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

Abstract

During myogenesis, proliferating myoblasts withdraw from the cell cycle, acquire an apoptosis-resistant phenotype, and differentiate into myotubes. Previous studies indicate that myogenic induction of the cyclindependent kinase inhibitor p21 results in an inhibition of apoptotic cell death in addition to its role as a negative cell cycle regulator. Here we demonstrate that the protein encoded by the Akt proto-oncogene is induced in C2C12 cells during myogenic differentiation with a corresponding increase in kinase activity. In differentiating cultures, expression of dominant-negative forms of Akt increase the frequency of cell death whereas expression of wild-type Akt protects against death, indicating that Akt is a positive modulator of myocyte survival. Antisense oligonucleotides against p21 block cell cycle withdrawal, inhibit Akt induction, and enhance cell death in differentiating myocyte cultures. Adenovirus-mediated transfer of wild-type or constitutively active Akt constructs confer partial resistance to cell death under conditions where cell cycle exit is blocked by the antisense oligonucleotides. Collectively, these data indicate that cell cycle withdrawal facilitates the induction of Akt during myogenesis, promoting 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).

Apoptosis coincident with the differentiation of skeletal myoblasts is delayed by caspase 3 inhibition and abrogated by MEK-independent constitutive Ras signaling

“…39 Since signaling through the PI3-kinase pathway has been shown to be critical for normal skeletal myoblast survival/differentiation, 20,21,23,24 and Ras signaling abrogates differentiation, 29,30,37,38 we focused our attention on the Raf/MEK pathway which has been shown to be anti-apoptotic. 33 We have shown that signaling through MEK may play a role in the survival of myoblasts in GM since the abrogation of MEK signaling increases apoptosis in GM to almost the level observed when myoblasts are cultured in DM.…”

“…Myoblasts expressing constitutively active Ras (A2:H-Ras myoblasts) are differentiation-defective 29,30 ( Figure 4A) and apoptose at a level less than 1/10 that of the parental 23A2 myoblasts in either GM ( Figure 4B Signaling by Ras through either the PI3-kinase/Akt pathway 32 or the Raf/MEK/MAPK pathway has been implicated in cell survival. 33 Since PI3 kinase/Akt signaling has been implicated in the normal survival/differentiation of skeletal myoblasts, 20,21,23,24 we focused on the role of Raf/ MEK signaling in the increased survival of A2:H-Ras myoblasts. We have previously reported that PD 098059, a selective MEK inhibitor, does not affect the differentiation of skeletal myoblasts.…”

“…7 The ability to escape this apoptosis seems to be linked to cell cycle exit. 7,23,24 The molecules responsible for the apoptotic process have not been examined in myoblasts but have been extensively studied in other systems. Apoptosis is tightly regulated by a continually emerging plethora of molecular checks and balances designed to control the activation of a family of cysteine, aspartic acid specific proteases (caspases).…”

Chronic UVA irradiation of human HaCaT keratinocytes induces malignant transformation associated with acquired apoptotic resistance

“…In comparison, wortmannin (100 and 500 nM) and LY (10 mM) had no significant effect on the control cells (data not shown), while LY (20 mM) increased apoptosis of the control cells to about 72% after UVA exposure. When ULTH cells were infected with dominant-negative AKT (DN-AKT) (Fujio et al, 1999) or wild-type PTEN (Huang and Kontos, 2002) adenovirus vectors, more ULTH cells underwent apoptosis upon UVA exposure than did cells infected with the empty vector (EV) (Figure 7d and e). In contrast, no significant effect was observed with the control cells under the similar conditions (data not shown).…”

Genome‐wide examination of myoblast cell cycle withdrawal during differentiation

“…Hypophosphorylated retinoblastoma protein is also required for protection against apoptosis (Wang et al, 1997). Akt, a proto-oncogene product, functions downstream of p21 in promoting myocyte survival (Fujio et al, 1999).…”