Rapid effects of calciotropic hormones on female rat enterocytes: combined actions of 1,25(OH)2-vitamin D3, PTH and 17beta-estradiol on intracellular Ca2+ regulation.

1,25(OH)(2)-Vitamin D(3) [1,25(OH)(2)D(3)], PTH and 17beta-estradiol increase intracellular Ca(2+) levels ([Ca(2+)](i)) in rat enterocytes by stimulating inner Ca(2+) store mobilization and voltage-dependent Ca(2+) channels through non-genomic activation of second-messenger cascades. The participation of store-operated Ca(2+) (SOC) channels in 17beta-estradiol regulation of enterocyte [Ca(2+)](i) has also been suggested. The aim of this work was to investigate whether PTH and/or 17beta-estradiol exert additive or synergistic effects acting in concert with the classic intestinal calciotropic hormone 1,25(OH)(2)D(3). Fura-2-loaded rat duodenal cells were stimulated using rPTH (10 nM), 17beta-estradiol (0.1 nM) or 1,25(OH)(2)D(3) (0.1 nM). The resulting Ca(2+) signal was characterized by an almost immediate rise in [Ca(2+)](i) (within 30 s) rapidly reaching peak levels, followed by a plateau phase that remained sustained as long as the cells were exposed to the stimulus. The addition of PTH at the sustained phase induced by 1,25(OH)(2)D(3) or, conversely, the addition of the secosteroid after the PTH-induced effect, did not induce additional increases in [Ca(2+)](i). Simultaneous treatment with both hormones resulted in an elevation of [Ca(2+)](i) equivalent to the maximal level caused by either agonist alone, suggesting common components for [Ca(2+)]i stimulation by PTH and 1,25(OH)(2)D(3). Treatment with 17beta-estradiol at the sustained phase induced by 1,25(OH)(2)D(3) or, conversely, treatment with the secosteroid after the 17beta-estradiol effect, induced additional increments in [Ca(2+)](i) (58 % and 63 %, respectively). Simultaneous treatment of enterocytes with both steroids potentiated their individual effects to the same extent as when added sequentially, also indicative of additive actions mediated by different sources of calcium signaling cascades. Moreover, 17beta-estradiol failed to further increase the 1,25(OH)(2)D(3)-induced initial Ca(2+) elevation in Ca(2+)-free medium, thus suggesting that extracellular influx mechanisms rather than intracellular Ca(2+) mobilization account for estrogen potentiation of 1,25(OH)(2)D(3) modulation of [Ca(2+)](i) in duodenal cells.     

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.     

