Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous work from our laboratory demonstrated that 1,25(OH)2D3 rapidly stimulated hydrolysis of membrane polyphosphoinositides (PI) in rat colonocytes and in Caco-2 cells, generating the second messengers DAG and IP3. [Ca2+]i subsequently increased due to IP3-mediated release of intracellular Ca2+ stores, and to Ca2+ influx through a receptor-mediated Ca channel. Studies examining purified antipodal plasma membranes and experiments using Caco-2 cell monolayers found that 1,25(OH)2D3 influenced PI turnover only in the basolateral (BLM) and not brush border (BBM) membranes. Vitamin D analogues with poor affinity for the vitamin D receptor were found to effectively stimulate PI turnover, suggesting the presence of a unique vitamin D receptor in the BLM. Studies from our laboratory have demonstrated saturable, reversible binding of 1,25(OH)2 D3 to colonocyte BLM. Recently, we found that 1,25(OH)2D3 activated the tyrosine kinase c-src in colonocyte BLM by a heterotrimeric guanine nucleotide binding protein (G-protein)-dependent mechanism, with subsequent phosphorylation, translocation to the BLM, and activation of PI-specific phospholipase C gamma. Due to the rise in [Ca2+]i and DAG, two isoforms of protein kinase C (PKCalpha and PKCbeta2), but not other isoforms were activated by 1,25(OH)2D3 in rat colonocytes. Recent studies demonstrated that the seco-steroid translocated the beta2 isoform to the BLM, but not the BBM. In contrast, the alpha isoform did not translocate to either antipodal plasma membrane, but modulated IP3-mediated Ca2+ release from the endoplasmic reticulum. Preliminary studies have shown that 1,25(OH)2D3 also activated phosphatidylcholine phospholipase D (PLD) in Caco-2 cells, generating phosphatidic acid and contributing to the sustained rise in DAG. PLD stimulation occurred by both PKC-dependent and -independent mechanisms. Inhibitors of G-proteins, c-src, and PKC blunted the seco-steroid-mediated activation of PLD. Cells stably transfected with sense PKCalpha showed increased 1,25(OH)2D3-stimulated PLD activation, whereas transfectants with antisense PKCalpha had an attenuated response. In addition, 1,25(OH)2D3 also regulated PLD by activating the monomeric G-protein rho A by a mechanism independent of the G-protein/ c-src/PKC pathway.
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PMID:Rapid effects of 1,25(OH)2 vitamin D3 on signal transduction systems in colonic cells. 1032 82

