Regulation of Corneal Epithelial Barrier Function by Kruppel-like Transcription Factor 4

PURPOSE. Previously, the authors showed that KO-conditional null (Klf4CN) corneas display epithelial fragility. Here, they investigated the mechanism by which KLF4 regulates corneal epithelial barrier function. METHODS. Klf4CN mice were generated by breeding Le-Cre with KLF4-LoxP mice. Fluorescein staining was used to test the corneal barrier function. RT-PCR, immunoblots, and immunofluorescence were used to detect the expression of cell junctional proteins. The effect of Klf4 on promoter activities was measured by transient cotransfection assays. Trans-epithelial electrical resistance (TEER was used to measure the barrier-forming ability of control or anti-KLF4 siRNA-treated cells. RESULTS. Increased fluorescein staining and decreased tight junction protein Tjp1 expression demonstrated that the KOCN corneal epithelial barrier function is defective. Expression of desmosomal components Dsp, Dsg-1a, and Dsg-1b was downregulated in the I:OWN corneas, and their corresponding promoter activities were upregulated by Klf4 in transient cotransfection assays. Hemidesmosomal alpha 3- and beta 4-integrin levels were not affected even though there were fewer hemidesmosomes in the KOCN corneas. The basement membrane components laminin-alpha 5, -alpha 3, -beta 3, and -beta 1-1 were downregulated, suggesting that the disrupted basement membrane is responsible for fewer hemidesmosomes in the KOCN cornea. Tight junction proteins OCLN1 and TJP1 were downregulated in anti-KLF4 siRNA-treated cells, which failed to develop epithelial barrier function as measured by TEER. CONCLUSIONS. Klf4 contributes to corneal epithelial barrier function by upregulating the expression of functionally related subsets of cell junctional proteins and basement membrane components. (Invest Ophthalmol Sci. 2011;52:1762-1769) DOI:10.1167/iovs.10-6134