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G Sosne, S Hafeez, L Mrock, M Kurpakus-Wheater; Thymosin Beta 4 Upregulates Laminin-5 in Cultured Ocular Surface Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4202.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the effect of exogenous thymosin ß4 on ocular surface epithelial cell motility, laminin-5 production, and integrin expression. Methods: Cultured human corneal and conjunctival epithelial cells treated with a range of thymosin ß4 concentrations from 1 ng/ml to 5000 ng/ml were used in this study. As the control, cells were cultured without thymosin ß4-treatment. A classical Boyden chamber migration assay was employed to determine the effect of thymosin ß4 on conjunctival epithelial cell migration. Double-label immunofluorescence microscopy using antibodies to laminin-5 and integrin α6 was performed to determine the effect of thymosin ß4 on laminin-5 and integrin localization. Western blot analysis and densitometry was completed using antibodies to laminin γ2 chain to determine the effect of thymosin ß4 on laminin-5 production. Results: In cultured human conjunctival epithelial cells, the presence of thymosin ß4 significantly increased migration compared to controls. This result agrees with the previous observation of the first author using human corneal epithelial cells. Immunofluorescence microscopy demonstrated that although no differences in labeling intensity could be noted in control vs. thymosin ß4-treated cells, the localization patterns of laminin-5 did appear to be correlated to the presence of the factor. As the concentration of thymosin ß4 increased, the extracellular trail of laminin-5 localized more prominently to aggregates. Integrin α6 co-localization with laminin-5 was observed. To provide a more quantitiative analysis of laminin-5 content, Western blot analysis and densitometry of cell-extracellular matrix lysates was completed. Thymosin ß4-treatment resulted in increased laminin-5 production in both corneal and conjunctival epithelial cells. Conclusion: Thymosin ß4 promotes corneal and conjunctival epithelial cell migration in vitro. Our findings suggest that thymosin ß4 may promote cell migration via increased laminin-5 production and laminin-5/integrin interactions.
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