ccg-1423 and Fibrosis

Topics: Anilides; Animals; Benzamides; Cell Survival; Female; Fibrosis; Gene Expression Regulation; Intervertebral Disc; Intervertebral Disc Degeneration; Nuclear Proteins; Nucleus Pulposus; Rats; Rats, Sprague-Dawley; rho GTP-Binding Proteins; Signal Transduction; Trans-Activators; Transcription Factors

Epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells is related to the pathogenesis of subretinal fibrosis such as that associated with macular degeneration. The role of myocardin-related transcription factor A (MRTF-A) in EMT of RPE cells and subretinal fibrosis was investigated.. The migratory activity of human RPE-1 cells in culture was evaluated using a scratch assay. The subcellular distribution of MRTF-A in RPE-1 cells, as well as the extent of subretinal fibrosis in a mouse model, were determined by immunofluorescence analysis. Expression of α-smooth muscle actin (α-SMA), collagen type I (COL1), connective tissue growth factor (CTGF), and paxillin was examined by immunoblot analysis or reverse transcription and quantitative polymerase chain reaction analysis, whereas that of pro-matrix metalloproteinase-2 (MMP-2) was assessed by gelatin zymography.. The MRTF-A signaling inhibitor CCG-1423 suppressed RPE-1 cell migration in a concentration-dependent manner. Transforming growth factor-beta (TGF-β2) induced MRTF-A translocation from the cytoplasm to the nucleus of RPE-1 cells, and this effect was attenuated by CCG-1423. TGF-β2 up-regulated the abundance of α-SMA, paxillin, and pro-MMP-2 proteins as well as the amounts of α-SMA, COL1, and CTGF mRNAs in a manner sensitive to inhibition by CCG-1423. Finally, intravitreal injection of CCG-1423 markedly attenuated the development of subretinal fibrosis induced by photocoagulation in vivo.. Our results implicate MRTF-A in EMT of RPE cells and in the development of subretinal fibrosis in vivo, suggesting that MRTF-A is a potential therapeutic target for retinal diseases characterized by subretinal fibrosis.

Topics: Actins; Anilides; Animals; Benzamides; Cell Movement; Collagen Type I; Connective Tissue Growth Factor; Disease Models, Animal; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Female; Fibrosis; Fluorescent Antibody Technique, Indirect; Humans; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Real-Time Polymerase Chain Reaction; Retina; Retinal Pigment Epithelium; Trans-Activators

Ras homolog gene family, member A (RhoA)/Rho-associated coiled-coil forming protein kinase signaling is a key pathway in multiple types of solid organ fibrosis, including intestinal fibrosis. However, the pleiotropic effects of RhoA/Rho-associated coiled-coil forming protein kinase signaling have frustrated targeted drug discovery efforts. Recent recognition of the role of Rho-regulated gene transcription by serum response factor (SRF) and its transcriptional cofactor myocardin-related transcription factor A (MRTF-A) suggest a novel locus for pharmacological intervention.. Because RhoA signaling is mediated by both physical and biochemical stimuli, we examined whether pharmacological inhibition of RhoA or the downstream transcription pathway of MRTF-A/SRF could block intestinal fibrogenesis in 2 in vitro models.. In this study, we demonstrate that inhibition of RhoA signaling blocks both matrix-stiffness and transforming growth factor beta-induced fibrogenesis in human colonic myofibroblasts. Repression of alpha-smooth muscle actin and collagen expression was associated with the inhibition of MRTF-A nuclear localization. CCG-1423, a first-generation Rho/MRTF/SRF pathway inhibitor, repressed fibrogenesis in both models, yet has unacceptable cytotoxicity. Novel second-generation inhibitors (CCG-100602 and CCG-203971) repressed both matrix-stiffness and transforming growth factor beta-mediated fibrogenesis as determined by protein and gene expression in a dose-dependent manner.. Targeting the Rho/MRTF/SRF mechanism with second-generation Rho/MRTF/SRF inhibitors may represent a novel approach to antifibrotic therapeutics.

Topics: Anilides; Benzamides; Blotting, Western; Cell Adhesion; Cell Nucleus; Cells, Cultured; Colon; DNA-Binding Proteins; Extracellular Matrix; Fibrosis; Humans; Myofibroblasts; Oncogene Proteins, Fusion; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA, Messenger; Serum Response Factor; Signal Transduction; Trans-Activators; Transforming Growth Factor beta