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 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C.
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.
Schwartz
Z
Z
Department of Orthopaedics, The University of Texas Health Science Center at San Antonio, 78284, USA.
Sylvia
V L
VL
Luna
M H
MH
DeVeau
P
P
Whetstone
R
R
Dean
D D
DD
Boyan
B D
BD
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D013237:Stereoisomerism; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D020962:Heterotrimeric GTP-Binding Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010739:Phospholipase D; D010741:Phospholipases A; D011493:Protein Kinase C; D011509:Proteoglycans; D013431:Sulfates; D010738:Type C Phospholipases; D014807:Vitamin D; D000077191:Wortmannin