Inhibition of human corneal epithelial production of fibrotic mediator TGF-beta2 by basement membrane-like extracellular matrix.

PURPOSE

Transforming growth factor (TGF)-beta2 is a major epithelial mediator of fibrotic marker expression during corneal repair in mice. Production of TGF-beta2 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-beta2 protein, mRNA, and gene transcriptional promotion were characterized in cultured human corneal epithelial cells.

RESULTS

TGF-beta2 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-beta2; (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-beta2 autocrine feedback loop, as addition of exogenous TGF-beta2 increased p-Smad3 and restored TGF-beta2 mRNA levels. In addition, Matrigel's ability to reduce TGF-beta2 was not explained by its geometry, as TGF-beta2 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-beta2 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.