Effects of 1alpha,25-(OH)(2)D(3) on rat growth zone chondrocytes are mediated via cyclooxygenase-1 and phospholipase A(2).

Abstract

1alpha,25-(OH)(2)D(3) mediates its effects on growth zone chondrocytes via rapid membrane-associated events as well as through traditional nuclear receptor mechanisms. The membrane-associated signaling pathways include rapid production of diacylglycerol and activation of protein kinase C (PKC), as well as activation of phospholipase A(2) (PLA(2)), increased production of arachidonic acid, and increased production of prostaglandins. This study examined the roles of PLA(2) and cyclooxygenase (Cox) in the mechanism of action of 1alpha,25-(OH)(2)D(3) in these cells to determine whether one or both enzymes catalyze the rate limiting step and whether constitutive or inducible Cox is involved. Cultures were incubated with 1alpha,25-(OH)(2)D(3) for 9 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, growth zone chondrocytes expressed mRNAs for both Cox-1 and Cox-2 and neither Cox was modulated by 1alpha,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). The results showed that Cox-1 inhibition reduced the 1alpha,25-(OH)(2)D(3)-dependent effects on proliferation, differentiation, and matrix production, whereas inhibition of Cox-2 only had an effect on proliferation. The effects of Cox inhibition were not rate limiting, based on experiments in which PLA(2) was activated with melittin or inhibited with quinacrine. However, at least part of the action of 1alpha,25-(OH)(2)D(3) was regulated by metabolism of arachidonic acid to prostaglandins. This supports the hypothesis that 1alpha,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) as a rate-limiting step. PKC regulation may occur at multiple stages in the signal transduction cascade. J. Cell. Biochem. Suppl. 36: 32-45, 2001.