title: Identification of retinoic acid in a high content screen for agents that overcome the anti-myogenic effect of TGF-beta-1.
abstract: BACKGROUND
  Transforming 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.
  
  
  RESULTS
  A 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
  Retinoic 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.
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Chateen
authors.initials: C
authors.lastname: Krueger
authors.forename: F Michael
authors.initials: FM
authors.lastname: Hoffmann
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001549:Benzamides / Q000494:pharmacology; D002216:Captopril / Q000494:pharmacology; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004149:Dioxoles / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D051379:Mice; D024510:Muscle Development / Q000187:drug effects*; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D000077185:Resveratrol; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000378:metabolism; D013267:Stilbenes / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014212:Tretinoin / Q000494:pharmacology*; D014807:Vitamin D / Q000494:pharmacology; D014815:Vitamins / Q000494:pharmacology
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D000806:Angiotensin-Converting Enzyme Inhibitors; D000975:Antioxidants; D001549:Benzamides; D004149:Dioxoles; D018125:Receptors, Transforming Growth Factor beta; D051900:Smad3 Protein; D013267:Stilbenes; D053773:Transforming Growth Factor beta1; D014815:Vitamins; D014807:Vitamin D; D014212:Tretinoin; D002392:Catechin; D002216:Captopril; C045651:epigallocatechin gallate; D000077185:Resveratrol