Activation of Src kinase in skeletal muscle cells by 1, 1,25-(OH(2))-vitamin D(3) correlates with tyrosine phosphorylation of the vitamin D receptor (VDR) and VDR-Src interaction

The rapid effect of 1 alpha,25(OH(2))-vitamin D(3) [1 alpha, 25(OH(2))D(3)] on tyrosine kinase Src and its relationship to the vitamin D receptor (VDR) was investigated to further characterize the hormone signaling mechanism in chick muscle cells. Exposure of cultured myotubes to 1 alpha,25(OH(2))D(3) caused a time-dependent increase in Src activity, which was evident at 1 min (one-fold) and reached a maximum at 5 min (15-fold). Immunoblotting with anti-phosphotyrosine antibody of immunoprecipitated Src showed that the hormone decreased Src tyrosine phosphorylation state with maximal effects at 5 min. Using a database for protein consensus motifs we found a putative tyrosine phosphorylation site (amino acids 164-170: KTFDTTY) within the primary sequence of the chick VDR. When the myotube VDR was immunoprecipitated it appeared onto SDS-PAGE gels as a single band of 58 kDa recognized by an anti-phosphotyrosine antibody. Prior treatment of cells with (1)alpha,25(OH(2))D(3) significantly increased tyrosine phosphorylation of the VDR (two- to three-fold above basal levels). In agreement with Src being a SH2-domain containing protein involved in recognition of tyrosine-phosphorylated targets, immunoprecipitation with anti-Src antibody under native conditions followed by blotting with anti-VDR antibody, or using the antibodies in inverse order, showed that the VDR co-precipitates with Src, thus indicating the existence of a VDR/Src complex. Stimulation with the cognate VDR ligand significantly increased formation of the complex with respect to basal conditions. These results altogether provide the first evidence to date for 1 alpha,25(OH(2))D(3) activation involving Src association to tyrosine phosphorylated VDR.     

Sensitivity to growth suppression by 1alpha,25-dihydroxyvitamin D(3) among MCF-7 clones correlates with Vitamin D receptor protein induction

The antiproliferative effect of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) has been studied for a decade in diverse model systems, but the signalling pathways linking 1alpha,25(OH)(2)D(3) to cell cycle arrest remains unclear. In our attempt to establish a model system which would allow further identification of important players in the process of the 1alpha,25(OH)(2)D(3) imposed cell cycle arrest, we have isolated derivatives of the human breast cancer cell line MCF-7 and chosen two nearly 1alpha,25(OH)(2)D(3) resistant and two hypersensitive sub-clones. Investigation of cell cycle proteins regulated by 1alpha,25(OH)(2)D(3) in these clones indicates that activation of one component/pathway is responsible for the linkage between 1alpha,25(OH)(2)D(3) and growth arrest. Protein levels of the Vitamin D receptor (VDR) were elevated in sensitive cells upon 1alpha,25(OH)(2)D(3) treatment, whereas resistant clones were unable to induce VDR upon 1alpha,25(OH)(2)D(3) treatment. Our data show that VDR protein levels and the ability of a cell to induce VDR upon 1alpha,25(OH)(2)D(3) treatment correlate with the antiproliferative effects of 1alpha,25(OH)(2)D(3), and suggest that the level of VDR in cancer cells might serve as a prognostic marker for treatment of cancer with 1alpha,25(OH)(2)D(3) analogues.     

1Alpha,25-dihydroxyvitamin D3-mediated stimulation of steroid sulphatase activity in myeloid leukaemic cell lines requires VDRnuc-mediated activation of the RAS/RAF/ERK-MAP kinase signalling pathway

1Alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) stimulates the activity of steroid sulphatase (STS) in myeloid cells [Hughes et al., 2001, 2005]. This was attenuated by inhibitors of phospholipase D (PLD) (n-butanol, 2,3-diphosphoglyceric acid, C(2)-ceramide) and phosphatidate phosphohydrolase (PAP) (propranolol and chlorpromazine), but was unaffected by inhibitors of phospholipase C. The 1alpha,25(OH)(2)D(3)-induced STS activity was also attenuated by inhibitors of protein kinase Calpha and protein kinase Cdelta (Go 6976, HBDDE and rottlerin), but not by an inhibitor of protein kinase Cbeta (LY379196). Additionally, 1alpha,25(OH)(2)D(3)-induced STS activity was attenuated by inhibitors of RAS (manumycin A), RAF (GW5074), MEK (PD098059 and U1026) and JNK (SP600125), but not p38 (PD169316). 1alpha,25(OH)(2)D(3) produced a rapid and long lasting stimulation of the ERK-MAP kinase signalling cascade in HL60 myeloid leukaemic cells. This 'non-genomic' effect of 1alpha,25(OH)(2)D(3) blocked by pharmacological antagonists of nuclear vitamin D receptors (VDR(nuc)) and does not appear to require hetero-dimerisation with the retinoid-X receptor (RXR). Inhibitors of the Src tyrosine kinase (PP1), RAS (manumycin A), RAS-RAF interactions (sulindac sulphide and RAS inhibitory peptide), RAF (GW5074 or chloroquine), and protein kinase Calpha (HBDDE) abrogated the 1alpha,25(OH)(2)D(3)-stimulated increase in ERK-MAP kinase activity. Taken together, these results show that 1alpha,25(OH)(2)D(3)/VDR(nuc) activation of the RAS/RAF/ERK-MAP kinase signalling pathway plays an important role in augmenting STS activity in human myeloid leukaemic cell lines.     

