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Inhibition of Human Corneal Epithelial Production of Fibrotic Mediator TGF-ß2 by Basement Membrane–Like Extracellular Matrix

¹From the Evelyn F. and William L. McKnight Vision Research Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida; the ²Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey.

PURPOSE. Transforming growth factor (TGF)-ß2 is a major epithelial mediator of fibrotic marker expression during corneal repair in mice. Production of TGF-ß2 protein by cultured rabbit corneal epithelial cells is reduced by plating on a basement membrane–like extracellular matrix extract (Matrigel; BD Biosciences, Bedford, MA). The goal of the present study was to understand further the nature of Matrigel regulation.

METHODS. TGF-ß2 protein, mRNA, and gene transcriptional promotion were characterized in cultured human corneal epithelial cells.

RESULTS. TGF-ß2 production was inhibited by Matrigel at the level of mRNA accumulation and activity of the gene transcriptional promoter. This effect of Matrigel was not explained by (1) growth factor contaminants, as growth-factor reduced Matrigel also inhibited TGF-ß2; (2) independent matrix components, as the pure forms of the major ECM components laminin and collagen IV did not reproduce the effect; or (3) inhibition of a constitutive TGF-ß2 autocrine feedback loop, as addition of exogenous TGF-ß2 increased p-Smad3 and restored TGF-ß2 mRNA levels. In addition, Matrigel’s ability to reduce TGF-ß2 was not explained by its geometry, as TGF-ß2 production was not inhibited by plating cells on a synthetic nanofiber matrix with a three-dimensional topography similar to Matrigel. Matrigel caused a reduction of ezrin, a member of the ezrin-radixin-moesin (ERM) family, which plays a role in establishing polarity of epithelial cells in tissues through the Rho signaling pathway.

CONCLUSIONS. These findings indicate that Matrigel inhibits TGF-ß2 gene expression and point to a mechanism dependent on Matrigel composition and structure. The capacity of Matrigel to reduce ezrin is consistent with this idea and directs the focus of future studies toward the ERM/Rho pathway.