Purpose: The ocular surface, composed of the conjunctiva and the cornea, is essential for vision. Its integrity depends on numerous molecular and cellular processes such as proliferation, differentiation, apoptosis, adhesion, and extracellular matrix homeostasis, whose deregulation can induce ophthalmological pathologies. The Krüppel-like transcription factors (KLFs) family is made up of 15 C2H2 zinc-finger proteins involved in vertebrate development and able to control cell proliferation, growth, and differentiation. In order to better define their respective roles in the human ocular surface, we decided to determine their pattern of expression in ocular tissues. We then focused on the expression of KLF4 and some of its target genes to establish KLF4's biological activities in human ocular surface.
Methods: Firstly, total mRNA was extracted from human total cornea, conjunctiva, corneal epithelial cells (primary culture and established cell line), corneal keratocytes (primary culture), corneal endothelial cells (established cell line), and conjunctival epithelial cells (established cell line) and submitted to RT-PCR experiments in order to determine the expression patterns of the different KLFs. Secondly, KLF4 protein localization was visualized by immunofluorescence assays at tissue and cell levels. Finally, KLF4 target genes (endoglin, ornithine decarboxylase) mRNA expression levels were determined by semi-quantitative RT-PCR, after KLF4 transient transfection in human corneal epithelium (HCE) cells.
Results: We detected the presence of twelve transcripts of KLFs in the cornea (KLF2, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF10, KLF11, KLF12, KLF13, and KLF16) and eight in the conjunctiva (KLF2, KLF3, KLF4, KLF6, KLF7, KLF10, KLF11, and KLF12). Under our conditions, the transcripts encoding KLF1 and KLF9 were never detected. Specific expression patterns of each KLF were also determined for the major cellular components of the human cornea and conjunctiva. KLF4 immunolocalization assays indicated its presence in both the cytoplasmic and nuclear compartments of conjunctival and corneal cells. KLF4 transient overexpression in HCE cells down regulated both endoglin and ODC mRNA levels.
Conclusions: For the first time, we established the presence of a KLF network in the human ocular surface and illustrated the conservation of KLF4's biological properties in a corneal derived epithelial cell line.