Originally published In Press as doi:10.1074/jbc.M003864200 on May 17, 2000
J. Biol. Chem., Vol. 275, Issue 31, 23825-23833, August 4, 2000
B-crystallin Gene Induction and Phosphorylation by
MKK6-activated p38
A POTENTIAL ROLE FOR 
B-CRYSTALLIN AS A TARGET OF THE p38
BRANCH OF THE CARDIAC STRESS RESPONSE*
Holly E.
Hoover
,
Donna J.
Thuerauf,
Joshua J.
Martindale, and
Christopher C.
Glembotski§
From the SDSU Heart Institute and the Department of Biology, San
Diego State University, San Diego, California 92182
Received for publication, May 5, 2000
 |
ABSTRACT |
The MAPK kinase MKK6 selectively stimulates p38
MAPK and confers protection against stress-induced
apoptosis in cardiac myocytes. However, the events lying
downstream of p38 that mediate this protection are unknown. The small
heat shock protein, B-crystallin, which is expressed in only a few
cell types, including cardiac myocytes, may participate in
MKK6-mediated cytoprotection. In the present study, we showed that, in
cultured cardiac myocytes, expression of MKK6(Glu), an active
form of MKK6, led to p38-dependent increases in
B-crystallin mRNA, protein, and transcription. MKK6(Glu) also
induced p38-dependent activation of the downstream
MAPK-activated protein kinase, MAPKAP-K2, and the phosphorylation of
B-crystallin on serine-59. Initially, exposure of cells to the
hyperosmotic stressor, sorbitol, stimulated MKK6, p38, and MAPKAP-K2
and increased phosphorylation of B-crystallin on serine 59. However,
after longer times of exposure to sorbitol, the cells began to undergo apoptosis. This sorbitol-induced apoptosis was increased when p38 was
inhibited in a manner that would block B-crystallin induction and
phosphorylation. Thus, under these conditions, the activation of MKK6,
p38, and MAPKAP-K2 by sorbitol can provide a degree of protection
against stress-induced apoptosis. Supporting this view was the finding
that sorbitol-induced apoptosis was nearly completely blocked in cells
expressing MKK6(Glu). Therefore, the cytoprotective effects of MKK6 in
cardiac myocytes are due, in part, to phosphorylation of
B-crystallin on serine 59 and to the induction of B-crystallin gene expression.
| |
INTRODUCTION |
The response of cells to stress can determine the fate of tissues
and, ultimately, the fate of the organism. The initial response to most
stresses is the induction of intracellular reactions that foster the
preservation of cell viability and, thus, organ function. However, if
the stress persists, this protective response can be superseded by
intracellular events that result in apoptosis or necrosis, leading
eventually to deterioration of organ function.
The ability of the myocardium to mount a productive stress response is
critical for survival. Examples of stresses to which the heart can be
exposed in vivo include transient ischemia, cytokines and
chemokines, reactive oxygen species, or toxins (1). In the absence of a
fully functional stress response, these insults could rapidly lead to a
devastating loss of cardiac myocytes due to apoptosis and to a decline
in tissue function, emphasizing the importance of understanding the
cellular and molecular events governing the myocardial stress response.
A vital feature of the mechanism by which cardiac myocytes respond to
stress is the activation of the stress mitogen-activated protein
kinases (MAPKs),1 such as p38
MAPK (2-6). For example, p38 is activated in response to ischemia in
isolated hearts (7, 8). In cultured cardiac myocytes, p38 is activated
by agonists such as endothelin and phenylephrine (9-12), that are
known to evoke hypertrophic growth (13), an event that mimics the
compensatory phase of the myocardial stress response (14).
Additionally, p38 is stimulated in transgenic mouse models of
Ras-induced cardiac hypertrophy (15).
Like other MAPKs, p38 is activated after becoming dually phosphorylated
on a critical -Thr-Gly-Tyr- motif (16). This phosphorylation is
accomplished by an upstream kinase, or a MAPK kinase (MKK). Several
MKKs are known to activate p38, as well as some of the other MAPKs.
However, among the MAPKs, MKK3 and MKK6 are capable of activating only
p38 (17, 18). Initial studies of the role of MKK-activated p38 in the
heart showed that the expression of a constitutively activated form of
MKK6, MKK6(Glu), induced certain features of the stress response in
primary neonatal cardiac myocytes, such as sarcomeric stabilization
(12) and protection from sphingosine-induced apoptosis (19). Later
studies corroborated these results, demonstrating roles for both MKK6
and MKK3 in the cardiac stress response (11, 15).
The present study is focused on the mechanism by which MKK6 protects
cardiac myocytes from apoptosis, with a particular emphasis on the
small heat shock proteins (hsps). Among the best characterized of the
small hsps are hsp27 and B-crystallin. In response to stress, hsp27
is phosphorylated by kinases that lie downstream of p38, notably
MAPK-activated protein-K2 (MAPKAP-K2), MAPKAP-K3, and p38-reactive
kinase (20, 21). In endothelial and smooth muscle cells, the
phosphorylation of hsp27, which is believed to contribute to protection
against stress-induced cell death, correlates temporally with the
translocation of hsp27 to actin filaments, where it contributes to
stabilization of the cytoskeleton (22-25). Furthermore, overexpression
of hsp27 has been shown to foster cell survival, although protection
from apoptosis was not demonstrated (26).
In contrast to hsp27, which is expressed in nearly all cell types, a
structurally related, small hsp, B-crystallin, is expressed mainly
in the lens, in certain neurons, in skeletal muscle, and, surprisingly,
in very high levels in cardiac myocytes (27-30). Like hsp27,
B-crystallin in lens cells is post-transcriptionally modified by
phosphorylation, and in cultured human glioma cells (U373),
B-crystallin is phosphorylated in response to stressors such as
sorbitol (hyperosmotic) or arsenite (31). Recent studies have shown
that when U373 cells are treated with stressors known to activate p38
and MAPKAP-K2, B-crystallin is phosphorylated in a site-specific
manner on serines located at positions 19, 45, and 59 (32). The roles
and the kinases responsible for each phosphorylation event remain unknown.
Stresses such as ischemia, sorbitol, and arsenite activate p38 and
MAPKAP-K2 in the heart and in cultured cardiac myocytes (7, 8, 11, 33).
In comparison with stress-induced hsp27 translocation to actin
filaments in other cell types, in the isolated perfused heart, ischemic
stress induces B-crystallin translocation from a diffuse, cytosolic
localization to sarcomeres (34). This translocation was shown to
be temporally coordinated with increased B-crystallin
phosphorylation, although the identities of phosphorylated residues
have not been established (35). Since hsp27 translocates to and
stabilizes actin filaments in endothelial and smooth muscle cells (4,
23), the sarcomeric localization of B-crystallin could foster
myofilament stabilization in stressed cardiac myocytes. Since
myofilament disintegration takes place in cardiac myocytes during
hypoxia, which leads to apoptosis (36), the sarcomeric translocation of
B-crystallin could be important for protecting myocytes against
apoptosis. Consistent with this possibility is the finding that
overexpression of B-crystallin increased cardiac myocyte survival
during ischemia (26), although apoptosis was not assessed in that study.
