1,25(OH)(2) VITAMIN-D-3 ACTIVATES PKC-ALPHA IN CACO-2 CELLS - A MECHANISM TO LIMIT SECOSTEROID-INDUCED RISE IN [CA2+](I)

Our laboratory recently reported that 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+](i)), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)(2)D-3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)(2)D-3-stimulated increase in [Ca2+](i). As previously demonstrated by our laboratory, 1,25(OH)(2)D-3 caused a biphasic increase in [Ca2+](i), with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)(2)D-3-induced increase in the transient phase of [Ca2+](i). Preincubation of Caco-2 cells with 1,2-bis(2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) abolished both the rise in [Ca2+](i) and the increase in particulate-associated PKC-a stimulated by 1,25(OH)(2)D-3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)(2)D-3-stimulated rise in [Ca2+](i) but had no effect on the 1,25(OH)(2)D-3-induced change in Ins(1,4,5)P-3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+](i). These results indicate that 1,25(OH)(2)D-3, which increases [Ca2+](i) and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+](i), at a step distal to Ins(1,4,5)P-3 accumulation in Caco-2 cells.