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)

The effect of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) on vitamin D receptors (VDRs) was studied in MDBK cells, a normal bovine renal epithelial cell line. 24 h treatment of MDBK cells with TPA resulted in down-regulation of VDR number, with no change in the binding affinity for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or approximate molecular weight determined by fast protein liquid chromatography (FPLC). TPA treatment also reduced the level of calbindin D-28K, a vitamin D-dependent renal protein. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), an inactive phorbol ester, did not affect either 1,25(OH)2D3 binding or calbindin D-28K levels. TPA elicited a significant decrease in membrane-associated protein kinase C (PKC) activity which coincided with the reduction in VDR number and calbindin D-28K. These data support a link between TPA, PKC activity and vitamin D actions in kidney.
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PMID:TPA decreases 1,25(OH)2D3 binding and calbindin D-28K in renal (MDBK) cells. 131 89

To determine whether 1,25-dihydroxycholecalciferol [1,25(OH)2D3] affects protein kinase C (PKC) activity in kidney, as has been demonstrated in HL-60 cells we measured 1,25(OH)2D3 binding, PKC activity and PKC immunoreactivity in Madin Darby bovine kidney (MDBK) cells, a normal renal epithelial cell line derived from bovine kidney. Our data demonstrate that MDBK cells exhibit specific high affinity binding for 1,25(OH)2D3, indicating the presence of the vitamin D receptor (VDR). Treatment of MDBK cells with 1,25(OH)2D3 for 24 h increased membrane PKC activity and immunoreactivity. The effect of 1,25(OH)2D3 was dose-dependent, with a peak effect observed at 10(-7)M 1,25(OH)2D3. The 1,25(OH)2D3 induced increase in membrane PKC was paralleled by a comparable decrease in cytosolic PKC activity and amount. Although time course studies were consistent with a VDR mediated effect of 1,25(OH)2D3 on PKC protein synthesis, total PKC activity was not increased by 1,25(OH)2D3, suggesting an effect on PKC translocation or localization. These results suggest that 1,25(OH)2D3 modulates PKC mediated events in kidney, a classic target for this steroid hormone.
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PMID:1,25(OH)2D3 increases membrane associated protein kinase C in MDBK cells. 131 48

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.
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PMID:Evidence for the functional involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 in bone. 132 1

The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino acid sequence of human VDR (hVDR) reveals the conservation of three potential sites of phosphorylation by protein kinase C (PKC)--namely, Ser-51, Ser-119, and Ser-125. Immunoprecipitated extracts derived from a rat osteoblast-like osteosarcoma cell line that contains the VDR in high copy number were incubated with the alpha, beta, and gamma isozymes of PKC, and VDR proved to be an effective substrate for PKC-beta, in vitro. When hVDR cDNAs containing single, double, and triple mutations of Ser-51, Ser-119, and Ser-125 were expressed in CV-1 monkey kidney cells, immunoprecipitated and phosphorylated by PKC-beta, in vitro, the mutation of Ser-51 selectively abolished phosphorylation. Furthermore, when transfected CV-1 cells were treated with phorbol 12-myristate 13-acetate, a PKC activator, phosphorylation of wild-type hVDR was enhanced, whereas that of the Ser-51 mutant hVDR was unaffected. Therefore, Ser-51 is the site of hVDR phosphorylation by PKC, both in vitro and in vivo. To evaluate the functional role of Ser-51 and its potential phosphorylation, hVDR-mediated transcription was tested using cotransfection with expression plasmids and a reporter gene that contained a vitamin D response element. Mutation of Ser-51 markedly inhibited transcriptional activation by the vitamin D hormone, suggesting that phosphorylation of Ser-51 by PKC could play a significant role in vitamin D-dependent transcriptional activation. Therefore, the present results link the PKC signal transduction pathway of growth regulation and tumor promotion to the phosphorylation and function of VDR.
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PMID:Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function. 165 68

