1,25(OH)₂ vitamin D₃ activates PKC-α in Caco-2 cells: A mechanism to limit secosteroid-induced rise in [Ca²⁺](i)

Our laboratory recently reported that 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] 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 α- and ζ-isoforms of PKC, with minimally detectable amounts of PKC-β and -ε by Western blotting. 1,25(OH)₂D₃ and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-α, but not PKC-ζ. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-α, but not PKC-ζ. 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)₂D₃-stimulated increase in [Ca²⁺](i). As previously demonstrated by our laboratory, 1,25(OH)₂D₃ 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)P₃] 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)₂D₃-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-acetoxy-methyl ester (BAPTA- AM) abolished both the rise in [Ca²⁺](i) and the increase in particulate- associated PKC-α stimulated by 1,25(OH)₂D₃. Moreover, downregulation of PKC-α by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)₂D₃-stimulated rise in [Ca²⁺](i) but had no effect on the 1,25(OH)₂D₃-induced change in Ins(1,4,5)P₃ concentration. Furthermore, in these PKC-α downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca²⁺](i). These results indicate that 1,25(OH)₂D₃, which increases [Ca²⁺](i) and diacylglycerol, activates PKC-α, but not PKC-ζ. The α-isoform, in turn, limits the secosteroid- stimulated rise in [Ca²⁺](i), at a step distal to Ins(1,4,5)P₃ accumulation in Caco-2 cells.