Laminin reduces expression of the human α6 integrin subunit gene by altering the level of the transcription factors Sp1 and Sp3

PURPOSE. Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after corneal injury, which is later replaced by the secretion of laminin (LM). Laminin is recognized by receptors (alpha 6 beta 1 and alpha 6 beta 4) that belong to the integrin family. The authors characterized the regulatory influence exerted by laminin on the Sp1/Sp3-mediated alpha 6 gene promoter function. METHODS. Recombinant plasmids bearing the CAT reporter gene, fused to various segments from the alpha 6 promoter, were transfected into rabbit corneal epithelial cells (RCECs) grown on untreated or on LM-coated culture plates or into Sp1-deficient Drosophila Schneider cells. Expression and DNA binding of Sp1/Sp3 was monitored with the use of Western blot and electrophoretic mobility shift assays (EMSAs), respectively. DNA target sites for Sp1/Sp3 in the alpha 6 gene promoter were identified in vitro by DNAse I footprinting. Binding of Sp1 and of a few other transcription factors was also examined in vivo by chromatin immunoprecipitation (ChIP) assays. Expression of the alpha 6 mRNA in RCECs grown on LM-coated culture plates was also assessed by Northern blot and RT-PCR analyses. RESULTS. Transfection experiments provided evidence that both Sp1 and Sp3 positively influence alpha 6 promoter activity in Sp1/Sp3-deficient SL2 Schneider cells. Two GC-rich target sites for Sp1/Sp3 (a proximal and a distal site) were identified in the alpha 6 promoter by DNAse I footprinting. Binding of Sp1/Sp3 was further validated in vivo by ChIP. Transfections conducted into RCECs grown on LM-coated culture plates resulted in repression of the activity directed by the alpha 6 promoter. This LM-mediated negative regulatory influence also translated into a similar reduction in the expression of the endogenous alpha 6 mRNA as revealed by both RT-PCR and Northern blot analyses. Most of all, results from EMSA and Western blot analyses suggested that the LM-mediated repression of the alpha 6 promoter activity results in part from the proteolytic cleavage of Sp1/Sp3. CONCLUSIONS. Unlike FN, which functions as an activator of alpha 5 gene transcription, LM repressed transcription, directed by the alpha 6 gene promoter, by altering the nuclear levels of Sp1 and Sp3. Reappearance of LM in the basement membrane after repair of the corneal damage is therefore expected to substantially contribute to the final steps of this process by influencing the degree to which genes that encode protein products requested for cell adhesion and migration, such as integrin genes, are expressed during corneal wound healing.