Recent studies have shown that 24R, 25-(OH)2D3 mediates its effects on growth plate chondrocytes via membrane receptors. This study examined the roles of phospholipase A2 (PLA2) and cyclooxygenase (Cox) in the mechanism of action of 24R,25-(OH)2D3 in resting zone chondrocytes in order to determine whether the activity of one or both enzymes provides a regulatory checkpoint 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)2D3 for 90 min to measure PKC or for 24 h to measure physiological responses ([3H]-thymidine incorporation, alkaline phosphatase-specific activity, [35S]-sulfate incorporation). Based on RT-PCR and Northern blot analysis, resting zone chondrocytes express mRNAs for both Cox-1 and Cox-2. Levels of mRNA for both proteins were unchanged from control levels after a 24-h incubation with 24R,25-(OH)2D3. To examine the role of Cox, the cultures were also treated with resveratrol (a specific inhibitor of Cox-1), NS-398 (a specific inhibitor of Cox-2), or indomethacin (a general Cox inhibitor). Cox-1 inhibition resulted in effects on proliferation, differentiation, and matrix production typical of 24R,25-(OH)2D3. In contrast, inhibition of Cox-2 had no effect, indicating that 24R, 25-(OH)2D3 exerts its effects via Cox-1. Inhibition of Cox-1 also blocked 24R, 25-(OH)2D3-dependent increases in PKC. Activation of PLA2 with melittin inhibited 24R, 25-(OH)2D3-dependent stimulation of PKC, and inhibition of PLA2 with quinacrine stimulated PKC in response to 24R,25(OH)2D3. Inclusion of resveratrol reduced the melittin- dependent inhibition of PLA2 and caused an increase in quinacrine-stimulated PLA2 activity. Metabolism of arachidonic acid to leukotrienes is not involved in the response to 24R,25-(OH)2D3 because inhibition of lipoxygenase had no effect. The effect of 24R,25(OH)2D3 was specific because 24S, 25-(OH)2D3, the biologically inactive stereoisomer, failed to elicit a response from the cells. These results support the hypothesis that 24R,25-(OH)2D3 exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulation of PLA2. PKC regulation may occur at multiple stages in the signal transduction cascade.
|Original language||English (US)|
|Number of pages||12|
|Journal||Journal of Cellular Physiology|
|Publication status||Published - Jan 29 2000|
ASJC Scopus subject areas
- Clinical Biochemistry
- Cell Biology