1,25(OH)2D3 regulates protein kinase C activity through two phospholipid-dependent pathways involving phospholipase A2 and phospholipase C in growth zone chondrocytes

We have previously shown that 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study nas to identify the signal transduction pathway used by 1,25(OH)(2)D-3 to stimulate PKC activation, Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation, Treatment of GC cultures, with 1,25(OH)(2)D-3 elicited a dose-dependent increase in both inositol-1,4,5-trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D, Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity, Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)(2)D-3-treated GCs, indicating that neither G(i) nor G(s) proteins were involved, Phospholipase A(2) inhibitors, quinacrine, OEPC (selective for secretory phospholipase A(2)), and AACOCF(3) (selective for cytosolic phospholipase A(2)), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A(2) activators melittin and mastoparan increased PKC activity in GC cultures, Arachidonic acid and prostaglandin E-2, two downstream products of phospholipase A(2) action, also increased PKC activity, These results indicate that 1,25(OH)(2)D-3-dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A(2).