1,25(OH)2 vitamin D3 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 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 alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 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)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-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha 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-alpha 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-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)P3 accumulation in Caco-2 cells.
Bissonnette
M
M
Department of Medicine, University of Chicago, Illinois 60637.
Tien
X Y
XY
Niedziela
S M
SM
Hartmann
S C
SC
Frawley
B P
BP
Roy
H K
HK
Sitrin
M D
MD
Perlman
R L
RL
Brasitus
T A
TA
D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D004789:Enzyme Activation; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000145:classification / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium