Expression of the alpha4 integrin subunit gene promoter is modulated by the transcription factor Pax-6 in corneal epithelial cells.
OBJECTIVE
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 alpha4 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 alpha4 integrin subunit is expressed in primary cultures of rabbit corneal epithelial cells (RCECs) and to test whether Pax-6 binds the alpha4 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 alpha4 subunit in RCECs. Expression of Pax6 was investigated by immunoblot analysis. Binding of PAX6 to the alpha4 gene promoter was tested in electrophoretic mobility shift assays (EMSAs). The regulatory influence exerted by Pax6 on the alpha4 promoter was studied by transfections in RCECs.


RESULTS
Expression of alpha4 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 alpha4 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 alpha4.1 element from the alpha4 basal promoter in vitro.


CONCLUSIONS
These results provide evidence that the integrin subunit alpha4 and Pax-6 are coexpressed in RCECs and raise the possibility that Pax-6 directly regulates the expression of the alpha4 gene during corneal wound healing.
Zaniolo
Karine
K
Oncology and Molecular Endocrinology Research Center, Centre Hospitalier Universitaire de Québec and Laval University, Ste-Foy, Canada.
Leclerc
Steeve
S
Cvekl
Ales
A
Vallières
Luc
L
Bazin
Richard
R
Larouche
Kathy
K
Guérin
Sylvain L
SL
D000818:Animals; D015153:Blotting, Western; D002452:Cell Count; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005136:Eye Proteins; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology*; D018398:Homeodomain Proteins / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D039441:Integrin alpha4 / Q000235:genetics* / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D021122:Protein Subunits; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; D039441:Integrin alpha4