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Johannes Menzel-Severing, Matthias Zenkel, Naresh Polisetti, Angelika Mössner, Elisabeth Sock, Friedrich E Kruse, Ursula Schlotzer-Schrehardt; SOX9 as a potential regulator of limbal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):488.
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© ARVO (1962-2015); The Authors (2016-present)
We previously demonstrated preferential expression of HMG box transcription factor genes SOX9 and SOX10 in limbal epithelial cells (Menzel-Severing et al., ARVO Annual Meeting 2013). Here, we examine potential involvement of this gene family in limbal stem cell maintenance and/or differentiation.
RNA from human basal limbal and corneal epithelial cells was obtained by combining laser capture microdissection (LCM) and mRNA-amplification as previously described. Quantitative real-time PCR (qPCR) was used to perform a complete screen of SOX factor expression in these specimens (n=5). SoxE group mRNA expression was assessed in cultured limbal epithelial cells (LECs) at various stages of differentiation. Co-expression of SoxE factors with progenitor or differentiation markers of LECs was assessed by immunofluorescent staining of cryosectioned limbal tissue both in the resting state and following activation of wound healing. Using electroporation, siRNA-mediated down-regulation of SOX9-expression in cultured LECs was achieved, and the effects on potential target gene expression were monitored by qPCR.
Of all SOX transcription factors, those of the SoxF group (SOX7, 17, 18) and those of the SoxE group (SOX8, 9, 10) were consistently expressed at higher levels in limbal compared to corneal specimens obtained by LCM. In the SoxE group, mRNA expression was strongest for SOX9, and weakest for SOX8. This same pattern was observed in cultured LECs. Here, SOX9 expression was increased upon differentiation. Immunofluorescence showed preferential localisation of all SoxE factors to limbal basal cell nuclei. While Sox9 co-localised with putative progenitor markers including Keratin 15, N-Cadherin, and p63alpha in limbal basal cell clusters, Sox10 was expressed mainly in associated MelanA-positive melanocytes. Activation of wound healing in an organ culture model led to increased staining for SOX9 in all corneal epithelial cells. Following RNAi-mediated knock-down of SOX9, cultured LECs up-regulated expression of cornea-specific differentiation marker gene Keratin 3.
SOX9 has been identified as a master regulator of transcription in a number of epithelial stem cell compartments in mammals. Strong expression of SOX9 specifically in limbal basal epithelium together with its upregulation under differentiation and wound healing conditions may indicate its significance for regulating the limbal epithelial cell phenotype.
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