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 D₃ (1,25(OH)₂D₃) 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 was to identify the signal transduction pathway used by 1,25(OH)₂D₃ 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)₂D₃ 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)₂D₃-treated GCs, indicating that neither G(i) nor G(s) proteins were involved. Phospholipase A₂ inhibitors, quinacrine, OEPC (selective for secretory phospholipase A₂), and AACOCF₃ (selective for cytosolic phospholipase A₂), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A₂ activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E₂, two downstream products of phospholipase A₂ action, also increased PKC activity. These results indicate that 1,25(OH)₂D₃-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 A₂.