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J.V. Jester, J. Huang; TGFß Induced Myofibroblast Differentiation in Rabbit Corneal Keratocytes Requires p38 but Not ERK1/2 Signaling . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2305.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous studies show that inhibition of PDGF or integrin signaling blocks TGFß induced myofibroblast differentiation without affecting TGFß receptor mediated SMAD signaling. The purpose of this study was to identify possible alternative signal transduction pathways that become activated during myofibroblast differentiation which lead to expression of alpha smooth muscle actin (α–SMA). Methods: Primary rabbit corneal keratocytes and serum starved immortalized human corneal fibroblasts were treated with 1 ng/ml TGFß in the presence or absence of inhibitors to PI–3Kinase (LY294002), ERK1/2 (PD98059) and p38 kinase (SB203580). Myofibroblast differentiation was then assessed by immunocytochemistry and western blotting for α–SMA. Cell proliferation was assessed by Ki67 staining and cell counting. Immunopreciptation and western blotting using antibodies specific for p38 assessed phosphorylation of p38. Results: Inhibition of ERK1/2 using PD98059 at 40 uM completely blocked cell cycle entry induced by TGFß after 3 days in culture but had no effect on cell spreading or expression of α–SMA. Treatment of keratocytes with LY294002 showed a dose dependent inhibition of α–SMA expression and cell cycle entry. LY294002 also showed a dose dependent reduction in the number of keratocytes while treatment with LY294002 alone showed no effect suggesting that inhibition of PI–3K induces apoptosis of activated and differentiating corneal keratocytes and acted downstream of myofibroblast differentiation. Keratocytes treated with SB203580, however, showed a dose dependent decrease in expression of α–SMA that was completely inhibited by 30 uM without affecting cell proliferation. Furthermore, treatment of TGFß–modified myofibroblasts with SB203480 significantly reversed myofibroblast differentiation. Immunoprecipitation experiments showed phosphorylation of p38 beginning at 2 hours after TGFß treatment that was inhibited by SB203580. Conclusions: These data indicate that myofibroblast differentiation involves a p38 mediated signal transduction cascade. Furthermore, cell proliferation is not required for myofibroblast differentiation. Overall these finding suggest new approaches to controlling myofibroblast differentiation in vivo.
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