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 αB-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, Bcl-xL) was not altered during myogenesis. We also demonstrate that ectopic expression of αB-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 αB-crystallin mutant is partly impaired in its cytoprotective function, whereas a pseudophosphorylation αB-crystallin mutant that mimics stress-induced phosphorylation is completely devoid of anti-apoptotic activity. Finally, we demonstrate that αB-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 αB-crystallin is a novel negative regulator of myogenic apoptosis that directly links the differentiation program to apoptosis resistance. 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 αB-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, Bcl-xL) was not altered during myogenesis. We also demonstrate that ectopic expression of αB-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 αB-crystallin mutant is partly impaired in its cytoprotective function, whereas a pseudophosphorylation αB-crystallin mutant that mimics stress-induced phosphorylation is completely devoid of anti-apoptotic activity. Finally, we demonstrate that αB-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 αB-crystallin is a novel negative regulator of myogenic apoptosis that directly links the differentiation program to apoptosis resistance. heat shock protein growth medium fetal bovine serum phosphate-buffered saline differentiation medium monoclonal antibody green fluorescent protein tumor necrosis factor 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 (1Wang J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (464) Google Scholar). 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 muscle-specific transcriptional regulator MyoD (2Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Crossref PubMed Scopus (1092) Google Scholar, 3Parker S.B. Eichele G. Zhang P. Rawls A. Sands A.T. Bradley A. Olson E.N. Harper J.W. Elledge S.J. Science. 1995; 267: 1024-1027Crossref PubMed Scopus (1025) Google Scholar). p21 expression in myoblasts promotes cell cycle withdrawal and confers resistance to apoptosis through its actions on its downstream target, the retinoblastoma (RB) protein (1Wang J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (464) Google Scholar, 4Wang J. Guo K. Wills K.N. Walsh K. Cancer Res. 1997; 57: 351-354PubMed Google Scholar). 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 (5Fujio Y. Guo K. Mano T. Mitsuuchi Y. Testa J.R. Walsh K. Mol. Cell. Biol. 1999; 19: 5073-5082Crossref PubMed Scopus (188) Google Scholar, 6Jiang B.H. Aoki M. Zheng J.Z., Li, J. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081Crossref PubMed Scopus (228) Google Scholar, 7Lawlor M.A. Feng X. Everding D.R. Sieger K. Stewart C.E. Rotwein P. Mol. Cell. Biol. 2000; 20: 3256-3265Crossref PubMed Scopus (63) Google Scholar). 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 (8Dominov J.A. Dunn J.J. Miller J.B. J. Cell Biol. 1998; 142: 537-544Crossref PubMed Scopus (82) Google Scholar). Nevertheless, given the complexity of the apoptotic cell death apparatus (9Cryns V.L. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar), it seems likely that other genes play important roles in myogenic apoptosis. One particularly intriguing candidate is αB-crystallin, a member of the small heat shock protein (HSP)1 family that also includes αA-crystallin, Hsp27, Hsp20, Hsp22, myotonic dystrophy protein kinase-binding protein (MKBP)/HspB2, and HspB3 (10Clark J.I. Muchowski P.J. Curr. Opin. Struct. Biol. 2000; 10: 52-59Crossref PubMed Scopus (220) Google Scholar, 11Sugiyama Y. Suzuki A. Kishikawa M. Akutsu R. Hirose T. Waye M.M. Tsui S.K. Yoshida S. Ohno S. J. Biol. Chem. 2000; 275: 1095-1104Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 12Benndorf R. Sun X. Gilmont R.R. Biederman K.J. Molloy M.P. Goodmurphy C.W. Cheng