The mechanisms responsible for the tissue-restricted expression of
B-crystallin are largely unknown. However, the B-crystallin promoter possesses a serum response element (SRE) (38) that is
homologous to SREs in other stress-induced cardiac genes
(e.g. ANF (39)). Thus, it is plausible
that B-crystallin expression is induced in concert with other
SRE-containing cardiac genes in response to stress and that the
increased levels of B-crystallin contribute to myocardial
protection. Moreover, it is possible that stress-mediated activation of
the p38 pathway can induce the selective, site-specific phosphorylation
of B-crystallin and that this phosphorylation may be critical for
optimal B-crystallin-mediated cytoprotection. These hypotheses were
tested in the present study using a well characterized cultured cell
model system for evaluating the response of cardiac myocytes to stress
and activated MKK6.
| |
MATERIALS AND METHODS |
Cell Culture
Primary ventricular myocytes were prepared from 1-4-day-old
Harlan Sprague-Dawley rats as described (12, 39). Briefly, hearts were
dissected in DMEM/air; the apical two-thirds of the ventricles were
dissected away from the atria. After mincing and washing the ventricles
twice with air-compatible DMEM, cells were isolated by multiple rounds
of 10-min-long tissue dissociation with 0.001% trypsin. After each
incubation with trypsin, the supernatant was added to an equal volume
of DMEM/F-12 (1:1) containing 20% fetal bovine serum, and all of the
supernatants were combined. Plastic wells were treated for at least
1 h with 5 ng of fibronectin/ml of DMEM/F-12 (1:1). Myocytes,
which, in some experiments, were derived from the nonadherent cells in
a 1-h preplating on untreated plastic dishes, were plated finally in
DMEM/F-12 (1:1) containing 10% fetal bovine serum for approximately
16 h. After this time, cultures were washed with DMEM/F-12 (1:1)
and then incubated in DMEM/F-12 (1:1) containing bovine serum albumin
at 1 g/liter (minimal medium) with or without any test agents for the
indicated times.
Northern Analysis
Northern analyses were performed as described previously (39),
with minor modifications. RNA was isolated from 3 × 106 myocytes using RNAzol B (Tel-Test, Inc., Friendswood,
TX) as described by the manufacturer. Five µg of each sample were
fractionated on a 1% agarose/formaldehyde gel and then transferred to
Hybond Nylon membrane (Amersham Pharmacia Biotech). Membranes were
cross-linked and then incubated with radioactive probes in QuikHyb
(Stratagene, Inc., Madison, WI). The B-crystallin probe was prepared
by digesting a rat B-crystallin cDNA (cLens/pBluescript(
) from
Dr. J. Piatigorsky, National Institutes of Health) with
EcoRI/HindIII to generate a 730-bp fragment of
the B-crystallin cDNA. The glyceraldehyde-3-phosphate dehydrogenase probe was prepared by digesting a
glyceraldehyde-3-phosphate dehydrogenase cDNA (ATCC 78105) with
EcoRI to generate a 1.2-kilobase pair fragment. cDNA
fragments were then labeled with [-32P]dCTP, using
Klenow fragment and standard protocols.
Western Analyses
B-crystallin--
Cultures composed of approximately
106 myocytes were lysed in 100 µl of Laemmli sample
buffer supplemented with 0.1 mM sodium o-vanadate, 10 µg/ml aprotinin, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM
p-nitrophenyl phosphate, and 10 µg/ml leupeptin and boiled
for 5 min. Eighty µl of each lysate were analyzed for phosphospecific
B-crystallin, and 5 µl of each lysate were analyzed for total
B-crystallin. Proteins were resolved on a 15% SDS-PAGE and
transferred to a nitrocellulose membrane in methanol transfer buffer at
60 V for 5 h or 30 V overnight. Membranes were blocked for 30 min
in 5% nonfat milk dissolved in TBS-Tween (0.01%) at room temperature
and then probed with rabbit affinity-purified B-crystallin antisera
specific for phosphorylation at serine 19, 45, or 59. All
B-crystallin antisera were generously provided by Dr. Kanefusa Kato
(Institute for Developmental Research, Japan). The B-crystallin
phosphoserine-specific antisera were used at 1 µg/ml for
phosphoserine 19 and at 0.5 µg/ml for phosphoserines 45 and 59; the
antisera were diluted into 5% nonfat milk TBS-Tween. Blots were
incubated with this mixture for 2 h at room temperature. Some
blots were incubated with an affinity-purified B-crystallin antiserum raised against the C terminus of B-crystallin, which was
used at a final concentration of 0.1 µg/ml. This antiserum recognizes
all phosphorylated and nonphosphorylated forms of B-crystallin, thus
serving as a measure of the total amount of B-crystallin in a
sample. Following incubation with the B-crystallin antisera, blots
were washed for 30 min in TBS-Tween and then incubated in a 1:2000
dilution of anti-rabbit IgG horseradish peroxidase (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) followed by washing for 30 min in
TBS-Tween. Visualization of immune complexes were carried out by an
enhanced chemiluminescence method using enhanced chemiluminescence
Western blotting detection reagents (NEN Life Science Products)
according to the manufacturer's instructions.
p38 and Phospho-p38--
Cultures composed of approximately
2 × 106 myocytes were lysed in 100 µl of
supplemented Laemmli sample buffer (see above), boiled for 5 min, and
then submitted to 10% SDS-PAGE and transferred to a nitrocellulose
membrane (see above). Western analyses were performed as described
above with 1:1000 dilutions of antisera specific for either phospho-p38
(New England BioLabs, Beverly, MA; catalog no. 9211S). Blots were
subsequently stripped with 6.25 mM Tris, 2% SDS, and 100 mM 2-mercaptoethanol for 30 min at 50 °C, washed for
1 h in TBS-Tween, and reprobed with 1:1000 dilution of p38
antibody (Stressgen Biotechnologies Corp., Victoria, Canada; catalog
no. KAP-MAOO9E) for normalization.
Transfection by Electroporation
Immediately following the final dissociation step, myocardial
cells were resuspended in serum-free DMEM:F-12 (1:1). Between 5 and
12 × 106 cells were combined with the indicated
amounts of plasmids in a total volume of 300 µl of DMEM/F-12 (1:1).
The total amount of plasmid used for each electroporation in one
experiment was equalized using pCMV6. Cells were electroporated at 500 V, 25 microfarads, and 100 ohms in a 0.2-cm gap electroporation cuvette (Bio-Rad) using a Gene Pulser II (Bio-Rad). Under these conditions, only cardiac myocytes are transfected (39, 40). For reporter assays,
1.5 × 106 cells were plated per 24-mm well, whereas
4.5 × 106 myocytes per 35-mm well were plated to
perform kinase assays.
