Cell Cycle Withdrawal Promotes Myogenic Induction of Akt, a Positive Modulator of Myocyte Survival

During myogenesis, proliferating myoblasts irreversibly withdraw from the cell cycle and differentiate into myotubes. The cyclin-dependent kinase (CDK) inhibitor p21 and the retinoblastoma protein (pRb) appear to be critical in establishing the postmitotic state during myogenesis (55). p21 is markedly induced in differentiating C2C12 cells and in 10T1/2 fibroblasts that are induced to differentiate following transformation with MyoD (23, 24, 40, 42). Bromodeoxyuridine-labeling experiments have shown that upregulation of p21 correlates with the initiation of cell cycle exit, an early event in the myogenic differentiation pathway (4). Myocytes lacking pRb, a downstream target of CDK inhibitors, are incapable of irreversible cell cycle exit upon differentiation (41, 46). The transcription of muscle-specific genes can be inhibited by the forced expression of cyclins and CDKs, or E2F1, and this inhibition is largely reversed by the expression of constitutively active mutants of pRb (22). It is reported that the myocyte differentiation and cell cycle-regulatory functions of pRb and E2F1 require different domains within these proteins (22, 48). A number of early studies described embryonic muscle precursor cells that undergo temporally regulated disintegration (reviewed in reference 21), a process that has more recently been referred to as programmed cell death or apoptosis. In previous studies, we found that a significant fraction of myoblasts undergo apoptosis during the differentiation of the C2C12 myogenic cell line, while differentiated C2C12 myotubes are relatively resistant to apoptosis (56, 57). Coimmunolocalization experiments with temporal markers of myogenesis revealed that acquisition of the apoptosis-resistant phenotype coincided with induction of the p21 CDK inhibitor but not with the appearance of myogenin, an earlier marker of myogenic differentiation (4). In addition, forced expression of the CDK inhibitors p21 or p16 blocked apoptosis during C2C12 differentiation (56, 57). The effects of CDK inhibitors on myocyte proliferation and survival are likely determined by their ability to modulate the state of pRb phosphorylation and cell growth. Consistent with this hypothesis, the CC42 pRb-deficient myogenic cell line undergoes a relatively high frequency of apoptosis during differentiation (56). These pRb−/− myocytes display a normal time course of p21 induction during differentiation, and forced expression of the p21 or p16 CDK inhibitors has no effect on the frequency of apoptosis. However, forced expression of pRb suppresses apoptosis in both pRb−/− and wild-type cell lines during differentiation. Consistent with these observations, transgenic mice expressing low levels of pRb display substantial cell death in muscle masses occurring prior to the onset of terminal differentiation (59). In these mice, surviving myocytes accumulate large polyploid nuclei, indicating a defect in the permanent withdrawal from the cell cycle. Collectively, these studies suggest that cell cycle activity markedly influences the susceptibility of differentiating myoblasts to apoptosis. However, the mechanisms by which perturbations in cell cycle activity induce apoptosis are essentially unknown for any cell type. Akt is a proto-oncogene encoding a serine-threonine kinase whose amino terminus contains a pleckstrin homology (PH) domain (53). Various extracellular stimuli activate Akt through the phosphoinositide 3-kinase (PI 3-kinase) pathway (12, 20, 30). The lipid products of the PI 3-kinase reaction may activate Akt either by binding to the Akt PH domain (19, 33) or by activating a protein kinase that phosphorylates Akt (34, 52). Activation of Akt inhibits apoptosis induced by growth factor withdrawal or irradiation in neural cells, fibroblasts, and lymphocytes (11, 25). Recently, it has been shown that Akt phosphorylates the proapoptotic proteins Bad and caspase 9 leading to their inactivation and cell survival (8, 13, 43). In the present study, we demonstrate that Akt protein and kinase activities are markedly upregulated during myogenic differentiation. We also show that myocytes die at a high frequency when cell cycle exit is blocked by treatment with antisense oligonucleotides to p21 mRNA. Under these conditions, endogenous Akt induction is suppressed, but forced Akt expression protects mitotic cells from death under conditions that promote myogenic differentiation. Thus, we propose that myocyte survival can be controlled through the ability of cell cycle activity to modulate Akt induction during myogenesis.