The Transcriptional Activity Directed by the Human alpha5 Integrin Gene Promoter is Determined through an Sp1/NF1/Ap1 Composite Element in Primary Cultured Rabbit Corneal Epithelial Cells

Abstract

Abstract: : Purpose: High level of expression for the α5 integrin subunit has been reported to occur in corneal epithelial cells in response to the massive secretion of FN that is typically observed upon corneal injury. We recently demonstrated that the activity directed by the α5 promoter could be induced by fibronectin (FN) in a dose–dependent manner when rabbit corneal epithelial cells (RCECs) are grown on FN–coated culture dishes. FN–responsiveness was shown to be mediated through the binding of phosphorylated Sp1 to a target site located between α5 promoter positions –53 to –64. A detailed examination of the α5 promoter sequence revealed the presence of putative binding sites for the transcription factors nuclear factor 1 (NF1) and AP1 in the immediate vicinity of the –53/–64 Sp1 site between positions –67/–71 and –45/–51, respectively. Here, we investigated the respective regulatory influence of each of these transcription factors binding site in primary cultured RCECs.Methods: Expression of Sp1, AP1 and NF1 in RCECs was investigated by electrophoretic mobility shift assays (EMSA). Binding of all three transcription factors to their respective α5 target site was demonstrated either in vitro by EMSAs or in vivo by ligation–mediated PCR (LM–PCR), or both. Finally, the regulatory influence exerted by these transcription factors on the α5 promoter was evaluated by site–directed mutagenesis experiments conducted in RCECs. Results: RCECs express Sp1, AP1 and NF1 and all three transcription factors were found to bind the α5 promoter both in vivo and in vitro. Site–directed mutagenesis indicated that both Ap1 and Sp1 possess the ability to positively influence α5 promoter function in vitro whereas NF1 apparently acts as a strong repressor in a similar context. Conclusion: These results therefore suggest that expression of the α5 integrin subunit gene is likely dictated by very subtle alterations in the cellular ratio of these transcription factors in primary cultured RCECs.

Keywords: transcription factors • gene/expression • cornea: epithelium