Calcium signaling in cancer and vitamin D

Calcium signals induced by the Ca(2+) regulatory hormone 1,25(OH)(2)D(3) may determine the fate of the cancer cell. We have shown that, in breast cancer cell lines, 1,25(OH)(2)D(3) induces a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) by depleting the endoplasmic reticulum (ER) Ca(2+) stores via inositol 1,4,5-trisphosphate receptor/Ca(2+) release channel and activating Ca(2+) entry from the extracellular space via voltage-insensitive Ca(2+) channels. In normal cells, 1,25(OH)(2)D(3) triggered a transient Ca(2+) response via activation of voltage-dependent Ca(2+) channels, which were absent in breast cancer cells. The normal cells, but not breast cancer cells, expressed the Ca(2+) binding/buffering protein calbindin-D(28k) and were capable of buffering [Ca(2+)](i) increases induced by a mobilizer of the ER Ca(2+) stores, thapsigargin, or a Ca(2+) ionophore, ionomycin. The 1,25(OH)(2)D(3)-induced sustained increase in [Ca(2+)](i) in breast cancer cells was associated with induction of apoptotic cell death, whereas the transient [Ca(2+)](i) increase in normal cells was not. The forced expression of calbindin-D(28k) in cytosol or increase in the cytosolic Ca(2+) buffering capacity with the cell-permeant Ca(2+) buffer BAPTA prevented induction of apoptosis with 1,25(OH)(2)D(3) in cancer cells. The sustained increase in [Ca(2+)](i) in breast cancer cells was associated with activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the fluorogenic peptide substrates. Selective inhibition of the Ca(2+) binding sites of mu-calpain decreased apoptotic indices in the cancer cells treated with 1,25(OH)(2)D(3), thapsigargin, or ionomycin. The mu-calpain activation preceded expression/activation of caspase-12, and calpain was required for activation/cleavage of caspase-12. Certain non-calcemic vitamin D analogs (e.g., EB 1089) triggered a sustained [Ca(2+)](i) increase, activated Ca(2+)-dependent apoptotic proteases, and induced apoptosis in breast cancer cells in a fashion similar to that of 1,25(OH)(2)D(3). The 1,25(OH)(2)D(3)-induced transient Ca(2+) response in normal mammary epithelial cells was not accompanied by activation of mu-calpain and caspase-12. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH)(2)D(3): increase in [Ca(2+)](i)-->mu-calpain activation-->caspase-12 activation-->apoptosis. Our results support the hypothesis that 1,25(OH)(2)D(3) directly activates this apoptotic pathway by inducing a sustained increase in [Ca(2+)](i). Differences of Ca(2+) regulatory mechanisms in cancer versus normal cells seem to allow 1,25(OH)(2)D(3) and vitamin D analogs to induce Ca(2+)-mediated apoptosis selectively in breast cancer cells. Thus, deltanoids may prove to be useful in the treatment of tumors susceptible to induction of Ca(2+)-mediated apoptosis.     

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts

The rapid, non-genomic actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(-/-) osteoblasts isolated from wild-type and VDR null mice to study the increase in intracellular calcium ([Ca(2+)](i)) and activation of protein kinase C (PKC) induced by 1,25(OH)(2)D(3). Within 1 min of 1,25(OH)(2)D(3) (100 nM) treatment, an increase of 58 and 53 nM in [Ca(2+)](i) (n = 3) was detected in VDR(+/+) and VDR(-/-) cells, respectively. By 5 min, 1,25(OH)(2)D(3) caused a 2.1- and 1.9-fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated-MBP(4-14) in VDR(+/+) and VDR(-/-) osteoblasts. The 1,25(OH)(2)D(3)-induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)(2)D(3) treatment resulted in the same degree of translocation of PKC-alpha and PKC-delta, but not of PKC-zeta, from cytosol to plasma membrane in both VDR(+/+) and VDR(-/-) cells. These experiments demonstrate that the 1,25(OH)(2)D(3)-induced rapid increases in [Ca(2+)](i) and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)(2)D(3) in osteoblasts.     

Hormonal regulation of [Ca(2+)](i) in periosteal-derived osteoblasts: effects of parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandin E(2)

The effects of hormonal modulators of osteoblast function, parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandins on [Ca(2+)](i) in periosteal-derived osteoblasts from rat femurs have been investigated. Our results show that application of parathyroid hormone PTH (10(-5) M) and prostaglandin E(2) (PGE(2)) (4 microM) result in a rapid heterogeneous elevation in [Ca(2+)](i) that, in the case of PTH, is dependent on both extracellular and intracellular sources of calcium. Variable responses to treatments have been found within populations of cells. The PGE(2) response is dose dependent. Treatment with 1,25(OH)(2)D(3) (10(-8) M) induces a brief (60-90 sec) elevation in [Ca(2+)](i) that is almost totally abolished in EGTA-buffered Ca(2+)-free medium. Interactive effects of multiple hormone treatments have been observed. Pretreatment with 1,25(OH)(2)D(3) results in near-total inhibition of the PTH and PGE(2) responses. In conclusion, modulation of [Ca(2+)](i) appears to play a role not only in the direct effects of osteotropic hormones on osteoblasts but also in the synergistic and antagonistic effects between circulating hormones.     

