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D. Kenchegowda, S. Swamynathan, S. K. Swamynathan; Regulation of Gastrokine-1, Uroplakin-1B and Uroplakin-3B Promoter Activities by KLF4, KLF5 and Oct1. Invest. Ophthalmol. Vis. Sci. 2010;51(13):706.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we found that the expression of gastrokine (GKN1), uroplakin (UPK)-1B and -3B, which help maintain the epithelial integrity, is downregulated in the Klf4 conditional null (Klf4CN) cornea. In the present study, we have examined the role of KLF4 in epithelial barrier formation and characterized the regulation of GKN1, UPK1B and UPK3B promoter activities by KLF4, KLF5, and Oct1.
Expression levels of GKN1, UPK1B and UPK3B in the wild type (WT) and Klf4CN corneas were compared by Q-RT-PCR. Influence of KLF4, KLF5 and Oct1 on GKN1, UPK1B and UPK3B promoter activities was tested by transient co-transfection assays in NCTC human keratinocytes. Anti-Klf4 shRNA was used to suppress the expression of Klf4. Trans-epithelial electrical resistance (TEER) was used as a measure of barrier forming ability of human corneal epithelial (HCE) cells in culture.
Downregulation of GKN1, UPK1B and UPK3B in the Klf4CN corneas was confirmed by Q-RT-PCR. Activity of the -479/+16 bp GKN1 proximal promoter fragment was stimulated 2.6-, 28.4- and 2.8-fold respectively, by pCI-KLF4, pCI-KLF5 and pCI-Oct1. The -479/+16 bp GKN1 promoter activity was stimulated upon co-transfection with KLF4 and KLF5 (18.9-fold), or KLF4 and Oct1 (6.9-fold), or KLF5 and Oct1 (67.8-fold), or KLF4, KLF5 and Oct1 (24.2-fold). Activity of UPK1B and UPK3B promoter fragments of different lengths was stimulated by 10- to 50-fold upon co-transfection with pCI-KLF4. Anti-KLF4 shRNA treated HCE cells showed significant reduction in the TEER (26 to 65 Ω) compared to WT (428 Ω) and control shRNA treated cells (404 to 430 Ω).
GKN1 proximal promoter activity is stimulated the most by KLF5, followed by Oct1 and KLF4. KLF5 and Oct1 have a synergistic effect on GKN1 promoter activity. Barrier forming ability of HCE cells is compromised upon downregulation of Klf4. Thus, KLF4, along with KLF5 and Oct1, contributes to the corneal epithelial integrity by regulating the expression of GKN1, UPK1B and UPK3B.
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