1,25-dihydroxyvitamin D3 and 12-O-tetradecanoyl phorbol 13-acetate cause differential activation of Ca2+-dependent and Ca2+-independent isoforms of protein kinase C in rat colonocytes

Abstract

Considerable evidence that alterations in protein kinase C (PKC) are intimately involved in important physiologic and pathologic processes in many cells, including colonie epithelial cells, has accumulated. In this regard, phorbol esters, a class of potent PKC activators, have been found to induce a number of cellular events in normal or transformed colonocytes. In addition, our laboratory has demonstrated that the major active metabolite of vitamin D3, 1,25(OH)2D3, also rapidly (seconds-minutes) activated PKC and increased intracellular calcium in isolated rat colonocytes. These acute responses, however, were lost in vitamin D deficiency and partially restored with the in vivo repletion of 1,25(OH)2D3. The Ca2+-independent or novel isoforms of PKC expressed in the rat colon and the isoform-specific responses of PKC to acute treatment with phorbol esters or 1,25(OH)2D3 have not been previously characterized. Moreover, the effects of vitamin D status on PKC isoform expression, distribution, and response to agonists are also unknown. In the present experiments, in addition to PKC-α, rat colonocytes were found to express the novel isoforms δ, ∈, and ζ by Western blotting using isoform-specific PKC antibodies. The tumor-promoting phorbol ester, 12-0-tetra-decanoyl phorbol 13-acetate, caused time- and concentration-dependent translocations of all these isoforms except PKC-ζ. In vitamin D deficiency, there were no alterations in colonie PKC isoform expression but significant changes in the subcellular distribution of PKC-α, -δ, and -ζ. Acute treatment of colonocytes from D-sufficient, but not D-deficient, rats with 1,25(OH)2D3 caused a rapid transient redistribution of only PKC-α from the soluble to the particulate fraction. The alterations in PKC isoform distribution and PKC-α responsiveness to 1,25(OH)2D3 in vitamin D deficiency were partially, but significantly, restored with 5-7 d in vivo repletion of this secosteroid. Both 12-O-tetradecanoyl phorbol 13-acetate and 1,25(OH)2D3 activated endogenous PKC, as assessed by inhibition of myristoylated alanine-rich C kinase substrate back-phosphorylation by exogenous PKC. These studies indicate that PKC-α, -δ, and/or -∈ likely mediate important phorbol ester-stimulated events described in the rat colon. In contrast, PKC-α is implicated in the rapid (s-min) PKC-dependent events initiated by 1,25(OH)2D3 in rat colonocytes.