H. Andrews P.C. Welsh M.J. J. Biol. Chem. 2001; 276: 26753-26761Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). With the exception of αA-crystallin, all of the small HSPs are abundantly expressed in muscle tissue where they function as molecular chaperones that facilitate refolding of non-native proteins (10Clark J.I. Muchowski P.J. Curr. Opin. Struct. Biol. 2000; 10: 52-59Crossref PubMed Scopus (220) Google Scholar, 11Sugiyama Y. Suzuki A. Kishikawa M. Akutsu R. Hirose T. Waye M.M. Tsui S.K. Yoshida S. Ohno S. J. Biol. Chem. 2000; 275: 1095-1104Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Structurally, each of the small HSPs contains a highly conserved α-crystallin domain flanked by largely non-conserved amino and carboxyl termini. We postulated that αB-crystallin might be an important regulator of apoptosis during myogenesis for a number of reasons. First, the expression of αB-crystallin is induced early during skeletal myogenesis in vivo and in vitroand is regulated by MyoD (11Sugiyama Y. Suzuki A. Kishikawa M. Akutsu R. Hirose T. Waye M.M. Tsui S.K. Yoshida S. Ohno S. J. Biol. Chem. 2000; 275: 1095-1104Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 13Dubin R.A. Gopal-Srivastava R. Wawrousek E.F. Piatigorsky J. Mol. Cell. Biol. 1991; 11: 4340-4349Crossref PubMed Google Scholar, 14Gopal-Srivastava R. Piatigorsky J. Mol. Cell. Biol. 1993; 13: 7144-7152Crossref PubMed Google Scholar, 15Benjamin I.J. Shelton J. Garry D.J. Richardson J.A. Dev. Dyn. 1997; 208: 75-84Crossref PubMed Scopus (73) Google Scholar). Second, αB-crystallin and the related small heat shock protein Hsp27 confer resistance to apoptosis induced by a wide range of stimuli (16Mehlen P. Kretz-Remy C. Preville X. Arrigo A.P. EMBO J. 1996; 15: 2695-2706Crossref PubMed Scopus (519) Google Scholar, 17Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 18Ray P.S. Martin J.L. Swanson E.A. Otani H. Dillmann W.H. Das D.K. FASEB J. 2001; 15: 393-402Crossref PubMed Scopus (206) Google Scholar). Indeed, we have recently demonstrated that αB-crystallin negatively regulates TNF-α- and DNA damage-induced apoptosis by a novel mechanism; αB-crystallin inhibits the activation of caspase-3, a key pro-apoptotic protease (17Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Third, a missense mutation of αB-crystallin (R120G) has been shown to cause an autosomal dominant myopathy characterized by the disruption of myofibrils and the accumulation of aggregates of desmin and αB-crystallin in degenerating muscle cells (19Vicart P. Caron A. Guicheney P., Li, Z. Prevost M.C. Faure A. Chateau D. Chapon F. Tome F. Dupret J.M. Paulin D. Fardeau M. Nat. Genet. 1998; 20: 92-95Crossref PubMed Scopus (975) Google Scholar). Biochemically, the R120G mutant is severely compromised in its chaperone activity (20Bova M.P. Yaron O. Huang Q. Ding L. Haley D.A. Stewart P.L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6137-6142Crossref PubMed Scopus (343) Google Scholar,21Perng M.D. Muchowski P.J. van Den I.P., Wu, G.J. Hutcheson A.M. Clark J.I. Quinlan R.A. J. Biol. Chem. 1999; 274: 33235-33243Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Fourth, mice with targeted deletion of the αB-crystallin gene, and the adjacent HspB2 gene, develop a progressive myopathy (22Brady J.P. Garland D.L. Green D.E. Tamm E.R. Giblin F.J. Wawrousek E.F. Invest. Ophthalmol. Vis. Sci. 2001; 42: 2924-2934PubMed Google Scholar). These findings suggest that αB-crystallin may promote muscle survival during differentiation and in response to stress. In this report, we demonstrate that the expression of αB-crystallin is selectively induced in surviving C2C12 myoblasts at an early stage in their differentiation in vitro. Moreover, we show that ectopic expression of αB-crystallin, but not Hsp27, is sufficient to inhibit differentiation-induced myoblast apoptosis. We also demonstrate that the myopathy-causing R120G