Expression Levels and Implications of Klf6 in the Promoter Activity of Genes Affected in Keratoconus Cornea

Abstract

Abstract: : Purpose: In keratoconus corneas, levels of degradative enzymes including lysosomal acid phosphatase (LAP) and cathepsin B (CB) are elevated; and those of the inhibitors alpha-1 proteinase inhibitor (a1PI) and alpha 2-macroglobulin (a2M) are reduced, especially in the epithelial layer. Sp1 transcription factor has been described to be increased in keratoconus cornea and involved in the promoter activity of these genes. Human Kruppel-like factor 6 (hKLF6) has been demonstrated to be expressed in human epithelial corneal cells, and to regulate same promoter regions than Sp1 (using GC boxes). In this way, we studied the mRNA expression levels of KLF6 in normal and keratoconus cornea, and the implications of hKLF6 in regulation of these genes affected in keratoconus . Methods: In order to check the expression of human KLF6 mRNA levels, semi-quantitiative RT-PCR assays (Light Cycler) were used. Immunohistological assays were used to determine localization of KLF6 in normal and keratoconus cornea. DNA segments containing flanking promoter sequences of the LAP, CB, a1PI and a2M genes were ligated into the secreted alkaline phosphatase (SEAP) reporter gene vector. These constructs, along with the pSV beta-galactosidase (BGAL) control vector, were transfected into cultured human corneal epithelial, stromal, embryonic pulmonary (MRC5) and COS-7 cells. Cotransfection with hKLF6 expression vector was performed in parallel. SEAP and BGAL activities were assayed. Results: Abnormal Sp1 expression levels were found (as previously described) in keratoconus cornea. No modification could be found for KLF6 in term of tissular localization between normal and keratonconus cornea. Alterations of KLF6 expression had to be confirmed in keratoconus cornea. In corneal epithelial cells, modifications of a1PI promoter activities could be detected by the transient transfection and expression of hKLF6. We also demonstrated a collaborative effect of KLF6 and Sp1 on the expression of a1PI Conclusions: We demonstrated for the first time the implications of KLF6 in physiopathology of keratoconus in collaboration with Sp1.

Keywords: cornea: epithelium • keratoconus • transcription factors