3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Increase Is Essential for Rat Muscle Differentiation CHIARA MARTINI, 1 LAURA TRAPANI, 1 LAURA NARCISO, 2 MARIA MARINO, 1 ANNA TRENTALANCE, 1 AND VALENTINA PALLOTTINI 1 * 1 Department of Biology, University of ‘‘Roma Tre,’’ Rome, Italy 2 Department of Environment and Primary Prevention, Istituto Superiore di Sanita`, Rome, Italy 3-Hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoAR) is the key and rate-limiting enzyme of cholesterol biosynthetic pathway. Although HMG-CoAR activity has already been related to the differentiation of some cellular lines there are no studies that analyze the role of HMG-CoAR, and the pathway it is involved with in a fully characterized muscle differentiation model. Thus, the aim of this work is to evaluate such role and delineate the pathway involved in foetal rat myoblasts (L6) induced to differentiate by insulin—a standard and feasible model of the myogenic process. The results obtained by biochemical and morphological approaches demonstrate that (i) HMG-CoAR increase is crucial for differentiation induction, (ii) p21waf, whose increase is a necessary requisite for differentiation to occur, rises downstream HMG-CoAR activation, (iii) the main role of p38/MAPK as key regulator also for HMG-CoAR. Pathologies characterized by muscle degeneration might benefit from therapeutic programmes committed to muscle function restoration, such as modulation and planning myoblast differentiation. Thus, the important role of HMG-CoAR in muscular differentiation providing new molecular basis for the control of muscle development can help in the design of therapeutic treatment for diseases characterized by the weakening of muscular fibers and aging-related disorders (sarcopenia). J. Cell. Physiol. 220: 524–530, 2009. ß 2009 Wiley-Liss, Inc. Myogenic differentiation is a process that begins with the commitment of mononucleated precursors to withdraw from the cell cycle, and continues with their planned progression towards specific cell types. Myogenesis can therefore be seen as a two-state process consisting of commitment and progression of myoblasts, both requiring the interplay of positive and negative regulatory signals. As they elongate, myoblasts align with each other, guided in this by mutual membrane recognition. Such alignment is followed by cell fusion, and by the formation of long and striated multinucleated myotubes (Mermelstein et al., 2007). One of the major intracellular signaling pathways activated during the differentiation of myogenic cell lines is p38/mitogen-activated protein kinase (MAPK). By modifying p38 activity in myoblasts, the pathway has been proved essential for the expression of muscle-specific genes. p38 affects the activities of transcription factors of the MyoD and MEF2 families, and participates in the remodeling of chromatin at the level of specific muscle-regulatory regions. p38 cooperates with the myogenic transcription factors in the activation of a subset of late-transcribed genes, thus contributing to the progressive expression of genes during differentiation. p38/MAPK signaling has been also described as an inductor of the expression of the cyclin-dependent inhibitor p21waf in myoblasts, supporting p38’s role in regulating cell cycle withdrawal of myoblasts at the G1 stage. Such withdrawal is considered a necessary condition for differentiation to occur (Keren et al., 2006). 3-Hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoAR) is the key and rate-limiting enzyme of the mevalonate pathway that leads to the production of cholesterol and isoprenoids (Goldstein and Brown, 1990). The enzyme is subjected to short- and long-term regulations. Short-term modulation is carried out by phosphorylation/ dephosphorylation mechanisms operated, respectively, by AMP activated kinase (AMPK) (Hardie, 1992; Fisslthaler et al., 2007) and Protein Phosphatase 2 A (PP2A) (Gaussin et al., 1997). Long-term regulation concerns the modulation of HMG-CoAR protein levels by several factors, which include Sterol Regulatory Elements Binding Protein (SREBP) and INSulin Induced Gene (INSIG)—both able to affect enzyme transcription and degradation (Goldstein et al., 2006). It has been demonstrated that, some HMG-CoAR end-products derivatives of mevalonate such as farnesyl- pyrophosphate (FPP) and geranyl-pyrophosphate (GGPP) are essential compounds for survival, proliferation, and differentiation of cells through the activation of small GTPases, such as Ras and RhoA (Allal et al., 2000; Prendergast and Oliff, 2000; van de Donk et al., 2005). RhoA, in particular, has been shown to trigger the myogenic potential of mouse embryo fibroblasts, to accelerate myoblast differentiation and to promote actin gene expression through serum response factor activation (Castellani et al., 2006). Moreover, it has been demonstrated that HMG-CoAR activity inhibiting statines Mevinolin and Mevastatin are able to inhibit myoblast fusion by impairing the synthesis of fusogenic cell surface N-linked glycoproteins, probably by affecting the synthesis of dolichol phosphate-oligosaccharides that are required as intermediates in N-linked glycoprotein biosynthesis (Belo et al., 1993). Contract grant sponsor: University of Roma Tre, CLAR 2007– 2008. *Correspondence to: Valentina Pallottini, Department of Biology, University of Roma Tre, Viale Marconi, 446-00146 Rome, Italy. E-mail: vpallott@uniroma3.it Received 12 January 2009; Accepted 30 March 2009 Published online in Wiley InterScience (www.interscience.wiley.com.), 22 April 2009. DOI: 10.1002/jcp.21810 ORIGINAL ARTICLE 524 Journal of Journal of Cellular Physiology Cellular Physiology ß 2009 WILEY-LISS, INC.