Transfection by Adenovirus
For adenovirus experiments, cultures were preplated for 1.5 h, and then, following culturing of the myocytes overnight in DMEM/F-12
containing 10% fetal bovine serum (see above), they were infected with
AdV-Con, AdV-MKK6(Glu), AdV-p38(AGF) for 5 h, as described
previously (41).
Plasmids
MKK6--
pcDNA3 FLAG-MKK6(wt), which codes for wild type
human MKK6, was derived by mutating pcDNA3 FLAG-MKK6(Glu) (see
below) from Glu208/Glu212 to
Ser208/Ser212.
MKK6(Glu)--
pcDNA3 FLAG-MKK6 (Glu) codes for activated
human MKK6 (17) and was obtained from R. J. Davis (University of
Massachusetts, Worcester, MA).
p38(AGF)--
pcDNA3 FLAG-p38 was obtained originally
from J. Han (The Scripps Research Institute, San Diego, CA). Using
site-directed mutagenesis (QuickChange, Stratagene, Inc., Madison, WI),
Thr181 and Tyr183 of p38 were converted to
Ala and Phe, respectively.
pGEX-6P-1/3XHA p38
2
(K53R)--
pGEX-6P-1/3XHA p382 (K53R)
was derived from p382(wt) (B. Stein, Signal
Pharmaceuticals, San Diego, CA) via polymerase chain reaction primers
with 5'-(EcoRI) + 3XHA tag and 3'-ter(NotI).
B-crystallin Promoter/Luciferase--
The murine
B-crystallin promoters, 426 to +44 and 164 to +44, in
chloramphenicol acetyltransferase constructs were obtained from Dr. J. Piatigorsky (38). These were recloned into the Luciferase construct,
pGL2 (Promega Corp., Madison, WI), by subcloning into the
BamHI site of pBluescriptII KS+ (Stratagene) and
subsequently cloning into pGL2 using EcoRI and
HindIII sites.
MKK6 Kinase Assays
Myocytes were transfected by electroporation with 30 µg of
pcDNA3 FLAG-MKK6(wt), as described above, using 9 × 106 cells per transfection. Transfected myocytes were
plated, and after 48 h in minimal media, cultures were treated
with 400 mM sorbitol or 0.5 mM arsenite for 5 min and then extracted in 1 ml of lysis buffer containing 20 mM Tris, pH 7.6, 20 mM -glycerophosphate, 250 mM NaCl, 3 mM EGTA, 3 mM EDTA,
0.5% Nonidet P-40, 0.1 mM sodium o-vanadate, 10 µg/ml aprotinin, 2 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, 1 mM
p-nitrophenyl phosphate, and 10 µg/ml leupeptin. The cell
debris was removed by brief centrifugation. Lysates were incubated
overnight at 4 °C with 4 µg of anti-FLAG M2 antibody (Sigma) per
sample. Forty µl of a protein G-Sepharose slurry were then added to
each sample and incubated for 60 min at 4 °C. The Sepharose pellet
was washed twice with lysis buffer and once with preincubation buffer
(30 mM HEPES, pH 7.4, 10 mM MgCl2,
20 mM -glycerophosphate, 1 mM DTT). Kinase
reactions were performed using 2 µg of GST-3XHA p382
(see below) per reaction as substrate in a final volume of 30 µl of
kinase buffer (30 mM HEPES, pH 7.4, 10 mM
MgCl2, 20 mM -glycerophosphate, 1 mM DTT, 20 µM ATP, and 6 µM
[
-32P]ATP (5000 Ci/mmol). After 20 min at 30 °C,
the reactions were terminated by the addition of Laemmli sample buffer,
and the phosphorylation level of substrate proteins was evaluated by
SDS-PAGE followed by autoradiography and phosphor image analyses.
Recombinant Protein Expression and Purification
A kinase-inactive form of human p382
(p382(K53R)), which retains the native sites that are
phosphorylated by MKK6 (Thr181 and Tyr183), was
used as the substrate for MKK6 kinase assays. To prepare this protein,
the plasmid, pGEX-6P-1/3XHA p382(K53R), was transformed into BL-21-competent cells (Stratagene, Inc., Madison, WI; catalog no.
200133). Cultures were grown in 2× YTA medium with 100 µg/ml ampicillin for 12-15 h until A600 = 2.0. Protein expression was induced with 0.2 mM
isopropyl-1-thio--D-galactopyranoside (Sigma; catalog
no. I-5502), at 30 °C and with shaking at 150 rpm for 6 h.
Cells were the collected by centrifugation at 6000 × g
for 10 min and then resuspended in 50 ml of buffer G (1×
phosphate-buffered saline, 50 mM EDTA, 5 mM
benzamidine, 100 µM phenylmethylsulfonyl fluoride) per
liter of culture. The resuspended cells were sonicated using a Branson
450 sonifier, at setting 5 for 15-30 s. Triton X-100 was added to
0.1% after sonication, and the sonicate was then centrifuged at
12,000 × g for 20 min. Recombinant GST-3XHA p382(K53R) was then purified from the crude cell extract
using a Bulk GST Purification Module (Amersham Pharmacia Biotech.;
catalog no. 27-4570-01), essentially as described in the accompanying protocol. Briefly, 1 ml of 50% glutathione-Sepharose was added per 100 ml of sonicate and incubated on ice for 1-2 h. The Sepharose was
pelleted at 1000 × g for 5 min, washed three times
with cold 1× phosphate-buffered saline, and eluted with 10 mM reduced glutathione (in 50 mM Tris-HCl, pH
8) in fractions of 1 ml/500 µl of Sepharose. The eluted protein was
dialyzed overnight at 4 °C in 2 liters of dialysis buffer (100 mM Tris, pH 7; 200 mM NaCl; 200 µM EDTA; 2 mM DTT). The purification of
p382(K53R) was verified by SDS-PAGE followed by staining
with Coomasie Blue, coupled with Western analyses using an anti-HA
antiserum. Final protein concentration was determined using the DC
Protein Assay (Bio-Rad; catalog no. 500-0116).
MAPKAP-K2 Kinase Assays
Cultures were extracted in 400 µl of buffer A (50 mM Tris (pH 7.5), 1 mM EDTA, 1 mM
EGTA, 1% Triton X-100, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 50 mM NaF, 0.5 mM sodium o-vanadate, 0.1% 2-mercaptoethanol,
0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin,
and 1 µg/ml leupeptin). Following removal of debris by
centrifugation, MAPKAP-K2 was immunoprecipitated using 1.5 µg of
anti-MAPKAP-K2 (Upstate Biotechnology, Inc., Lake Placid, NY; catalog
no. 06-534) and submitted to a kinase assay using [-32P]ATP and hsp27 as the substrates, as described by
the manufacturer's protocol. Labeled hsp27 was then resolved by 12%
SDS-PAGE, and the gel was then submitted to phosphor image analyses.
