Add time:07/19/2019         Source:sciencedirect.com

1α,25-(OH)2D3 regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)2D3 regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase 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 24R,25-(OH)2D3 and determined the role of phospholipases D, C, and A2 as well as G-proteins in mediating the effects of vitamin D3 metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)2D3. Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)2D3. This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)2D3 on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1α,25-(OH)2D3-dependent PKC. Inhibition of PI-PLC blocked the 1α,25-(OH)2D3-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA2 with melittin inhibited basal and 24R,25-(OH)2D3-stimulated PKC in RC cells and stimulated basal and 1α,25-(OH)2D3-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)2D3 on PKC, whereas GDPβS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1α,25-(OH)2D3 regulates PKC in GC cells via PI-PLC and PLA2, but not PC-PLC or PLD, whereas 24R,25-(OH)2D3 regulates PKC in RC cells via PLD2.

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