The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin ␣ 5 ␤ 1 . In this work, we determined whether the activity directed by the ␣ 5 gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/␣ 5 promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediating FN responsiveness was shown to bear a perfect inverted repeat that we designated the fibronectin-responsive element (FRE). Analyses in electrophoretic mobility shift assays provided evidence that Sp1 is the predominant transcription factor binding the FRE. Its DNA binding affinity was found to be increased when RCEC are grown on FN-coated dishes. The addition of the MEK kinase inhibitor PD98059 abolished FN responsiveness suggesting that alteration in the state of phosphorylation of Sp1 likely accounts for its increased binding to the ␣ 5 FRE. The FRE also proved sufficient to confer FN responsiveness to an otherwise unresponsive heterologous promoter. However, site-directed mutagenesis indicated that only the 3 half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its ␣ 5 ␤ 1 integrin activates a signal transduction pathway that results in the transcriptional activation of the ␣ 5 gene likely through altering the phosphorylation state of Sp1.Corneal wounds account for a substantial proportion of all visual disabilities and medical consultations for ocular problems in North America. They can be superficial with damage limited to the epithelium or associated with a deeper involvement of the epithelial basement membrane and of the stromal lamella. Severe recurrent and persistent corneal wounds are most commonly secondary to ocular diseases and damage such as recurrent erosion, mild chemical burns, superficial herpetic infections, neuroparalytic cornea, autoimmune diseases, and stromal ulcerations due to viral or bacterial infections or to severe burns (1). Despite currently available treatments, many of these corneal wounds persist for weeks and months or else recur frequently and can progress to corneal perforation.Tissue repair requires cell migration, proliferation, and adhesion. Cell adhesion and migration in turn require extracellular matrix (ECM) 1 synthesis and assembly. ECM is a complex, cross-linked structure of proteins and polysaccharides. It organizes the geometry of normal tissues. Fibronectin (FN) is an ECM adhesion protein identified as a potential wound healing agent because of its cell attachment, migration, differentiation, and orientation properties (for a review see Refs. 2-4). In the unwounded rat eye, FN is observed by immunohistological staining at the level of the corneal epithelium basement membrane (5-7). Shortly after corneal injury, the basal cells that border the injured area and stromal keratocytes start producing massive amounts of FN (5,[8]...

These results provide support to the hypothesis that the strong reduction in the activity of the alpha5 promoter when RCECs reach a high cell density is the consequence of a reduced expression of Sp1/Sp3 under such cell culture conditions.

Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes.

These results provide evidence that the integrin subunit alpha4 and Pax-6 are coexpressed in RCECs and raise the possibility that Pax-6 directly regulates the expression of the alpha4 gene during corneal wound healing.

Procedures used for investigating DNA-protein interactions, such as the electrophoretic mobility shift assay (EMSA) or DNasel footprinting, require that exogenous nucleic acids (or synthetic equivalents) be added to the reaction mixture to prevent or reduce the nonspecific interaction of nuclear proteins with the labeled probe of choice, especially when proteins are obtained from crude nuclear extracts. One of the most potent, and likely the most widely used, non-specific competitor is the synthetic polymer poly(dI-dC).poly(dI-dC). Its addition to the reaction mixture prior to crude nuclear proteins has unquestionably proven very efficient in reducing nonspecific interactions by facilitating detection of the complexes of interest. However, in certain instances, the use of crude extracts alone does not provide adequate answers and the need to further enrich such extracts becomes absolutely necessary. In this study, we provide evidence that amounts of poly(dI-dC).poly(dI-dC) well below those currently described in the literature substantially impair, or even totally prevent, the detection of specific DNA-protein complexes in EMSA when enriched, gel-fractionated or commercially purified nuclear proteins are used, therefore indicating the need to precisely optimize the amount of such a competitor in DNA-protein interaction studies.