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 (alpha6beta1 and alpha6beta4) that belong to the integrin family. The authors characterized the regulatory influence exerted by laminin on the Sp1/Sp3-mediated alpha6 gene promoter function. METHODS: Recombinant plasmids bearing the CAT reporter gene, fused to various segments from the alpha6 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 alpha6 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 alpha6 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 alpha6 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 alpha6 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 alpha6 promoter. This LM-mediated negative regulatory influence also translated into a similar reduction in the expression of the endogenous alpha6 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 alpha6 promoter activity results in part from the proteolytic cleavage of Sp1/Sp3. CONCLUSIONS: Unlike FN, which functions as an activator of alpha5 gene transcription, LM repressed transcription, directed by the alpha6 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.
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 (alpha6beta1 and alpha6beta4) that belong to the integrin family. The authors characterized the regulatory influence exerted by laminin on the Sp1/Sp3-mediated alpha6 gene promoter function. METHODS: Recombinant plasmids bearing the CAT reporter gene, fused to various segments from the alpha6 promoter, were transfected into rabbitcorneal 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 alpha6 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 alpha6 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 alpha6 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 alpha6 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 alpha6 promoter. This LM-mediated negative regulatory influence also translated into a similar reduction in the expression of the endogenous alpha6 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 alpha6 promoter activity results in part from the proteolytic cleavage of Sp1/Sp3. CONCLUSIONS: Unlike FN, which functions as an activator of alpha5 gene transcription, LM repressed transcription, directed by the alpha6 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.
Authors: Danielle L Peacock Brooks; Luciana P Schwab; Raisa Krutilina; Deanna N Parke; Aarti Sethuraman; David Hoogewijs; Alexandra Schörg; Lauren Gotwald; Meiyun Fan; Roland H Wenger; Tiffany N Seagroves Journal: Mol Cancer Date: 2016-03-22 Impact factor: 27.401