Identification of Retinoic Acid in a High Content Screen for Agents that Overcome the Anti-Myogenic Effect of TGF-Beta-1

Abstract

BackgroundTransforming growth factor beta 1 (TGF-β1) is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. When neutralizing antibodies to TGF-β1 or the angiotensin II inhibitor losartan were used to reduce TGF-β1 signaling, muscle morphology and function were restored in mouse models of Marfan Syndrome and muscular dystrophy. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-β1.Methodology/Principal FindingsA high-content cell-based assay was developed in a 96-well plate format that detects the expression of myosin heavy chain (MHC) in C2C12 cells. The assay was used to quantify the dose-dependent responses of C2C12 cell differentiation to TGF-β1 and to the TGF-β1 Type 1 receptor kinase inhibitor, SB431542. Thirteen agents previously described as promoting C2C12 differentiation in the absence of TGF-β1 were screened in the presence of TGF-β1. Only all-trans retinoic acid and 9-cis retinoic acid allowed a maximal level of C2C12 cell differentiation in the presence of TGF-β1; the angiotensin-converting enzyme inhibitor captopril and 10 nM estrogen provided partial rescue. Vitamin D was a potent inhibitor of retinoic acid-induced myogenesis in the presence of TGF-β1. TGF-β1 inhibits myoblast differentiation through activation of Smad3; however, retinoic acid did not inhibit TGF-β1-induced activation of a Smad3-dependent reporter gene in C2C12 cells.Conclusions/SignificanceRetinoic acid alleviated the anti-myogenic effect of TGF-β1 by a Smad3-independent mechanism. With regard to the goal of improving muscle regeneration and function in individuals with muscle disease, the identification of retinoic acid is intriguing in that some retinoids are already approved for human therapy. However, retinoids also have well-described adverse effects. The quantitative, high-content assay will be useful to screen for less-toxic retinoids or combinations of agents that promote myoblast differentiation in the presence of TGF-β1.

In vitro differentiation of functional human skeletal myotubes in a defined system

“…Sonic Hedgehog is found to be a cell-autonomous factor for the survival, myogenesis, and migration of muscle precursors during development [43, 44] . Similarly, retinoic acid is known to enhance myogenesis [45] . In general, all the factors included in our differentiation medium 2 have demonstrated some positive effect on muscle differentiation, maturation and/ or function, which may explain why the myotubes in this study developed more mature characteristics.…”

Retinoid and TGF-β Families: Crosstalk in Development, Neoplasia, Immunity, and Tissue Repair

“…In embryonic stem cells, ATRA induces the expression of Foxa1, which acts as a pioneer transcription factor, binding and poising chromatin for intersection with the TGF-β-induced SMAD signaling, and cooperates with the latter in de novo activation of α-fetoprotein gene expression 17 . In C2C12 mouse myoblasts, both ATRA and 9 cis -retinoic acid are potent inhibitors of the anti-myogenic effect of TGF-β1, likely through a SMAD-independent mechanism 18 .…”

Stem cells for cardiac regeneration and possible roles of the transforming growth factor-β superfamily

“…However, using the myoblast cell line C2C12, TGF-β 1 has also been shown to act as a negative regulator for myogenesis (52) . In this response, Smad3 activation was mediated.…”