Preparation of Recombinant Adenovirus
AdV-p38AGF and AdV-MKK6(Glu)--
The AdEasy system was used for
preparing recombinant adenoviral strains using previously described
methods (41, 42). Briefly, human MKK6(Glu) and human p38(AGF) were
polymerase chain reaction-amplified from the parent templates (see
above) such as to create restriction sites that would facilitate
cloning into pAdTrack-CMV, an adenoviral shuttle vector that harbors
CMV-driven green fluorescent protein, and a CMV-flanked multiple
cloning site for the insertion of the gene of interest. Polymerase
chain reaction-amplified p38(AGF) and MKK6(Glu) were cloned into the
EcoRI and NotI sites of pGEX-6P-1 (Amersham
Pharmacia Biotech), which served as a shuttle cloning vector.
pGEX-6P-1/p38(AGF) and pGEX-6P-1/MKK6(Glu) were then digested with
BamHI and NotI, and the resulting products of
interest were cloned into the BglII and NotI
sites of pAdTrack-CMV to create pAdTrack-CMV-p38(AGF) and
pAdTrack-CMV-MKK6(Glu). pAdTrack-CMV-p38(AGF) or
pAdTrack-CMV-MKK6(Glu) was linearized and then co-transformed with the adenoviral vector, pAdEasy-1, into Escherichia coli
strain BJ5183. This strain of E. coli allows for homologous
recombination of pAdEasy-1 and the pAdTrack-CMV shuttle vector
containing the gene of interest. Recombinants were selected on
kanamycin and screened by restriction digestion with PacI.
Recombinant plasmids were then retransformed into E. coli
DH5 for propagation purposes. Recombinant adenoviral plasmids were
linearized with PacI and then transfected into 293 human
embryonic kidney cells, using LipofectAMINE® (Life Technologies,
Inc.). Transfection efficiency was determined by observing green
fluorescent protein fluorescence, as described previously (41). The
recombinant viruses were then harvested 7-10 days
postinfection. Viral titers were determined by observing green
fluorescent protein fluorescence of primary neonatal cardiac myocytes;
the minimum quantity of viral stock that afforded 100% transfection
efficiency was selected for the experiments in this study.
Reporter Assays
-Galactosidase--
Following the appropriate time in
culture, each well of myocytes was washed twice with phosphate-buffered
saline and then lysed in 500 µl of ice-cold lysis buffer (25 mM Gly-Gly, pH 7.8, 15 mM MgSO4, 4 mM EDTA, 0.25% Triton X-100) containing 1 mM
DTT. The cell debris was removed by centrifugation. To measure
-galactosidase activity, 200 µl of the cell extract was added to
400 µl of -galactosidase buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1 mM MgSO4) containing 1 mg/ml chlorophenol
red--D-galactopyranoside and 50 mM
-mercaptoethanol. The reaction was incubated for 2 h at
37 °C, after which absorbance was measured at 570 nm.
Luciferase--
To measure luciferase activity, 100 µl of
buffer (25 mM Gly-Gly, pH 7.8, 15 mM
MgSO4, 4 mM EGTA, 45 mM
KPO4 pH 7.8, 1 mM DTT, 0.3 mM
D-luciferin, 3 mM ATP) were added to 100 µl
of cell lysate. Light emission of each sample was measured by an
Optocomp II luminometer for 10 s. The relative luciferase
activities were determined by dividing the relative luciferase activity
by the relative -galactosidase activity.
Apoptosis
DNA Laddering--
Assessment of apoptosis was performed by DNA
laddering as described previously (19). Briefly, each sample began with
a 100-mm culture containing approximately 8 × 106
cardiac myocytes. The cells from each culture dish were lysed in a
digestion buffer containing 100 mM NaCl, 10 mM
Tris (pH 8.0), 25 mM EDTA, 0.5% SDS, 0.4 mg/ml proteinase
K (added fresh); scraped; pipetted several times; and incubated at
55 °C for 5-14 h. Samples were then extracted twice with
phenol and once with chloroform/isoamyl alcohol, and then DNA was
isopropyl alcohol-precipitated and washed once with 80% ethanol and
allowed to air-dry. The DNA pellet was then dissolved in a buffer
containing 10 mM Tris (pH 7.9), 10 mM
MgCl2, 50 mM NaCl, 1 mM DTT and
then digested for 2 h at 37 °C with RNase A (3 mg/ml). These
samples were then extracted once with phenol and once with
chloroform/isoamyl alcohol and then ethanol-precipitated. The DNA
pellets were then washed with 80% ethanol, air-dried and dissolved in
a small volume of Tris/EDTA. The absorbance of the sample was
determined and identical amounts of each sample were then fractionated
on a 2% agarose gel.
| |
RESULTS |
MKK6(Glu) Induces B-Crystallin Expression in Cardiac
Myocytes--
To determine whether the B-crystallin gene is induced
upon stimulation of p38 by MKK6, an activated form of MKK6, MKK6(Glu), was expressed in primary neonatal rat ventricular myocytes (NRVMCs) using adenovirus (AdV)-mediated gene transfer, followed by Northern and
Western analyses of B-crystallin mRNA and protein levels. Previous studies have demonstrated that using AdV-mediated gene transfer, a transfection efficiency of 100% can be achieved in NRVMCs
(41). Compared with control cells, cultures expressing MKK6(Glu)
displayed a 6.3-fold increase in the quantity of B-crystallin mRNA, which was reduced to 2.8-fold upon treatment with the
membrane-permeable, p38-specific inhibitor SB203580 (Fig.
1A). Western analysis showed that cells expressing MKK6(Glu) displayed a 2.2-fold increase over
control in the quantity of B-crystallin protein, which was reduced
to control levels upon treatment of the cells with SB203580 (Fig.
1A).
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Fig. 1.
Effects of MKK6(Glu) on
B-Crystallin mRNA, protein, and promoter.