mutant is partly impaired in its cytoprotective function, whereas a pseudophosphorylation αB-crystallin mutant that mimics stress-induced phosphorylation is completely defective in its anti-apoptotic function. Finally, we show that αB-crystallin negatively regulates differentiation-induced myoblast apoptosis by inhibiting the proteolytic activation of caspase-3. Overall, our findings demonstrate for the first time that the small HSP αB-crystallin is a novel negative regulator of myogenic apoptosis that directly links the differentiation program to apoptosis resistance. Murine C2C12 cells were maintained in growth medium (GM): Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS). To induce differentiation, cells were washed twice in phosphate-buffered saline (PBS) and transferred to differentiation medium (DM): Dulbecco's modified Eagle's medium supplemented with 0.5% FBS. Whole cell lysates were prepared and analyzed by immunoblotting as described (23Cryns V.L. Bergeron L. Zhu H., Li, H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) using the following Abs: αB-crystallin (StressGen), Hsp27 (StressGen), tubulin (Sigma), desmin (Sigma), p21 (Oncogene Science), M2 FLAG (Sigma), FLIPL(kindly provided by Dr. H. Perlman), XIAP (BD PharMingen), and Bcl-xL (BD PharMingen). The FLAG-tagged, wild-type human αB-crystallin and Hsp27 cDNAs have been described previously (17Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). The R120G mutant αB-crystallin cDNA was made using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions with the following oligonucleotide primers: 5′-CTCCAGGGAGTTCCACGGGAAATACCGGATCCC-3′ and 5′-GGGATCCGGTATTTCCCGTGGAACTCCCTGGAG-3′. The triple pseudophosphorylation mutant αB-crystallin cDNA (S19E,S45E,S59E, abbreviated 3XSE) was made using this same site-directed mutagenesis kit by sequentially altering each of these serine residues using the following primers: 5′-CCCTTCTTTCCTTTCCACGAACCCAGCCGCCTCTTTGAC-3′ and 5′-GTCAAAGAGGCGGCTGGGTTCGTGGAAAGGAAAGAAGGG-3′ (S19E), and and The αB-crystallin cDNA amino and its amino was using the following primers: and The was with and and into a modified in a FLAG was of the were by DNA C2C12 cells were on to in and transiently with of or wild-type or mutant αB-crystallin or the myoblasts were with of and of αB-crystallin, Bcl-2, wild-type or were using (Invitrogen) according to the manufacturer's a C2C12 cells were washed and transferred to for an to their by To expressed C2C12 cells were prepared as To the αB-crystallin, C2C12 cells were to on in washed twice in and transferred to for were in for at and for at with αB-crystallin in to the protein or with FLAG M2 to the expressed with the cells were washed in and with to and for at a that the of was for at by a antibody to The of apoptotic cells was by by as (17Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, Y. Chen F. R. M. Green K. Cryns Cell 2001; PubMed Scopus Google Scholar). In each at cells were and were in was by a To to αB-crystallin might play a in apoptosis during we its expression in C2C12 myoblasts that were induced to differentiation by a characterized in (1Wang J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (464) Google Scholar). C2C12 myoblasts were in 0.5% for and the expression of αB-crystallin was analyzed by shown in the expression of αB-crystallin was induced in myoblasts of to and in a the a critical during myoblasts differentiate into multinucleated myotubes and become resistant to apoptosis and J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (464) Google Scholar). Indeed, the myogenic induction of αB-crystallin expression that of a muscle-specific protein that is of the of myogenic S.J. R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google G. C. F. Scholar), and a of cell cycle exit and apoptosis resistance (1Wang J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (464) Google Scholar). In