REGULATION OF PROTEIN-KINASE-C BY TRANSFORMING GROWTH-FACTOR BETA(1) IN RAT COSTOCHONDRAL CHONDROCYTE CULTURES

Transforming growth factor beta (TGF-beta) regulates the proliferation and differentiation of chondrocytes; however, the mechanism of TGF-be ta signal transduction remains unclear. We examined whether the respon se to TGF-beta is mediated by protein kinase C activity in chondrocyte s at different stages of maturation. The aims were to examine the effe ct of recombinant human TGF-beta(1) (rhTGF-beta(1)) on protein kinase C in rat costochondral chondrocyte cultures; determine the major isofo rm present; assess the involvement of phospholipase C or tyrosine kina ses; determine whether genomic or nongenomic pathways are involved; an d test whether these mechanisms differ as a function of the stage of c ell maturation. Dose-dependent increases in protein kinase C activity were observed in confluent, fourth-passage cultures of rat costochondr al growth zone and resting zone chondrocytes treated with rhTGF-beta(1 ). In growth zone cells, elevated activity was observed at 12 h and de creased markedly by 24 h. In resting zone cells, elevated activity was observed at 9 h, maximum stimulation occurred at 12 h, and activity r eturned to baseline levels after 48 h. Immunoprecipitation studies sho wed protein kinase C alpha is the major isoform present in both untrea ted and treated cells. Neither the phospholipase C inhibitor, U73122, nor the tyrosine kinase inhibitor, genistein, significantly reduced th e protein kinase C response to rhTGF-beta(1). Actinomycin D and cycloh eximide, inhibitors of transcription and translation, produced dose-de pendent inhibition of rhTGF-beta(1) stimulated protein kinase C activi ty in both resting zone and growth zone chondrocytes. The time course of activation and insensitivity to U73122 suggest that phospholipase C -mediated events are not involved in rhTGF-beta(1) stimulation of prot ein kinase C in costochondral chondrocytes. Similarly, because geniste in had no effect, tyrosine kinases are not implicated. Rather, the red uction in protein kinase C activity observed when rhTGF-beta(1) is adm inistered along with actinomycin D or cycloheximide indicates that new gene expression and protein synthesis are required for the response. These results indicate that the effect of rhTGF-beta(1) is mediated by protein kinase C; however, it is very slow and may require new protei n kinase C production, perhaps via a cytokine cascade. Moreover, the c lassic mechanism of activation of protein kinase C by phospholipase C was not found, suggesting a novel mechanism of activation. Finally, th e effects of rhTGF-beta(1) on protein kinase C are dependent on the st ate of cell maturation with respect to onset and duration of response.