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J. Biol. Chem., Vol. 277, Issue 41, 38731-38736, October 11, 2002
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B-crystallin Negatively
Regulates Apoptosis during Myogenic Differentiation by Inhibiting
Caspase-3 Activation*
From the Center for Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
Received for publication, February 21, 2002, and in revised form, July 3, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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
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 muscle-specific 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 In this report, we demonstrate that the expression of Cell Culture and Reagents--
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.
Western Blot Analyses--
Whole cell lysates were prepared and
analyzed by immunoblotting as described (23) using the following Abs:
Construction of FLAG Epitope-tagged cDNAs--
The
FLAG-tagged, wild-type human Transfection of C2C12 Cells--
C2C12 cells were grown on glass
coverslips to 40% confluence in GM and transiently transfected with 1 µg of control vector (pEGFPN1, CLONTECH) or
pcDNA3-FLAG plasmid containing wild-type or mutant Indirect Immunofluorescence and Apoptosis Assays--
To detect
ectopically expressed cDNAs, C2C12 cells were prepared as above. To
detect the endogenous Induction of Ectopic Expression of
To delineate the domains of We have demonstrated that We have also demonstrated that We have also reported for the first time structure-function analyses of
the anti-apoptotic domains of Furthermore, the novel anti-apoptotic function of 
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, Bcl-xL) 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin, a member of
the small heat shock protein
(HSP)1 family that also
includes A-crystallin, Hsp27, Hsp20, Hsp22, myotonic dystrophy
protein kinase-binding protein (MKBP)/HspB2, and HspB3 (10-12).
With the exception of A-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 (10, 11).
Structurally, each of the small HSPs contains a highly conserved
-crystallin domain flanked by largely non-conserved amino and
carboxyl termini. We postulated that B-crystallin might be an
important regulator of apoptosis during myogenesis for a number of
reasons. First, the expression of B-crystallin is induced early
during skeletal myogenesis in vivo and in vitro
and is regulated by MyoD (11, 13-15). Second, B-crystallin and the
related small heat shock protein Hsp27 confer resistance to apoptosis
induced by a wide range of stimuli (16-18). Indeed, we have recently
demonstrated that B-crystallin negatively regulates TNF-- and DNA
damage-induced apoptosis by a novel mechanism; B-crystallin inhibits
the activation of caspase-3, a key pro-apoptotic protease (17).
Third, a missense mutation of B-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
B-crystallin in degenerating muscle cells (19). Biochemically, the
R120G mutant is severely compromised in its chaperone activity (20,
21). Fourth, mice with targeted deletion of the B-crystallin gene,
and the adjacent HspB2 gene, develop a progressive myopathy (22). These
findings suggest that B-crystallin may promote muscle survival
during differentiation and in response to stress.
B-crystallin
is selectively induced in surviving C2C12 myoblasts at an early stage
in their differentiation in vitro. Moreover, we show that
ectopic expression of B-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 B-crystallin mutant that mimics stress-induced phosphorylation is
completely defective in its anti-apoptotic function. Finally, we show
that B-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 B-crystallin is a novel negative regulator of myogenic
apoptosis that directly links the differentiation program to apoptosis resistance.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-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).
B-crystallin and Hsp27 cDNAs have
been described previously (17). The R120G mutant B-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 B-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),
5'-CCGACGTCTACTTCCCTGGAACCCTTCTACCTTCGGCC-3' and
5'-GGCCGAAGGTAGAAGGGTTCCAGGGAAGTAGACGTCGG-3' (S45E),
5'-CCTTCCTGCGGGCACCCGAATGGTTTGACACTGGACTC-3' and
5'-GAGTCCAGTGTCAAACCATTCGGGTGCCCGCAGGAAGG-3' (S59E). The
B-crystallin cDNA encoding amino acids 1-161 and lacking
its carboxyl-terminal 14 amino acids (
C) was PCR-amplified using
the following primers: 5'-GGCCGAATTCATGGACATCGCCATCCACCAC-3' and
5'-GGCCCTCGAGGATGGGAATGGTGCGCTCAGGGCC-3'. The PCR product was
then digested with EcoRI and XhoI and cloned into
a modified pcDNA3 vector in which a sequence encoding FLAG was
inserted upstream of the multiple cloning site. All sequences were
confirmed by automated DNA sequencing.
