24,25-(OH)(2)D-3 REGULATES PROTEIN-KINASE-C THROUGH 2 DISTINCT PHOSPHOLIPID-DEPENDENT MECHANISMS

We have previously shown that 24,25-(OH)(2)D-3 plays a major role in r esting zone (RC) chondrocyte differentiation and that this vitamin D m etabolite regulates protein kinase C (PKC). The aim of the present stu dy was to identify the signal transduction pathway used by 24,25-(OH)( 2)D-3 to stimulate PKC activation. Confluent, fourth passage RC cells from rat costochondral cartilage were used to evaluate the mechanism o f PKC activation. Treatment of RC cultures with 24,25(OH)(2)D-3 for 90 min produced a dose-dependent increase in diacylglycerol (DAC). Addit ion of R59022, a diacylglycerol kinase inhibitor, significantly increa sed PKC activity in cultures treated with 24,25-(OH)(2)D-3. Addition o f dioctanoylglycerol (DOG) to plasma membranes isolated from RC increa sed PKC activity 447-fold. Addition of pertussis toxin or cholera toxi n to control cultures elevated basal PKC activity. When added together with 10(-9) M 24,25-(OH)(2)D-3, there was an additive effect on PKC a ctivity but in cultures treated with 10(-8) M 24,25-(OH)(2)D-3, only t he hormone-dependent stimulation of PKC was observed. The phospholipas e C inhibitor, U73-122, had no effect on PKC activity, indicating that the DAG produced in response to 24,25-(OH)(2)D-3 is not derived from phosphatidylinositol. Addition of the tyrosine kinase inhibitor, genis tein, also had no effect on 24,25-(OH)(2)D-3-stimulated PKC, further s upporting the hypothesis that phospholipase C is not involved in the m echanism and that phospholipase D is responsible for the increase in D AG production. Phospholipase A(2) inhibitors, quinacrine and AACOCF3, and the cyclooxygenase inhibitor indomethacin increased PKC activity i n the RC cultures. Exogenous PGE(2), one of the downstream products of phospholipase A(2) action, inhibited PKC activity. These results sugg est that 24,25-(OH)(2)D-3 regulates PKC activity by two distinct phosp holipid-dependent mechanisms: production of DAG via phospholipase D an d inhibition of the production of PGE(2) via inhibition of phospholipa se A(2) and cyclooxygenase. (C) 1996 Wiley-Liss, Inc.