1,25-Dihydroxyvitamin D3 and 12-O-tetradecanoyl phorbol 13-acetate cause differential activation of Ca(2+)-dependent and Ca(2+)-independent isoforms of protein kinase C in rat colonocytes.
Considerable evidence that alterations in protein kinase C (PKC) are intimately involved in important physiologic and pathologic processes in many cells, including colonic 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 Ca(2+)-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-alpha, rat colonocytes were found to express the novel isoforms delta, epsilon, and zeta by Western blotting using isoform-specific PKC antibodies. The tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate, caused time- and concentration-dependent translocations of all these isoforms except PKC-zeta. In vitamin D deficiency, there were no alterations in colonic PKC isoform expression but significant changes in the subcellular distribution of PKC-alpha, -delta, and -zeta. 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-alpha from the soluble to the particulate fraction. The alterations in PKC isoform distribution and PKC-alpha 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-alpha, -delta, and/or -epsilon likely mediate important phorbol ester-stimulated events described in the rat colon. In contrast, PKC-alpha is implicated in the rapid (s-min) PKC-dependent events initiated by 1,25(OH)2D3 in rat colonocytes.
Bissonnette
M
M
Department of Medicine, University of Chicago, Illinois 60637.
Wali
R K
RK
Hartmann
S C
SC
Niedziela
S M
SM
Roy
H K
HK
Tien
X Y
XY
Sitrin
M D
MD
Brasitus
T A
TA
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003106:Colon / Q000187:drug effects / Q000201:enzymology*; D015870:Gene Expression / Q000187:drug effects; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D011493:Protein Kinase C / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012016:Reference Values; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014808:Vitamin D Deficiency / Q000201:enzymology
D007527:Isoenzymes; D011493:Protein Kinase C; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium