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Che Connon, Roanne Jones, James Foster, ; Localisation of Yap/Taz in corneal epithelia: a marker of mechano-sensitivity and role in epithelial homeostasis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2582.
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To investigate the cellular location of the nuclear transcription factor Yap/Taz in limbal and central corneal epithelial cells and relate this expression to changes in corneal stiffness centripetally across the ocular surface. Yap/Taz has recently been found to be an important regulator of mechanotransduction. We believe that changes in substrate stiffness occur across the cornea and that a centripetal stiffness gradient drives both differentiation and migration of epithelial cells form the limbus towards the central cornea forming an important structural component of ocular surface homeostasis.
The localisation of Yap/Taz, CK3, CK14 and ZO-1 across the ocular surface of bovine corneas was examined by immunohistochemistry. Limbal stem cells were isolated form fresh bovine corneas and expanded upon type I collagen gels of differing stiffness for 14 days. The localisation of Yap/Taz, CK3, CK14 and ZO-1 was examined by immunohistochemistry within these corneal constructs. Furthermore, the transcription levels of Yap/Taz, CK3 and ABCG2 were quantified by QPCR.
Across the healthy bovine cornea Yap/Taz was predominately expressed cytoplasmically within the limbus, where as in central corneal epithelial cells Yap/Taz was retained within the nucleus. Isolated limbal epithelial cells expanded upon the more compliant collagen gels showed significantly less gene expression of Yap/Taz, which was predominately cytoplasimic at the protein level, whereas more nuclear expression was seen within epithelial cells expanded upon the stiffer collagen gels. This corresponded with more cells expressing cytokeratin 3 and ZO-1 and less cytokeratin 14 and ABCG2 at the gene and protein level.
The nuclear to cytoplasmic expression ratio of Yap/Taz between limbal and central epithelial cells supports the notion of a centripetal stiffness gradient across the corneal surface. The suitability of YAP/Taz as a marker of mechanosensitivity (substrate stiffness) in corneal epithelial cells was successfully demonstrated by use of collagen gels as cell substrates with differing stiffness. The presence of a centripetal stiffness gradient across the cornea is likely to underpin new directions in corneal wound healing and our understanding of ocular surface homeostasis.
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