Purchase this article with an account.
F Chiambaretta, L Blanchon, B Rabier, WW Kao, J Liu, B Dastugue, D Rigal, V Sapin; Regulation of Human Keratin 12 Gene Expression by hKLF6, a Human Krüppel-Like Transcription Factor . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3197.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: The Keratin12 (K12) protein is essential for corneal epithelium integrity. Due to the presence of GC motifs (a target sequence recognized by Krüppel-like transcription factors) in the human K12 promoter and the already described role of Krüppel-like factor 6 (KLF6) in regulating keratin genes expression, we tested the potential implications of KLF6 (never explored in mammalian eye physiology) in the corneal regulation of the Keratin 12 gene transcription. Method: In order to check the expression of human KLF6 mRNA and protein in five human cornea, RT-PCR, Western blotting and immunohistochemistry experiments were used. Same experimental strategy was used to explore human corneal epithelial cells in 20 patients (undergoing epithelial ablation for photorefractive keratectomy with an excimer laser) and HCE cell line. We therefore explored the KLF6 protein ability to modulate K12 promoter activity in HCE cell line using transient transfection with KLF6 expression plasmid, and (beta Gal and CAT) reporter gene assays. Gel shift assay was performed to confirm the physical interaction between KLF6 protein and specific sequences of K12 promoter. Results: We first demonstrated the presence of KLF6 transcripts and proteins in human total corneal extract: a 323 bp PCR product was generated by RT-PCR and Western Blot revealed a single 46kDa band corresponding to KLF6. Immunohistochemical experiments showed a positive staining specifically present in the epithelium corneal layer. KLF6 transcripts and proteins were also specifically present in corneal epithelium cells in our 20 patients and HCE cell line. Transient transfection of KLF6 showed significant transactivation of K12 promoter in HCE cells (dose dependent manner). Gel shift assay showed a physical interaction between KLF6 protein and a specific sequence of K12 promoter. Conclusion: We demonstrated for the first time the expression of KLF6 in human corneal epithelial cells, and its role in regulation of K12 gene expression.
This PDF is available to Subscribers Only