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(Investigative Ophthalmology and Visual Science. 2004;45:1692-1704.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0908
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Expression of the {alpha}4 Integrin Subunit Gene Promoter Is Modulated by the Transcription Factor Pax-6 in Corneal Epithelial Cells

Karine Zaniolo,1 Steeve Leclerc,1 Ales Cvekl,2,3 Luc Vallières,1 Richard Bazin,2 Kathy Larouche,1 and Sylvain L. Guérin1,4

1From the Oncology and Molecular Endocrinology Research Center and the 4Unit of Ophthalmology, Centre Hospitalier Universitaire de Québec and Laval University, Ste-Foy, Québec; and the 2Departments of Ophthalmology and Visual Sciences and 3Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York.

PURPOSE. Expression of several membrane-bound integrins is thought to be altered during corneal wound healing as a consequence of the massive secretion of fibronectin occurring during this process. Examination of the {alpha}4 integrin subunit gene promoter revealed the presence of three putative binding sites for the transcription factor Pax-6 expressed in the basal cells of the corneal epithelium during corneal wound healing. This study was undertaken to investigate whether the {alpha}4 integrin subunit is expressed in primary cultures of rabbit corneal epithelial cells (RCECs) and to test whether Pax-6 binds the {alpha}4 gene promoter and regulates its transcriptional activity.

METHODS. Both flow cytometry and immunocytochemical analyses, along with an antibody-directed receptor interference assay, were used to examine expression of the {alpha}4 subunit in RCECs. Expression of Pax6 was investigated by immunoblot analysis. Binding of PAX6 to the {alpha}4 gene promoter was tested in electrophoretic mobility shift assays (EMSAs). The regulatory influence exerted by Pax6 on the {alpha}4 promoter was studied by transfections in RCECs.

RESULTS. Expression of {alpha}4 was detected at both the mRNA and protein levels. Pax-6 was expressed in a cell-density–dependent manner in RCECs and altered the activity of the {alpha}4 promoter by interacting with multiple sites in both the promoter and 5'-flanking sequences. Pax-6 was also identified as the major protein component from the Bp5 complex, one of five protein complexes reported to bind the {alpha}4.1 element from the {alpha}4 basal promoter in vitro.

CONCLUSIONS. These results provide evidence that the integrin subunit {alpha}4 and Pax-6 are coexpressed in RCECs and raise the possibility that Pax-6 directly regulates the expression of the {alpha}4 gene during corneal wound healing.




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