A, cultured cardiac myocytes were infected with either
AdV-Con or AdV-MKK6(Glu) and treated with or without 5 µM
SB203580, as indicated and as described under "Materials and
Methods." Culture extracts were then analyzed for B-crystallin or
glyceraldehyde dehydrogenase (GDH) mRNA by Northern
blotting or for B-crystallin protein by Western blotting, as
described under "Materials and Methods." Each treatment was carried
out on either three (Northern) or two (Western) identical cultures, and
the Northern and Western blot results, as obtained by phosphor image
analyses, are shown at the top. Following densitometeric
quantitation using ImageQuant software (Molecular Dynamics), the mean
intensities of the bands in the blots from each treatment ± S.E.
were determined and are shown as a bar graph at
the bottom. The B-crystallin mRNA band intensities
were normalized to the glyceraldehyde-3-phosphate dehydrogenase band
intensities. This experiment was replicated at least three times; the
results shown are from one representative experiment. * and  ,
p < 0.05 different from all other values as determined
by ANOVA followed by Neuman Keuls post hoc analysis of
variance. B, cultured cardiac myocytes were transfected by
electroporation with BC(426)/Luc, pCH110 (-galactosidase), and
various quantities of pcDNA3-MKK6(Glu), as indicated. Following
48 h in minimal medium, cell extracts were analyzed for
luciferase and -galactosidase activities. Relative luciferase
(Rel Luc) values are luciferase/galactosidase for
each treatment, shown as -fold of the control (control = no
pcDNA3-MKK6(Glu)). The data are presented as the mean of three
identically treated cultures ± S.E. This experiment was repeated
at least three times; one representative experiment is shown. *,
p < 0.05 different from control (0 MKK6(Glu)) as
determined by ANOVA followed by Neuman Keuls post hoc
analysis of variance. C, cultures were transfected by
electroporation with BC(426)/Luc, pCH110 (-gal) with or without
pcDNA3-MKK6(Glu) or pcDNA3-p38AGF and treated with or without 5 µM SB203580, as indicated. Following 48 h in minimal
medium, extracts were analyzed for luciferase and
-galactosidase activities. Relative luciferase (Rel Luc)
values are luciferase/galactosidase values for each treatment, shown as
-fold of the control (control = no pcDNA3-MKK6(Glu)). Data are
presented as the mean of three identically treated cultures ± S.E. This experiment was repeated at least three times; one
representative experiment is shown. * and , p < 0.05 different from all other values as determined by ANOVA followed by
Neuman Keuls post hoc analysis of variance.
|
|
To evaluate whether increased transcription contributed to
MKK6-inducible B-crystallin expression, a reporter construct
comprised of 426 nucleotides of the mouse B-crystallin 5'-flanking
sequence and promoter driving firefly luciferase
(BC(426)/Luc) was prepared. When NRVMCs were co-transfected
by electroporation with BC(426)/Luc, along with a construct encoding
MKK6(Glu), there was an increase in the amount of luciferase activity,
which correlated with the quantity of MKK6(Glu) (Fig. 1B).
At the highest dose of MKK6(Glu) tested, there was an approximate
5.5-fold increase in luciferase expression. MKK6(Glu)-inducible
BC(426)/Luc was significantly inhibited by SB203580 or by
co-transfecting a dominant-interfering form of p38 (Fig.
1C). Taken together, these results demonstrated that in
cardiac myocytes the expression of MKK6(Glu), which among the MAPK
kinases has been shown to activate only p38 (12), increased the levels
of B-crystallin transcription, mRNA, and protein in a
p38-dependent manner. This is consistent with roles for
B-crystallin gene induction in MKK6/p38-mediated protection from apoptosis.
Site-selective Phosphorylation of B-crystallin in Response to
MKK6--
Antisera that bind selectively to B-crystallin that is
phosphorylated on either serine 19, 45, or 59 (32) were used to
determine the extent of phosphorylation at these sites in cells
expressing MKK6(Glu). In control NRVMCs, B-crystallin was poorly
phosphorylated on serine 19; however, phosphorylation on serines 45 and
59 was clearly detectable (Fig.
2A, top). The
extent of phosphorylation of all three of these B-crystallin serine
residues increased in cells expressing MKK6(Glu). However, relative to
serine 45 and serine 59, phosphorylation at serine 19 was nearly
undetectable, even in MKK6(Glu)-expressing cells. Accordingly,
phosphorylation of B-crystallin at serine 19 was not pursued in
further detail in this study. Expression of MKK6(Glu) increased
phosphorylation at serines 45 and 59 by about 2- and 6.5-fold,
respectively (Fig. 2A). The phosphorylation of
B-crystallin at both serine 45 and 59 observed in
MKK6(Glu)-expressing cells was reduced to control levels or below
control levels upon incubation with SB203580, indicating a requirement
for p38. The activation of both p38 and MAPKAP-K2 in
MKK6(Glu)-expressing cells (Fig. 2B) was consistent with the
hypothesis that p38-activated kinases, such as MAPKAP-K2, are
responsible for B-crystallin phosphorylation in response to MKK6.
SB203580, which interacts with the ATP binding site of p38 and, thus,
inhibits its kinase activity, did not block the ability of MKK6(Glu) to
phosphorylate p38, as expected. However, the kinase activity of
endogenous MAPKAP-K2, which was stimulated by about 9-fold in
MKK6(Glu)-expressing cells, was inhibited completely by SB203580 (Fig.
2B). Thus, the coordinate reduction of MKK6(Glu)-activated MAPKAP-K2 and B-crystallin phosphorylation at serines 45 and 59 by
SB203580 is consistent with the premise that in cardiac myocytes,
B-crystallin serves as a target for p38-activated MAPKAP-K2 and that
the phosphorylation of B-crystallin may contribute to the
antiapoptotic effects of MKK6.
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Fig. 2.
Effects of MKK6(Glu) on
B-crystallin phosphorylation and on the activities
of p38 and MAPKAP-K2. A, phospho- B-crystallin.
Cultured cardiac myocytes were infected with either AdV-Con or
AdV-MKK6(Glu) and treated with or without 5 µM SB203580,
as indicated and as described under "Materials and Methods."
Culture extracts were then analyzed for B-crystallin
phospho-Ser19, B-crystallin phospho-Ser45,
B-crystallin phospho-Ser59, or total B-crystallin by
Western blotting with the appropriate antisera, as described under
"Materials and Methods." Each treatment was carried out on two
identical cultures. The phosphor image results are shown at the
top, and the quantitated results of each blot, as determined
using ImageQuant software (Molecular Dynamics), are shown at the
bottom as the mean densities of phospho-Ser19,
phospho-Ser45, or
phospho-Ser59/B-crystallin total and are
compared with the control (AdV-Con without SB203580). Data are
presented as the mean of two identically treated cultures, ± S.D. This
experiment was replicated at least three times; the results shown are
from one representative experiment. *, p < 0.05 different from control (no MKK6(Glu)) as determined by ANOVA followed
by Neuman Keuls post hoc analysis of variance. B,
p38 and MAPKAP-K2 activities. Cultured cardiac myocytes were infected
with either AdV-Con or AdV-MKK6(Glu) and treated with or without 5 µM SB203580, as indicated and as described under
"Materials and Methods." Culture extracts were then analyzed for
phospho-p38 and total p38 by Western blotting and for MAPKAP-K2 kinase
activity as described under "Materials and Methods." Each treatment
was carried out on three identical cultures. The phosphor image results
are shown at the top, and the quantitated results of each
blot, as determined using ImageQuant software (Molecular Dynamics), are
shown as the mean density ± S.E. compared with the control
(AdV-Con without SB203580). The phospho-p38 band intensities were
normalized to total p38. The data are presented as the mean of three
identically treated cultures. This experiment was replicated at least
three times; the results shown are from one representative experiment.