We have recently identified a membrane vitamin D receptor (mVDR) specific for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and shown that it mediates the rapid activation of protein kinase C (PKC) in growth zone chondrocytes (GCs). In this study, we examine the role of the 1, 25(OH)2D3-mVDR in chondrocyte physiology and provide evidence for the existence of a specific membrane receptor for 24, 25-dihydroxyvitamin D3 (24,25(OH)2D3-mVDR). Fourth-passage cultures of growth plate chondrocytes at two distinct stages of endochondral development, resting zone (RC) and growth zone (GC) cells, were used to assess the role of the mVDR in cell proliferation, PKC activation, and proteoglycan sulfation. To preclude the involvement of the nuclear vitamin D receptor (nVDR), we used hybrid analogs of 1, 25(OH)2D3 with <0.1% affinity for the nVDR (2a, 1alpha-CH2OH-3beta-25D3; 3a, 1alpha-CH2OH-3beta-20-epi-22-oxa-25D3; and 3b, 1beta-CH2OH-3alpha-20-epi-22-oxa-25D3). To determine the involvement of the mVDR, we used an antibody generated against the highly purified 1,25(OH)2D3 binding protein from chick intestinal basolateral membranes (Ab99). Analog binding to the mVDR was demonstrated by competition with [3H]1,25(OH)2D3 using matrix vesicles (MVs) isolated from cultures of RC and GC cells. Specific recognition sites for 24,25(OH)2D3 in RC MVs were demonstrated by saturation binding analysis. Specific binding of 24,25(OH)2D3 was also investigated in plasma membranes (PMs) from RC and GC cells and GC MVs. In addition, we examined the ability of Ab99 to block the stimulation of PKC by analog 2a in isolated RC PMs as well as the inhibition of PKC by analog 2a in GC MVs. Like 1,25(OH)2D3, analogs 2a, 3a, and 3b inhibit RC and GC cell proliferation. The effect was dose dependent and could be blocked by Ab99. In GC cells, PKC activity was stimulated maximally by analogs 2a and 3a and very modestly by 3b. The effect of 2a and 3a was similar to that of 1, 25(OH)2D3 and was blocked by Ab99, whereas the effect of 3b was unaffected by antibody. In contrast, 2a was the only analog that increased PKC activity in RC cells, and this effect was unaffected by Ab99. Analog 2a had no effect on proteoglycan sulfation in RC cells, whereas analogs 3a and 3b stimulated it and this was not blocked by Ab99. Binding of [3H]1,25(OH)2D3 to GC MVs was displaced completely with 1,25(OH)2D3 and analogs 2a, 3a, and 3b, but 24, 25(OH)2D3 only displaced 51% of the bound ligand. 24,25(OH)2D3 displaced 50% of [3H]1,25(OH)2D3 bound to RC MVs, but 2a, 3a, and 3b displaced <50%. Scatchard analysis indicated specific binding of 24, 25(OH)2D3 to recognition sites in RC MVs with a Kd of 69.2 fmol/ml and a Bmax of 52.6 fmol/mg of protein. Specific binding for 24, 25(OH)2D3 was also found in RC and GC PMs and GC MVs. GC membranes exhibited lower specific binding than RC membranes; MVs had greater specific binding than PMs in both cell types. 2a caused a dose-dependent increase in PKC activity of RC PMs that was unaffected by Ab99; it inhibited PKC activity in GC MVs, and this effect was blocked by Ab99. The results indicate that the 1, 25(OH)2D3 mVDR mediates the antiproliferative effect of 1,25(OH)2D3 on chondrocytes. It also mediates the 1,25(OH)2D3-dependent stimulation of PKC in GC cells, but not the 2a-dependent increase in RC PKC activity, indicating that 24,25(OH)2D3 mediates its effects through a separate receptor. This is supported by the failure of Ab99 to block 2a-dependent stimulation of PKC in isolated PMs. The data demonstrate for the first time the presence of a specific 24, 25(OH)2D3 mVDR in endochondral chondrocytes and show that, although both cell types express mVDRs for 1,25(OH)2D3 and 24,25(OH)2D3, their relative distribution is cell maturation-dependent.
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PMID:Physiological importance of the 1,25(OH)2D3 membrane receptor and evidence for a membrane receptor specific for 24,25(OH)2D3. 1035 93

Side-chain modified vitamin D analogs including 20-Epi-22-oxa-24a,26a,27a-trihomo-1alpha,2 5-dihydroxyvitamin D3 (KH1060), and 1,24-dihydroxy-22-ene-24-cyclopropyl-vitamin D3 (MC903) were originally designed to aid in the treatment of hyperproliferative disorders including psoriasis and cancer. Here we demonstrate that these analogs, as well as the 6-cis-locked conformer, 1alpha,25-dihydroxy-lumisterol3 (JN) prime NB4 cells for monocytic differentiation. Previously, the action of MC903 and KH1060 was presumed to be mediated by the nuclear vitamin D receptor (VDRnuc). Differentiation in response to all analogs was shown to be inhibited by 1beta,25-dihydroxyvitamin D3 (HL), the antagonist to the nongenomic activities of 1,25D3. These data suggest that although MC903 and KH1060 may bind the VDRnuc, that the differentiative activities of these agents requires nongenomic signaling pathways. Here we show that 1alpha,25(OH)2-d5-previtamin D3 (HF), JN, KH1060, and MC903 induce expression of PKC alpha and PKC delta and translocation of both isoforms to the particulate fraction, and PKC alpha to the nuclear fraction. The full differentiation response with combinations of analogs and TPA was inhibited 50% by the membrane permeable Ca2+ chelator, 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) or calpain inhibitor I. These data demonstrate that intracellular free calcium and the calcium-dependent protease, calpain play critical roles in monocytic differentiation. Intracellular calcium appears to be most critical in the 1,25D3-priming stage of differentiation, while calpain is essential in the TPA maturation response.
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PMID:Vitamin D analogs, 20-Epi-22-oxa-24a,26a,27a,-trihomo-1alpha,25(OH)2-vitamin D3, 1,24(OH)2-22-ene-24-cyclopropyl-vitamin D3 and 1alpha,25(OH)2-lumisterol3 prime NB4 leukemia cells for monocytic differentiation via nongenomic signaling pathways, involving calcium and calpain. 1049 38

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts/stromal cells are involved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracellular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in calcium concentration in culture medium also induced osteoclast formation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-tetra(acetoxymethyl)ester, an intracellular calcium chelator, suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast formation in response to ionomycin in cocultures with these stromal cell lines. OPG strongly inhibited osteoclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL mRNA expression, which, in turn, stimulates osteoclast formation.
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PMID:Intracellular calcium and protein kinase C mediate expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in osteoblasts. 1110 86

