Transforming growth factor β (TGF‐β) regulates the proliferation and differentiation of chondrocytes; however, the mechanism of TGF‐β signal transduction remains unclear. We examined whether the response to TGF‐β is mediated by protein kinase C activity in chondrocytes at different stages of maturation. The aims were to examine the effect of recombinant human TGF‐β1 (rhTGF‐β1) on protein kinase C in rat costochondral chondrocyte cultures; determine the major isoform present; assess the involvement of phospholipase C or tyrosine kinases; determine whether genomic or nongenomic pathways are involved; and test whether these mechanisms differ as a function of the stage of cell maturation. Dose‐dependent increases in protein kinase C activity were observed in confluent, fourth‐passage cultures of rat costochondral growth zone and resting zone chondrocytes treated with rhTGF‐β1. In growth zone cells, elevated activity was observed at 12 h and decreased markedly by 24 h. In resting zone cells, elevated activity was observed at 9 h, maximum stimulation occurred at 12 h, and activity returned to baseline levels after 48 h. Immunoprecipitation studies showed protein kinase C α is the major isoform present in both untreated and treated cells. Neither the phospholipase C inhibitor, U73122, nor the tyrosine kinase inhibitor, genistein, significantly reduced the protein kinase C response to rhTGF‐β1. Actinomycin D and cycloheximide, inhibitors of transcription and translation, produced dose‐dependent inhibition of rhTGF‐β1 stimulated protein kinase C activity 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‐β1 stimulation of protein kinase C in costochondral chondrocytes. Similarly, because genistein had no effect, tyrosine kinases are not implicated. Rather, the reduction in protein kinase C activity observed when rhTGF‐β1 is administered 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‐β1 is mediated by protein kinase C; however, it is very slow and may require new protein kinase C production, perhaps via a cytokine cascade. Moreover, the classic mechanism of activation of protein kinase C by phospholipase C was not found, suggesting a novel mechanism of activation. Finally, the effects of rhTGF‐β1 on protein kinase C are dependent on the state of cell maturation with respect to onset and duration of response.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Orthopedics and Sports Medicine