TGFβ Induced Myofibroblast Differentiation of Rabbit Keratocytes Requires Synergistic TGFβ, PDGF and Integrin Signaling

Abstract

There is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes which undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast. Both in vivo and in vitro studies have shown this transition to be dependent, in part, on transforming growth factor beta (TGFβ). In many fibroblastic cells autocrine production of platelet derived growth factor (PDGF) is known to mediate the growth up-regulation by TGFβ. In this study, blocking antibodies to PDGF significantly reduced by 80% (P<0.025) the TGFβ1 stimulated cell cycle entry of serum-free cultured rabbit corneal keratocytes. AntiPDGF treatment also markedly reduced the TGFβ1-induced intracellular actin filament re-organization, fibronectin fibril assembly, and focal contact formation as well as reducing by 80% the expression of α-smooth muscle (α-SM) specific isoform of actin characteristic of myofibroblast differentiation. Although PDGF treatment of quiescent keratocytes produced an activated, fibroblastic cell type, PDGF stimulated keratocytes exhibited the same temporal, myofibroblastic differentiation response to TGFβ1 as did quiescent keratocytes. Furthermore, blocking TGFβ1 induction of myofibroblast differentiation with the Arg–Gly–Asp containing peptide, GRGDdSP, for 3 days followed by allowing progression of myofibroblast differentiation by removing GRGDdSP did not change the temporal response or tyrosine phosphorylation cascade (2–72hr) leading to myofibroblast differentiation. Nor did PDGF treatment of keratocytes reverse the RGD blockade of TGFβ1 induced myofibroblast differentiation. Overall these cumulative findings indicate that myofibroblast differentiation in the rabbit corneal keratocyte requires synergistic growth factor/integrin signaling involving TGFβ, PDGF, and the fibronectin receptor. Additionally, the similar TGFβ1 temporal response of PDGF-stimulated compared to naı̈ve keratocytes suggests that myofibroblast differentiation does not require transition through a fibroblast phenotype.