The role of protein kinase C in vitamin D-mediated effects in kidney.

Abstract

The role of the calcium/phospholipid-dependent serine/threonine kinase Protein Kinase C (PKC), in the effects of vitamin D on kidney was studied. Madin Darby Bovine Kidney (MDBK) cells, a normal epithelial-like cell line, were found to express the Vitamin D Receptor (VDR) and the vitamin D-dependent calcium binding protein Calbindin D-28K (CaBP D-28K). In MDBK cells, 1,25(OH)$\sb2\rm D\sb3$ increased PKC activity in a time- and dose-dependent manner as measured by two different phosphorylation assays. This activation appeared to result from translocation of PKC from the cytosol to the membrane and was accompanied by an increase in immunoreactive PKC in the membrane. PKC was also activated by short term exposure of MDBK cells to TPA, whereas PKC activity was completely down-regulated by long term exposure to TPA. Down-regulation of PKC activity was accompanied by a loss of immunoreactive PKC. The phorbol ester analogue 4$\alpha$PDD had no effect on PKC activity or amount. Concurrent with activation of PKC 1,25(OH)$\sb2\rm D\sb3$ homologously up-regulated the VDR and increased total immunoreactive CaBP D-28K in MDBK cells. In contrast, down-regulation of PKC activity in TPA treated cells was associated with decreased expression of the VDR and CaBP D-28K. The phorbol ester analogue 4$\alpha$PDD, which had no effect on PKC, did not affect the expression of the VDR or CaBP D-28K. Short term TPA treatment, which activated PKC, increased CaBP D-28K without altering VDR levels. The divergent effects of 1,25(OH)$\sb2\rm D\sb3$ and TPA were associated with differential regulation of PKC isozymes. Treatment of MDBK cells with 1,25(OH)$\sb2\rm D\sb3$ increased membrane association of PKC $\alpha,$ induced nuclear translocation of PKC $\beta$ and had no effect on PKC $\zeta.$ In contrast, long term treatment of MDBK cells with TPA induced down-regulation of PKC $\alpha,$ nuclear translocation of PKC $\beta,$ and decreased PKC $\zeta.$ Nuclear translocation of PKC $\beta$ by 1,25(OH)$\sb2\rm D\sb3$ treatment was accompanied by an increase in phosphorylation of endogenous nuclear proteins. However nuclear translocation of PKC $\beta$ by TPA treatment did not affect phosphorylation of endogenous nuclear proteins. The data have been incorporated into a model based on the hypothesis that the VDR could be a nuclear substrate for PKC $\beta$ and CaBP D-28K could be a cytosolic substrate for PKC $\alpha.$ This model predicts a novel role for PKC-dependent phosphorylations in the renal actions of 1,25(OH)$\sb2\rm D\sb3.$