Calcitriol (1,25-dihydroxyvitamin D3) induces differentiation and inhibits proliferation of human promyelocytic leukemia cells. The mechanisms involved in the regulation of these processes are not clearly understood. Previous studies have shown that calcitriol mediates cell differentiation not only by interaction with nuclear vitamin D receptor, but also by numerous rapid, membrane--mediated effects. Since in the light of past studies, involvement of raf/MEK1,2/erk1,2 signal transduction pathway in calcitriol-induced cell differentiation was questionable, another attempt was undertaken in this study in order to investigate the problem. PD 98059, the specific inhibitor of MEK1 and MEK2 was found to inhibit calcitriol-induced monocytic differentiation of HL-60 cells. This finding proves that activation of the raf/MEK1,2/erk1,2 signal transduction pathway is essential for monocytic differentiation of human leukemia cells. The results reported in this paper suggest that inhibition of protein kinase C, which upstream regulates activation of erk1 and erk2, may be bypassed during the process of calcitriol-induced leukemia cell differentiation.
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PMID:Evidence that activation of MEK1,2/erk1,2 signal transduction pathway is necessary for calcitriol-induced differentiation of HL-60 cells. 1129 87

In this study, the interrelationship between signal transduction pathways and 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] action was examined in UMR106 osteoblastic cells. Treatment of these cells with 8-bromo-cAMP (1 mM) resulted in an upregulation of the vitamin D receptor (VDR) and an augmentation in the induction by 1,25(OH)2D3 of 25(OH)D3 24-hydroxylase [24(OH)ase] and osteopontin (OPN) mRNAs as well as gene transcription. Transfection with constructs containing the vitamin D response element devoid of other promoter regulatory elements did not alter the cAMP-mediated potentiation, suggesting that cAMP-enhanced transcription is due, at least in part, to upregulation of VDR. Treatment with phorbol ester [12-O-tetradecanoyl-phorbol-13-acetate (TPA) 100 nM], an activator of protein kinase C, significantly enhanced 1,25(OH)2D3-induced OPN mRNA and transcription but had no effect on VDR or on 24(OH)ase mRNA or transcription. Studies using OPN promoter constructs indicate that TPA-enhanced OPN transcription is mediated by an effect on the OPN promoter separate from an effect on VDR. Thus interactions with signal transduction pathways can enhance 1,25(OH)2D3 induction of 24(OH)ase and OPN gene expression, and, through different mechanisms, changes in cellular phosphorylation may play a significant role in determining the effectiveness of 1,25(OH)2D3 on transcriptional control in cells expressing skeletal phenotypic properties.
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PMID:Interrelationship between signal transduction pathways and 1,25(OH)2D3 in UMR106 osteoblastic cells. 1140 34

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha,25(OH)(2)D(3). Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha,25(OH)(2)D(3) in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha,25(OH)(2)D(3) during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha,25(OH)(2)D(3), PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha,25(OH)(2)D(3) was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1alpha,25(OH)(2)D(3) membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha,25(OH)(2)D(3)-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1alpha,25(OH)(2)D(3) in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G(i)/G(s) had no effect. However, inhibition of G(q) blocked the effect of 1alpha,25(OH)(2)D(3). The rapid effect of 1alpha,25(OH)(2)D(3) also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1alpha,25(OH)(2)D(3) at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.
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PMID:1alpha,25(OH)2D3 regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis. 1180

Vitamin D is a secosteroid that is metabolically activated and degraded through the actions of three cytochrome P450 hydroxylase enzymes. Bioactivation occurs through the sequential actions of cytochromes P450C25 and P450C1, resulting in synthesis of the pleiotropic hormone 1,25-dihydroxyvitamin D (1,25VD), which regulates over 60 genes whose actions include those associated with calcium homeostasis and immune responses as well as cellular growth, differentiation, and apoptosis. Inactivation of 1,25VD occurs by C23/C24 oxidation pathways that are catalyzed by the multifunctional cytochrome P450C24 enzyme. Both P450C1 and P450C24 are highly regulated enzymes whose differential expression is controlled in response to numerous cellular modulatory agents such as parathyroid hormone (PTH), calcitonin, interferon gamma, calcium, phosphorus, and pituitary hormones as well as the secosteroid hormone 1,25VD. Most thoroughly studied at the molecular level are the actions of PTH to upregulate P450C1 gene expression and 1,25VD to induce the expression of P450C24. The regulatory action of PTH is mediated through the protein kinase A pathway and involves the phosphorylation of transcription factors that function at the proximal promoter of the P450C1 gene. The upregulation of P450C24 by 1,25VD has both a rapid nongenomic and a slower genomic component that are functionally linked. The rapid response involves protein kinase C and mitogen-activated protein kinase (MAPK) pathways that direct the phosphorylation of nuclear transcription factors. The slower genomic actions are linked to the binding of 1,25VD to the vitamin D receptor (VDR) and the interaction of the VDR-1,25VD complex with its heterodimer partner retinoid-X-receptor and associated coactivators. The regulatory complex is assembled on vitamin D response elements in the proximal promoter of the P450C24 gene and functions to increase the transcription rate.
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PMID:Hydroxylase enzymes of the vitamin D pathway: expression, function, and regulation. 1205 41

