Mechanisms Determining Epithelial Basement Membrane Inhibition of the Fibrotic Repair Response in Cornea

Abstract

Abstract: : Purpose: Refractive surgical procedures such as LASIK and epi–LASIK are designed to limit the fibrotic repair response, thought to be mediated by an epithelial–stromal interaction. We recently demonstrated that the corneal epithelium stimulates fibrotic marker expression by releasing TGF–beta2 into the corneal stroma (Stramer et al., 2003, IOVS). We also reported that release of TGF–beta2 in vivo is inhibited by the presence of an intact basement membrane and in vitro by plating cells on a mixture of basement membrane components (matrigel). The goal of the current study was to define further the molecular mechanisms for this inhibition. Methods: Human corneal epithelial cells explanted from corneal rims were plated on matrigel or the purified basement membrane component, laminin–1. The next day, cells were left untreated or treated with TGF–beta2. Three days after plating, TGF–beta2 promoter activity was assayed using a beta–lactamase reporter vector driven by the TGF–beta2 promoter. Intracellular TGF–beta2 mRNA levels were quantified by quantitative Reverse Transcription–Polymerase Chain Reaction (qRT–PCR). Results:As is the case for many cytokines, TGF–betas are known to stimulate expression of their own genes via an autocrine feedback loop. One mechanism for matrigel inhibition of TGF–beta2 release may be binding of TGF–beta2, preventing its ability to signal cells. We found that TGF–beta2 promoter activity and mRNA levels were reduced (2.8 +/– 0.5 fold and 1.2 +/– 0.2 fold, respectively) when cells were plated on matrigel as opposed to plating on laminin. These results indicate that laminin is insufficient to mediate the matrigel effect. TGF–beta2 treatment increased TGF–beta2 promotion (2.4 +/– 0.3 fold) in cells plated on laminin, but not in cells plated on matrigel, consistent with autocrine loop inhibition. Conclusions: The results suggest that inhibition of TGF–beta2 release from epithelial cells by matrigel is regulated at the level of the transcriptional promoter and support the concept that matrigel inhibits TGF–beta2 release by inhibiting the TGF–beta2 autocrine feedback loop. It appears that the laminin component of matrigel is not sufficient to inhibit TGF–beta2 transcriptional promoter activity. Supported by NIH grants R01 EY09828 and P30 EY14081, and an unrestricted grant from Research to Prevent Blindness. MEF holds the Walter G. Ross Chair in Ophthalmic Research.

Keywords: cornea: epithelium • extracellular matrix • gene/expression