Transforming growth factor-β1 regulation of growth zone chondrocytes is mediated by multiple interacting pathways

Transforming growth factor beta 1 (TGF-β1) affects growth plate chondrocytes through Smad-mediated mechanisms and has been shown to increase protein kinase C (PKC). This study determined if PKC mediates the physiological response of rat costochondral growth zone (GC) chondrocytes to TGF-β1; if the physiological response occurs via type II or type III TGF-β receptors, and, if so, which receptor mediates the increase in PKC; and the signal transduction pathways involved. Treatment of confluent GC cells with TGF-β1 stimulated [³H]thymidine and [³⁵S]sulfate incorporation as well as alkaline phosphatase (ALPase) and PKC specific activities. Inhibition of PKC with chelerythrine, staurosporine, or H-7 caused a dose-dependent decrease in these parameters, indicating that PKC signaling was involved. TGF-β1-dependent PKC and the physiological response of GC cells to TGF-β1 was reversed by anti-type II TGF-β receptor antibody and soluble type II TGF-β receptor, showing that TGF-β1 mediates these effects through the type II receptor. The increase in [³H]thymidine incorporation and ALPase specific activity were also regulated by protein kinase A (PKA) signaling, since the effects of TGF-β1 were partially blocked by the PKA inhibitor H-8. The mechanism of TGF-β1 activation of PKC is through phospholipase A₂ (PLA₂) and not through phospholipase C (PLC). Arachidonic acid increased PKC in control cultures and was additive with TGF-β1. Prostanoids are required, as indomethacin blocked the effect of TGF-β1, and Cox-1, but not Cox-2, is involved. TGF-β1 stimulates prostaglandin E₂ (PGE₂) production and exogenous PGE₂ stimulates PKC, but not as much as TGF-β1, suggesting that PGE₂ is not sufficient for all of the prostaglandin effect. In contrast, TGF-β1 was not regulated by diacylglycerol; neither dioctanoylglycerol (DOG) nor inhibition of diacylglycerol kinase with R59022 had an effect. G-proteins mediate TGF-β1 signaling at different levels in the cascade. TGF-β1-dependent increases in PGE₂ levels and PKC were augmented by the G protein activator GTPγS, whereas inhibition of G-protein activity via GDPβS, pertussis toxin, or cholera toxin blocked stimulation of PKC by TGF-β1, indicating that both G_i and G_s are involved. Inhibition of PKA with H-8 partially blocked TGF-β1-dependent PKC, suggesting that PKA inhibition on the physiological response was via PKA regulation of PKC signaling. This indicates that multiple interacting signaling pathways are involved: TGF-β1 stimulates PLA₂ and prostaglandin release via the action of Cox-1 on arachidonic acid. PGE₂ activates the EP2 receptor, leading to G-protein-dependent activation of PKA. PKA signaling results in increased PKC activity and PKC signaling regulates proliferation, differentiation, and matrix synthesis.