Studies of the relationship between PTH structure and function in the activation of protein kinases have revealed that different regions within the biologically active PTH-(1-34) peptide are responsible for different functions. The first two N-terminal amino acids are required for plasma membrane adenylyl cyclase stimulation, and the C-terminal region 29-32 is necessary for the translocating activity of protein kinase C. In the present study, we explored the structure-function relationship of human (h) PTH in the regulation of the vitamin D receptor (VDR) in osteoblast-like cells (ROS 17/2.8). VDR-rich cytosol extract was prepared after the confluent cells were incubated with different hPTH fragments for 16 h. hPTH-(1-34) at concentrations of 10(-9)-10(-7) M caused a dose-dependent decrease in VDR content from a control level of 70.2 +/- 2.2 fmol/mg protein to 62.1 +/- 3.3 (-16%) at 10(-9) M, 52.3 +/- 5.3 (-25.5%; P < 0.02) at 10(-8) M, and 45.5 +/- 3.5 fmol/mg protein (-35.3%; P = 0.001) at 10(-7) M (n = 6). hPTH-(1-31) also decreased VDR content from 65.5 +/- 3.6 to 55.2 +/- 7.9 (-19.5%) at 10(-9) M, 44.3 +/- 5.8 (-32.4%; P < 0.05) at 10(-8) M, and 40.6 +/- 3.2 fmol/mg protein (-38.9%; P < 0.05) at 10(-7) M (n = 6). Incubation of ROS 17/2.8 cells with 0.5 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] led to up-regulation of VDR content by 340-370% of the control value. hPTH-(1-34) decreased the VDR up-regulatory effect of 1,25-(OH)2D3 from 340% to 230% of the control value at 10(-8) M (P < 0.0001) and 170% of the control value (P < 0.0001) at 10(-7) M, respectively (n = 6). hPTH-(1-31) also decreased the receptor up-regulatory effect of 1,25-(OH)2D3 from 370% to 286% (P < 0.02) at 10(-8) M and 220% (P < 0.002) at 10(-7) M, respectively (n = 6). hPTH-(3-34) and -(13-34) at concentrations of 10(-9)-10(-7) M did not decrease VDR content in either the absence or presence of 1,25-(OH)2D3. Quantitation of VDR messenger RNA by reverse transcription-polymerase chain reaction showed that PTH-(1-34) and -(1-31) at 10(-7) M, but not PTH-(3-34) and -(13-34), inhibited ROS 17/2.8 cell VDR gene expression in both the absence and presence of 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure-function relationship of human parathyroid hormone in the regulation of vitamin D receptor expression in osteoblast-like cells (ROS 17/2.8). 764 79

1 alpha,25-Dihydroxyvitamin D3 (10(-12) M to 10(-8) M) caused a dose dependent increase in PKC activity in the solubilized membrane fractions of cultured human keratinocytes and in the cytosolic fractions of cultured human fibroblasts. Maximum activity was induced by 1 alpha,25-dihydroxyvitamin D3 at 24 h. Sphingosine, which is believed to inhibit PKC mediated biological responses, blunted 1 alpha,25(OH)2D3's inducement of PKC activity in both keratinocytes and fibroblasts. Identical hormone treatment of vitamin D receptor deficient fibroblasts did not increase PKC activity. Treatment of keratinocytes and fibroblasts with 1 beta,25-dihydroxyvitamin D3, which is believed to be ineffective in inducing genomic responses, did not induce PKC activity.
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PMID:Increased PKC activity in cultured human keratinocytes and fibroblasts after treatment with 1 alpha, 25-dihydroxyvitamin D3. 775 71

The regulation of vitamin D receptor (VDR) abundance in MC3T3-E1 mouse osteoblasts and UMR 106-01 rat osteosarcoma cells by rat PTH 1-34, human PTH-related protein 1-34, and agents that activate specific signal transduction pathways was studied. Treatment of these cells with forskolin (FSK) caused up-regulation of VDR, whereas treatment with phorbol esters suppressed VDR levels. PTH or PTH-related protein treatment induced a 2- to 3-fold increase in VDR, which was equivalent to that elicited by FSK in UMR 106-01 cells but less than the FSK-induced increase (approximately 8-fold) in MC3T3-E1 cells. PTH treatment of MC3T3-E1 cells resulted in an approximately 3-fold increase in VDR levels with maximum stimulation occurring at 10(-9) M PTH after 4 h of treatment. In UMR 4-7 cells, a subclone of UMR 106-01 cells that express cAMP resistance due to regulated expression of a mutant form of the type 1 regulatory subunit of the cAMP-dependent protein kinase A (PKA), the up-regulation of VDR abundance due to FSK and PTH treatment was mostly prevented. Pretreatment of MC3T3-E1 cells with staurosporine, an inhibitor of PKC, resulted in an approximately 3-fold increase in basal VDR levels but did not enhance the PTH-mediated up-regulation of VDR. Collectively, these data suggest that the increase in VDR abundance observed in these target cells is mainly due to the activation of the PKA signal transduction pathway. Treatment of UMR 106-01 cells with PTH for 4 h before exposure of the cells to 1,25-dihydroxyvitamin D3 resulted in a 2-fold increase in the induction of 25-hydroxyvitamin D3-24 hydroxylase messenger RNA. Thus, exposure of target cells to PTH augments their response to 1,25-dihydroxyvitamin D3 due to up-regulation of VDR abundance.
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PMID:Regulation of 1,25-dihydroxyvitamin D3 receptors by parathyroid hormone in osteoblastic cells: role of second messenger pathways. 783 3

