was inhibited (30-70% inhibition) by 25 microM of the protein kinase C (PKC) inhibitors 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG) and sphingosine without affecting basal osteocalcin synthesis. 1,25(OH)2D3-stimulated osteocalcin secretion by nontransformed isolated fetal rat osteoblasts was also inhibited (30-55%) by AMG. Also, AMG inhibited 10(-9) M 1,25(OH)2D3-induced up-regulation of vitamin D receptor in ROS 17/2.8 cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) did not cause an increase in osteocalcin secretion, while only a small increase in cellular content of osteocalcin in ROS 17/2.8 cells was observed. Addition of PMA together with 1,25(OH)2D3 did not change the response to 1,25(OH)2D3. The PKC inhibitors were not toxic for the cells. 1,25(OH)2D3 did not stimulate diacylglycerol production in ROS 17/2.8 cells up to 5 min after administration. However, 4- and 24-h incubation with 10 nM 1,25(OH)2D3 increased phorbol ester binding in ROS 17/2.8 cells. 1,25(OH)2D3 potently stimulated bone resorption after 3 and 6 days of culture in fetal mouse long bones and calvaria. Both the PKC inhibitors AMG (25 microM) and staurosporine (50 nM) strongly inhibited (60-86% inhibition) 1,25(OH)2D3-stimulated bone resorption without affecting basal 45Ca release. These effects were not due to a cytotoxic effect of both PKC inhibitors. Nor is it likely that the effects of AMG and staurosporine are due to inhibition of cell proliferation as hydroxyurea did not affect 1,25(OH)2D3-stimulated bone resorption. The inhibition of 1,25(OH)2D3-stimulated bone resorption by PKC inhibitors suggests that besides osteocalcin synthesis PKC is also involved in other responses of 1,25(OH)2D3 in bone. 1,25(OH)2D3 does not directly activate PKC via an increase in diacylglycerol production but more likely via an increase in PKC. Together, the present study demonstrates a functional involvement of PKC in the action of 1,25(OH)2D3 in bone and bone cells which may have consequences for the development of 1,25(OH)2D3 analogs, e.g. with less hypercalcemic and relatively more antiproliferative activity.     

The effects of 1alpha,24(S)-dihydroxyvitamin D(2) analog on cancer cell proliferation and cytokine expression

It is well established that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, plays a role in regulating proliferation and differentiation of cells, in addition to its classic function in mineral homeostasis. Recent studies have also provided evidence for the involvement of 1alpha,25(OH)(2)D(3) in regulating the immune system. However, therapeutic application of 1alpha,25(OH)(2)D(3) to hyperproliferative diseases such as cancer, or for immunologic purposes, is thwarted by its hypercalcemic activity. In order to overcome this obstacle, analogs of 1alpha,25(OH)(2)D(3) have been produced that exhibit decreased hypercalcemic activity while retaining the growth and immunologic regulating properties. In the present study, the efficacy of 1alpha,24(S)-dihydroxyvitamin D(2) (1alpha,24(S)(OH)(2)D(2)), a vitamin D(2) analog, in restraining cell proliferation was compared to that of 1alpha,25(OH)(2)D(3). In parallel studies, cancer cell lines were grown in increased concentrations (10(-10)-10(-7) M) of each compound for various incubation periods (1-4 days). Growth was assessed by measuring [(3)H]thymidine incorporation. The results revealed that 1alpha,24(S)(OH)(2)D(2) significantly inhibits proliferation to an extent similar to that observed for 1alpha,25(OH)(2)D(3). Moreover, incubating the human leukemia cell line, HL-60, with 1alpha,24(S)(OH)(2)D(2) resulted in an induction of differentiation of these promyelomonocyte cells into monocyte-macrophage-like cells, in a manner similar to that observed with 1alpha,25(OH)(2)D(3). Using a Western procedure, it was also shown that 1alpha,24(S)(OH)(2)D(2) like 1alpha,25(OH)(2)D(3) enhances the expression of vitamin D receptors (VDR) in the rat osteosarcoma cell line, ROS 17/2.8. The expression of tumor necrosis factor (TNF) alpha (TNF-alpha) in human peritoneal macrophages (HPM) obtained from uremic patients treated with continuous ambulatory peritoneal dialysis (CAPD) was found to be regulated by 1alpha,25(OH)(2)D(3) as well as by 1alpha,24(S)(OH)(2)D(2). Incubations of HPM with 1alpha,25(OH)(2)D(3) or 1alpha,24(S)(OH)(2)D(2), have inhibited the expression of TNF-alpha on both mRNA and protein levels. These results suggest that 1alpha,25(OH)(2)D(3) has a role in controlling the rate of inflammation in the peritoneal cavity of CAPD treated patients. Since 1alpha,24(S)(OH)(2)D(2) does not cause hypercalcemia, the present results encourage the possible use of this vitamin D(2) analog in the treatment of cancer and hyper-inflammatory diseases.     

