The Regulatory Influence of Type IV Collagen, Laminin and Fibronectin on the 5 Integrin Subunit Gene Promoter Activity in Rabbit Corneal Epithelial Cells

Abstract

Purpose:: Damage to the corneal epithelium results in the rapid secretion of fibronectin (FN), a transitory component from the extracellular matrix that support epithelial cells adhesion and migration during corneal wound healing. In contrast, type IV collagen (CIV) and laminin (LM), two major constituents of the basement membrane, temporarily disappears until the denuded area is completely covered and then sequentially reappears beneath the newly reconstructed epithelium. In response to the massive secretion of FN, expression of the α5ß1 FN binding integrin increases in the basal cells of the regenerating epithelium to promote their adhesion and migration on the FN matrix. We recently showed that FN positively influences the activity directed by the α5 promoter when rabbit corneal epithelial cells (RCECs) are grown on FN-coated culture dishes. FN-responsiveness was essentially mediated by the binding of the transcription factor Sp1 to a target site, designated the FRE, that also bears putative binding sites for the transcription factors NFI and AP-1. Here, we investigated the respective regulatory influence of each of these binding sites and evaluated the influence exerted by FN, CIV and LM on the α5 promoter activity in RCECs.

Methods:: Binding of Sp1, AP-1 and NFI was examined in vitro by EMSA or in vivo by chromatin immunoprecipitation (ChIP). Their regulatory influence was evaluated by site-directed mutagenesis. The influence exerted by FN, CIV and LM was investigated by the transfection of α5-CAT recombinant constructs into RCECs grown on matrix-coated plates. EMSA and Western blot analyses were conducted to monitor the influence of FN, CIV and LM on both the expression and DNA binding properties of Sp1, NFI and AP-1.

Results:: All three transcriptions factors were found to bind the α5 promoter both in vivo and in vitro. Both AP-1 and Sp1 positively influenced promoter function whereas NF1 acted as a repressor. CIV exerted a strong positive influence on the α5 promoter at a low cell density whereas it functioned negatively at a high cell density. At either cell densities, FN and LM exerted positive and negative influences, respectively, on this promoter activity. The expression, DNA binding and regulatory influences of Sp1, AP-1 and NF1 were found to be differently affected by CIV, LM and FN.

Conclusions:: FN, CIV and LM apparently exert their regulatory influence (positive or negative) on the α5 promoter by altering the level to which Sp1, AP-1 and NFI are expressed in RCECs.

Keywords: gene/expression • extracellular matrix • wound healing