Calcium as a mediator of 1,25-dihydroxyvitamin D3-induced apoptosis

Cellular calcium has been implicated in induction of apoptosis. We have shown that 1,25(OH)(2)D(3)-induced apoptosis is associated with a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from depletion of the endoplasmic reticulum (ER) Ca(2+) stores and activation of the voltage-insensitive Ca(2+) entry pathway [1,25-Dihydroxyvitamin D(3), intracellular Ca(2+) and apoptosis in breast cancer cells, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D: Chemistry, Biology and Clinical Applications of the Steroid Hormone, University of California, Riverside, 1997, pp. 473-474; Vitamin D and intracellular calcium, in: P. Quinn, V. Kagan (Eds.), Subcellular Biochemistry: Fat-Soluble Vitamins, Plenum Press, New York, 1998, pp. 271-297; 1,25-Dihydroxyvitamin D(3) and calcium signaling, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D Endocrine System: Structural, Biological, Genetic and Clinical Aspects, University of California, Riverside, 2000, pp. 715-718; 1,25-Dihydroxyvitamin D(3) triggers calcium-mediated apoptosis in breast cancer cells, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D Endocrine System: Structural, Biological, Genetic and Clinical Aspects, University of California, Riverside, 2000, pp. 399-402; Endocrine 9 (1998) 321]. This study was undertaken to investigate mechanism of 1,25(OH)(2)D(3)-induced apoptosis in breast cancer cells and compare effects of the hormone on Ca(2+) and apoptosis in cancer and normal human mammary epithelial cells. The treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D(3) induced a sustained increase in [Ca(2+)](i) and activated the Ca(2+)-dependent proapoptotic proteases, micro-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the calpain substrate. The selective inhibition of Ca(2+) binding sites of micro-calpain decreased apoptotic indices in the 1,25(OH)(2)D(3)-treated cells. 1,25(OH)(2)D(3) did not induce apoptosis in normal human mammary epithelial cells (HMECs), as evaluated by DNA fragmentation (TUNEL), loss of the plasma membrane asymmetry (Annexin V assay) and morphological criteria. In these cells, 1,25(OH)(2)D(3) triggered a transient Ca(2+) response, which was not accompanied by the calpain and caspase activation. HMEC, but not MCF-7 cells expressed the Ca(2+) binding protein calbindin-D(28k) and buffered Ca(2+) increases induced by a Ca(2+) ionophore ionomycin. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH)(2)D(3): increase in [Ca(2+)](i) -->micro-calpain activation --> caspase-12 activation --> apoptosis. Our findings also imply that differences of Ca(2+) regulatory mechanisms in breast cancer versus normal mammary epithelial cells underlay resistance of normal cells and susceptibility of cancer cells to 1,25(OH)(2)D(3)-induced Ca(2+)-mediated apoptosis.     

Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells

The active form of vitamin D(3) (1,25(OH)(2)D(3)) induces an increase in the intracellular free calcium ([Ca(2+)](i)) and caspase-independent cell death in human breast cancer cells. Here we show that the treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D(3) or its chemotherapeutic analog, EB 1089, releases Ca(2+) from the endoplasmic reticulum. The increase in [Ca(2+)](i) was associated with the activation of a calcium-dependent cysteine protease, mu-calpain. Interestingly, ectopic expression of a calcium-binding protein, calbindin-D(28k), in MCF-7 cells not only attenuated the elevation in [Ca(2+)](i) and calpain activation, but also reduced death triggered by vitamin D compounds. Similarly, the inhibition of calpain activity by structurally unrelated chemical inhibitors increased the survival of the cells and reduces the amount of annexin V-positive cells. Despite the complete absence of effector caspase activation, transmission electron microscopy of MCF-7 cells treated with 1,25(OH)(2)D(3) or EB 1089 revealed apoptosis-like morphology characterized by the condensed cytoplasm, nuclei, and chromatin. Overall, these results suggest that calpain may take over the role of the major execution protease in apoptosis-like death induced by vitamin D compounds. Thus, these compounds may prove useful in the treatment of tumors resistant to therapeutic agents dependent on the classical caspase cascade.     

