Kruppel-like factor 6 (KLF6) affects the promoter activity of the alpha1-proteinase inhibitor gene.
OBJECTIVE
Keratoconus is a progressive disease that thins and scars the cornea. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of inhibitors alpha1-proteinase inhibitor (alpha1-PI) and alpha2-macroglobulin (alpha2-M) are reduced. The present study explored the possible involvement in keratoconus of Krüppel-like factor 6 (KLF6), a transcription factor previously described to be essential for the integrity of the corneal epithelium. The transcript and proteins level of KLF6 and its action in regulating the genes affected in keratoconus were examined in this study.


METHODS
Semiquantitative RT-PCR, Western blot analysis, immunofluorescence and in situ hybridization were used to investigate the expression of KLF6 mRNA and protein in normal and keratoconus corneas. Modulation by KLF6 of the promoter activity of alpha1-PI, LAP, cathepsin B, and alpha2-M genes was studied after transient transfection of KLF6 expression plasmid into corneal epithelial cells using promoter-reporter gene assays. Chromatin immunoprecipitation (ChIP) assays were performed to confirm the interactions between KLF6 and promoters of the genes affected in keratoconus.


RESULTS
A global increased expression of the transcription factor KLF6 in terms of mRNAs and proteins was observed in total cornea and/or the epithelium in a substantial number of the keratoconus specimens. The promoter activity of the human alpha1-PI gene was suppressed by expression of KLF6 in corneal epithelial cells. The ChIP assay confirmed a physical interaction between KLF6 and the alpha1-PI promoter.


CONCLUSIONS
Transcription factor KLF6 downregulates the alpha1-PI gene in corneal epithelial cells and may thereby be involved in keratoconus.
Chiambaretta
Frédéric
F
Department of Ophthalmology, University Hospital, Centre Hospitalier Universitaire (CHU), Clermont-Ferrand, France.
Nakamura
Hiroshi
H
De Graeve
Fabienne
F
Sakai
Hiroshi
H
Marceau
Geoffroy
G
Maruyama
Yasuhiro
Y
Rigal
Danièle
D
Dastugue
Bernard
B
Sugar
Joël
J
Yue
Beatrice Y J T
BY
Sapin
Vincent
V
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002648:Child; D015536:Down-Regulation; D019573:Epithelium, Corneal / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D017403:In Situ Hybridization; D007640:Keratoconus / Q000235:genetics*; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors / Q000502:physiology*; D008875:Middle Aged; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016335:Zinc Fingers / Q000502:physiology; D000515:alpha 1-Antitrypsin / Q000235:genetics*
C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D000515:alpha 1-Antitrypsin