Control of osteoblast growth and development can be characterized from receptor mediated events to nuclear messengers controlling gene transcription. From this analysis it is possible to formulate a model to explain the reciprocal relationship between growth and differentiation as well as differential cytokine modulation of osteoblast function. Central to this model are putative tissue specific transcriptional switches (possibly of the bHLH gene superfamily) that may repress proliferation and permit the regulation of mature osteoblast phenotypic characteristics. This model proposes that in post-mitotic differentiated osteoblasts, tissue specific transcription factors determine the capacity to express osteoblastic characteristic, whereas receptor activated signalling cascades, namely, cAMP/protein kinase A, receptor serine/threonine kinase, and vitamin D receptor-dependent pathways, regulate mature osteoblast-specific gene expression. Activated differentiation switches also may feedback to transcriptionally repress proliferation. Conversely, in preosteoblasts, in which differentiation switches are turned off, distinct signalling cascades involving tyrosine kinases, PKC, and calcium/calmodulin regulate proliferation. Proliferating preosteoblasts also exhibit negative modulation of maturation either through inactivation of putative tissue-specific transcription factors and/or through AP-1 dependent phenotype suppression of genes expressed in mature osteoblast. Thus, the final outcome of transcriptional regulation of osteoblast function results from complex interactions between signalling pathways and permissive differentiating transcription factors. Though many aspects of this model remain speculative and require confirmation, it serves as a useful conceptual framework to further investigate the differential control of osteoblast proliferation and differentiation that may lead to improved pharmacologic ways to manipulate bone formation in vivo.
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PMID:Molecular to pharmacologic control of osteoblast proliferation and differentiation. 796 62

In this paper we demonstrate the existence of specific, high affinity 17-beta estradiol binding in MDBK cells (a normal non-transformed renal cell line). Scatchard analysis revealed binding characteristics typical of the estrogen receptor (ER). Only unlabelled 17-beta estradiol, diethyl stilbestrol and estrone effectively competed with [3H] 17-beta estradiol for binding, other steroids did not compete. Short term TPA treatment of MDBK cells with TPA increased PKC activity and immunoreactivity and caused a transient increase in 17-beta estradiol binding, while longer term treatment with TPA decreased PKC activity and immunoreactivity. The inactive phorbol ester 4 alpha PDD was without effect on PKC activity and the ER. TPA did not affect the affinity of the ER for the nucleus nor did it increase degradation of the receptor. We hypothesize that the renal ER may be a substrate for PKC and its properties can be modified by PKC similarly to the vitamin D receptor.
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PMID:Modulation of a renal estrogen receptor by protein kinase C. 808 31

Effects of protein kinase C (PKC) inhibitor and activator on 1,25(OH)2D3-induced gene expression were examined in rat intestinal epithelial cells, IEC-6 cells. A potent PKC inhibitor, H-7 (20 microM), completely abated 1,25(OH)2D3-induced 24-hydroxylase gene expression at 3 and 6 h. The effect of H-7 was dose dependent with IC50 around 5 microM. Other protein kinase inhibitors, HA-1004 and H-89 (20 microM), had no effects. Furthermore, the activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) potentiated the effect of 1,25(OH)2D3 by 1 h. TPA appeared to exert its effect at a transcriptional step, since mRNA stability was not affected by TPA treatment. At 3 h after the treatment of the cells with H-7 and TPA, vitamin D receptor (VDR) contents estimated by 3H-1,25(OH)2D3 binding capacity were 72.4 and 63.2% of vehicle-treated cells without significant changes of binding affinities, suggesting that the effect of H-7 and TPA was not the result of changes in VDR content or its binding affinity. In conclusion, PKC is involved in 1,25(OH)2D3-induced 24-hydroxylase gene expression in IEC-6 cells between 1,25(OH)2D3-VDR binding and VDR-induced gene transactivation.
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PMID:Protein kinase C is involved in 24-hydroxylase gene expression induced by 1,25(OH)2D3 in rat intestinal epithelial cells. 808 98


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