B-crystallin
or Hsp27. For the co-transfection experiments (Fig. 3D),
myoblasts were co-transfected with 0.4 µg of pEGFPN1 and 0.8 µg of
plasmids containing B-crystallin, p21, Bcl-2, wild-type Akt, or
Hsp27. Transfections were performed using LipofectAMINE Plus reagent
(Invitrogen) according to the manufacturer's instructions. After a
24-h incubation, C2C12 cells were washed and transferred to DM for an
additional 24 h prior to their analysis by immunofluorescence.
B-crystallin, C2C12 cells were grown to 40%
confluence on glass coverslips in GM, washed twice in PBS, and
transferred to DM for 0-72 h. Cells were then fixed in 100% methanol
for 2 min at 
20 °C and incubated for 2 h at 37 °C with
B-crystallin mAb (1:500 dilution in PBS) to detect the endogenous
protein or with FLAG M2 mAb (Sigma, 1:500) to detect the ectopically
expressed proteins. After incubation with the primary Ab, cells were
washed in PBS and incubated with fluorescein-conjugated goat
affinity-purified Ab to mouse IgG (ICN Pharmaceuticals, 1:20) and 10 µg/ml Hoechst 33258 (Sigma) for 30 min at 37 °C. For
co-immunofluorescence studies, a polyclonal Ab that recognizes the
active, large subunit of caspase-3 (CM1, BD PharMingen, 1:250) was used
for 2 h at 37 °C followed by a rhodamine-conjugated antibody to
rabbit IgG (1:200). The percentage of apoptotic cells was determined by
scoring fragmented/condensed nuclei by fluorescence microscopy as
detailed (17, 24). In each experiment, at least 200 cells were counted,
and experiments were performed in triplicate. Statistical significance
was assessed by a two-tailed, paired Student's t test.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin Expression during an Early Stage of
Myogenic Differentiation--
To begin to determine whether
B-crystallin might play a role in regulating apoptosis during
myogenesis, we examined its expression in C2C12 myoblasts that were
induced to undergo differentiation by mitogen withdrawal, a well
characterized in vitro model (1). C2C12 myoblasts were grown
in DM containing 0.5% FBS for 0-72 h, and the expression of
B-crystallin was analyzed by immunoblotting. As shown in Fig.
1A, the expression of
B-crystallin was rapidly induced in myoblasts within 4 h of
transfer to DM and increased in a time-dependent manner
throughout the entire 72-h interval, a critical period during which
myoblasts differentiate into multinucleated myotubes and become
resistant to apoptosis (see Fig. 2 and
Ref. 1). Indeed, the myogenic induction of B-crystallin expression preceded that of desmin, a muscle-specific intermediate filament protein that is one of the earliest markers of myogenic commitment (25,
26), and p21, a mediator of cell cycle exit and apoptosis resistance
(1). In contrast, the expression of Hsp27, a closely related small HSP,
was transiently increased at a later time point (18-24 h), its
expression falling below baseline levels at 48-72 h in DM.
Furthermore, the expression levels of other anti-apoptotic proteins
(XIAP, FLIPL, and Bcl-xL) were not
significantly altered during myogenic differentiation (Fig.
1B). Interestingly, the induction of B-crystallin
expression observed in C2C12 myoblasts during differentiation was
similar to that observed in these cells in response to heat shock at
44 °C for 1 h (Fig. 1C).
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Fig. 1.
Induction of
B-crystallin expression during an early stage of
myogenic differentiation in C2C12 myoblasts. A and
B, murine C2C12 myoblasts were cultured in differentiation
medium (DM) containing 0.5% FBS for 0-72 h, and whole cell lysates
were then analyzed by immunoblotting as detailed under
"Experimental Procedures." C, C2C12 myoblasts
were heat-shocked at 44 °C for 1 h, transferred to 37 °C for
the indicated time, and the expression of B-crystallin was
determined by immunoblotting as described under
"Experimental Procedures."
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Fig. 2.
B-crystallin is preferentially
expressed in surviving myoblasts during myogenesis. C2C12
myoblasts were grown in DM for 0-72 h, and then cells were examined
for B-crystallin expression by indirect immunofluorescence and
scored for apoptotic (condensed/fragmented) nuclei as described under
"Experimental Procedures." A, representative
photomicrographs showing B-crystallin expression (upper
panels) and nuclear morphology (lower panels, the
nuclei corresponding to B-crystallin-positive myoblasts are
indicated by arrows). B, the data represent the
mean ± S.E. of three independent experiments (*,
p < 0.05 for B-negative versus
B-positive cells at each time point).
