24R,25-(OH)2D3 mediates its membrane receptor-dependent effects on protein kinase C and alkaline phosphatase via phospholipase A2 and cyclooxygenase-1 but not cyclooxygenase-2 in growth plate chondrocytes
✍ Scribed by Z. Schwartz; V.L. Sylvia; F. Del Toro; R.R. Hardin; D.D. Dean; B.D. Boyan
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 308 KB
- Volume
- 182
- Category
- Article
- ISSN
- 0021-9541
No coin nor oath required. For personal study only.
✦ Synopsis
Recent studies have shown that 24R,25-(OH) 2 D 3 mediates its effects on growth plate chondrocytes via membrane receptors. This study examined the roles 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 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) 2 D 3 for 90 min to measure PKC or for 24 h to measure physiological responses ([ 3 H]-thymidine incorporation, alkaline phosphatase-specific activity, [ 35 S]-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) 2 D 3 . 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) 2 D 3 . In contrast, inhibition of Cox-2 had no effect, indicating that 24R,25-(OH) 2 D 3 exerts its effects via Cox-1. Inhibition of Cox-1 also blocked 24R,25-(OH) 2 D 3 -dependent increases in PKC. Activation of PLA 2 with melittin inhibited 24R,25-(OH) 2 D 3 -dependent stimulation of PKC, and inhibition of PLA 2 with quinacrine stimulated PKC in response to 24R,25-(OH) 2 D 3 . Inclusion of resveratrol reduced the melittin-dependent inhibition of PLA 2 and caused an increase in quinacrine-stimulated PLA 2 activity. Metabolism of arachidonic acid to leukotrienes is not involved in the response to 24R,25-