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Stephanie Gillespie, Audrey M Bernstein; Targeting alpha v integrins promotes regenerative healing in the cornea. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1273.
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© ARVO (1962-2015); The Authors (2016-present)
Scarring and fibrotic disease result from the persistence of myofibroblasts characterized by high cell-surface expression of alpha v integrins, which activate the fibrotic factor TGFβ leading to pathological cell adhesion and fibrotic matrix secretion. We propose that increasing ubiquitin-mediated intracellular degradation of alpha v integrins prevents scarring and induces regenerative healing.
Experiments were performed in supplemented serum-free media on collagen. Transient transfection in primary human corneal cells (HCFs) with USP10 or control cDNA was performed using Amaxa Nucleofection. Standard lentiviral infection using immortalized htert-HCFs was used to generate a USP10 overexpressing cell line. TGFβ activity was measured by co-culturing USP10 cells with htert-SMAD-luciferase/GFP reporter cells and Bright Glo Luciferase Reagent. Detection of a-SMA and FN-EDA was by RT-PCR, Western blot, and immunocytochemistry. Porcine corneas, control or wounded by keratectomy, were treated with control or USP10 siRNA and cultured for 2 weeks prior to histological examination for fibrotic markers. USP10 deubiqiuintion of integrin β-chains was examined by immunoprecipitation of integrins in Ubiquitin-FLAG overexpressing htert-HCFs and Western blot for FLAG.
Through genetic screening (RNA seq), we determined that corneal stromal myofibroblasts overexpressed a subset of deubiquitinating enzymes (DUBs), which remove ubiquitin from proteins, saving them from degradation. We found that overexpression of the DUB, USP10 produced the fibrotic profile: a) significantly increased αv/β5/β3/β1 protein levels (2.0-3.9 fold), b) activated TGFβ (70% increase), c) increased FN-EDA and alpha smooth muscle actin (α-SMA) gene (1.4-2.1 fold) and protein expression (2.6-5.1 fold), as well as promoting organization of FN-EDA fibrils and α-SMA stress fibers. Silencing USP10 in corneal organ culture prevented induction of fibrotic markers and promoted regenerative healing in the epithelium and stroma. Mechanistically, we found that USP10 deubiquitinated integrin β-chains leading to decreased degradation and increased recycling to the cell surface.
This novel mechanism puts DUB expression at the head of the cascade that regulates alpha v integrin cell-surface abundance and fibrosis, and suggests USP10 as a novel anti-fibrotic target.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
USP10 overexpression induces an increase in FN-EDA (2.7 fold) compared to control.
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