B-crystallin Is Preferentially Expressed in Surviving Myoblasts
during Myogenesis--
If the expression of B-crystallin confers
resistance to myogenic apoptosis, B-crystallin should be selectively
expressed in surviving myoblasts. To examine this hypothesis, C2C12
cells were transferred to DM for 0-72 h, and cells were then
simultaneously examined for B-crystallin expression by
immunofluorescence and for the induction of apoptosis by nuclear
morphology; cells with condensed or fragmented nuclei were scored as
apoptotic. As shown in Fig. 2A, the vast majority of C2C12
cells expressing B-crystallin had intact nuclei (the nuclei
corresponding to B-crystallin-positive myoblasts are indicated by
arrows in the lower panels) even after 72 h
in DM, whereas virtually all of the apoptotic myoblasts with fragmented/condensed nuclei lacked B-crystallin. These results are
presented quantitatively in Fig. 2B. Indeed,
B-crystallin-negative myoblasts had dramatically higher
rates of differentiation-induced apoptosis than
B-crystallin-positive cells at 24, 48, and 72 h after transfer
to DM. This striking inverse correlation between the induction of
apoptosis and the expression of B-crystallin suggests that
B-crystallin may promote myoblast survival during differentiation.
B-crystallin Protects Myoblasts from
Differentiation-induced Apoptosis--
We next wanted to determine
whether ectopic expression of B-crystallin was sufficient
to protect myoblasts from differentiation-induced apoptosis. C2C12
cells were transiently transfected with empty vector or FLAG-tagged
cDNAs encoding wild-type B-crystallin or Hsp27. After overnight
incubation, cells were transferred to DM for 24 h. Transfected
cells were then identified by immunofluorescence with an anti-FLAG mAb,
and the percentage of transfected cells with apoptotic nuclei was
determined. As shown in Fig.
3A, ectopic expression of
wild-type B-crystallin potently inhibited differentiation-induced myoblast apoptosis. In contrast, Hsp27 did not significantly inhibit differentiation-induced myoblast apoptosis, thereby underscoring the
specificity of the role of B-crystallin in regulating this process.
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Fig. 3.
Ectopic expression of
B-crystallin protects myoblasts from
differentiation-induced apoptosis. A, C2C12 myoblasts
were transiently transfected with empty vector or FLAG-tagged wild-type
B-crystallin or Hsp27 cDNAs as described under "Experimental
Procedures." B, C2C12 cells were similarly transfected
with empty vector or cDNAs encoding either wild-type or mutant
B-crystallin (R120G, a carboxyl-terminal deletion (C) or a
triple pseudophosphorylation mu- tant (S19E,S45E,S59E) labeled 3XSE). In both A and
B, after overnight incubation, cells were transferred to DM
for 24. Transfected cells were identified by indirect
immmunofluorescence using an anti-FLAG mAb, and the percentage of
transfected cells with apoptotic nuclei was scored as detailed under
"Experimental Procedures." In A, B, and
D, the data represent the mean ± S.E. of three
independent experiments (*, p < 0.02). C,
immunoblot of C2C12 cells transiently transfected with empty vector or
FLAG-tagged wild-type or mutant (R120G, C, or 3XSE) B-crystallin
or Hsp27 cDNAs. The ectopically expressed proteins were detected
with a FLAG mAb as described under "Experimental Procedures."
D, B-crystallin confers a similar degree of protection
against differentiation-induced apoptosis as p21. C2C12 cells were
transiently co-transfected with pEGFPN1 or excess plasmid containing
B-crystallin, p21, Bcl-2, wild-type Akt, or Hsp27, and apoptosis was
scored in GFP-positive cells 24 h after transfer to DM as
described under "Experimental Procedures."
B-crystallin that mediate
its anti-apoptotic actions, we examined the ability of several well defined B-crystallin mutants to inhibit differentiation-induced myoblast apoptosis. Specifically, we used the following mutant constructs: (i) the myopathy-causing R120G missense mutation, which
severely impairs its chaperone activity (19-21); (ii) a
pseudophosphorylation triple mutant (S19E,S45E,S59E, abbreviated
3XSE) in which each of the three amino-terminal Ser residues that are
phosphorylated in response to stress has been altered to a Glu residue
to mimic phosphorylation (27, 28); and (iii) a truncated
B-crystallin, which lacks its carboxyl-terminal tail (C), a
region which mediates substrate binding and stabilizes the protein (10,
29). These mutant proteins were expressed at levels comparable with
those of wild-type B-crystallin and Hsp27 in transiently transfected C2C12 myoblasts (see Fig. 3C). As shown in Fig.
