Myostatin Inhibits Myogenesis and Promotes Adipogenesis in C3H 10T(1/2) Mesenchymal Multipotent Cells

0013-7227/05/$15.00/0 Printed in U.S.A. Endocrinology 146(8):3547–3557 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2005-0362 Myostatin Inhibits Myogenesis and Promotes Adipogenesis in C3H 10T(1/2) Mesenchymal Multipotent Cells Jorge N. Artaza, Shalender Bhasin, Thomas R. Magee, Suzanne Reisz-Porszasz, Ruoquin Shen, Nigel P. Groome, Meerasaluh M. Fareez, and Nestor F. Gonzalez-Cadavid Division of Endocrinology Metabolism and Molecular Medicine (J.N.A., S.B., S.R.-P., R.S., N.F.G.-C.), RCMI DNA Molecular Core, Charles R. Drew University of Medicine and Science, Los Angeles, California 90059; Department of Urology (T.R.M., N.F.G.-C.), University of California, Los Angeles School of Medicine, LABioMed Research Institute at HarborUCLA Medical Center, Torrance, California 90502; and School of Biological and Molecular Sciences (N.P.G., M.M.F.), Oxford Brookes University, Headington Campus, Oxford OX3 0BP, United Kingdom Inactivating mutations of the mammalian myostatin gene are associated with increased muscle mass and decreased fat mass; conversely, myostatin transgenic mice that overexpress myostatin in the skeletal muscle have decreased muscle mass and increased fat mass. We investigated the effects of recombinant myostatin protein and antimyostatin antibody on myogenic and adipogenic differentiation of mesenchymal multipotent cells. Accordingly, 10T(1/2) cells were incubated with 5ⴕ-azacytidine for 3 d to induce differentiation and then treated with a recombinant protein for myostatin (Mst) carboxy terminal 113 amino acids or a polyclonal anti-Mst antibody for 3, 7, and 14 d. Cells were also cotransfected with a Mst cDNA plasmid expressing the full-length 375-amino acid protein (pcDNA-Mst375) and the silencer RNAs for either Mst (pSil-Mst) or a random sequence (pSil-RS) for 3 or 7 d, and Mst expression was determined. Adipogenesis was evaluated by quantitative image analysis of fat cells before and after oil- T RANSGENIC MICE THAT overexpress recombinant myostatin (Mst) protein in the skeletal muscle have lower skeletal muscle mass and higher whole body fat mass, in comparison with wild-type controls (1). Similarly, the mice implanted with cells engineered to overexpress Mst experience significant cachexia (2). In general, Mst expression is higher under conditions in which the content of muscle mass is reduced (3). Conversely, null mutations of Mst gene in knockout mice and in double-muscle cattle are associated with hypermuscularity and decreased fat mass (4 – 6). Thus, Mst gene expression is an important modulator of body composition in experimental animals. A recent report of a child with an inactivating mutation of the Mst gene that was associated with hypermuscularity and First Published Online May 5, 2005 Abbreviations: ALK, Activin receptor-like kinase; AZCT, 5⬘-azacytidine; BMP, bone morphogenic protein; C/EBP, CCAAT/enhancer binding protein; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; MHC-II, myosin heavy chain type II; Mst, myostatin; Mst-113, Mst carboxy terminal 113 amino acids; pcDNA-Mst-375, cDNA-expressing mouse Mst full-length 375-amino acid sequence under a cytomegalovirus promoter; Sca-1, stem cell antigen or ataxin-1; si, silencer. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. red-O staining, immunocytochemistry of adiponectin, and Western blot for CCAAT/enhancer binding protein-␣. Myogenesis was estimated by quantitative image analysis-immunocytochemistry for MyoD (Myo differentiation protein), myogenin, and myosin heavy chain type II, or by Western blot for myogenin. 5ⴕ-azacytidine-mediated differentiation induced endogenous full-length Mst expression. Recombinant Mst carboxy terminal 113 amino acids inhibited both early and late markers of myogenesis and stimulated both early and late markers of adipogenesis, whereas the antibody against Mst exerted the reverse effects. Myogenin levels at 7 d after transfection of pcDNA-Mst375 were reduced as expected and elevated by pSil-Mst, which blocked efficiently Mst375 expression. In conclusion, myostatin promotes the differentiation of multipotent mesenchymal cells into the adipogenic lineage and inhibits myogenesis. (Endocrinology 146: 3547–3557, 2005) decreased fat mass further supports the role of Mst in the regulation of body composition (7). Significantly, in this child, as well as in the Mst knockout mice, fat mass, as measured by the weight or size of fat pads, was reduced in association with inactivating mutations of the Mst gene; in other experimental paradigms, fat mass is increased when Mst is overexpressed (5, 8 –10). The mechanisms by which Mst regulates body composition are not fully understood. Both the full-length and the 110-amino acid carboxy terminal Mst peptide have been shown to inhibit protein synthesis and satellite cell proliferation, in the latter case by blocking satellite cell entry into the cell cycle (11). However, these effects of Mst on satellite cells do not explain easily the observed changes in fat mass in Mst null and transgenic mice (1, 4 – 6). It is also difficult to reconcile these effects with a putative mechanism of preadipocyte activation, particularly because Mst has been reported to inhibit in vitro the adipogenic conversion of 3T3 preadipocytes (9) and of C3H 10T(1/2) cells undergoing bone morphogenic protein (BMP)-induced adipogenesis (12). Therefore, we considered the alternative interpretation that Mst acts upstream of the committed cell stage, e.g. on multipotent cell differentiation into myogenic and adipogenic lineages. Multipotent cells have the ability to undergo in vitro differentiation into multiple cell lineages according to the type 3547 3548 Endocrinology, August 2005, 146(8):3547–3557 of incubation medium and/or the factors that are added to induce specific differentiation programs, in contrast to progenitor cells that are already committed to a single cell lineage or terminally differentiated cells (13–15). One of the multipotent cell lines widely used to study myogenic, and to a certain extent adipogenic differentiation, is the C3H 10T(1/2) cell, a mesenchymal fibroblast-like cell line of embryonic origin that upon incubation with 5⬘-azacytidine can form in regular Dulbecco’s medium myotubes and adipocytes (16, 17) and in more specialized media can evolve into osteogenic or myofibroblast cell lineages (18). Transfection of the untreated C3H 10T(1/2) with certain key lineage factors, such as BMP (19), TGF␤ (20), or MyoD (17), can trigger highly efficient specific differentiation programs selected by the forced expression of these proteins. We used C3H 10T(1/2) cells as our experimental model because these cells have been used widely to study the mechanisms that modulate myogenic and adipogenic differentiation and the molecular pathways of lineage determination. We hypothesized that Mst promotes the commitment (...truncated)