Abstract
Purpose :
Myocardin-related transcription factor (MRTF) has been implicated as a key signaling molecule involved in transdifferentiation of retinal pigment epithelial (RPE) cells into myofibroblasts, which play a critical role in development of fibrosis. The purpose of this study was to examine the role of Rho signaling in MRTF function and myofibroblast transdifferentiation of RPE cells.
Methods :
Transforming growth factor-beta2 (TGF-b2) was used to stimulate primary cultured porcine RPE cells. Confocal microscopy was utilized to examine immunocytochemically stained MRTF-A. Myofibroblast markers, alpha-smooth muscle actin (aSMA) and tropomyosin-1 (TPM1), was examined by western blot analyses. Myofibroblast function was assessed by collagen hydrogel contraction. The following inhibitors were used: CCG-1423, cell permeable C3 transferase, and Y27632 for inhibition of MRTF signaling, Rho and ROCK, respectively.
Results :
TGF-b2 stimulation induced MRTF-A nuclear localization and myofibroblast differentiation, characterized by significantly increased myofibroblast marker expression as well as enhanced hydrogel contraction. CCG-1423 prevented induction of these myofibroblastic traits by TGF-b2, confirming the critical role of MRTF in myofibroblast transdifferentiation. Rho inhibitor suppressed MRTF-A nuclear localization, and significantly reduced myofibroblast marker expression as well as hydrogel contraction. In contrast, ROCK inhibition had little effect on TGF-b2 induced MRTF localization or myofibroblast differentiation, and significantly reduced hydrogel contraction only at a higher concentration.
Conclusions :
Our data show Rho, but not its downstream effector ROCK, is involved in the regulation of MRTF localization and function. Further understanding of mechanisms involved in Rho activation as well as signaling linking Rho to MRTF nuclear localization may reveal novel targets for therapeutic intervention for fibrotic complications in which RPE cells play a significant role such as proliferative vitreoretinopathy or exudative age-related macular degeneration.
This is a 2021 ARVO Annual Meeting abstract.