Thymosin Beta 4 Stimulated Corneal Laminin-5 Production Is Not Mediated by TGF-Beta

Abstract

Abstract: : Purpose: Previously, we found that thymosin ß₄ promotes laminin-5 production in conjunctival epithelial cells. The purpose of this project is to test the hypothesis that thymosin ß₄ increases corneal epithelial cell laminin-5 production and to determine whether it occurs through a TGF-beta (TGF-ß) related pathway. Methods: Human corneal epithelial cells at 50-60% confluence were cultured with and without thymosin ß₄ (1000ng/ml) for 24 hours. RT-PCR, ELISA, and Western blot analysis were used to determine laminin-5 γ2 chain and TGF-ß1 gene transcript and protein levels. In vitro, cells were treated with thymosin ß₄ or TGF-ß1 in the presence or absence of neutralizing antibody to TGF-ß1. Cell lysates were subjected to Western blot analysis to determine laminin-5 γ2 chain protein expression levels. In vivo, TGF-ß1 knockout mice were subjected to a 1-mm epithelial scrape wound and treated topically with either PBS or thymosin ß₄ (5µg/5µl PBS) (n = 5 per group). After partial healing, RT-PCR was used to determine gene transcript levels for laminin-5. Results: Thymosin ß₄ treatment increased gene and protein levels of laminin-5 γ2 chain and TGF-ß1. Inhibiting TGF-ß1 with neutralizing antibody resulted in a significant up-regulation of laminin-5 γ2 chain protein in corneal epithelial cells, regardless of the presence or absence of exogenous TGF-ß1 or thymosin ß₄. TGF-ß1 knockout mice demonstrated higher laminin-5 γ2 chain gene transcript levels compared to wild-type mice. Thymosin ß₄ treatment increased gene transcription levels for laminin-5 γ2 chain in both wild-type (1.5x) and knockout mice (>2x). Conclusions: Thymosin ß₄ treatment of cultured human corneal epithelial cells or of corneal epithelium in vivo following wounding results in increased laminin-5 γ2 chain and TGF-ß1 expression. Since this increase in laminin is observed in TGF-ß1 knockout mice and in the presence of TGF-ß1 neutralizing antibodies, we conclude that thymosin ß₄ does not increase laminin-5 γ2 chain expression via a TGF-ß1 related pathway.

Keywords: cornea: epithelium • extracellular matrix • cytokines/chemokines