Krüppel-Like Factor 4 Contributes to Corneal Epithelial and Epidermal Barrier Functions by Upregulating Dsp, Dsg-1a and Dsg-1b Promoter Activities

Abstract

Purpose: : Desmosomes are multi-protein cell adhesion complexes that provide structural integrity and help resist shearing forces in epithelial tissues. In the Klf4-conditional null (Klf4CN) mouse, we showed previously that the fragile corneal epithelium contains fewer desmosomes and the genes encoding different desmosomal components are significantly downregulated. Here, we investigated the effect of downregulation of KLF4 on cell adhesion and migration and whether Klf4 regulates promoter activity of 3 desmosomal genes (Dsp, Dsg-1a and Dsg-1b).

Methods: : Microarray and Q-RT-PCR were employed to measure the relative levels of desmosomal transcripts. Immunoblots and immunohistochemistry were used to measure the relative amounts of desmoplakin (Dsp) and desmoglein (Dsg). Effect of downregulation of KLF4 on cell adhesion and migration was tested using stable cell lines expressing anti-KLF4 shRNA. Mouse Dsp, Dsg-1a and Dsg-1b promoters were cloned in pGL3-Basic vector and tested by transient co-transfection with pCMV-Klf4 in human corneal epithelial (HCE) and human skin keratinocyte (NCTC) cell lines.

Results: : Microarray and Q-RT-PCR showed that different desmosomal transcripts are significantly downregulated in Klf4CN compared to WT corneas. Immunoblots showed that the levels of Dsp-1, Dsp-2, and Dsg are reduced to 35%, 22%, and 58% respectively, in the Klf4CN compared to WT corneas. Immunohistochemistry confirmed this reduction. Human cell lines wherein KLF4 was downregulated using anti-KLF4 shRNA showed decreased adhesion and increased mobility in in vitro binding and mobility assays, respectively. Mouse Dsp, Dsg-1a and Dsg-1b promoter activities were upregulated by Klf4in transient co-transfection assays in both HCE and NCTC cell lines.

Conclusions: : Expression of desmosomal components is downregulated in the Klf4CN cornea, consistent with Klf4CN corneal epithelial fragility. Downregulation of KLF4 resulted in decreased cell-matrix binding and increased cell migration in human cell lines. We conclude that Klf4 contributes to the development and maintenance of the corneal epithelial and epidermal barrier function by upregulating Dsp, Dsg-1a and Dsg-1b promoter activities.

Keywords: cell adhesions/cell junctions • gene/expression • cornea: epithelium