The Small Heat Shock Protein αB-crystallin Negatively Regulates Apoptosis during Myogenic Differentiation by Inhibiting Caspase-3 Activation

Abstract

Myoblasts respond to growth factor deprivation either by differentiating into multinucleated myotubes or by undergoing apoptosis; hence, the acquisition of apoptosis resistance by myogenic precursors is essential for their development. Here we demonstrate that the expression of the small heat shock protein ␣B-crystallin is selectively induced in C2C12 myoblasts that are resistant to differentiation-induced apoptosis, and we show that this induction occurs at an early stage in their differentiation in vitro. In contrast, the expression of several known anti-apoptotic proteins (FLIP, XIAP, Bclx L ) was not altered during myogenesis. We also demonstrate that ectopic expression of ␣B-crystallin, but not the closely related small heat shock protein Hsp27, renders C2C12 myoblasts resistant to differentiation-induced apoptosis. Furthermore, we show that the myopathy-causing R120G ␣B-crystallin mutant is partly impaired in its cytoprotective function, whereas a pseudophosphorylation ␣B-crystallin mutant that mimics stress-induced phosphorylation is completely devoid of anti-apoptotic activity. Finally, we demonstrate that ␣B-crystallin negatively regulates apoptosis during myogenesis by inhibiting the proteolytic activation of caspase-3, whereas the R120G and pseudophosphorylation mutants are defective in this function. Taken together, our findings indicate that ␣B-crystallin is a novel negative regulator of myogenic apoptosis that directly links the differentiation program to apoptosis resistance.During skeletal muscle development, a subset of proliferating myoblasts exit the cell cycle and become resistant to apoptosis; these surviving myoblasts fuse to form multinucleated myotubes and differentiate into mature myocytes, whereas apoptosis-sensitive myoblasts are eliminated (1). Although the acquisition of apoptosis resistance by myogenic precursors is a critical event in their differentiation, only a few genes that regulate this process have been identified. One such gene is the cyclin-dependent kinase inhibitor p21 whose expression is induced at an early stage of myogenesis by the skeletal musclespecific transcriptional regulator MyoD (2, 3). p21 expression in myoblasts promotes cell cycle withdrawal and confers resistance to apoptosis through its actions on its downstream target, the retinoblastoma (RB) protein (1, 4). The anti-apoptotic kinase Akt is also induced during skeletal muscle development and promotes the survival of differentiating myoblasts, although its anti-apoptotic mechanism(s) in muscle is unclear (5-7). Finally, Bcl-2, a protein that inhibits many of the mitochondrial events in apoptosis, is transiently expressed in myogenic precursors and promotes their clonal expansion (8). Nevertheless, given the complexity of the apoptotic cell death apparatus (9), it seems likely that other genes play important roles in myogenic apoptosis.One particularly intriguing candidate is ␣B-crystallin, a member of the small heat shock protein (HSP) 1 family that also includes ␣A-crystallin, Hsp27, Hsp20, Hsp22, myotoni...

Human αA- and αB-crystallins bind to Bax and Bcl-XS to sequester their translocation during staurosporine-induced apoptosis

“…Previous studies 73,74 have shown that both mutants displayed decreased protection against apoptosis induced by staurosporine and other stress conditions. To explore the possible mechanisms for the decreased protection, we have created the two mutants using wild-type HaA and HaB cDNAs and PCR amplification procedure.…”

“…The differential antiapoptotic mechanism was further illustrated in a recent study in which aB-crystallin but not Hsp27 was found capable of repressing differentiation-induced caspase-3 activation. 74 The results from both GST pulldown assay ( Figure 4) and coimmunoprecipication-linked Western blot analysis with antibodies against aA/B-crystallin and Bcl-X S (Figure 5a and d) suggest that HaB seems to have slightly stronger affinity to Bax and Bcl-X S than HaA does. However, this is not consistent with the coimmunoprecipitation-linked Western blot analysis with anti-Bax antibody (Figure 5c).…”

“…[39][40][41][42][43][44][45][46][62][63][64][65][66][67][68][69][70][71][72][73][74] One of the most important functions is the ability to protect cell from induced apoptosis. 39-46 a-Crystallin and Hsp27 are closely related family members and protect cells from apoptosis induced by a large numbers of stress factors.…”

The lens epithelium: Focus on the expression and function of the α-crystallin chaperones

“…This is correlated with reduction in cell proliferation and mitosis inhibition . For many cell types and stressors, αB-crystallin inhibits apoptosis by preventing the auto proteolytic maturation of caspase-3 (Arrigo et al, 2002;Kamradt et al, 2002). αA-crystallin may also enhance survival by interacting with anti-apoptotic factors Bax and Bcl-xs (Liu et al, 2004).…”

Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy

“…Some sHSPs have crucial roles in neuronal apoptosis and muscle function 3,22 . R116C in αA-crystallin diminishes its protective ability against stress-induced lens epithelial cell apoptosis 23 , and αB-crystallin negatively regulates apoptosis by inhibiting caspase-3 activation 24 . HSPB2 associates specifically with and activates the myotonic dystrophy protein kinase, which causes myotonic dystrophy (OMIM 160900; ref.…”