Identification of Candidate KLF4 Target Genes Reveals the Molecular Basis of the Diverse Regulatory Roles of KLF4 in the Mouse Cornea

¹ Ophthalmology, Univ. Pittsburgh School of Medicine , 203 Lothrop Street Room 1025, Pittsburgh, Pennsylvania, 15213, United States
² National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
³ Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, Maryland, United States

^* To whom correspondence should be addressed. E-mail: swamynathansk{at}upmc.edu.

	Abstract

Purpose: Kruppel-like factor KLF4 plays a crucial role in the development and maintenance of the mouse cornea. Here, we have compared the wild type (WT) and Klf4-conditional null (Klf4CN) corneal gene expression patterns to understand the molecular basis of the Klf4CN corneal phenotype. Methods: Expression of more than 22,000 genes in 10 WT and Klf4CN corneas was compared by microarrays, analyzed using BRB ArrayTools and validated by Q-RT-PCR. Transient cotransfections were employed to test if KLF4 activates the aquaporin-3, Aldh3a1 and TKT promoters. Results: Scatter plot analysis identified 740 and 529 genes up- and down-regulated by more than 2-fold, respectively, in the Klf4CN corneas. Cell cycle activators were upregulated while the inhibitors were downregulated, consistent with the increased Klf4CN corneal epithelial cell proliferation. Desmosomal components were downregulated, consistent with the Klf4CN corneal epithelial fragility. Downregulation of aquaporin-3, detected by microarray, was confirmed by immunoblot and immunohistochemistry. Aquaporin-3 promoter activity was stimulated 7-10 fold by cotransfection with pCI-KLF4. Corneal crystallins Aldh3A1 and TKT were downregulated in the Klf4CN cornea and their respective promoter activities were upregulated 16- and 9-fold by pCI-KLF4 in co-transfections. Expression of epidermal keratinocyte differentiation markers was affected in the Klf4CN cornea. While the cornea specific keratin-12 was downregulated, most other keratins were upregulated, suggesting hyperkeratosis. Conclusions: We have identified functionally diverse candidate KLF4 target genes, revealing the molecular basis of the diverse aspects of the Klf4CN corneal phenotype. These results establish KLF4 as an important node in the genetic network of transcription factors regulating the corneal homeostasis.

Key Words: corneal development, corneal edema, corneal epithelium, transcription factors, gene expression, knockout animals