Identification of Candidate Klf4 Target Genes Reveals the Molecular Basis of the Diverse Regulatory Roles of Klf4 in the Mouse Cornea

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Abstract
Purpose. Krüppel-like factor 4 (Klf4) plays a crucial role in the development and maintenance of the mouse cornea. In the current study, wild-type (WT) and Klf4-conditional null (Klf4CN) corneal gene expression patterns were examined, to gain understanding of 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 (National Cancer Institute, Bethesda, MD) and validated by Q-RT-PCR. Transient cotransfections were used to test whether Klf4 activates the aquaporin-3, Aldh3a1, and TKT promoters. results. Scatterplot analysis identified 740 and 529 genes up- and downregulated by more than twofold, respectively, in the Klf4CN corneas. Cell cycle activators were upregulated, whereas 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- to 10-fold by cotransfection with pCI-Klf4. The 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 cotransfections. The expression of epidermal keratinocyte differentiation markers was affected in the Klf4CN cornea. Although the cornea-specific keratin-12 was downregulated, most other keratins were upregulated, suggesting hyperkeratosis. conclusions. Functionally diverse candidate Klf4 target genes were identified, 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.