Abstract
Inhibitor of differentiation protein-2 (Id2) is a dominant negative helix-loop-helix (HLH) protein, and a positive regulator of proliferation, in various cells. The N-terminal region of Id2 contains a consensus cdk2 phosphorylation sequence SPVR, which may be involved with the induction of apoptosis, at least in myeloid 32d.3 cells. However, the role of Id2 phosphorylation at serine 5 in skeletal muscle cells is unknown. The objective of this study was to determine if the phosphorylation of Id2 at serine 5 alters its cellular localization and its role in apoptosis in C2C12 myoblasts. Overexpression of wild type Id2 decreased MyoD protein expression, which corresponded to the increased binding of Id2 to basic HLH proteins E47 and E12. Bromodeoxyuridine incorporation was significantly decreased by the overexpression of phospho-ablated Id2 (S5A); conversely, overexpression of wild type Id2 increased cellular proliferation. The subcellular localization of Id2 and phospho-mimicking Id2 (S5D) were predominantly nuclear compared to S5A. The decreased nuclear localization of S5A corresponded to a decrease in cellular proliferation, and an increase in apoptosis. These data suggest that unphosphorylated Id2 is primarily localized in the cytosol, where it is growth suppressive and potentially pro-apoptotic. These results imply that reducing unphosphorylated Id2 may improve the pool of myoblasts available for differentiation by increasing proliferation and inhibiting apoptosis.
Highlights
Myogenesis requires that muscle precursor cells, or myoblasts, undergo proliferation, followed by cell-cycle exit
To determine if the apoptotic role of Inhibitor of differentiation protein-2 (Id2) is determined by the phosphorylation status of this protein, C2C12 myoblasts were transfected with Id2:green fluorescent protein (GFP), S5A:GFP, or S5D:GFP
Overexpression of Id2:F, S5A:F, and S5D:F resulted in a significant increase of caspase 3 activity compared to CON and empty flag vector (EV):F (Figure 2D); caspase 3 activity was significantly higher in S5A:F transfected myoblasts than all other groups
Summary
Myogenesis requires that muscle precursor cells (e.g. satellite cells), or myoblasts, undergo proliferation, followed by cell-cycle exit. This is followed by myogenic differentiation, and cell fusion into multinucleated myotubes myofibers. Loss of myoblasts during the initial stages of myogenesis limit or prevent muscle growth or in aging, may contribute to sarcopenia by reducing the ability to replace muscle mass during normal protein turnover [1]. While the majority of muscle precursor cells/myoblasts exit the cell cycle and undergo terminal differentiation during myogenesis and muscle repair/growth, ,30% of differentiating myoblasts undergoes cell death during differentiation [2,3]. The mechanism by which the majority of muscle cells undergo caspase-dependent differentiation but escapes caspase-dependent or caspase-independent apoptosis is not clear
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