Myocardin‐Related Transcription Factor Plays a Role in Myofibroblast Transdifferentiation of Retinal Pigment Epithelial Cells

Abstract

Retinal pigmented epithelial (RPE) cells have been implicated in fibrotic scarring of the retina. Past studies have shown transforming growth factor (TGF)‐beta plays a critical role in this process by inducing myofibroblast transdifferentiation. Myocardin‐related transcription factor (MRTF) has been implicated to play a key role in TGF‐beta initiated signaling in several fibrotic diseases, and thus, we examined its role in an in vitro model using small molecule inhibitors.Primary cultured porcine RPE cells were cultured for 3 days in media containing TGF‐beta2 (10ng/ml) in the presence or absence of the following inhibitors: TGF‐beta receptor inhibitor SB431542 (10 micro M), MRTF signaling inhibitors CCG100602 and CCG203971 (25 microM), and CCG1423 (10 microM). Staining of actin cytoskeleton and alpha‐smooth muscle actin (SMA) were performed using phalloidin and anti‐alpha‐SMA antibody, respectively. For the collagen matrix contraction assay, an in vitro model for fibrosis, cells were cultured three days on type 1 collagen gels, which were subsequently released from 24 well plates and photographed to evaluate contraction by measuring gel size. These same gels with attached cells were then lysed by RIPA buffer and used for Western blot analysis of alpha‐SMA, and tropomyosin1.TGF‐beta treatment induced myofibroblast transdifferentiation as evidenced by expression of alpha‐SMA incorporated prominent stress fibers and the associated significant increase in contractility. This phenotypic change was completely blocked by the TGF‐beta receptor inhibitor SB431542. Similarly, MRTF inhibitors CCG100602, CCG203671, and CCG1423 prevented cytoskeletal changes as well as the enhanced collagen matrix contraction induced by TGF‐beta. Interestingly, MRTF inhibitors did not significantly alter alpha‐SMA expression, but reduced expression of tropomyosin 1, a MRTF target that has been implicated to play a role in alpha‐SMA incorporation into stress fiber.These data strongly implicate that MRTF plays a key role in myofibroblast transdifferentiation of RPE cells downstream of TGF‐beta receptor activation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.