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G. Sosne, L. Xu, L. Prach, L. Mrock, H.K. Kleinman, J. Letterio, L.D. Hazlett, M. Kurpakus-Wheater; Thymosin Beta 4 Stimulated Corneal Laminin-5 Production Is Not Mediated by TGF-Beta . Invest. Ophthalmol. Vis. Sci. 2003;44(13):861.
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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.
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