contrast, the expression of Hsp27, a closely related small was transiently at a time its expression at in Furthermore, the expression of other anti-apoptotic proteins and Bcl-xL) were not altered during myogenic differentiation the induction of αB-crystallin expression in C2C12 myoblasts during differentiation was to that in these cells in response to heat shock at for is expressed in surviving myoblasts during myogenesis. C2C12 myoblasts were in for and cells were for αB-crystallin expression by and for apoptotic as described αB-crystallin expression and the to myoblasts are by the the of for cells at each time the expression of αB-crystallin confers resistance to myogenic apoptosis, αB-crystallin be selectively expressed in surviving To this C2C12 cells were transferred to for and cells were for αB-crystallin expression by and for the induction of apoptosis by cells with or were as shown in the of C2C12 cells αB-crystallin to myoblasts are in the in whereas all of the apoptotic myoblasts with These are in Indeed, myoblasts of differentiation-induced apoptosis cells at and to the induction of apoptosis and the expression of αB-crystallin that αB-crystallin may promote myoblast survival during We to ectopic expression of αB-crystallin was myoblasts differentiation-induced apoptosis. C2C12 cells were transiently with or cDNAs wild-type αB-crystallin or cells were transferred to for cells were by with an and the of cells with apoptotic was shown in ectopic expression of wild-type αB-crystallin differentiation-induced myoblast apoptosis. In contrast, Hsp27 not inhibit differentiation-induced myoblast apoptosis, the of the of αB-crystallin in this To the of αB-crystallin that its anti-apoptotic we the of several αB-crystallin mutants to inhibit differentiation-induced myoblast apoptosis. we the following mutant the myopathy-causing R120G missense severely its chaperone activity (19Vicart P. Caron A. Guicheney P., Li, Z. Prevost M.C. Faure A. Chateau D. Chapon F. Tome F. Dupret J.M. Paulin D. Fardeau M. Nat. Genet. 1998; 20: 92-95Crossref PubMed Scopus (975) Google Scholar, M.P. Yaron O. Huang Q. Ding L. Haley D.A. Stewart P.L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6137-6142Crossref PubMed Scopus (343) Google Scholar, M.D. Muchowski P.J. van Den I.P., Wu, G.J. Hutcheson A.M. Clark J.I. Quinlan R.A. J. Biol. Chem. 1999; 274: 33235-33243Abstract Full Text Full Text PDF PubMed Scopus (166) Google a pseudophosphorylation triple mutant (S19E,S45E,S59E, abbreviated 3XSE) in each of the residues that are in response to has been altered to a to phosphorylation H. K. H. T. K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, H. K. Y. D. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google and a αB-crystallin, its a and the protein (10Clark J.I. Muchowski P.J. Curr. Opin. Struct. Biol. 2000; 10: 52-59Crossref PubMed Scopus (220) Google D. M.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). These mutant proteins were expressed at with of wild-type αB-crystallin and Hsp27 in transiently C2C12 myoblasts shown in the R120G and mutants were partly impaired in their to myoblasts differentiation-induced apoptosis with wild-type In contrast, the triple pseudophosphorylation mutant was completely devoid of anti-apoptotic activity. stress-induced phosphorylation of these residues the of the of αB-crystallin H. K. Y. D. