Glycogen Synthase Kinase 3β Suppresses Myogenic Differentiation through Negative Regulation of NFATc3

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 3␤ (GSK-3␤) 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-3␤ 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-3␤ 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-3␤. In addition, GSK-3␤ inhibition restored myogenic differentiation following calcineurin blockade, which suggested the involvement of NFAT. GSK-3␤-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-3␤. Overexpression of NFATc3 increased muscle gene promoter transactivation, which was abolished by co-expression of wild type GSK-3␤. Finally, stimulation of muscle gene expression observed following GSK-3␤ 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-3␤ through a transcriptional mechanism that depends on NFATc3. Inhibition of GSK-3␤ may be a potential strategy in prevention or treatment of muscle atrophy.Maintenance of muscle mass is critical for health, and loss of skeletal muscle mass compromises human physical condition and survival in chronic diseases, such as chronic obstructive pulmonary disease (1, 2). Restoring lost muscle mass is important for improving quality of life and ultimately disease prognosis (3). To restore muscle mass and improve muscle function in various diseases conditions, a better understanding of the molecular mechanisms of skeletal muscle (re)growth is required. Skeletal muscle differentiation is a critical element of certain types of postnatal growth of the skeletal musculature and is mainly dependent on satellite cells (quiescent myoblasts), which upon activation proliferate, differentiate, and fuse with existing muscle fibers or with each other to form new myofibers (4). Myoblast fusion allows additional muscle growth by myonuclear accretion, beyond the limitations imposed by the myonuclear domain (i.e. the maximal cytoplasm/nucleus ratio) (5).The protein kinase GSK-3 2 is ubiquitously expressed, and, although 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). Recent studies have ide...

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Cooperation between Classical and Alternative NF-κB Pathways Regulates Proinflammatory Responses in Epithelial Cells

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Cyclosporin A inhibits the growth of neonatal MHC-expressing myotubes independent of NFATc1 and NFATc3 in the mechanically overloaded soleus muscle of mice

“…Treatment with CsA did not affect the percentage of fibers expressing NFATc3 in the muscle. In addition, the expression pattern of GSK3, a major regulator of NFATc3 signaling [31], was not altered in the soleus muscle by CsA treatment. Furthermore, the localization of NFATc1 did not correspond to that in neonatal MHC-positive myotubes (Figure 5), although NFATc1-immunoreactivity was observed in some mononuclear cells.…”

“…NFATc3 is known to be regulated by GSK-3 [31]. More GSK-3 would affect the functional role of NFATc3, even if a similar amount of NFATc3 existed in the mechanically overloaded soleus muscle in both placebo-treated and CsA-treated mice.…”