Abstract
Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Keywords: 480 cornea: basic science •
482 cornea: epithelium •
500 differentiation