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.
Highlights
Transforming growth factor beta 1 (TGF-b1) plays a prominent role in regulating a variety of cellular functions including cell migration, cell proliferation, apoptosis, differentiation, immunosuppression, inflammation, tumor-suppression, and angiogenesis [1,2]
In order to screen for agents that overcome the anti-myogenic effect of TGF-b1 on C2C12 differentiation, an assay was established that quantified C2C12 cell differentiation as indicated by expression of myosin heavy chain (MHC)
We noted that differentiation of C2C12 cells in the 96-well plate was sensitive to many variables in the assay including cell passage number, cell density, media conditions and time
Summary
Transforming growth factor beta 1 (TGF-b1) plays a prominent role in regulating a variety of cellular functions including cell migration, cell proliferation, apoptosis, differentiation, immunosuppression, inflammation, tumor-suppression, and angiogenesis [1,2]. TGF-b1 inhibits expression of two key transcriptional mediators of muscle cell differentiation, MyoD and myogenin [8]. Smad binds to and interferes with the myogenic transcription factor MEF2 to prevent muscle-specific gene expression [10]. Increased expression of the inhibitory Smad, Smad, promotes myogenesis [11] Another TGF-b family member, myostatin, is a potent inhibitor of muscle differentation and growth [12,13]. Transforming growth factor beta 1 (TGF-b1) is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-b1
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