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.     

Nongenomic action of 1 alpha,25(OH)(2)-vitamin D3. Activation of muscle cell PLC gamma through the tyrosine kinase c-Src and PtdIns 3-kinase.

We have previously demonstrated that the steroid hormone 1 alpha,25(OH)(2)-vitamin D(3)[1 alpha,25(OH)(2)D(3)] stimulates the production of inositol trisphosphate (InsP(3)), the breakdown product of phosphatidylinositol 4,5-biphosphate (PtdInsP(2)) by phospholipase C (PtdIns-PLC), and activates the cytosolic tyrosine kinase c-Src in skeletal muscle cells. In the present study we examined whether 1 alpha,25(OH)(2)D(3) induces the phosphorylation and membrane translocation of PLC gamma and the mechanism involved in this isozyme activation. We found that the steroid hormone triggers a significant phosphorylation on tyrosine residues of PLC gamma and induces a rapid increase in membrane-associated PLC gamma immunoreactivity with a time course that correlates with that of phosphorylation in muscle cells. Genistein, a tyrosine kinase inhibitor, blocked the phosphorylation of PLC gamma. Inhibition of 1 alpha,25(OH)(2)D(3)-induced c-Src activity by its specific inhibitor PP1 or muscle cell transfection with an antisense oligodeoxynucleotide directed against c-Src mRNA, prevented hormone stimulation of PLC gamma tyrosine phosphorylation. The isozyme phosphorylation is also blocked by both wortmannin and LY294002, two structurally different inhibitors of phosphatidyl inositol 3-kinase (PtdIns3K), the enzyme that produces PtdInsP(3) known to activate PLC gamma isozymes specifically by interacting with their SH2 and pleckstrin homology domains. The hormone also increases the physical association of c-Src and PtdIns3K with PLC gamma and induces a c-Src-dependent tyrosine phosphorylation of the p85 regulatory subunit of PtdIns3K. The time course of hormone-dependent PLC gamma phosphorylation closely correlates with the time course of its redistribution to the membrane, suggesting that phosphorylation and redistribution to the membrane of PLC gamma are two interdependent events. 1 alpha,25(OH)(2)D(3)-induced membrane translocation of PLC gamma was prevented to a great extent by c-Src and PtdIns3K inhibitors, PP1 and LY294002. Taken together, the present data indicates that the cytosolic tyrosine kinase c-Src and PtdIns 3-kinase play indispensable roles in 1 alpha,25(OH)(2)D(3) signal transduction cascades leading to PLC gamma activation.     

Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells

Vitamin D(3) compounds are currently in clinical trials for human breast cancer and offer an alternative approach to anti-hormonal therapies for this disease. 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active form of vitamin D(3), induces apoptosis in breast cancer cells and tumors, but the underlying mechanisms are poorly characterized. In these studies, we focused on the role of caspase activation and mitochondrial disruption in 1alpha,25(OH)(2)D(3)-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of 1alpha,25(OH)(2)D(3) on MCF-7 cells was compared with that of tumor necrosis factor alpha, which induces apoptosis via a caspase-dependent pathway. Our major findings are that 1alpha,25(OH)(2)D(3) induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species. Moreover, we show that Bax translocation and mitochondrial disruption do not occur after 1alpha,25(OH)(2)D(3) treatment of a MCF-7 cell clone selected for resistance to 1alpha,25(OH)(2)D(3)-mediated apoptosis. These mitochondrial effects of 1alpha,25(OH)(2)D(3) do not require caspase activation, since they are not blocked by the cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks 1alpha,25(OH)(2)D(3)-mediated events downstream of mitochondria such as poly(ADP-ribose) polymerase cleavage, external display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to 1alpha,25(OH)(2)D(3)-mediated cell death is caspase-independent.     

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

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

Epigenetic repression of transcription by the Vitamin D3 receptor in prostate cancer cells

Normal prostate epithelial cells are acutely sensitive to the antiproliferative action of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), whilst prostate cancer cell lines and primary cultures display a range of sensitivities. We hypothesised that key antiproliferative target genes of the Vitamin D receptor (VDR) were repressed by an epigenetic mechanism in 1alpha,25(OH)(2)D(3)-insensitive cells. Supportively, we found elevated nuclear receptor co-repressor and reduced VDR expression correlated with reduced sensitivity to the antiproliferative action of 1alpha,25(OH)(2)D(3). Furthermore, the growth suppressive actions of 1alpha,25(OH)(2)D(3) can be restored by co-treatment with low doses of histone deacetylation inhibitors, such as trichostatin A (TSA) to induce apoptosis. Examination of the regulation of VDR target genes revealed that co-treatment of 1alpha,25(OH)(2)D(3) plus TSA co-operatively upregulated GADD45alpha. Similarly in a primary cancer cell culture, the regulation of appeared GADD45alpha repressed. These data demonstrate that prostate cancer cells utilise a mechanism involving deacetylation to suppress the responsiveness of VDR target genes and thus ablate the antiproliferative action of 1alpha,25(OH)(2)D(3).     

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.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.