Abstract
Purpose :
Corneal scarring is driven by the persistence of pathological myofibroblasts in healing tissue. Pathological myofibroblasts are characterized by an increase in cell-surface integrins, increasing their adherence to extracellular matrix, and facilitating local TGFβ 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. Yeast two-hybrid screening using USP10 as bait revealed a high confidence interaction with both G3BP1 and 2 proteins. G3BP1/2 have a known function related to stress granule formation but their role in corneal wounding has not been investigated.
Methods :
A yeast two-hybrid screen was carried out by Hybrigenics (France). USP10/G3BP1/2 association in cells was determined by proximity ligation assay (PLA) in primary human corneal fibroblasts (HCFs) and in corneal mouse tissue (C57BL/6, Jackson Labs). Mouse cornea were wounded with 0.5M NaOH for 1 min and compared to control, unwounded corneas. Transfection of siRNA was by Lonza Nucleofection followed by a Fibronectin (FN) Recycling Assay. Protein expression was detected by western blot. Ubiquant ELISA was used to quantify ubiquitination on integrins.
Results :
G3BP1 and 2 proteins are expressed in HCFs. PLA between USP10 and G3BP1 (p<0.01) and G3BP2 (p<0.05) in HCFs support the yeast 2-hybrid data. The frequency of interaction increased by 2-fold with TGFβ1 treatment (p< 0.0001 and p<0.001, respectively). PLA signal for USP10/G3BP2 in mouse wounded cornea was non-significant 24 hours after wounding compared to control, however, the interaction increased 2.8-fold 72 hours after wounding (p<0.0001), suggesting that the USP10/G3BP2 axis is involved in wound healing. Mechanistically, we found that concurrent knockdown of G3BP1/2 in HCFs with targeting siRNA was anti-fibrotic, with reduced αvβ5 integrin accumulation (increased ubiqutination, 3.6-fold p<0.01) and reduced FN recycling (35.0% decrease in cell surface accumulation, p<0.001).
Conclusions :
Our data support a role for G3BP1/2 proteins in wound healing. G3BP1/2 may be novel anti-scarring targets for the cornea.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.