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Christopher Murphy, Sara Thomasy, Vijaykrishna Raghuanthan, Christopher Reilly, Paul Russell; The Mechanotransducers YAP and TAZ are Modulated by Substratum Compliance in Corneal Stromal Cells and during Corneal Wound Healing. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5979.
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© ARVO (1962-2015); The Authors (2016-present)
Biophysical cues profoundly influence corneal cellular behavior including processes integral to corneal wound healing such as migration and proliferation. Mechanotransduction or the translation of external biomechanical cues into internal biochemical events remains poorly understood in corneal cells. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have been identified as cellular mechanotransducers. Thus, the purposes of this study were 1) to determine the effects of substratum stiffness on expression of YAP/TAZ in human corneal fibroblasts (HCF) and myofibroblasts and 2) to determine the effects of corneal wounding on expression and localization of YAP/TAZ.
Primary HCFs were cultured on polyacrylamide substrates of differing stiffness (10 or 85 kPa) or on tissue culture plastic (TCP; >1GPa) in serum-containing media with 0 or 10 ng/ml TGFβ-1. Expression of YAP, TAZ and α-smooth muscle actin (αSMA) to confirm fibroblast to myofibroblast transition was determined by qPCR. Rabbits underwent epithelial debridement only or in combination with phototherapeuic keratectomy (PTK; 6 mm diameter, 100 μm deep) on the right eye (OD); the left eye (OS) served as a control. Rabbits were euthanized 6 days later and the globes were removed and fixed in formalin. Immunohistochemistry for YAP/TAZ was performed.
In the presence of 10 ng/ml TGFβ-1, TAZ expression was significantly greater on the more compliant substrates in comparison to TCP. Interestingly, αSMA was markedly decreased on the 10 kPa substrate versus 85 kPa or TCP. In normal cornea, YAP/TAZ were predominantly localized to the epithelial cell cytoplasm and minimally detected in normal stroma. Following PTK, YAP/TAZ were markedly localized in the epithelial cell nuclei and robust expression in anterior stromal cells was observed. Similar results were observed with epithelial debridement only except the depth of effect on anterior stromal cells was much less.
Substratum compliance markedly alters TGFβ-1 induced transformation of fibroblasts to myofibroblasts with stiffer substrates promoting transformation and decreasing TAZ expression. YAP/TAZ expression and localization are markedly altered during corneal wound healing. Further studies are required to determine the role of YAP/TAZ in corneal cellular mechanotransduction and wound healing.
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