Identification of candidate Klf4 target genes reveals the molecular basis of the diverse regulatory roles of Klf4 in the mouse cornea

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.

Figure 1

Scatter plot analysis of 6333 genes passing the filter described in Materials and Methods. Expression of 529 genes was downregulated and 740 genes upregulated by more than two folds in Klf4CN compared to the wild type corneas. The expression of about 80% of the genes (5064 genes) passing the filter remained relatively stable with less than 2-fold change.

Figure 2

A. Expression levels of different desmosomal components in the wild type and Klf4CN corneas. The values shown are log transformed. B. Validation of downregulation of genes encoding desmosomal components in the Klf4CN compared to the wild type corneas, by semi-quantitative RT-PCR. M, Molecular weight markers; WT, wild type; CN, Klf4 conditional null.

Figure 3

Aquaporin-3 expression is reduced in the Klf4CN relative to the wild type cornea. A. Q-RT-PCR comparison of expression levels of Aqp3 in the wild type and Klf4CN corneas. B. Immunoblot analysis of Aqp3 levels in total proteins extracted from wild type and Klf4CN corneas. The blot probed with anti-Aqp3 antibody (on the left) was stripped of the antibody and re-probed with anti-actin antibody (on the right) to ensure equal loading of proteins. C. Immunohistochemistry with anti-Aqp3 antibody. Images in the top row show nuclei stained with DAPI, while those in the bottom row show fluorescence coming from the secondary antibody bound to the primary anti-Aqp3 antibody. The expression of Aqp3 localized to the epithelial and endothelial cell membranes in the wild type (bottom, left), is reduced in the Klf4CN cornea (bottom, center). The sections processed in a similar manner without the primary antibody served as controls (bottom, right). D. Aquaporin-3 promoter is stimulated by KLF4 in cultured human corneal epithelial cells cotransfected with Aqp3-Luc and pCI-KLF4.

Figure 4

Expression of corneal crystallins Aldh3a1 and TKT is regulated by KLF4 A. Comparison of expression of Aldh3a1 and TKT in the wild type (WT) and Klf4 conditional null (Klf4CN) cornea, as detected by microarray. B. Q-RT-PCR comparison of expression of Aldh3a1 and TKT in WT and Klf4CN corneas. C. Aldh3a1 and TKT promoter activities are stimulated by KLF4 in Cos-7 cells upon cotransfection with pCI-KLF4.

Figure 5

A. Expression levels of different keratins in the wild type and Klf4CN corneas. The values shown are log transformed. B. Validation of upregulation of genes encoding different keratin genes in the Klf4CN compared to the wild type corneas, by semi-quantitative RT-PCR. M, Molecular weight markers; WT, wild type; CN, Klf4 conditional null.