3B, the R120G and C mutants were partly impaired in their
ability to protect myoblasts from differentiation-induced
apoptosis compared with wild-type B-crystallin. In contrast,
the 3XSE triple pseudophosphorylation mutant was completely devoid of
anti-apoptotic activity. Because stress-induced phosphorylation of
these Ser residues triggers the dissociation of the large oligomeric
complexes of B-crystallin (28), our findings strongly suggest that
the anti-apoptotic function of B-crystallin is tightly linked to its
oligomerization state. Importantly, as demonstrated in Fig.
3D, the degree of apoptosis inhibition conferred by
B-crystallin was similar to that of p21, a previously described
regulator of myocyte survival (1), whereas neither Bcl-2 nor wild-type
Akt significantly inhibited myogenic apoptosis under these conditions.
B-crystallin Inhibits Myogenesis-induced Caspase-3
Activation--
Because we have previously demonstrated that
B-crystallin negatively regulates TNF-- and DNA damage-induced
apoptosis by inhibiting the activation of caspase-3 (17), we wanted to
determine whether B-crystallin inhibited myogenic apoptosis by the
same mechanism. To this end, we transferred C2C12 myoblasts to DM for 72 h and examined cells for expression of B-crystallin and
active caspase-3; the latter was detected using an antibody that
specifically recognizes the large subunit of activated
caspase-3 (and does not detect pro-caspase-3). As shown in Fig.
4A, the vast majority of C2C12
cells that expressed B-crystallin (left panel) had
non-apoptotic nuclear morphology (middle panel, the nuclei
corresponding to B-crystallin-positive cells are indicated by
arrows, some of which are multinucleated) and lacked active
caspase-3 (right panel), whereas only a very small
percentage of cells that expressed active caspase-3 also expressed
B-crystallin (see Fig. 4B). Moreover, as demonstrated in
Fig. 4C, transient transfection of wild-type B-crystallin, but not Hsp27, was sufficient to inhibit
myogenesis-induced caspase-3 activation, again underscoring the
specificity of our observations. In contrast, each of the
B-crystallin mutants (R120G, C, and 3XSE) was impaired in its
ability to inhibit caspase-3 activation; the 3XSE mutant was most
severely impaired, consistent with its complete loss of anti-apoptotic
function. Taken together, these findings strongly suggest that
B-crystallin inhibits myogenic apoptosis by disrupting caspase-3
activation.
View larger version (23K):
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Fig. 4.
B-crystallin inhibits
myogenesis-induced caspase-3 activation. A and
B, C2C12 myoblasts were transferred to DM for 72 h and
then examined for expression of B-crystallin and the active large
subunit of caspase-3 as described under "Experimental Procedures."
A, representative photomicrograph showing the expression of
B-crystallin (left panel), nuclear morphology
(middle panel, the nuclei corresponding to
B-crystallin-positive cells are indicated by arrows), and
active caspase-3 (right panel). B, cells that
stained positively for active caspase-3 were scored for their
expression of B-crystallin. The data represent the mean ± S.E.
of three independent experiments (*, p < 0.01).
C, ectopic expression of wild-type B-crystallin inhibits
myogenesis-induced caspase-3 activation. C2C12 myoblasts were
transiently transfected with empty vector or FLAG-tagged cDNAs
encoding wild-type or mutant B-crystallin (R120G, a
carboxyl-terminal deletion (C), a triple pseudophosphorylation
mutant 3XSE), or Hsp27. After overnight incubation, C2C12 myoblasts
were transferred to DM for 24 h, and transfected (FLAG-positive)
cells were scored for expression of active caspase-3 as detailed under
"Experimental Procedures." The data represent the mean ± S.E.
of three independent experiments (*, p < 0.02).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin is a novel negative
regulator of myogenic apoptosis; its expression is selectively induced
in surviving myoblasts during an early stage of their differentiation
in vitro. Indeed, the B-crystallin promoter contains a
canonical skeletal muscle-specific E-box element that binds MyoD family
members and accounts for the early expression of B-crystallin during
skeletal myogenesis (11, 13-15). In this way, B-crystallin provides
a direct and previously unrecognized link between the myogenic
differentiation program and the acquisition of apoptosis resistance,
the latter event being a critical step in myogenesis because
apoptosis-sensitive myogenic precursors are eliminated (1). Although a
few other regulators of myogenic apoptosis have been described (1,
5-8), B-crystallin is the first HSP to be implicated in this
process. Interestingly, we observed that Hsp27, a protein ~40%
identical to B-crystallin (10, 11), was only transiently induced
during myogenic differentiation and did not confer resistance to
differentiation-induced apoptosis, thereby indicating that there is
considerable specificity among HSPs with respect to their role in
myogenic cell death. Moreover, our finding that several other
anti-apoptotic proteins (XIAP, FLIPL, and
Bcl-xL) were not induced during skeletal myogenesis indicates that the apoptosis resistance acquired by myogenic precursors represents the activation of a specific subset of anti-apoptotic proteins. However, the long term survival of a subset of
B-crystallin-negative myoblasts indicates that other anti-apoptotic
proteins must also contribute to the apoptosis resistance of mature myocytes.
