1,25(OH)(2)D-3 REGULATES PROTEIN-KINASE-C ACTIVITY THROUGH 2 PHOSPHOLIPID-DEPENDENT PATHWAYS INVOLVING PHOSPHOLIPASE-A(2) 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 use d by 1,25(OH)(2)D-3 to stimulate PKC activation, Confluent, fourth pas sage GC cells from costochondral cartilage were used to evaluate the m echanism 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-trisphos phate and diacylglycerol (DAG) production, suggesting a role for phosp holipase C and potentially for phospholipase D, Addition of dioctanoyl glycerol 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) inhi bitors, quinacrine, OEPC (selective for secretory phospholipase A(2)), and AACOCF(3) (selective for cytosolic phospholipase A(2)), and the c yclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A(2) activators melittin and mastoparan increased PKC a ctivity in GC cultures, Arachidonic acid and prostaglandin E-2, two do wnstream products of phospholipase A(2) action, also increased PKC act ivity, These results indicate that 1,25(OH)(2)D-3-dependent stimulatio n of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and prod uction of arachidonic acid via phospholipase A(2).