Expression of the alpha(5) integrin subunit gene promoter is positively regulated by the extracellular matrix component fibronectin through the transcription factor Sp1 in corneal epithelial cells in vitro

The accumulation of fibronectin (FN) in response to corneal epithelium inju ry has been postulated to turn on expression of the FN-binding integrin alp ha (5)beta (1). In this work, we determined whether the activity directed b y the ct, gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/alp ha (5) promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediati ng FN responsiveness was shown to bear a perfect inverted repeat that we de signated the fibronectin-responsive element (FRE). Analyses in electrophore tic mobility shift assays provided evidence that Spl is the predominant tra nscription 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 MER kinase inhibitor PD98059 abolished FN responsiveness suggesting that alter ation in the state of phosphorylation of Spl likely accounts for its increa sed binding to the a, FRE. The FRE also proved sufficient to confer FN resp onsiveness to an otherwise unresponsive heterologous promoter. However, sit e-directed mutagenesis indicated that only the 3 ' half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its a lpha (5)beta (1) integrin activates a signal transduction pathway that resu lts in the transcriptional activation of the alpha (5) gene likely through altering the phosphorylation state of Sp1.