*, p < 0.05 different from no MKK6(Glu), as determined
by ANOVA followed by Neuman Keuls post hoc analysis of
variance.
|
|
Stressors Activate MKK6, p38, MAPKAP-K2, and B-crystallin
Phosphorylation in Cardiac Myocytes--
In most cell types, the MAPK
pathways are activated in response to various stresses and are thus
thought to serve important roles in the cellular stress response (43).
Accordingly, we determined whether treating myocardial cells with two
well characterized stressors, sorbitol and arsenite, activated the
MKK6/p38/MAPKAP-K2 pathway in a manner that could lead to
B-crystallin phosphorylation. Either sorbitol or arsenite
coordinately induced the activities of MKK6, p38, and MAPKAP-K2, from
approximately 8- to 15-fold (Fig. 3,
A-C). Importantly, the activation of MAPKAP-K2 was blocked by SB203580 (Fig. 3C), indicating that under these
conditions, p38 serves as the primary activator of MAPKAP-K2 in
response to sorbitol or arsenite.
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Fig. 3.
Effects of sorbitol and arsenite on MKK6,
p38, and MAPKAP-K2. A, MKK6. Cultured cardiac myocytes
were transfected by electroporation with pcDNA3 FLAG-MKK6(wt).
Forty-eight h later, they were treated for 5 min with or without 400 mM sorbitol or 0.5 mM arsenite. Following
immunoprecipitation of FLAG-MKK6 from culture extracts, MKK6 kinase
assays were carried out using p382(K53R) as the
substrate and SDS-PAGE separation of products, as described under
"Materials and Methods." The phosphor image of the region of the
gel containing p382 is shown at the top, and
the digital analyses of those images are shown in the bar
graph at the bottom as the mean density ± S.E. The data are presented as the mean of three identically treated
cultures. B, p38. Cardiac myocytes were cultured identically
to those used in A, except they were not transfected. After
48 h, they were treated for 5 min with or without 400 mM sorbitol or 0.5 mM arsenite, and the levels
of phospho-p38 (P-p38) and total p38 were determined by
Western blotting, as described under "Materials and Methods" and as
in Fig. 2. The image of the p38 region of the blot is shown at the
top. The relative intensities of each phospho-p38 band were
divided by the relative intensities of each total p38 band to normalize
for slight differences in total p38 levels between the cultures. The
means ± S.E. of the relative intensities of phospho-p38/p38 for
each treatment are shown in the bar graph at the
bottom. The data are presented as the mean of three
identically treated cultures. C, MAPKAP-K2. Cardiac myocytes
were cultured identically to those used in A, except they
were not transfected. After 48 h, they were treated for 10 min
with or without 400 mM sorbitol or 0.5 mM
arsenite with or without SB203580, and then endogenous MAPKAP-K2 was
immunoprecipitated from culture extracts, and MAPKAP-K2 kinase assays
were carried out using hsp27 as the substrate, as described under
"Materials and Methods." The image of the region of the blot where
hsp27 migrates is shown at the top of the panel.
The mean intensity of each hsp27 band ± S.E. for each treatment
is shown in the bar graph at the
bottom. The data are presented as the mean of three
identically treated cultures. *, p < 0.05 different
from control as determined by ANOVA followed by Neuman Keuls post
hoc analysis of variance.
|
|
Sorbitol and arsenite also induced the site-specific phosphorylation of
B-crystallin on serines 45 and 59 by 2.5- and 10-fold, respectively
(Fig. 4). Interestingly, B-crystallin
phosphorylation on serine 45 in response to sorbitol was very sensitive
to the cell-permeable inhibitor of the ERK MAPK pathway,
PD980590, but not to SB203580 (Fig.
5A). In contrast,
B-crystallin phosphorylation at serine 59 was not inhibited by
PD098059 but was completely blocked by SB203580 (Fig.
5B). Thus, the enhancement of the phosphorylation of
B-crystallin on serine 45 was relatively small and appeared to
require ERK, while sorbitol-mediated phosphorylation of B-crystallin on serine 59 was relatively high and appeared to require p38. This
site-selective phosphorylation was consistent with the relatively low
ERK activation and high p38 activation observed in response to sorbitol
or arsenite (Fig. 5C).
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Fig. 4.
Effects of sorbitol and arsenite on
B-crystallin phosphorylation. Cultured cardiac
myocytes were treated for 10 min with or without 400 mM
sorbitol or 0.5 mM arsenite, as indicated and as in Fig. 3.
Culture extracts were then analyzed for B-crystallin
phospho-Ser45, B-crystallin phospho-Ser59,
or total B-crystallin by Western blotting, as described under
"Materials and Methods." Phospho-Ser45 and
phospho-Ser59 analyses were carried out on separate
cultures; the B-crystallin total was determined for each.
The phosphor image of the region of the blot where B-crystallin
migrates is shown at the top. The mean intensity of each
phospho-B-crystallin band/total B-crystallin ± S.D. for
each treatment is shown in the bar graph at the
bottom. Data are presented as the mean of three identically
treated cultures. *, p < 0.05 different from control
as determined by ANOVA followed by Neuman Keuls post hoc
analysis of variance.
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Fig. 5.
Effects of inhibiting p38 or ERK on sorbitol-
and arsenite-mediated B-crystallin
phosphorylation. Cultures were treated for 30 min with or without
SB203580 (5 µM) or PD098059 (5 µM) and then for 10 min with or without 400 mM sorbitol
or 0.5 mM arsenite with or without SB203580 or
PD098059, as indicated. A,
phospho-Ser45. Culture extracts were analyzed for
B-crystallin phospho-Ser45 or B-crystallin total (not
shown) by Western blotting, as described under "Materials and
Methods." B, phospho-Ser59. Culture extracts
were analyzed for B-crystallin phospho-Ser59 or total
B-crystallin (not shown) by Western blotting, as described under
"Materials and Methods." The phosphor image of the region of the
blot where B-crystallin migrates is shown at the top of
each panel. The mean intensity of each
phospho-B-crystallin band/total B-crystallin ± S.D. for
each treatment is shown in the bar graph at the
bottom. The data are presented as the mean ± S.D. of
three identically treated cultures. C, ERK and p38. The
abilities of sorbitol and arsenite to increase levels of phospho-p38
were determined as described in the legend to Fig. 3. Phospho-ERK
assays were carried out as described under "Materials and Methods."