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), our findings suggest that the anti-apoptotic function of αB-crystallin is to its as demonstrated in the of apoptosis by αB-crystallin was to that of a previously described regulator of survival (1Wang J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (464) Google Scholar), whereas wild-type Akt myogenic apoptosis these we have previously demonstrated that αB-crystallin negatively regulates TNF-α- and DNA damage-induced apoptosis by inhibiting the activation of (17Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), we to αB-crystallin myogenic apoptosis by the same To this we transferred C2C12 myoblasts to for and cells for expression of αB-crystallin and the was using an antibody that the of not shown in the of C2C12 cells that expressed αB-crystallin the to cells are of are and whereas only a small of cells that expressed also expressed αB-crystallin Moreover, as demonstrated in of wild-type αB-crystallin, but not Hsp27, was sufficient to inhibit the of our In contrast, each of the αB-crystallin mutants and 3XSE) was impaired in its to inhibit the mutant was severely with its of anti-apoptotic function. Taken together, these findings suggest that αB-crystallin inhibits myogenic apoptosis by We have demonstrated that αB-crystallin is a novel negative regulator of myogenic apoptosis; its expression is selectively induced in surviving myoblasts during an early stage of their vitro. Indeed, the αB-crystallin contains a skeletal muscle-specific that MyoD family and for the early expression of αB-crystallin during skeletal myogenesis (11Sugiyama Y. Suzuki A. Kishikawa M. Akutsu R. Hirose T. Waye M.M. Tsui S.K. Yoshida S. Ohno S. J. Biol. Chem. 2000; 275: 1095-1104Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 13Dubin R.A. Gopal-Srivastava R. Wawrousek E.F. Piatigorsky J. Mol. Cell. Biol. 1991; 11: 4340-4349Crossref PubMed Google Scholar, 14Gopal-Srivastava R. Piatigorsky J. Mol. Cell. Biol. 1993; 13: 7144-7152Crossref PubMed Google Scholar, 15Benjamin I.J. Shelton J. Garry D.J. Richardson J.A. Dev. Dyn. 1997; 208: 75-84Crossref PubMed Scopus (73) Google Scholar). In this αB-crystallin a and previously the myogenic differentiation program and the acquisition of apoptosis the event a critical in myogenesis apoptosis-sensitive myogenic precursors are eliminated (1Wang J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (464) Google Scholar). Although a few other of myogenic apoptosis have been described (1Wang J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (464) Google Y. Guo K. Mano T. Mitsuuchi Y. Testa J.R. Walsh K. Mol. Cell. Biol. 1999; 19: 5073-5082Crossref PubMed Scopus (188) Google Scholar, 6Jiang B.H. Aoki M. Zheng J.Z., Li, J. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081Crossref PubMed Scopus (228) Google Scholar, 7Lawlor M.A. Feng X. Everding D.R. Sieger K. Stewart C.E. Rotwein P. Mol. Cell. Biol. 2000; 20: 3256-3265Crossref PubMed Scopus (63) Google Scholar, J.A. Dunn J.J. Miller J.B. J. Cell Biol. 1998; 142: 537-544Crossref PubMed Scopus (82) Google Scholar), αB-crystallin is the first HSP to be in this we that Hsp27, a protein to αB-crystallin (10Clark J.I. Muchowski P.J. Curr. Opin. Struct. Biol. 2000; 10: 52-59Crossref PubMed Scopus (220) Google Scholar, 11Sugiyama Y. Suzuki A. Kishikawa M. Akutsu R. Hirose T. Waye M.M. Tsui S.K. Yoshida S. Ohno S. J. Biol. Chem. 2000; 275: 1095-1104Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), was only transiently induced during myogenic differentiation and not confer resistance to differentiation-induced apoptosis, that is HSPs with to their in myogenic cell Moreover, our that several other anti-apoptotic proteins and Bcl-xL) were not induced during skeletal myogenesis that the apoptosis resistance by myogenic precursors the activation of a subset of anti-apoptotic the survival of a subset of myoblasts that other anti-apoptotic proteins also to the apoptosis resistance of mature We have also demonstrated that αB-crystallin myogenic apoptosis by inhibiting the activation of caspase-3. is with our findings in cells that αB-crystallin inhibits activation by its proteolytic (17Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). In contrast, Hsp27 not inhibit myogenic differentiation-induced activation or apoptosis. that the anti-apoptotic of these closely related small HSPs are likely to be Indeed, we have demonstrated previously that Hsp27 not directly inhibit the proteolytic of (17Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Hsp27 has been to inhibit apoptosis by to and the of to the J.M. C. P. L. C. S. Arrigo A.P. G. C. Nat. Cell Biol. 2000; PubMed Scopus Google Scholar), a of and (9Cryns V.L. