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Regulation of protein kinase C by transforming growth factor β 1 in rat costochondral chondrocyte cultures
Author(s) -
Sylvia V.L.,
Mackey S.,
Schwartz Z.,
Schuman L.,
Gomez R.,
Boyan B.D.
Publication year - 1994
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650090921
Subject(s) - chondrocyte , biology , transforming growth factor beta , protein kinase c , tyrosine kinase , kinase , endocrinology , cycloheximide , protein kinase a , medicine , signal transduction , microbiology and biotechnology , protein biosynthesis , biochemistry , in vitro
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‐β 1 (rhTGF‐β 1 ) 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‐β 1 . 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‐β 1 . Actinomycin D and cycloheximide, inhibitors of transcription and translation, produced dose‐dependent inhibition of rhTGF‐β 1 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‐β 1 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‐β 1 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‐β 1 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‐β 1 on protein kinase C are dependent on the state of cell maturation with respect to onset and duration of response.