An Involucrin Promoter AP1 Transcription Factor Binding Site Is Required for Expression of Involucrin in the Corneal Epithelium In Vivo

Abstract

PURPOSE. Cell division of corneal limbal stem cells gives rise to transient amplifying cells that ultimately differentiate to form the multilayered corneal epithelium. The mechanisms that regulate changes in gene expression during this process are not well understood. In the present study, the involucrin gene was used as a model to study this regulation. METHODS. Regulation of human involucrin gene expression and promoter activity was assessed using in vivo transgenic mouse models and cultured primary human corneal epithelial cells. RESULTS. Human involucrin (hINV) is a structural protein that is selectively expressed in differentiating corneal epithelial cells. The results reveal that an activator protein one (AP1) DNAbinding site is essential for appropriate basal and stimulusdependent hINV promoter activity. Mutation of this site, AP1-5, results in a loss of hINV gene expression in the corneal epithelium in vivo and in cultured corneal epithelial cells. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1 and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased AP1 factor (Fra-1 and JunB) binding to the hINV gene AP1-5 response element. CONCLUSIONS. These findings point to an essential role for AP1 transcription factors, acting through a distal regulatory region AP1-5 element, in the regulation of involucrin gene expression during corneal epithelial cell differentiation. (Invest Ophthalmol Vis Sci.

Suppression of AP1 Transcription Factor Function in Keratinocyte Suppresses Differentiation

“…Involucrin is a keratinocyte differentiation marker and is known to be increased by AP1 transcription factor signaling [25], [47], [49]–[51]. Control and TAM67-FLAG expressing keratinocytes were harvested and the level of hINV protein and mRNA was measured.…”

Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells

“…Abnormal development and/or defective maintenance of the cornea lead to severe defects in vision (Klintworth, 2003; Vincent et al, 2005). The involvement of various transcription factors in regulating the corneal development has been intensely studied (Adhikary et al, 2005a; Adhikary et al, 2005b; Chiambaretta et al, 2002; Chiambaretta et al, 2006; Davis et al, 2003; Dwivedi et al, 2005; Francesconi et al, 2000; Hough and Piatigorsky, 2004; Lambiase et al, 2005; Nakamura et al, 2004; Nakamura et al, 2005; Sivak et al, 2000; Sivak et al, 2004; Swamynathan et al, 2008; Swamynathan et al, 2007; Ueta et al, 2005). In spite of this progress, knowledge of the genetic network of transcription factors required for maturation and maintenance of the cornea and other components of the ocular surface remains incomplete.…”

Conditional Deletion of the Mouse Klf4 Gene Results in Corneal Epithelial Fragility, Stromal Edema, and Loss of Conjunctival Goblet Cells

“…Relatively little is known about the genetic network of transcription factors required for embryonic morphogenesis, postnatal maturation, and maintenance of cornea (1,2,10,12,20,23,26,32,39,46,47,55,59,60,65). Serial analysis of gene expression identified KLF4, a member of the Krüppel-like transcription factor (KLF) subfamily of Cys2-His2 zinc finger proteins, as one of the most highly expressed transcription factors in both 9-day-old and 6-week-old mouse cornea (50).…”