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N. Sundarraj, E. Guerriero, J. Chen; MAPK Pathways in the Regulation of b-FGF and TGF-beta1 Induced Keratocyte Activation in vitro. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2751.
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Corneal stromal keratocytes cultured in serum-free media can be activated to wound healing phenotypes with b-FGF or TGF-beta1. The goal of the present study was to evaluate the signaling mechanisms that regulate the phenotypic changes associated with growth factor-induced keratocyte activation.
Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum free (SF) medium. They were treated with b-FGF/heparin or TGF-beta1 with or without inhibitors of JNK, ERK and p38 (SP600125, PD-098059 and SB-203580, respectively). Specific phenotypic changes were analyzed by immunohistochemistry and western blotting, and the relative abundance of specific mRNAs was estimated by quantitative RT-PCR.
Rabbit corneal stromal cells cultured in serum free (SF) medium expressed alpha3(IV) collagen, keratan sulfate proteoglycans (KSPGs: lumican and keratocan), and corneal crystallins including ALDH1. Activation of keratocytes with b-FGF/heparin or TGF-beta1 resulted in downregulation of keratocan, lumican, ALDH1 and alpha3(IV) collagen, and upregulation of tenascin-C and fibronectin mRNAs and proteins. TGF-beta1 also induced expression of alpha-smooth muscle actin (SMA). TGF-beta1- and b-FGF- promoted expression of tenascin-C and fibronectin and TGF-beta1-promoted expression of alpha-SMA were inhibited by the inhibition of ERK, JNK or p38. However, bFGF or TGF-beta1-induced down regulation of alpha3(IV) collagen was prevented by inhibition of ERK but not of JNK or p38 signaling. Growth factor-induced downregulation of keratocan and ALDH1 was prevented by the inhibition of JNK but not ERK or p38 signaling pathways.
Phenotypic changes associated with growth factor-induced corneal stromal activation are differentially regulated by activation of one or more MAPK pathways, namely, JNK, ERK and p38 signaling pathways. All three MAPKs regulate TGF-beta1- and b-FGF-induced expression tenascin-C and fibronectin and TGF-beta1- induced expression of alpha-SMA. However, the loss in the expression of alpha3(IV) collagen, and that of KSPGs and ALDH1 are differentially regulated by ERK and JNK signaling pathways, respectively.
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