THE UBIQUITIN PATHWAY IN CORNEAL SCARRING

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 DESCRIPTION (provided by applicant): Persistence of myofibroblasts (Mfs) in a healing corneal wound leads to scarring and vision loss. Mfs arise by activation of resident keratocytes and bone marrow-derived fibrocytes, which produce excessive extracellular matrix (ECM) and overly contracted tissue. Stromal Mfs are characterized by excess cell surface expression of integrin αvβ5, which increases cell adhesion and activates the fibrotic growth factor, TGFβ generating fibrosis. Thus, understanding the regulation of αvβ5 may be key to preventing persistent Mfs. Unbiased screening of corneal Mfs revealed that the gene expression of the deubiquitinase (DUB), USP10 was increased, implicating ubiquitin removal as a possible mechanism for αvβ5 accumulation. When we overexpressed USP10 we found a posttranslational increase in αvβ5 protein and fibrotic markers (αSMA and fibronectin-EDA). In corneal organ culture, USP10 increased concomitantly with αSMA, integrin αvβ5, and FNEDA; Conversely USP10 knockdown with targeted USP10 siRNA after wounding reduced these markers to control levels. In the following 3 specific aims we now propose to examine the mechanisms underlying the USP10 function in antiscarring therapy using primary human corneal Mfs, ex-vivo porcine organ culture and in-vivo rabbit studies. In Specific Aim 1 we will test whether USP10 binding to and deubiquitination of αvβ5 results in αvβ5 accumulation on the cell surface, inducing integrin-mediated TGFβ signaling and downstream FAK/AKT/FNEDA (antiapoptotic) signaling that leads to persistent Mfs. In Specific Aim 2 we will test whether interaction with p120RasGap and G3BP1 (shown to bind to integrin and USP10 in other systems) drives integrin αvβ5/USP10 to the cell surface. Furthermore, we will test whether the increased USP10-mediated recycling (reduced degradation) of the internalized integrin/ECM complex contributes to fibrotic ECM accumulation and impacts focal adhesion turnover and cell migration. In Specific Aim 3 we will silence USP10 gene expression in rabbits after wounding by anterior corneal keratectomy. The effect on Mf persistence, corneal clarity and thickness, as well as other fibrotic endpoints will be quantified. Together, our proposed studies will provide a rigorous test that will determine the value of DUB USP10 as a potential novel target for corneal antiscarring therapy.