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Audrey Bernstein, Lingyan Wang, Stephanie Gillespie; An intracellular degradation pathway implicated in corneal scarring. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3911.
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Scarring in the cornea can lead to vision loss. Corneal haze and stromal fibrosis is the result of the persistence of myofibroblasts (Mfs), which excessively contract tissue and secrete fibrotic extracellular matrix. We recently reported that corneal Mfs are characterized by cell surface accumulation of integrin αvβ5 resulting from decreased integrin degradation. An agnostic screening approach (RNA-seq) was used to identify possible molecular mechanisms.
Methods: Primary human and porcine corneal fibroblasts were cultured. Gene expression was quantified by RNA-seq, alpha-smooth muscle actin (α-SMA) and integrin expression by Western blotting and RT-PCR, and protein complex formation by co-IP. Transfection of cDNA was by Nucleofection. Mfs were detected with antibody to α-SMA. The EDA-type of fibronectin (FN-EDA) was imaged by confocal microscopy and quantified with Leica software. A recycling assay quantified recycling of integrins. Porcine corneas (wounded by keratectomy and control) were cultured for 2 weeks prior to histological examination. USP10 expression was quantified with Image J software.
RNA-seq screening revealed that Mfs have increased gene expression of a subset of de-ubiquitinases (Dubs). Dubs remove ubiquitin from proteins, saving them from degradation thereby stabilizing protein levels. We found that the Dub, ubiquitin specific peptidase 10 (USP10), controls integrin αvβ5 protein levels and is increased in Mfs. This fits our recent finding that Mf differentiation occurs when integrin αvβ5 ubiquitination and degradation is reduced. Furthermore, over-expression of USP10 cDNA led to a 3-fold increase in integrin αvβ5 protein expression (with no increase in RNA expression) and a 70% increase in recycling of integrin αvβ5 to the cell surface (because of reduced degradation). USP10 over-expression also induced fibrotic proteins, a 2-fold increase in α-SMA, a 4.4-fold increase in FN-EDA with organization of extracellular FN-EDA when USP10 is over-expressed but not in control. Co-IP confirmed that USP10 is in a complex with integrin αvβ5. Finally, keratectomy generated stromal Mfs with a 6.8-fold increase in USP10 expression.
Mfs have increased USP10 expression and experimental USP10 over-expression induced fibrotic markers. Cellular degradation systems and USP10 in particular may be a novel anti-scarring target.
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