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Andrew Phillips, Edward F Boumil, Nileyma Castro, Audrey M Bernstein; Corneal scarring: Decreased intracellular degradation of integrins promotes matrix recycling. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4668.
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© ARVO (1962-2015); The Authors (2016-present)
After wounding, the formation of scar tissue in the corneal stroma is associated with the persistence of pathological myofibroblasts, which are characterized in part by an increase in cell surface integrin expression. Our previous studies demonstrated that after wounding, protection of integrins from intracellular proteolysis pathways shifted the balance of integrin homeostasis resulting in a return of integrins to the cell surface with subsequent accumulation. Specifically, we found that the deubiquitinase USP10 removes ubiquitin from αv-integrins β1 and β5, and this activity is increased after wounding. Since integrins bind to and internalize extracellular matrix (ECM), we have tested if ECM accumulation can result from a decrease in intracellular integrin/ECM degradation and a concomitant recycling of the matrix.
Recycling assay: Human Corneal Fibroblasts or Human Foreskin Fibroblasts were transfected with USP10 cDNA or control vector cDNA by Nucleofection (Lonza) and then loaded with biotin tagged fibronectin (FN) in supplemented serum free media for 3 hours. Cells were detached with Trypsin to ensure that no extracellular FN remained after passage (flow cytometry). After 48 hours in culture, the cells were treated with sodium azide and blocking solution. A streptavidin-488 antibody was used to detect extracellular (recycled) biotin-tagged FN and cells were imaged by confocal (Zeiss LSM780).
In cells transfected with USP10 cDNA compared to control cDNA, the recycled extracellular FN-containing puncta were a) significantly larger (5-fold, p<0.001), b) with increased average fluorescence intensity (1.8-fold, p<0.01) and c) increased average number of puncta per image (1.6-fold, p<0.05). Flow cytometry was used to confirm that FN was absent from the cell surface after trypsinization and therefore did not contribute to the recycled FN that was quantified.
Organized extracellular FN is increased when USP10 is overexpressed. This suggests that reduced integrin degradation after wounding may be a newly identified mechanism for the characteristic accumulation of ECM in scar tissue.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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