Protein kinase C activates store-operated Ca(2+) channels in human glomerular mesangial cells

Store-operated Ca(2+) channels (SOC) are expressed in cultured human mesangial cells and activated by epidermal growth factor through a pathway involving protein kinase C (PKC). We used fura-2 fluorescence and patch clamp experiments to determine the role of PKC in mediating the activation of SOC after depletion of internal stores by thapsigargin. The measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) revealed that the thapsigargin-induced Ca(2+) entry pathway was abolished by calphostin C, a protein kinase C inhibitor. The PKC activator, phorbol 12-myristate 13-acetate (PMA), promoted a Ca(2+) influx that was significantly attenuated by calphostin C and La(3+) but not by diltiazem. Neither PMA nor calphostin C altered the thapsigargin-induced initial transient rise in [Ca(2+)](i). In cell-attached patch clamp experiments, the thapsigargin-induced activation of SOC was potentiated by PMA and abolished by both calphostin C and staurosporine. However, SOC was unaffected by thapsigargin when clamping [Ca(2+)](i) with 1,2-bis (o-Aminophenoxy)ethane-N,N,N',N'tetraacetic acid tetra(acetoxymethyl)ester. In the absence of thapsigargin, PMA and phorbol 12, 13-didecanoate evoked a significant increase in NP(O) of SOC, whereas calphostin C did not affect base-line channel activity. In inside-out patches, SOC activity ran down immediately upon excision but was reactivated significantly after adding the catalytic subunit of 0.1 unit/ml of PKC plus 100 microm ATP. Neither ATP alone nor ATP with heat-inactivated PKC rescued a rundown of SOC. Metavanadate, a general protein phosphatase inhibitor, also enhanced SOC activity in inside-out patches. Bath [Ca(2+)] did not significantly affect the channel activity in inside-out patch. These results indicate that the depletion of Ca(2+) stores activates SOC by PKC-mediated phosphorylation of the channel proteins or a membrane-associated complex.     

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.     

Activation of RAF-1 through Ras and protein kinase Calpha mediates 1alpha,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells

We have previously shown that stimulation of proliferation of avian embryonic muscle cells (myoblasts) by 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)) is mediated by activation of the mitogen-activated protein kinase (MAPK; ERK1/2). To understand how 1alpha,25(OH)(2)D(3) up-regulates the MAPK cascade, we have investigated whether the hormone acts upstream through stimulation of Raf-1 and the signaling mechanism by which this effect might take place. Treatment of chick myoblasts with 1alpha,25(OH)(2)D(3) (1 nm) caused a fast increase of Raf-1 serine phosphorylation (1- and 3-fold over basal at 1 and 2 min, respectively), indicating activation of Raf-1 by the hormone. These effects were abolished by preincubation of cells with a specific Ras inhibitor peptide that involves Ras in 1alpha,25(OH)(2)D(3) stimulation of Raf-1. 1alpha,25(OH)(2)D(3) rapidly induced tyrosine de-phosphorylation of Ras-GTPase-activating protein, suggesting that inhibition of Ras-GTP hydrolysis is part of the mechanism by which 1alpha,25(OH)(2)D(3) activates Ras in myoblasts. The protein kinase C (PKC) inhibitors calphostin C, bisindolylmaleimide I, and Ro 318220 blocked 1alpha,25(OH)(2)D(3)-induced Raf-1 serine phosphorylation, revealing that hormone stimulation of Raf-1 also involves PKC. In addition, transfection of muscle cells with an antisense oligodeoxynucleotide against PKCalpha mRNA suppressed serine phosphorylation by 1alpha,25(OH)(2)D(3). The increase in MAPK activity and tyrosine phosphorylation caused by 1alpha,25(OH)(2)D(3) could be abolished by Ras inhibitor peptide, compound PD 98059, which prevents the activation of MEK by Raf-1, or incubation of cell lysates before 1alpha,25(OH)(2)D(3) exposure with an anti-Raf-1 antibody. In conclusion, these results demonstrate for the first time in a 1alpha,25(OH)(2)D(3) target cell that activation of Raf-1 via Ras and PKCalpha-dependent serine phosphorylation plays a central role in hormone stimulation of the MAPK-signaling pathway leading to muscle cell proliferation.     

