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

1 alpha ,25-(OH)(2)D-3 mediates its effects on growth zone chondrocytes via rapid membrane-associated events as well as through traditional nuclear re ceptor 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 ex amined the roles of PLA(2) and cyclooxygenase (Cox) in the mechanism of act ion of 1 alpha ,25-(OH)(2)D-3 in these sells to determine whether one or bo th enzymes catalyze the rate limiting step and whether constitutive or indu cible Cox is involved. Cultures were incubated with 1 alpha ,25-(OH)(2)D-3 for 9 min to measure PKC or for 24 h to measure physiological responses ([H -3]-thymidine incorporation, alkaline phosphatase specific activity, [S-35] -sulfate incorporation). Based on RT-PCR and Northern blot analysis, growth zone chondrocytes expressed mRNAs for both Cox-1 and Cox-2, and neither Co w was modulated by 1 alpha ,25-(OH)(2)D-3. To examine the role of Cox, the cultures were also treated with resveratrol ia specific inhibiter of Cox-1) , NS-398 (a specific inhibitor oi Cox-2, or indomethacin (a general Cos inh ibitor). The results showed that Cox-1 inhibition reduced the 1 alpha ,25-( OH)(2)D-3-depenclent 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 experiment s in which PLA(2) was activated with melittin or inhibited with quinacrine. However, at least part of the action of 1 alpha ,25-(OH)(2)D-3 was regulat ed by metabolism of arachidonic acid to prostaglandins. This supports the h ypothesis that 1 alpha ,25-(OH)(2)D-3 exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulati on of PLA2 as a rate-limiting step. PKC regulation may occur at multiple st ages in the signal transduction cascade. (C) 2001 Wiley-Liss, Inc.