*, p < 0.05 different from control as determined by
ANOVA followed by Neuman Keuls post hoc analysis of
variance.
|
|
The Role of p38 in Sorbitol-induced Apoptosis--
Long term
exposure to sorbitol or arsenite leads to apoptosis in most cell types
(43-45). Thus, it is possible that the initial activation of the p38
pathway, and, ultimately, B-crystallin phosphorylation, which occur
within minutes of exposure to these stressors, represent an attempt by
cells to mount a protective response. To test this possibility, we
evaluated the effects of inhibiting the p38 pathway on sorbitol-induced
apoptosis in cardiac myocytes. As measured by DNA laddering, the
initiation of apoptosis in cultured cardiac myocytes required
approximately 5 h of exposure to 400 mM sorbitol (Fig.
6A). Compared with control (0 h of sorbitol), 5 or 8 h of sorbitol increased band intensity in
the DNA ladders by about 2- and 6-fold, respectively (Fig.
6B). Consistent with the hypothesis that MKK6-activated p38
can afford protection from apoptosis was the finding that SB203580
increased DNA band intensity in sorbitol-treated cultures by as much as
2-fold (Fig. 6, A and B).
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Fig. 6.
Effects of p38 on sorbitol-induced
apoptosis. A, cultured cardiac myocytes were treated
with 400 mM sorbitol for the times shown with or without
SB203580. DNA was then extracted and fractionated on an agarose gel,
stained with ethidium bromide, and photographed. L, 100-bp
DNA ladder; 100 bp is the fastest migrating band. B, the
intensity of the 400-bp band (lowest) from each sample shown
in A was assessed using ImageQuant software (Molecular
Dynamics). Each intensity relative to the control (no sorbitol or
SB203580) is shown in the bar graph.
C, triplicate cultures of cardiac myocytes were treated for
5 h with 400 mM sorbitol with or without SB203580 or
PD098059, as shown. DNA fragmentation was assessed as described
for A. D, the intensities of the 400-bp bands
from C were determined and plotted as means ± S.E. *,
p < 0.05 different from control; , p < 0.05 different from sorbitol or sorbitol plus PD098059, as
determined by ANOVA followed by Neuman Keuls post hoc
analysis of variance. E, cultured cardiac myocytes were
infected with or without AdV-Con, AdV-MKK6(Glu), or AdV-p38AGF and,
after 48 h, treated with or without 400 mM sorbitol
for 8 h. DNA fragmentation was assessed as described for
A. F, the intensity of the 400-bp band
(lowest) from each sample shown in E was assessed
using ImageQuant software (Molecular Dynamics). Each intensity relative
to the control (no sorbitol) is shown in the bar
graph. G, duplicate cultures of cardiac myocytes
were infected with or without AdV-Con or AdV-MKK6(Glu) and, after
48 h, treated with or without 400 mM sorbitol for 0, 12, or 24 h, as shown. DNA fragmentation was assessed as described
for A. H, the intensities of the 400-bp bands
from G were determined and plotted as means ± S.D. *,
p < 0.05 different from control (0 h sorbitol;
AdV-Con), as determined by ANOVA followed by Neuman Keuls post
hoc analysis of variance.
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|
In a second experiment, the effect of PD098059 on apoptosis
induced by 5 h of sorbitol treatment was compared with SB203580. As expected, sorbitol-mediated apoptosis was increased by inhibiting p38 with SB203580; however, PD098059 had no effect (Fig. 6,
C and D). Thus, treating cells in a manner known
to block ERK-mediated increases in phosphorylation of B-crystallin
on serine 45 had no effect on apoptosis, while treating cells in a
manner known to block only p38/MAPKAP-K2-mediated phosphorylation of
B-crystallin on serine 59 enhanced apoptosis.
To further demonstrate a protective role for p38 under these
conditions, sorbitol-induced apoptosis was assessed in cells expressing
either MKK6(Glu) or dominant negative p38. As expected, sorbitol
strongly induced DNA laddering in control cells, amounting to nearly
8-fold over control; however, there was no visible laddering in
MKK6(Glu)-expressing cells, even after 8 h of sorbitol treatment (Fig. 6, E and F). In fact, cells expressing
MKK6(Glu) were so refractory to apoptosis that after 24 h of
sorbitol treatment they displayed about 5-6-fold less DNA laddering,
as determined by band intensity, than control cells (Fig. 6,
G and H). As expected, overexpression of a
dominant-negative p38, p38AGF, resulted in no protection from apoptosis
(Fig. 6, E and F).
| |
DISCUSSION |
The reduction of heart function due to cardiac myocyte
apoptosis contributes to the progression of heart failure (47, 48), emphasizing the importance of identifying signaling pathways that regulate myocardial cell apoptosis. In this regard, recent attention has focused on p38 MAPK. In cardiac myocytes, it has been shown that
the activation of p38 by MKK3 leads to apoptosis, while activation
of p38 by MKK6 leads to protection from apoptosis (15, 19). The
mechanism of MKK6/p38-mediated cytoprotection remains largely
unknown. However, one hypothesis is that by activating p38, MKK6
induces the expression of cytoprotective proteins. In support of this
possibility are recent findings from our laboratory showing that
MKK6-activated p38 can stimulate NF
B, leading to the induction of
the gene for the cytokine interleukin-6, which is secreted from and has
cytoprotective effects on myocardial cells (41). This finding suggests
that some other cytokines and/or trophic factors, such as tumor
necrosis factor , cardiotrophin-1, and leukocyte inhibitory factor,
which are likely to be induced upon NF-B activation, also
participate in cytoprotection in response to stress-mediated MKK6 and
p38 activation.
Another possible mechanism by which MKK6 could protect cells from
apoptosis involves the small hsps, such as B-crystallin. The results
of the present study show that in cardiac myocytes, constitutively
active MKK6 can augment B-crystallin gene expression, and it can
induce the site-selective phosphorylation of B-crystallin on serine
59. Moreover, the blockade of B-crystallin gene induction and serine
59 phosphorylation by inhibitors of p38 was shown to augment myocyte
apoptosis. These results indicate that the activation of MKK6, p38, and
MAPKAP-K2 by stress probably represents a cellular response that is
mounted in an effort to provide protection from apoptosis.
The observation that MKK6-mediated increases in B-crystallin gene
expression contribute to cytoprotection is in accord with a previous
report that showed that overexpression of B-crystallin protects
cultured cardiac myocytes from ischemic injury, as measured by
cytosolic enzyme release (49). The mechanism of B-crystallin induction in the heart is unknown. However, since the 5'-flanking sequence of B-crystallin possesses an SRE, transcriptional induction is probably coordinate with that of other stress-activated cardiac genes that possess regulatory SREs, such as ANF. Two
SREs, located at 406 and 134 in the promoter-proximal region of the
ANF 5'-flanking sequence, have been shown to bind
serum response factor and to be required for both basal
ANF expression and ANF
transcriptional induction in response to MKK6-activated p38 (39, 50).
