1,25-DIHYDROXYVITAMIN D-3 AND 12-O-TETRADECANOYL PHORBOL 13-ACETATE CAUSE DIFFERENTIAL ACTIVATION OF CA2-DEPENDENT AND CA2+-INDEPENDENT ISOFORMS OF PROTEIN-KINASE-C IN RAT COLONOCYTES()

Considerable evidence that alterations in protein kinase C (PKC) are i ntimately involved in important physiologic and pathologic processes i n many cells, including colonic epithelial cells, has accumulated. In this regard, phorbol esters, a class of potent PKC activators, have be en found to induce a number of cellular events in normal or transforme d colonocytes. In addition, our laboratory has demonstrated that the m ajor active metabolite of vitamin D-3, 1,25(OH)(2)D-3, also rapidly (s econds-minutes) activated PKC and increased intracellular calcium in i solated rat colonocytes. These acute responses, however, were lost in vitamin D deficiency and partially restored with the in vivo repletion of 1,25(OH)(2)D-3. The Ca2+-independent or novel isoforms of PKC expr essed in the rat colon and the isoform-specific responses of PKC to ac ute treatment with phorbol esters or 1,25(OH)(2)D-3 have not been prev iously characterized. Moreover, the effects of vitamin D status on PKC isoform expression, distribution, and response to agonists are also u nknown. In the present experiments, in addition to PKC-alpha, rat colo nocytes were found to express the novel isoforms delta, epsilon, and z eta by Western blotting using isoform-specific PKC antibodies. The tum or-promoting phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate, cau sed time- and concentration-dependent translocations of all these isof orms except PKC-zeta. In vitamin D deficiency, there were no alteratio ns in colonic PKC isoform expression but significant changes in the su bcellular distribution of PKC-alpha, -delta, and -zeta. Acute treatmen t of colonocytes from D-sufficient, but not D-deficient, rats with 1,2 5(OH)(2)D-3 caused a rapid transient redistribution of only PKC-alpha from the soluble to the particulate fraction. The alterations in PKC i soform distribution and PKC-alpha responsiveness to 1,25(OH)(2)D-3 in vitamin D deficiency were partially, but significantly, restored with 5-7 d in vivo repletion of this secosteroid. Both 12-O-tetradecanoyl p horbol 13-acetate and 1,25(OH)(2)D-3 activated endogenous PKC, as asse ssed by inhibition of myristoylated alanine-rich C kinase substrate ba ck-phosphorylation by exogenous PKC. These studies indicate that pKC-a lpha, -delta, and/or -epsilon likely mediate important phorbol ester-s timulated events described in the rat colon. In contrast, PKC-alpha is implicated in the rapid (s-min) PKC-dependent events initiated by 1,2 5(OH)(2)D-3 in rat colonocytes.