This review discusses the regulation of growth plate chondrocytes by vitamin D(3). Over the past ten years, our understanding of how two vitamin D metabolites, 1alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3), exert their effects on endochondral ossification has undergone considerable advances through the use of cell biology and signal transduction methodologies. These studies have shown that each metabolite affects a primary target cell within the endochondral developmental lineage. 1alpha,25-(OH)(2)D(3) affects primarily growth zone cells, and 24R,25-(OH)(2)D(3) affects primarily resting zone cells. In addition, 24R,25-(OH)(2)D(3) initiates a differentiation cascade that results in down-regulation of responsiveness to 24R,25-(OH)(2)D(3) and up-regulation of responsiveness to 1alpha,25-(OH)(2)D(3). 1alpha,25-(OH)(2)D(3) regulates growth zone chondrocytes both through the nuclear vitamin D receptor, and through a membrane-associated receptor that mediates its effects via a protein kinase C (PKC) signal transduction pathway. PKCalpha is increased via a phosphatidylinositol-specific phospholipase C (PLC)-dependent mechanism, as well as through the stimulation of phospholipase A(2) (PLA(2)) activity. Arachidonic acid and its downstream metabolite prostaglandin E(2) (PGE(2)) also modulate cell response to 1alpha,25-(OH)(2)D(3). In contrast, 24R,25-(OH)(2)D(3) exerts its effects on resting zone cells through a separate, membrane-associated receptor that also involves PKC pathways. PKCalpha is increased via a phospholipase D (PLD)-mediated mechanism, as well as through inhibition of the PLA(2) pathway. The target-cell-specific effects of each metabolite are also seen in the regulation of matrix vesicles by vitamin D(3). However, the PKC isoform involved is PKCzeta, and its activity is inhibited, providing a mechanism for differential autocrine regulation of the cell and events in the matrix by these two vitamin D(3) metabolites.
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PMID:Differential regulation of growth plate chondrocytes by 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 involves cell-maturation-specific membrane-receptor-activated phospholipid metabolism. 1209 57

Expression of cytochrome P450 3A4 (CYP3A4) is induced by 1,25-dihydroxyvitamin D(3)(1,25(OH)(2)D(3)) in Caco-2 cells. However, since a typical vitamin D responsive element has not been found in the 5(')-flanking region of the CYP3A4 gene, the mechanism of 1,25(OH)(2)D(3)-induced CYP3A4 mRNA expression is poorly understood. In the present study, we demonstrated that vitamin D receptor (VDR) is a critical factor for the induction using the antisense oligonucleotide technique. In addition, we found that treatment of Caco-2 cells with the protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and the tyrosine kinase inhibitor, genistein, but not with the protein kinase A inhibitor, H-89, suppressed CYP3A4 mRNA induction by 1,25(OH)(2)D(3). The depletion of PKC by prolonged treatment with phorbol ester abolished the induction. On the other hand, protein kinase inhibitors used had no effects on the constitutive expression of VDR mRNA. Therefore, these observations suggest that 1,25(OH)(2)D(3)-induced CYP3A4 mRNA expression might be involved in phosphorylation events in addition to transcriptional regulation via VDR. However, 1,25(OH)(2)D(3) did not rapidly activate PKC in the Caco-2 cells used, while the treatment with staurosporine and GF109203X, but not genistein, decreased basal PKC activity by approximately 30% of the controls. Taken together, these findings suggest that the change in the phosphorylation state via PKC and tyrosine kinase might, at least in part, modulate 1,25(OH)(2)D(3)-induced CYP3A4 mRNA expression via VDR.
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PMID:Alteration of cellular phosphorylation state affects vitamin D receptor-mediated CYP3A4 mRNA induction in Caco-2 cells. 1214 48


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