Mechanistic Insight Into the Diverse Regulatory Roles of Krüppel-Like Transcription Factor KLF4 in the Mouse Cornea

Abstract

Purpose:: Conditional deletion of the widely expressed Krüppel-like factor KLF4, one of the most abundant transcription factors in the mouse cornea, resulted in corneal epithelial fragility, stromal edema, vacuolated corneal endothelium and lens, and loss of conjunctival goblet cells (Swamynathan et al. 2007. Mol. Cel. Biol. 27 (1): in press). In order to obtain mechanistic insight into the function of KLF4, we have investigated the KLF4 target genes in the mouse cornea.

Methods:: Total RNA from 5 different wild-type (WT) and Klf4 conditional null (Klf4CN) mouse corneas was isolated to probe expression levels of more than 39000 known and predicted transcripts in the mouse genome, using Affymetrix mouse 430 2.0 GeneChip microarrays. Results were normalized using median values and analyzed using "BRB Array Tools" developed by the Biometric Research Branch of the National Cancer Institute, NIH. Results of microarray analysis were validated by quantitative real time RT-PCR analysis of selected genes using total RNA from WT and Klf4CN corneas.

Results:: Expression of 18 selected genes in the wild-type and Klf4CN corneas measured by Q-RT-PCR was similar to that observed in the microarray analysis, validating the microarray results. Scatter plot analysis revealed that 830 genes were upregulated and 440 genes were downregulated by more than 2-fold in the Klf4CN corneas compared to the wild-type. Genes whose expression was severely affected in the Klf4CN corneas belonged to different functional subgroups, such as cell-cell interaction, adhesion, apoptosis, and regulation of gene expression. Genes involved in immune response and cell cycle regulation were mostly upregulated in the Klf4CN corneas. Even though most keratins were upregulated in the Klf4CN corneas indicating hyperkeratosis, the cornea specific keratin-12 was downregulated. Expression of corneal crystallins aldehyde dehydrogenase and transketolase was downregulated.

Conclusions:: We have identified several potential KLF4 target genes, whose functional diversity suggests that KLF4 regulates, either directly or indirectly, various aspects of mouse corneal physiology.

Keywords: cornea: epithelium • gene/expression • transcription factors