Regulation of protein kinase C by transforming growth factor β1 in rat costochondral chondrocyte cultures

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Abstract
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.
Funding Information
  • U.S. Public Health Service (DE-08603, DE-05937)
  • U.S. Department of Veterans Affairs/Department of Defense
  • U.S./Israel Binational Science Foundation