Regulation Of Involucrin Expression In Normal Human Corneal Epithelial Cells – A Role For Activator Protein One

Abstract

Abstract: : Purpose: Understanding the mechanisms that regulate differentiation–dependent gene expression in the human corneal epithelium is an important goal. In the present study, we use the involucrin gene as a model to study this regulation. Human involucrin (hINV) is a structural protein that is expressed in differentiating human corneal epithelial cells but not in other eye tissues. Methods: Regulation of human involucrin gene expression and promoter activity was monitored in cultures of normal human primary corneal epithelial cells. Normal corneal epithelial cells were obtained from the central corneal epithelium and cultured in serum–free medium. Early passage cultures were transfected with hINV promoter–luciferase reporter constructs. Cell extracts were prepared and monitored for luciferase activity as an index of promoter activation. Expression of the endogenous involucrin gene was monitored by measuring mRNA level and protein level. Parallel studies were performed using transgenic mice encoding various segments of the hINV promoter. Results: Our studies reveal that an activator protein one (AP1) DNA binding site is essential for appropriate basal and stimulus–dependent hINV promoter activity in cultured human corneal cells. Mutation of this site, AP1–1, results in a loss of hINV promoter activity. Gel mobility supershift analysis reveals interaction of the AP1 transcription factors, Fra–1, Fra–2 and JunB, with this element. Inhibition of AP1 transcription factor function using a dominant–negative form of AP1, also inhibits expression. Treatment with 12–0–tetradecanoylphorbol–13–acetate (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 AP1–1 response element. Parallel studies implicate the AP1–5 transcription factor binding site. Expression of the endogenous hINV gene is also increased by TPA treatment, as measured by effects on mRNA and protein level. Transgenic mouse studies show that the full–length hINV promoter drives differentiation–appropriate expression. Moreover, mutation of selected AP1 transcription factor binding sites in the hINV promoter completely inactivates hINV gene expression in the mouse corneal epithelium. Conclusions: These findings suggest that the involucrin gene provides an useful model for the study of corneal epithelial gene expresison, and point to an important role for AP1 transcription factors in this regulation.

Keywords: cornea: epithelium • transcription factors • transgenics/knock–outs