Abstract
Purpose :
Corneal scarring is driven by the persistence of myofibroblasts in healing tissue. Pathological myofibroblasts are characterized by an increase in cell-surface integrins, increasing their adherence to the ECM, promoting over-contraction of surrounding tissue, and facilitating local TGFβ1 release. After wounding, the deubiquitinase USP10 is upregulated in myofibroblasts and promotes an increase in expression of αv-integrins by protecting them from ubiquitin-mediated intracellular proteolysis. The purpose of this study is to elucidate how USP10 coordinates with the cytoskeleton to promote integrin accumulation under pathological conditions.
Methods :
Yeast two-hybrid screen was carried out by Hybrigenics (France). Human corneal fibroblasts (HCFs) were plated on collagen in supplemented serum-free media and stimulated with 2ng/ml TGFB1. USP10/Daam1 association was determined by proximity ligation assay (PLA, positive signal by immunocytochemistry demonstrates a <40nm interaction) and standard immunocytochemistry. Transfection of siRNA was by Lonza Nucleofection. Protein expression was detected by Western blot.
Results :
Yeast two-hybrid screening using USP10 as bait revealed a novel high confidence interaction with the formin protein Daam1, which plays a role in actin bundling and increased Daam1 expression is associated with fibrotic disease. In HCFs, by Western blot, TGFB1 treatment increased Daam1 expression by 1.8-fold +/- 0.1 (p<0.001) and by immunocytochemistry, USP10 colocalizes with Daam1 on α-SMA stress fibers (1.3+/-0.4-fold increase; p<0.001). Conversely, Daam1 and USP10 were diffuse throughout the cell in untreated cultures. TGFB1 treatment also significantly increased PLA between USP10 and Daam1 (2.0-fold increase in puncta compared to untreated cells, p<0.001) suggesting that they are in a complex together. siRNA knockdown of Daam1 reduced Daam1, αv, β1, and β5 integrin levels in TGFB1 treated cells (70.8%, 23.0%, 36.7%, and 30.3%, respectively).
Conclusions :
Our data suggest that Daam1/USP10 binding coordinates the actin cytoskeleton with the activity of USP10 on integrins to promote an increase in αv-integrin heterodimers observed in pathological myofibroblasts.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.