S100B Protein in Myoblasts Modulates Myogenic Differentiation Via NF-kB-Dependent Inhibition of MyoD Expression CLAUDIA TUBARO, 1 CATALDO ARCURI, 1 ILEANA GIAMBANCO, 1 AND ROSARIO DONATO 2 * 1 Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy 2 Department of Experimental Medicine and Biochemical Sciences and Istituto Interuniversitario di Miologia, University of Perugia, Perugia, Italy S100B, a Ca 2þ -binding protein of the EF-hand type, is expressed in myoblasts, the precursors of skeletal myofibers, and muscle satellite cells (this work). S100B has been shown to participate in the regulation of several intracellular processes including cell cycle progression and differentiation. We investigated regulatory activities of S100B within myoblasts by stable overexpression of S100B and by inhibition of S100B expression. Overexpression of S100B in myoblast cell lines and primary myoblasts resulted in inhibition of myogenic differentiation, evidenced by lack of expression of myogenin and myosin heavy chain (MyHC) and absence of myotube formation. S100B-overexpressing myoblasts showed reduced MyoD expression levels and unchanged Myf5 expression levels, compared with control myoblasts, and transient transfection of S100B-overexpressing myoblasts with MyoD, but not Myf5, restored differentiation and fusion in part. The transcriptional activity of NF-kB, a negative regulator of MyoD expression, was enhanced in S100B-overexpressing myoblasts, and blocking NF-kB activity resulted in reversal of S100B’s inhibitory effects. Yin Yang1, a transcriptional repressor that is induced by NF-kB (p65) and mediates NF-kB inhibitory effects on several myofibrillary genes, also was upregulated in S100B-overexpressing myoblasts. Conversely, silencing S100B expression in myoblast cell lines by RNA interference resulted in reduced NF-kB activity and enhanced MyoD, myogenin and MyHC expression and myotube formation. Thus, intracellular S100B might modulate myoblast differentiation by interfering with MyoD expression in an NF-kB-dependent manner. J. Cell. Physiol. 223: 270–282, 2010. ß 2010 Wiley-Liss, Inc. The Ca 2þ -binding protein of the EF-hand, S100B, has been shown to regulate a variety of intracellular activities by interacting with effector proteins (for review see Donato, 2001; Heizmann et al., 2002; Donato et al., 2009). S100B, a non- ubiquitous protein, is expressed in high abundance in astrocytes, melanocytes, adipocytes, and chondrocytes, and in smaller amounts in several other cell types including myoblasts. The human S100B gene maps to chromosome 21q22.3 with consequent augmented expression of S100B protein in Down syndrome. Effects of S100B within cells include regulation of cell cycle progression, Ca 2þ homeostasis, gene expression, protein phosphorylation, enzyme activity, differentiation and apoptosis, and modulation of the dynamics of cytoskeleton components (Donato, 2001; Marenholz et al., 2004; Donato et al., 2009). There is growing interest in the regulatory effects of S100B on cell cycle progression, differentiation and apoptosis because the protein is upregulated in the neoplastic counterpart of S100B expressing cells (which raises the possibility that S100B might play a role in tumorigenesis) and in reactive gliosis (which raises the possibility that S100B might play a role in the pathophysiology of neurodegenerative disorders and the inflammatory response in the brain) (Lin et al., 2004; Marenholz et al., 2004; Donato et al., 2009; Brozzi et al., 2009). Moreover, evidence has been presented that S100B might function as an intracellular modulator of cell differentiation (Arcuri et al., 2005; Saito et al., 2007; Brozzi et al., 2009). Myogenesis is a multistep process by which mesenchimal cells are committed to myoblasts: these then migrate to body sites of skeletal muscle formation and proliferate under the action of a number of extracellular factors operating via cell surface receptors, and eventually fuse with each other to form myotubes, the ultimate precursors of skeletal muscle fibers (Charge ´ and Rudnicki, 2004; Buckingham, 2006). Intracellular factors orchestrating this process are transcription factors, some of which are muscle-specific (e.g., Myf5, MyoD, myogenin, and MRF4), that represent the ultimate target of extracellular cues. A similar process takes place in the mature skeletal muscle Claudia Tubaro and Cataldo Arcuri contributed equally to this work. Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: Ministero dell’Universita ` e della Ricerca- University of Perugia; Contract grant number: PRIN 2004054293 (to RD). Contract grant sponsor: Association Franc ¸aise contre les Myopathies (Project 12992); Contract grant number: 12992 (to RD). Contract grant sponsor: Fondazione Cassa di Risparmio di Perugia; Contract grant number: 2004.0282.020_001 (to RD). Contract grant sponsor: Associazione Italiana per la Ricerca sul Cancro; Contract grant number: 1110 (to RD). *Correspondence to: Rosario Donato, Department Experimental Medicine and Biochemical Sciences, Sect. Anatomy, University of Perugia, Via del Giochetto C.P. 81 Succ. 3, 06122 Perugia, Italy. E-mail: donato@unipg.it Received 10 August 2009; Accepted 20 November 2009 Published online in Wiley InterScience (www.interscience.wiley.com.), 12 January 2010. DOI: 10.1002/jcp.22035 ORIGINAL ARTICLE 270 Journal of Journal of Cellular Physiology Cellular Physiology ß 2010 WILEY-LISS, INC.