1,25-dihydroxyvitamin D(3) stimulated protein kinase C phosphorylation of type VI adenylyl cyclase inhibits parathyroid hormone signal transduction in rat osteoblastic UMR 106-01 cells

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) treatment of osteoblastic cells was shown previously to attenuate Parathyroid hormone (PTH) response by inhibiting adenylyl cyclase (AC) activity. In this study, we have investigated the mechanism by which 1,25(OH)(2)D(3) inhibits AC in rat osteoblastic UMR 106-01 cells. 1,25(OH)(2)D(3) treatment inhibited both PTH and forskolin-stimulated AC activity by 25%-50% within 12 min in a concentration-dependent manner suggesting a direct inhibition of the AC enzyme. Treatment with 25(OH)D(3) had no effect on basal or stimulated AC activity. We determined the profile of AC subtypes expressed in UMR cells and found AC VI to be the dominant subtype accounting for 50% of AC mRNA. Since AC VI can be inhibited by protein kinase C (PKC) phosphorylation, we examined 1,25(OH)(2)D(3) activation of various PKC isoforms. 1,25(OH)(2)D(3) increased the membrane translocation of PKC-betaI, -delta, and -zeta with a concomitant increase in PKC activity. The translocation of PKC-betaI and -delta was blocked by the PLC inhibitor U73122 whereas that of PKC-zeta was abolished by the PI-3 kinase inhibitor wortmannin. The attenuation of cAMP production by 1,25(OH)(2)D(3) was antagonized by the PKC inhibitors Go6850, calphostin C, and wortmannin, but not by a calmodulin kinase II (CaMKII) inhibitor. Treatment with 1,25(OH)(2)D(3) for 20 min increased AC VI phosphorylation by 10.8-fold and this was blocked partially by Go6850 and partially by wortmannin but was unaffected by CaMKII inhibitor. These results demonstrate that 1,25(OH)(2)D(3) activation of PKC isoforms leads to phosphorylation of AC VI and inhibition of PTH-activation of this pathway in osteoblasts.     

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.     

Membrane mediated signaling mechanisms are used differentially by metabolites of vitamin D(3) in musculoskeletal cells

1 alpha,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) mediate their effects on chondrocytes and osteoblasts in part through increased activity of protein kinase C (PKC). For both cell types, 1 alpha,25(OH)(2)D(3) exerts its effects primarily on more mature cells within the lineage, whereas 24R,25(OH)(2)D(3) exerts its effects primarily on relatively immature cells. Studies using the rat costochondral cartilage growth plate as a model indicate that the two metabolites increase PKC activity by different mechanisms. In growth zone cells (prehypertrophic/upper hypertrophic cell zones), 1 alpha,25(OH)(2)D(3) causes a rapid increase in PKC that does not involve new gene expression. 1 alpha,25(OH)(2)D(3) binds its membrane receptor (1,25-mVDR), resulting in activation of phospholipase A(2) and the rapid release of arachidonic acid, as well as activation of phosphatidylinositol-specific phospholipase C, resulting in formation of diacylglycerol and inositol-1,4,5-tris phosphate (IP(3)). IP(3) leads to release of intracellular Ca(2+) from the rough endoplasmic reticulum, and together with diacylglycerol, the increased Ca(2+) activates PKC. PKC is then translocated to the plasma membrane, where it initiates a phosphorylation cascade, ultimately phosphorylating the extracellular signal-regulated kinase-1 and -2 (ERK1/2) family of MAP kinases (MAPK). PKC increases are maximal at 9 min, and MAPK increases are maximal at 90 min in these cells. By contrast, 24R,25(OH)(2)D(3) increases PKC through activation of phospholipase D in resting zone cells. Peak production of diacylglycerol via phospholipase D2 is at 90 min, as are peak increases in PKC. Some of the effect is direct on existing plasma membrane PKC, but most is due to new PKC expression; translocation is not involved. Arachidonic acid and its metabolites also play differential roles in the mechanisms, stimulating PKC in growth zone cells and inhibiting PKC in resting zone cells. 24R,25(OH)(2)D(3) decreases phospholipase A(2) activity and prostaglandin production, thereby overcoming this potential inhibitory component, which may account for the delay in the PKC response. Ultimately, ERK1/2 is phosphorylated. PKC-dependent MAPK activity transduces some, but not all, of the physiological responses of each cell type to its respective vitamin D metabolite, suggesting that the membrane receptor(s) and nuclear receptor(s) may function interdependently to regulate proliferation and differentiation of musculoskeletal cells, but different pathways are involved at different stages of phenotypic maturation.     

