24R,25-(OH)(2)D-3 mediates its membrane receptor-dependent effects on protein kinase C and alkaline phosphatase via phospholipase A(2) and cyclooxygenase-1 but not cyclooxygenase-2 in growth plate chondrocytes

Recent studies have shown that 24R,25-(OH)(2)D-3 mediates its effects on gr owth plate chondrocytes via membrane receptors. This study examined the rol es of phospholipase A(2) (PLA(2)) and cyclooxygenase (Cox) in the mechanism of action of 24R,25-(OH)(2)D-3 in resting zone chondrocytes in order to de termine whether the activity of one or both enzymes provides a regulatory c heckpoint in the signaling pathway resulting in increased protein kinase C (PKC) activity. We also determined whether constitutive or inducible Cox is involved. Cultures were incubated with 24R,25-(OH)(2)D-3 for 90 min to mea sure PKC or for 24 h to measure physiological responses ([H-3]-thymidine in corporation, alkaline phosphatase-specific activity, [S-35]-sulfate incorpo ration). Based on RT-PCR and Northern blot analysis, resting zone chondrocy tes express mRNAs for both Cox-1 and Cox-2. Levels of mRNA for both protein s were unchanged from control levels after a 24-h incubation with 24R,25-(O H)(2)D-3. To examine the role of Cox, the cultures were also treated with r esveralrol (a specific inhibitor of Cox-1), NS-398 (a specific inhibitor of Cox-2), or indomethacin (a general Cox inhibitor). Cox-1 inhibition result ed in effects on proliferation, differentiation, and matrix production typi cal of 24R25-(OH)(2)D-3. In contrast, inhibition of Cox-2 had no effect, in dicating that 24R,25-(OH)(2)D-3 exerts its effects via Cox-1. Inhibition of Cox-1 also blocked 24R,2 5-(OH)(2)D-3-dependent increases in PKC. Activati on of PLA(2) with melittin inhibited 24R,25-(OH)(2)D-3-dependent stimulatio n of PKC, and inhibition of PLA(2) with quinacrine stimulated PKC in respon se to 24R,25(OH)(2)D-3. Inclusion of resveratrol reduced the melittin-depen dent inhibition of PLA(2) and caused an increase in quinacrine-stimulated P LA(2) activity. Metabolism of arachidonic acid to leukotrienes is not invol ved in the response to 24R,25-(OH)(2)D-3 because inhibition of lipoxygenase had no effect. The effect of 24R,25(OH)(2)D-3 was specific because 24S,25- (OH)(2)D-3, the biologically inactive stereoisomer, failed to elicit a resp onse from the cells. These results support the hypothesis that 24R,25-(OH)( 2)D-3 exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulation of PLA(2). PKC regulation may occur at multiple stages in the signal transduction cascade. (C) 2000 Wile y-Liss, Inc.