B-crystallin antagonizes myogenic
apoptosis by inhibiting the activation of caspase-3. This observation
is consistent with our previous findings in cancer cells that
B-crystallin inhibits caspase-3 activation by disrupting its
proteolytic maturation (17). In contrast, Hsp27 did not inhibit
myogenic differentiation-induced caspase-3 activation or apoptosis.
This latter finding provides additional evidence that the
anti-apoptotic mechanisms of these closely related small HSPs are
likely to be quite distinct. Indeed, we have demonstrated previously
that Hsp27 does not directly inhibit the proteolytic maturation of
caspase-3 (17). Instead, Hsp27 has been reported to inhibit apoptosis
by binding to cytosolic cytochrome c and preventing the
recruitment of pro-caspase-9 to the apoptosome (30), a cytosolic
caspase-9 activating complex composed of cytochrome c,
APAF-1, and pro-caspase-9 (9). In this way, Hsp27 inhibits the
activation of caspase-9, the apical caspase in the mitochondrial apoptotic pathway (30), whereas B-crystallin only weakly inhibits caspase-9 activation (17). Recently, the mitochondrial apoptotic pathway has been reported to be inactive in skeletal muscle due to the
absence of APAF-1 in skeletal muscle (31), thereby potentially accounting for the inability of Hsp27 to inhibit myogenic apoptosis.
B-crystallin. We observed that the
myopathy-causing R120G mutation and deletion of its carboxyl-terminal tail partly suppressed the ability of B-crystallin to inhibit differentiation-induced caspase-3 activation and apoptosis, whereas a
pseudophosphorylation triple mutant that mimics stress-induced phosphorylation was severely impaired in these functions. Because each
of these mutations likely diminishes the chaperone activity of
B-crystallin (20, 21, 28, 29), but only the triple pseudophosphorylation mutant is defective in its ability to form large
500-kDa oligomers (28), these findings strongly suggest that
oligomerization of B-crystallin is essential for its cytoprotective actions, as has been demonstrated for Hsp27 in other systems (32).
B-crystallin
reported here and the impaired ability of the R120G mutant to inhibit
caspase-3 activation may provide new insights into the etiology of the
progressive myopathy caused by this mutation (19). Although the R120G
mutant is also compromised in its ability to stabilize desmin
intermediate filaments, thereby resulting in the accumulation of
aggregates of desmin and B-crystallin and the disruption of
myofibrils (19-21, 33), the defect in its ability to inhibit caspase-3
activation would likely sensitize muscle cells to stress-induced
apoptosis. Indeed, the observation that the R120G mutation in patients
or in transgenic mice causes muscle degeneration in early adulthood
(19, 33) suggests that the cumulative oxidative stress of years of
contractile activity is required to unmask the deleterious consequences
of this mutation in vivo. Finally, given the central role
that caspase-3 plays in the execution of apoptosis (9), our results
suggest that B-crystallin may participate broadly in the regulation
of muscle cell death.
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ACKNOWLEDGEMENTS |
|---|
We are indebted to Drs. Honglin Li, Harris Perlman, and Navdeep Chandel for the critical reading of the manuscript and to Dr. H. Perlman for antibodies.
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FOOTNOTES |
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* This work was supported in part by a grant from the Muscular Dystrophy Association (to V. L. C.), by National Institutes of Health Grants NS31957 (to V. L. C.) and 5T32-CA70085 (to M. C. K.), by institutional research grants to Northwestern University from the Howard Hughes Medical Institute (to V. L. C.), and by the Elizabeth Boughton Trust (to V. L. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of
Endocrinology, Tarry 15-755, Feinberg School of Medicine, Northwestern University, 303 East Chicago Ave., Chicago, IL 60611. Tel.:
312-503-0644; Fax: 312-908-9032; E-mail:
v-cryns@northwestern.edu.
Published, JBC Papers in Press, July 24, 2002, DOI 10.1074/jbc.M201770200
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ABBREVIATIONS |
|---|
The abbreviations used are: HSP, heat shock protein; GM, growth medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DM, differentiation medium; mAb, monoclonal antibody; GFP, green fluorescent protein; TNF, tumor necrosis factor.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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