Induction of tissue plasminogen activator secretion from rat heart microvascular cells by fM 1,25(OH)(2)D(3)

We investigated the effects of 1,25-dihydroxyvitamin D(3) [25(OH)(2)D(3)] on tissue plasminogen activator (tPA) secretion from primary cultures of rat heart microvascular cells. After an initial 5-day culture period, cells were treated for 24 h with 1,25(OH)(2)D(3) and several of its analogs. The results showed that 1,25(OH)(2)D(3) induced tPA secretion at 10(-10) to 10(-16) M. A less calcemic analog, Ro-25-8272, and an analog that binds the vitamin D receptor but is ineffective at perturbing Ca(2+) channels, Ro-24-5531, were approximately 10% as active as 1,25(OH)(2)D(3). An analog that binds the vitamin D receptor poorly but is an effective Ca(2+) channel agonist, Ro-24-2287, required approximately 10(-13) M to induce tPA secretion. Combinations of Ro-24-5531 and Ro-24-2287 were approximately as potent as 1,25(OH)(2)D(3). Treatment of the cells with BAY K 8644 or thapsigargin also increased tPA secretion, suggesting that increased cytosolic calcium concentration ([Ca(2+)]) induces tPA secretion. The results suggested that the sensitivity of the tPA secretory response of microvascular cells to 1,25(OH)(2)D(3) was due in part to generation of a vitamin D-depleted state in vitro and in part to synergistic effects of 1,25(OH)(2)D(3) on two different induction pathways of tPA release.     

Protein kinase C alpha modulates the Ca2+ influx phase of the Ca2+ response to 1alpha,25-dihydroxy-vitamin-D3 in skeletal muscle cells.

Treatment of chick skeletal muscle cells with 1alpha,25-dihydroxy-vitamin D3 [1alpha,25(OH)2D3] triggers a rapid and sustained increase in cytosolic Ca2+ ([Ca2+]i), which depends on Ca2+ mobilization from inner stores and extracellular Ca2+ entry. Fluorimetric analysis of changes in [Ca2+]i in Fura-2-loaded cells revealed that the hormone significantly stimulates the Ca2+ influx phase within the concentration range of 10(-12)-10(-6) M, with maximal effects (3.5-fold increase) at 10(-9) M 1alpha,25(OH)2D3. The effects of the sterol on the Ca2+ entry pathway were abolished by the PKC inhibitors bisindolylmaleimide and calphostin. We have recently shown that, in these cells, 1alpha,25(OH)2D3 activates and translocates PKC alpha to the membrane, suggesting that this isozyme accounts for PKC-dependent 1alpha,25(OH)2D3 modulation of Ca2+ entry. The role of PKC alpha was specifically addressed here using antisense technology. When the expression of PKC alpha was selectively knocked out by intranuclear microinjection of an antisense oligonucleotide against PKC alpha mRNA, the Ca2+ influx component of the response to 1alpha,25(OH)2D3 was markedly reduced (-60%). These results demonstrate that 1alpha,25(OH)2D3-induced activation of PKC alpha enhances extracellular Ca2+ entry partially contributing to maintainance of the sustained phase of the Ca2+ response to the sterol.