The effect of 24R,25-(OH)(2)D-3 on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1 alpha,25-(OH)(2)D-3 is mediated by phospholipase C

1 alpha ,25-(OH)(2)D-3 regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol specific ph ospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D-3 regulates PKC activity in resting zo ne (RC) cells, but PLC does not appear to be involved, suggesting that phos pholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24 R,25-(OH)(2)D-3 and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D-3 metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS c aused a dose-dependent inhibition of basal [H-3]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D-3. Wortmannin also inhibited basal alkaline phosphatase, activity and [S-35] -sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)( 2)D-3. This inhibitory effect of wortmannin was not seen in cultures treate d with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affect ed PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D-3 on this enzyme activity. Neithe r inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC act ivity. Wortmannin had no effect on basal PLD in GC cells, nor on 1 alpha ,2 5-(OH)(2)D-3-dependent PKC. Inhibition of PI-PLC blocked the 1 alpha ,25-(O H)(2)D-3-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R, 25-(OH )(2)D-3-stimulated PKC in RC cells and stimulated basal and 1 alpha ,25-(OH )(2)D-3-stimulated PKC in GC cells, but wortmannin had no effect on the mel ittin-induced changes in either cell type. Pertussis toxin modestly increas ed the effect of 24R,25-(OH)(2)D-3 on PKC, whereas GDP betaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1 alp ha ,25-(OH)(2)D-3 regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D-3 regulates PKC in RC cells via PLD2 . (C) 2001 Elsevier Science Inc. All rights reserved.