Regulation of Gene Expression During the Transition From Human Corneal Epithelial Cell Stem Cell to Differentiated Cell: A Role for AP1 Transcription Factors

Abstract

Abstract: : Purpose: Division of corneal epithelial stem cells gives rise to transient amplifying cells that ultimately differentiate to form the multilayered corneal epithelium. In the present study we use the involucrin gene as a model to study the regulation of gene expression during this progression. Human involucrin (hINV) is a structural protein that is absent from the limbal corneal epithelial stem cells, but is expressed in transient amplifying and differentiated cells. Methods: Involucrin gene expression and promoter activity were monitored in primary cultures of normal human corneal epithelial cells and in hINV promoter transgenic mice. Cells were transfected with hINV promoter–luciferase reporter constructs and cell extracts were prepared and monitored for luciferase activity as an index of promoter activation. Transcription factor binding was monitored using nuclear extracts and gel mobility shift analysis. Parallel studies utilized hINV promoter transgenic mice to monitor the effects of transcription factor binding site–specific mutations on gene expression in vivo. Results: Our studies identify an activator protein one (AP1) DNA binding site in the hINV gene upstream regulatory region that is essential for baseline and stimulus–associated hINV promoter activity. Mutation of this site, AP1–5, results in a lose of hINV promoter activity. In vivo studies, using hINV promoter transgenic mice, reveal that targeted mutation of AP1–5 causes a complete cessation of hINV gene expression in the corneal epithelium, confirming that this site is essential for in vivo expression. Gel mobility supershift analysis reveals interaction of the AP1 factors, Fra–1, Fra–2 and Jun–B, with the AP1–5 element. In addition, expression of dominant–negative c–jun inhibits AP1 site–dependent hINV expression. Moreover, treatment with TPA, a protein kinase c activator, increases hINV promoter activity, a response that correlates with increased nuclear AP1 factor level and AP1 factor binding to the hINV gene AP1–5 response element. Conclusions: These findings provide definitive evidence that AP1 factors are required for activation of gene expression during the transition form corneal stem cell to differentiated cell.

Keywords: cornea: basic science • cornea: epithelium