Regulation of corneal keratin-12 gene expression by the human Krüppel-like transcription factor 6.
OBJECTIVE
The keratin-12 (K12) protein is essential for the integrity of the corneal epithelium. This study was conducted to investigate the possible involvement of Krüppel-like factor 6 (KLF6) in the corneal regulation of K12 gene expression, in view of the presence of one KLF6 potential binding site in the human K12 promoter and the known role of KLF6 in regulating keratin gene expression.


METHODS
RT-PCR, Western blot analysis, and immunolocalization experiments were used to investigate the expression of KLF6 mRNA and protein in five human total corneas. The same experimental design was used to explore human corneal epithelial (HCE) cells in 20 patients and a HCE cell line. The ability of the KLF6 protein to modulate K12 promoter activity was studied in the HCE cell line, by transient transfections with a KLF6 expression plasmid and promoter-reporter gene assays. Gel-shift assays were performed to confirm the interactions between the KLF6 protein and specific sequences of the K12 promoter.


RESULTS
The presence of KLF6 transcripts and proteins in human total corneal extracts was demonstrated. Immunohistofluorescence experiments showed positive staining specifically present in the corneal epithelial layer. KLF6 transcripts and proteins were also present in corneal epithelial cells in 20 patients and the HCE cell line. Transient transfections of KLF6 showed statistical transactivation of the K12 promoter in HCE cells. The gel-shift assay showed a physical interaction between KLF6 and the K12 promoter.


CONCLUSIONS
The expression of KLF6 in HCE cells and its role in the regulation of K12 gene expression were demonstrated.
Chiambaretta
Frédéric
F
Department of Ophthalmology, University of Clermont-Ferrand, France.
Blanchon
Loïc
L
Rabier
Bénédicte
B
Kao
Winston W-Y
WW
Liu
Janice J
JJ
Dastugue
Bernard
B
Rigal
Danièle
D
Sapin
Vincent
V
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003315:Cornea / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007633:Keratins / Q000235:genetics*; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D008969:Molecular Sequence Data; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000502:physiology*; D014162:Transfection
C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D007633:Keratins