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Johannes Menzel-Severing, Matthias Zenkel, Friedrich Kruse, Ursula Schlotzer-Schrehardt; Transcription factor screening of limbal vs. corneal epithelium identifies distinct patterns for SOX-9 and peroxisome proliferator-activated receptor gamma (PPARG). Invest. Ophthalmol. Vis. Sci. 2013;54(15):994.
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© ARVO (1962-2015); The Authors (2016-present)
To identify transcriptional regulators potentially involved in limbal epithelial stem cell homeostasis and differentiation.
Limbal and central corneal epithelial specimens were obtained from four human post-mortem donor eyes using laser capture microdissection. RNA extracted from these specimens underwent linear amplification (MessageAmp II, Ambion). Samples were screened for differential expression of stem cell transcription factor genes using quantitative real-time PCR arrays (SABiosciences). Candidate genes were confirmed using specific real-time PCR hydrolysis probe assays (Roche) and immunohistochemistry of frozen corneal sections.
Of 86 genes screened, nine appeared to be differentially expressed, and were therefore investigated further (FOXP2, HOXA11, KLF2, SOX9, STAT3, WRN, MYC, DACH1, PPARG). Using individual assays, increased mRNA expression in limbal specimens was confirmed for SOX9 and PPARG. Immunohistochemistry showed nuclear localization of SOX-9 in limbal basal cells, whereas no specific staining was seen in central corneal epithelium. PPARG was detected within nuclei of basal epithelial cells of conjunctiva and central cornea, while perinuclear/cytoplasmic staining was observed in small, basal cell clusters at the limbus.
Transcription factor SOX-9 has been suggested to contribute to progenitor cell regulation in hair follicle, intestinal epithelium and the subependymal zone. Several reports have identified links to Wnt signaling transducer beta-catenin, for which a role in maintenance of limbal stem cells has been proposed. PPARG is a nuclear receptor associated with differentiation pathways of adipocytes and epithelial cells as well as growth inhibition of carcinoma cells. Nuclear export of PPARG is mediated by MAP2K1/MEK1, which invites to speculate about a possible upstream activity of the MAPK-pathway in putative limbal progenitors. Further research is warranted to elucidate the functional involvement of SOX-9 and PPARG in corneal epithelial cell differentiation, and to assess the usefulness of modulating their activity in the context of cell therapy.
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