ABSTRACT
1alpha,25-(OH)(2)D(3) 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)(2)D(3) 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)(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 caused a dose-dependent inhibition of basal [3H]-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 [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 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)(2)D(3) 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 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(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 1alpha,25-(OH)(2)D(3)-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)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,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.
Subject(s)
Chondrocytes/drug effects , Chondrocytes/enzymology , Phospholipase D/metabolism , Protein Kinase C/metabolism , Type C Phospholipases/metabolism , Vitamin D/analogs & derivatives , Vitamin D/pharmacology , Androstadienes/pharmacology , Animals , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Heterotrimeric GTP-Binding Proteins/antagonists & inhibitors , Heterotrimeric GTP-Binding Proteins/metabolism , Models, Biological , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phospholipase D/antagonists & inhibitors , Phospholipases A/metabolism , Proteoglycans/metabolism , Rats , Stereoisomerism , Substrate Specificity , Sulfates/metabolism , Type C Phospholipases/antagonists & inhibitors , WortmanninABSTRACT
Many of the effects of 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 on costochondral chondrocytes are mediated by the protein kinase C (PKC) signal transduction pathway. 1alpha,25-(OH)2D3 activates PKC in costochondral growth zone chondrocytes through a specific membrane receptor (1alpha,25-mVDR), involving rapid increases in diacylglycerol via a phospholipase C (PLC)-dependent mechanism. 24R,25-(OH)2D3 activates PKC in resting zone chondrocytes. Although diacylglycerol is increased by 24R,25-(OH)2D3, PLC is not involved, suggesting a phospholipase D (PLD)-dependent mechanism. Here, we show that resting zone and growth zone cells express mRNAs for PLD1a, PLD1b, and PLD2. Both cell types have PLD activity, but levels are higher in resting zone cells. 24R,25-(OH)2D3, but not 24S,25-(OH)2D3 or 1alpha,25-(OH)2D3, stimulates PLD activity in resting zone cells within 3 min via nongenomic mechanisms. Neither 1alpha,25-(OH)2D3 nor 24R,25-(OH)2D3 affected PLD in growth zone cells. Basal and 24R,25-(OH)2D3-stimulated PLD were inhibited by the PLD inhibitors wortmannin and EDS. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase), PKC, phosphatidylinositol-specific PLC (PI-PLC), and phosphatidylcholine-specific PLC (PC-PLC) had no effect on PLD activity. Thus, 24R,25-(OH)2D3 stimulates PLD, and PI 3-kinase, PI-PLC and PKC are not involved, whereas PLD is required for stimulation of PKC by 24R,25-(OH)2D3. Pertussis toxin, GDPbetaS, and GTPgammaS had no effect on 24R,25-(OH)2D3-dependent PLD when added to cell cultures, indicating that G-proteins are not involved. These data show that PKC activation in resting zone cells is mediated by PLD and suggest that a functional 24R,25-(OH)2D3-mVDR is required. The results also support the conclusion that the 24R,25-(OH)2D3-responsive PLD is PLD2, since this PLD isoform is G-protein-independent.
