PURPOSE: TGFbeta is the major mediator to induce myofibroblast differentiation in the corneal wound-healing process. Elevated cAMP can reduce TGFbeta-induced fibrosis in other tissues. This study was conducted to determine whether elevated cAMP can inhibit TGFbeta1-induced rabbit corneal keratocyte-myofibroblast transformation. METHODS: Primary isolated rabbit corneal keratocytes were cultured in serum-free medium. The effects of the adenylate cyclase agonist forskolin (FSK; 2 microM) on TGFbeta1 (5 ng/mL)-induced alpha-smooth muscle actin (alpha-SMA) expression was examined by immunofluorescence, flow cytometry, and immunochemistry 72 hours after treatment. The effects of TGFbeta+FSK on activated pSmad3, CREB binding protein (CBP), MAPKs, and RhoA were determined by coimmunoprecipitation and Western blot. RESULTS: FSK significantly reduced the myofibroblast phenotype and alpha-SMA expression induced by TGFbeta1 in rabbit corneal keratocytes. TGFbeta1 increased the phosphorylation of ERK and Smad3. TGFbeta1-induced alpha-SMA expression was reduced by MEK inhibition (U0126); however, the levels of pERK, pSmad3, or the extent of the interaction between pSmad3 and CBP induced by TGFbeta1 were not affected by FSK. TGFbeta1 also activated RhoA and ROCK (Y27632) inhibition reduced alpha-SMA expression. Activation of RhoA was significantly reduced by FSK. CONCLUSIONS: Raising cAMP by FSK treatment inhibits the TGFbeta1-induced corneal myofibroblast transformation and alpha-SMA expression and thereby provides a promising method to control corneal fibrosis. The data suggest that cAMP-dependent inhibition does not occur by altering Smads or MAPK signaling, but possibly by reducing the activation of RhoA.
PURPOSE:TGFbeta is the major mediator to induce myofibroblast differentiation in the corneal wound-healing process. Elevated cAMP can reduce TGFbeta-induced fibrosis in other tissues. This study was conducted to determine whether elevated cAMP can inhibit TGFbeta1-induced rabbit corneal keratocyte-myofibroblast transformation. METHODS: Primary isolated rabbit corneal keratocytes were cultured in serum-free medium. The effects of the adenylate cyclase agonist forskolin (FSK; 2 microM) on TGFbeta1 (5 ng/mL)-induced alpha-smooth muscle actin (alpha-SMA) expression was examined by immunofluorescence, flow cytometry, and immunochemistry 72 hours after treatment. The effects of TGFbeta+FSK on activated pSmad3, CREB binding protein (CBP), MAPKs, and RhoA were determined by coimmunoprecipitation and Western blot. RESULTS:FSK significantly reduced the myofibroblast phenotype and alpha-SMA expression induced by TGFbeta1 in rabbit corneal keratocytes. TGFbeta1 increased the phosphorylation of ERK and Smad3. TGFbeta1-induced alpha-SMA expression was reduced by MEK inhibition (U0126); however, the levels of pERK, pSmad3, or the extent of the interaction between pSmad3 and CBP induced by TGFbeta1 were not affected by FSK. TGFbeta1 also activated RhoA and ROCK (Y27632) inhibition reduced alpha-SMA expression. Activation of RhoA was significantly reduced by FSK. CONCLUSIONS: Raising cAMP by FSK treatment inhibits the TGFbeta1-induced corneal myofibroblast transformation and alpha-SMA expression and thereby provides a promising method to control corneal fibrosis. The data suggest that cAMP-dependent inhibition does not occur by altering Smads or MAPK signaling, but possibly by reducing the activation of RhoA.
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