Species difference exists in the effects of 1alpha,25(OH)(2)D(3) and its analogue 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD) on osteoblastic cells

The direct effect of 1alpha,25(OH)(2)D(3) on osteoblasts remains unclear. In this study, we evaluated the in vitro effects of 1alpha,25(OH)(2)D(3) and its analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD), on osteoblasts from three different species, i.e. bone marrow stromal cells from the Sprague-Dawley (SD) rat, from the C57BL/6 mouse, as well as human osteoblast NHOst cells and human osteosarcoma derived MG-63 cells. We found that in rat cells, both compounds increased cell proliferation, inhibited cell apoptosis and increased alkaline phosphatase (ALP) activity. In mouse cells, however, both compounds initiated cell apoptosis and inhibited ALP activity. In human cells, although cell proliferation was inhibited by both compounds, cell apoptosis was inhibited and ALP activity was enhanced. In each species, 2MD was much more potent than 1alpha,25(OH)(2)D(3). To summarize, species differences should be taken into account in studies of vitamin D effects. However, in all tested species - rat, mouse and human - 2MD is considerably more potent in its effects on osteoblastic cells in vitro than 1alpha,25(OH)(2)D(3).     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

1alpha,25(OH)2-vitamin D3 inhibits HGF synthesis and secretion from MG-63 human osteosarcoma cells

Several mesenchymally derived cells, including osteoblasts, secrete hepatocyte growth factor (HGF). 1alpha,25(OH)(2)-vitamin D(3) [1,25(OH)(2)D(3)] inhibits proliferation and induces differentiation of MG-63 osteoblastic cells. Here we show that MG-63 cells secrete copious amounts of HGF and that 1,25(OH)(2)D(3) inhibits HGF production. MG-63 cells also express HGF receptor (c-Met) mRNA, suggesting an autocrine action of HGF. Indeed, although exogenous HGF failed to stimulate cellular proliferation, neutralizing endogenous HGF with a neutralizing antibody inhibited MG-63 cell proliferation; moreover, inhibiting HGF synthesis with 1,25(OH)(2)D(3) followed by addition of HGF rescued hormone-induced inhibition of proliferation. Nonneutralized cells displayed constitutive phosphorylation of c-Met and the mitogen-activated protein kinases mitogen/extracellular signal-regulated kinase (MEK) 1 and extracellular signal-regulated kinase (Erk) 1/2, which were inhibited by anti-HGF antibody. Constitutive phosphorylation of Erk1/2 was also abolished by 1,25(OH)(2)D(3). Addition of HGF to MG-63 cells treated with neutralizing HGF antibody induced rapid phosphorylation of c-Met, MEK1, and Erk1/2. Thus endogenous HGF induces a constitutively active, autocrine mitogenic loop in MG-63 cells. The known antiproliferative effect of 1,25(OH)(2)D(3) on MG-63 cells can be accounted for by the concomitant 1,25(OH)(2)D(3)-induced inhibition of HGF production.     

Beta-1 integrins mediate substrate dependent effects of 1alpha,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. beta(1) integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1alpha,25(OH)(2)D(3) in a surface-dependent manner. To determine if beta(1) has a role in mediating osteoblast response, we silenced beta(1) expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human beta(1) to block ligand binding. beta(1)-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta(1), prostaglandin E(2), and osteoprotegerin in comparison with control cells. Moreover, beta(1)-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)(2)D(3). Anti beta(1) antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1alpha,25(OH)(2)D(3) on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta(1) plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)(2)D(3). The results also show that beta(1) mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)(2)D(3).     

Vitamin D and bone

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.