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Submitted on March 28, 2005
Accepted on April 25, 2005
Division of Endocrinology Metabolism and Molecular Medicine; RCMI DNA Molecular Core. Charles R. Drew University of Medicine and Science, Los Angeles, California, 90059; Department of Urology, UCLA School of Medicine; LABioMed Research Institute at Harbor-UCLA Medical Center, Torrance, California, 90502; School of Biological and Molecular Sciences. Oxford Brookes University, Oxford, UK
* To whom correspondence should be addressed. E-mail: joartaza{at}cdrewu.edu.
Inactivating mutations of the mammalian myostatin gene are associated with increased muscle mass and decreased fat mass; conversely myostatin transgenic mice that over-express myostatin in the skeletal muscle have decreased muscle mass and increased fat mass. We investigated the effects of recombinant myostatin protein and anti-myostatin antibody on myogenic and adipogenic differentiation of mesenchymal multi-potent cells. Accordingly, 10T(1/2) cells were incubated with AZCT for 3 days to induce differentiation, and then treated with a recombinant protein for Mst carboxy terminal 113 amino acids (Mst-113), or a polyclonal anti-Mst antibody for 3, 7 and 14 days. Cells were also co-transfected 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 days and Mst expression was determined. Adipogenesis was evaluated by QIA of fat cells before and after Oil-Red-O staining, ICC of adiponectin, and Western blot for C/EBP
. Myogenesis was estimated by QIA-ICC for MyoD, myogenin, and MHCII, or by Western blot for myogenin. AZCT-mediated differentiation induced endogenous full length Mst expression. Recombinant Mst-113 inhibited both early and late markers of myogenesis and stimulated both early and late markers of adipogenesis, while the antibody against Mst exerted the reverse effects. Myogenin levels at 7 days after transfection of pcDNA-Mst375 were reduced as expected, and elevated by pSil-Mst, that blocked efficiently Mst375 expression. In conclusion, myostatin promotes the differentiation of multi-potent mesenchymal cells into the adipogenic lineage, and inhibits myogenesis.
•
myosin heavy chain •
siRNA
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