Moreover, it is through the phosphorylation of a recently discovered
serum response factor binding partner, ATF6, that p38 is thought
to increase ANF transcription (50). Thus, it
is feasible that the SRE located at nucleotide 384 in the
B-crystallin 5'-flanking sequence, which, like the SREs in the
ANF gene, binds serum response factor (38), serves a
role in cardiac expression and p38 inducibility of B-crystallin. In
support of this hypothesis is a preliminary experiment where we showed
that truncating 426(BC)/Luc to 164(BC)/Luc resulted in an
approximately 200-fold reduction in MKK6(Glu)-inducible promoter
activity. Therefore, it seems likely that B-crystallin induction
depends on the ability of p38 to phosphorylate ATF6 and may not rely
directly upon p38-mediated activation of MAPKAP-K2.
The precise mechanism by which B-crystallin contributes to
protecting cardiac myocytes against apoptosis is not known; however, there are likely to be many pathways in which B-crystallin
participates. One observation that has captured a great deal of
attention is that during stresses that typically lead to apoptosis in
either isolated hearts or cultured cardiac myocytes, B-crystallin
translocates from a soluble, cytosolic location to the Z-bands of the
sarcomeres (34, 35, 51). It is possible that this translocation fosters sarcomeric stabilization, which provides a degree of resistance to apoptosis. This concept is supported by studies showing that a
collection of proteins, including -actinin, -actin, titin, nebulin, and Nsp11, localize to the Z-band, where they are believed to
be critical for proper sarcomere formation during early
myofibrilogenesis (52, 53). Moreover, the recent finding that a
naturally occurring point mutation in human B-crystallin, R120G, is
the cause of desmin-related myopathy, a disease where patients present
with symptoms similar to cardiomyopathy, supports a role for
B-crystallin in sarcomeric stabilization as well as protection from
apoptosis (54). Interestingly, this point mutation results in a form of B-crystallin that is less stable to heat denaturation and acts as a
poor chaperone and may thus contribute to the cardiomyopathic phenotype
(55).
The nature of the binding of B-crystallin to Z-bands remains to be
elucidated. However, it has been observed that B-crystallin binds
directly to portions of titin that are located near the Z-band (35). It
has also been shown that desmin can bind to B-crystallin in
vitro, prompting some speculation that B-crystallin may bind to
a portion of desmin that resides at or near the Z-bands (56). Another
potential B-crystallin binding partner is nebulette, a
cardiac-specific isoform of nebulin that is localized at the actin-Z-band junction (57).
The molecular mechanism underlying the translocation of B-crystallin
to Z-bands is unknown; however, there is evidence supporting a role for
phosphorylation. For example, the phosphorylation of B-crystallin
following myocardial stress is temporally correlated with its
translocation to the Z-bands (35). In fact, some of the stresses that
lead to this phosphorylation and translocation, such as
ischemia/reperfusion, are well known to activate p38 and MAPKAP-K2 in
the heart (7, 8, 22). These findings support the hypothesis that the
phosphorylation of B-crystallin on serine 59 by p38-activated
MAPKAP-K2 might play an important role in the mechanism by which
B-crystallin translocates from a diffuse cytosolic localization to
Z-bands. Moreover, the finding in the present study that inhibiting the
phosphorylation of B-crystallin on serine 59 leads to the
potentiation of apoptosis in response to stress, coupled with other
B-crystallin localization studies, suggests that it is the movement
of B-crystallin to the Z-bands that participates in protecting the
integrity of myofibrils during the cardiac stress response.
The importance of B-crystallin in the cellular stress response
extends beyond the heart. In addition to the lens, where
B-crystallin was first discovered and where it is believed to
protect from cataract formation (58), B-crystallin is also expressed
in skeletal muscle cells as well as some CNS cells, including
oligodendrocytes, astrocytes, and neurons (58). Interestingly, the same
mutation that results in desmin-related myopathy in humans also causes premature cataracts, which are believed to be the result of the inability of lens cells to respond to stresses, such as UV exposure (54). B-crystallin has also been implicated in several
neurodegenerative diseases. For example, the expression of
B-crystallin in astrocytes is considerably increased in Parkinson's
patients suffering from dementia and in Alzheimer's disease, where it
is believed to contribute to cellular mechanisms of adaptation to the
stress caused by the disease (60). B-crystallin has also been
implicated in multiple sclerosis, where it is thought to serve as the
autoantigen against which antibodies are expressed that lead to
eventual neurodegeneration (37). Thus, although B-crystallin
displays a tissue-restricted expression pattern, it appears to play
potentially important roles in providing protection against potentially
harmful stresses in a variety of cell types.
The quantity of B-crystallin expression in the heart is strikingly
high, amounting to as much as 5% of the total protein in cardiac
myocytes. This level of expression is equal only to the major
sarcomeric proteins that comprise the basic functional units of all
cardiac myocytes. Thus, it is apparent that in comparison with the
sarcomeric proteins, B-crystallin plays an important structural role
in the heart. However, in contrast to the sarcomeric proteins, the
localization of B-crystallin is clearly dynamically regulated in a
manner that allows it to transiently localize to myocyte structures
that require a molecular chaperone to maintain structure and cell
viability. It is probable that B-crystallin localization is
regulated in response to numerous signaling pathways in addition to the
p38/MAPKAP-K2 pathway studied in the present paper. Future studies
focused on determining the precise biochemical mechanisms governing the
localization of B-crystallin to the cellular machinery that is
critical for mounting a protective stress response will reveal new
information about this important molecular chaperone. Moreover, such
studies will provide a better understanding of how to devise molecular
approaches aimed at exploiting the cytoprotective roles of
B-crystallin to treat myocardial pathologies.
| |
ACKNOWLEDGEMENTS |
We thank Lo Vang for expert technical
assistance and Drs. R. J. Davis, J. Han, K. Kato, J. Piatigorsky,
B. Stein, and B. Vogelstein for various reagents used in this study.
| |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants NS/HL-25073, HL-46345, HL-56861, and HL-63975 and by
graduate student fellowships from the SDSU Heart Institute and Isis
Pharmaceuticals, Inc. (Carlsbad CA) (to R. C. and A. L.).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.
These two authors contributed equally to this work.
§
To whom correspondence should be addressed: SDSU Heart Institute
and the Department of Biology, San Diego State University, San Diego,
CA 92182. Tel.: 619-594-2959; Fax: 619-594-6200; E-mail: cglembotski@sunstroke.sdsu.edu.
Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.M003864200
| |
ABBREVIATIONS |
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
MKK, MAPK kinase;
hsp, heat shock
protein;
MAPKAP, MAPK-activated protein kinase;
SRE, serum response
element;
DMEM, Dulbecco's modified Eagle's medium;
bp, base pair(s);
PAGE, polyacrylamide gel electrophoresis;
TBS, Tris-buffered
saline;
CMV, cytomegalovirus;
NRVMC, neonatal rat ventricular myocyte;
AdV, adenovirus;
ANOVA, analysis of variance.
| |
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TOP
ABSTRACT
INTRODUCTION
