Inhibition of human corneal epithelial production of fibrotic mediator TGF-beta2 by basement membrane-like extracellular matrix.
OBJECTIVE
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.
LaGier
Adriana J
AJ
Evelyn F. and William L. McKnight Vision Research Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA.
Yoo
Sonia H
SH
Alfonso
Eduardo C
EC
Meiners
Sally
S
Fini
M Elizabeth
ME
D001672:Biocompatible Materials / Q000494:pharmacology*; D002460:Cell Line; D003094:Collagen / Q000494:pharmacology*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004338:Drug Combinations; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000473:pathology; D005109:Extracellular Matrix; D005355:Fibrosis / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D015151:Immunoblotting; D007797:Laminin / Q000494:pharmacology*; D011509:Proteoglycans / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053781:Transforming Growth Factor beta2 / Q000235:genetics*; D000099149:Ezrin
D001672:Biocompatible Materials; D003094:Collagen; D003598:Cytoskeletal Proteins; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D012333:RNA, Messenger; D053781:Transforming Growth Factor beta2; D000099149:Ezrin; C059485:matrigel