Expression of the Gene Encoding Integrin Subunit Alpha5 Is Positively Regulated by the Transcription Factors Sp1/Sp3 During Wound Healing of Rabbit Corneal Epithelial Cells

Abstract

Abstract: : Purpose:Expression of the α5ß1 FN integrin is well recognized in the corneal epithelium and was postulated to increase during wound healing. However, no study has ever been conducted in order to investigate how the α5 integrin subunit gene is regulated at the transcriptional level during this process. Here, we examined expression of the α5 subunit at the surface of rabbit corneal epithelial cells (RCECs) and correlated the results with the activity directed by the promoter of the α5 gene in confluent and subconfluent RCECs, a model that mimic wound healing. Methods:Expression of the α5 subunit was assessed by immunofluorescence and FACS analyses. The regulatory elements required to direct expression of the α5 gene were identified by transfecting RCECs with recombinant plasmids bearing various lengths from the human α5 gene promoter fused to the CAT reporter gene. Binding of Sp1/Sp3 to the α5 promoter was assessed by electrophoretic mobility shift assays (EMSAs). Endogenous levels of Sp1/Sp3 were assessed by Western blot and supershift analyses. The regulatory influence exerted by Sp1/Sp3 on the α5 promoter was evaluated by co-transfection in Sp1-deficient Drosophila SL-2 Schneider cells. Results:Both confluent and subconfluent RCECs were found to express substantial amounts of the membrane-bound α5 subunit. The activity directed by the α5 promoter was found to be affected by cell density: strong promoter activity was observed in subconfluent RCECs whereas a dramatic repression was obtained in confluent cells. The α5 promoter sequence located between positions -92 to -132, which is required in order to reach maximal promoter function, was shown to bind Sp1/Sp3 in EMSAs. Co-transfection experiments in Schneider cells confirmed the positive regulatory influence of Sp1 on the -92 to -132 α5 promoter segment. Most of all, both EMSA and Western blot analyses demonstrated the expression of moderate levels of Sp1/Sp3 in sub-confluent but not in confluent RCECs. Conclusion:These results provide support to the hypothesis that the dramatic reduction in the activity of the α5 promoter when RCECs reach a high cell density is the consequence of a reduced expression of Sp1/Sp3 under such cell culture conditions.

Keywords: 372 cornea: epithelium • 417 gene/expression • 605 transcription factors