Repression of the Alpha6 Integrin Gene Promoter by the ECM Component Laminin

Abstract

Abstract: : Purpose: Injury to the corneal epithelium induces a massive secretion of fibronectin (FN) within the first few hours following the eye damage. During the wounding process, FN secretion is progressively turned off while secretion of laminin (LM) increases. These extracellular matrix components are recognized by membrane–bound receptors that belong to the integrin family. Both the α6ß1 and α6ß4 integrins recognize LM as their ligand. The promoter from the α6 gene has been recently cloned and shown to bear multiple binding sites for members of the Sp1 family of transcription factors. In this study, we characterized the regulatory influence exerted by Sp1 and Sp3 on both the promoter and 5’ flanking region of the α6 gene when primary cultures of rabbit corneal epithelial cells (RCECs) are grown in the presence of LM. Methods: Recombinant plasmids bearing the CAT reporter gene fused to various segments from the α6 promoter were transfected into both RCECs grown on untreated or LM–coated culture plates, or into Sp1–deficient Drosophila Schneider cells. Expression and DNA binding of Sp1/Sp3 was monitored by Western blot and electrophoretic mobility shift assays (EMSAs), respectively. DNA target sites for Sp1/Sp3 in the α6 promoter and 5’ flanking sequence were identified by DNaseI footprinting analyses. Experiments in northern blot and RT–PCR were conducted to evaluate α6 mRNA expression in RCECs grown on LM–coated culture plates. Results: Transfections conducted into Drosophila cells provided evidence that Sp1 and Sp3 act in an additive manner on the activity driven by the α6 promoter. Two Sp1/Sp3 target sites were identified in the α6 promoter by DNaseI footprinting analyses. Transfections conducted into RCECs grown on LM–coated culture plates resulted in a dose–dependent repression in the activity directed by the α6 promoter that also vary in strength in a cell density–dependent manner. Most of all, expression of α6 mRNA diminished dramatically when RCECs are grown on LM–coated plates. Repression of the α6 promoter activity was suggested to result from the proteolytic cleavage of Sp1/Sp3 when cells are grown on LM, as revealed by both EMSA and Western blot analyses. Conclusions: Taken together, these results suggest that the signal transduced by the binding of LM to its integrin receptor subunit α6, triggers the activation of one (or more) unknown protease(s) that control the level to which Sp1/Sp3 are to be expressed in RCECs.

Keywords: wound healing • gene/expression • signal transduction