Abstract
Skeletal muscle atrophy is a prominent and disabling feature in many chronic diseases. Prevention or reversal of muscle atrophy by stimulation of skeletal muscle growth could be an important therapeutic strategy. Glycogen synthase kinase 3beta (GSK-3beta) has been implicated in the negative regulation of skeletal muscle growth. Since myogenic differentiation is an essential part of muscle growth, we investigated if inhibition of GSK-3beta is sufficient to stimulate myogenic differentiation and whether this depended on regulation of the transcription factor nuclear factor of activated T-cells (NFAT). In both myogenically converted mouse embryonic fibroblasts and C2C12 myoblasts, deficiency of GSK-3beta protein (activity) resulted in enhanced myotube formation and muscle-specific gene expression during differentiation, which was reversed by reintroduction of wild type but not kinase-inactive (K85R) GSK-3beta. In addition, GSK-3beta inhibition restored myogenic differentiation following calcineurin blockade, which suggested the involvement of NFAT. GSK-3beta-deficient mouse embryonic fibroblasts or myoblasts displayed enhanced nuclear translocation of NFATc3 and elevated NFAT-sensitive promoter transactivation, which was reduced by reintroducing wild type, but not K85R GSK-3beta. Overexpression of NFATc3 increased muscle gene promoter transactivation, which was abolished by co-expression of wild type GSK-3beta. Finally, stimulation of muscle gene expression observed following GSK-3beta inhibition was strongly attenuated in NFATc3-deficient myoblasts, indicating that this response requires NFATc3. Collectively, our data demonstrate negative regulation of myogenic differentiation by GSK-3beta through a transcriptional mechanism that depends on NFATc3. Inhibition of GSK-3beta may be a potential strategy in prevention or treatment of muscle atrophy.
Highlights
The protein kinase GSK-32 is ubiquitously expressed, and, it was originally identified as a suppressor of glycogen synthase [6], GSK-3 has been implicated in a myriad of metabolic and signaling pathways [7]
Overexpression of ca NFATc3 stimulated muscle-specific gene expression but was insensitive to GSK-3 (Fig. 6C). These results show that muscle gene expression during myogenic differentiation is promoted by NFATc3, which in turn is negatively regulated by GSK-3
Stimulation of Muscle Gene Expression during Myogenic Differentiation by GSK-3 Inhibition Requires NFATc3—we investigated whether stimulation of myogenic differentiation by inhibition of GSK-3 is dependent on NFATc3 signaling
Summary
Cell Culture—The murine skeletal muscle cell line C2C12 obtained from the American Type Culture Collection (ATCC number CRL1772) was cultured in growth medium (GM), composed of low glucose Dulbecco’s modified Eagle’s medium (DMEM) containing antibiotics (50 units/ml penicillin and 50 g/ml streptomycin) and 9% (v/v) fetal bovine serum (FBS) (all from Invitrogen), or differentiation medium (DM), which contained low glucose DMEM with 0.5% heat-inactivated FBS and antibiotics. Woodgett (Ontario Cancer Institute, Toronto, Canada) and cultured in GM or DM, which contained high glucose DMEM with 0.5% heat-inactivated FBS and antibiotics Both cell types were grown on Matrigel (BD Biosciences)-coated (1:50 in DMEM) dishes as described previously [27]. Nitrocellulose blots were washed in PBS-Tween 20 (0.05%), followed by overnight incubation (4 °C) with a polyclonal antibody specific for either GSK-3 1:1000 (catalog number 9315; Cell Signaling, Danvers, MA) or NFATc3 1:500 (catalog number sc-8321; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a monoclonal antibody ␣-tubulin 1:500 Cells were grown on Matrigel-coated 60-mm dishes, and after 72 h in differentiation medium, cells were washed twice in PBS (room temperature), fixed in methanol and stained in May-Grunwald Giemsa (Sigma) according to the manufacturer’s instructions. Images were taken at a ϫ400 magnification, using a fluorescent microscope connected to a digital DXM 1200F camera, both from Nikon (Kanagawa, Japan)
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