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar). In this Hsp27 inhibits the activation of the in the mitochondrial apoptotic J.M. C. P. L. C. S. Arrigo A.P. G. C. Nat. Cell Biol. 2000; PubMed Scopus Google Scholar), whereas αB-crystallin only inhibits activation (17Kamradt M.C. Chen F. Cryns V.L. J. Biol. Chem. 2001; 276: 16059-16063Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). the mitochondrial apoptotic has been to be in skeletal muscle to the of in skeletal muscle M. T. K. S. Cell 1999; PubMed Scopus Google Scholar), for the of Hsp27 to inhibit myogenic apoptosis. We have also for the first time of the anti-apoptotic of We that the myopathy-causing R120G mutation and deletion of its partly the of αB-crystallin to inhibit differentiation-induced activation and apoptosis, whereas a pseudophosphorylation triple mutant that mimics stress-induced phosphorylation was severely impaired in these each of these likely the chaperone activity of αB-crystallin (20Bova M.P. Yaron O. Huang Q. Ding L. Haley D.A. Stewart P.L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6137-6142Crossref PubMed Scopus (343) Google Scholar, M.D. Muchowski P.J. van Den I.P., Wu, G.J. Hutcheson A.M. Clark J.I. Quinlan R.A. J. Biol. Chem. 1999; 274: 33235-33243Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, H. K. Y. D. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, D. M.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), but only the triple pseudophosphorylation mutant is defective in its to form H. K. Y. D. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), these findings suggest that of αB-crystallin is essential for its cytoprotective as has been demonstrated for Hsp27 in other T. M. Preville X. A. G. C. C. M. Arrigo A.P. J. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, the novel anti-apoptotic function of αB-crystallin and the impaired of the R120G mutant to inhibit activation may into the of the progressive myopathy by this mutation (19Vicart P. Caron A. Guicheney P., Li, Z. Prevost M.C. Faure A. Chateau D. Chapon F. Tome F. Dupret J.M. Paulin D. Fardeau M. Nat. Genet. 1998; 20: 92-95Crossref PubMed Scopus (975) Google Scholar). Although the R120G mutant is also compromised in its to desmin in the accumulation of aggregates of desmin and αB-crystallin and the disruption of myofibrils (19Vicart P. Caron A. Guicheney P., Li, Z. Prevost M.C. Faure A. Chateau D. Chapon F. Tome F. Dupret J.M. Paulin D. Fardeau M. Nat. Genet. 1998; 20: 92-95Crossref PubMed Scopus (975) Google Scholar, M.P. Yaron O. Huang Q. Ding L. Haley D.A. Stewart P.L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6137-6142Crossref PubMed Scopus (343) Google Scholar, M.D. Muchowski P.J. van Den I.P., Wu, G.J. Hutcheson A.M. Clark J.I. Quinlan R.A. J. Biol. Chem. 1999; 274: 33235-33243Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, X. H. R. A.M. M. J. T. J. Res. 2001; PubMed Scopus Google Scholar), the in its to inhibit activation likely muscle cells to stress-induced apoptosis. Indeed, the that the R120G mutation in or in mice muscle in early (19Vicart P. Caron A. Guicheney P., Li, Z. Prevost M.C. Faure A. Chateau D. Chapon F. Tome F. Dupret J.M. Paulin D. Fardeau M. Nat. Genet. 1998; 20: 92-95Crossref PubMed Scopus (975) Google Scholar, X. H. R. A.M. M. J. T. J. Res. 2001; PubMed Scopus Google Scholar) that the of of activity is to the of this mutation in Finally, given the that in the of apoptosis (9Cryns V.L. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar), our suggest that αB-crystallin may in the of muscle cell We are to Li, and for the critical of the and to Dr. H. for