p38 MAPK and NF-
B Collaborate to Induce Interleukin-6 Gene
Expression and Release
EVIDENCE FOR A CYTOPROTECTIVE AUTOCRINE SIGNALING PATHWAY IN A
CARDIAC MYOCYTE MODEL SYSTEM*
Rian
Craig
,
Andrea
Larkin,
Amy M.
Mingo,
Donna J.
Thuerauf,
Catherine
Andrews,
Patrick M.
McDonough, 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, December 6, 1999, and in revised form, March 15, 2000
 |
ABSTRACT |
In cardiac myocytes, the stimulation of p38 MAPK
by the MAPKK, MKK6, activates the transcription factor, NF-
B, and
protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to
IB kinase (IKK)-
and stimulated its abilities to phosphorylate IB and to activate NF-B. MKK6(Glu) induced
NF-B-dependent interleukin (IL)-6 transcription and IL-6
release in a p38-dependent manner. IL-6 protected
myocardial cells against apoptosis. Like IL-6, TNF-
, which activates
both NF-B and p38, also induced p38-dependent IL-6
expression and release and protected myocytes from apoptotis. While
TNF- was relatively ineffective, IL-6 activated myocardial cell
STAT3 by about 8-fold, indicating a probable role for this transcription factor in IL-6-mediated protection from apoptosis. TNF--mediated IL-6 induction was inhibited by a kinase-inactive form
of the MAPKKK, TGF- activated protein kinase (Tak1), which is known
to activate p38 and NF-B in other cell types. Thus, by stimulating
both p38 and NF-B, Tak1-activating cytokines, like TNF-, can
induce IL-6 expression and release. Moreover, the myocyte-derived IL-6
may then function in an autocrine and/or paracrine fashion to augment
myocardial cell survival during stresses that activate p38.
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INTRODUCTION |
Cytokines derived from either infiltrating cells, such as
macrophages, or sometimes from cells comprising the tissue itself, play
important roles in the wound healing process (1). Since some injuries,
such as myocardial infarction, are frequently life-threatening, a
better understanding of the roles of cytokines in tissues such as the
heart is critical. Following a myocardial infarction there is an
accumulation of tumor necrosis factor
(TNF),1 interleukin
(IL)-1, and IL-6 at or near the affected region (2-5). Previously,
production of these cytokines in the heart was attributed to
infiltrating macrophages or leukocytes and endothelial cells
(e.g. see Ref. 6). However, recent findings have
demonstrated the expression of all three of these cytokines in cardiac
myocytes following ischemic stress (7-9). Moreover, certain cytokines are thought to promote cardiac tissue recovery after a brief ischemic insult (4, 9-11). Accordingly, there is renewed interest in studying
the possible beneficial effects of cytokines in the heart as well as
elucidating the signal transduction pathways and mechanisms responsible
for their induction.
The induction of most cytokine genes requires activation of the
transcription factor, nuclear factor (NF)-B (12-15). NF-B is
activated and cytokine expression is increased in rat hearts submitted
to ischemic stress; interestingly, these events appear to require the
mitogen-activated protein kinase (MAPK), p38 (16). Additionally, in
cultured cardiac myocytes, NF-B-dependent reporter gene
expression is activated following the selective stimulation of p38 by
its upstream activator, MKK6 (17). Moreover, this p38-dependent NF-B activation contributes to protecting
cultured myocardial cells from undergoing apoptosis (17). However,
while p38 and NF-B are both important for the maintenance of heart function following stress, neither the mechanism by which p38 activates
NF-B nor the role of p38-activated NF-B in myocardial cell
survival are well understood. Accordingly, the present study was
undertaken to begin addressing these questions.
Many signaling pathways can interact with each other through
biochemical cross-talk; however, such interactions between p38 and
NF-B are not well understood. Like the other MAPK family members,
p38 is part of a cascade of kinases. One of the best studied activators
of p38 is the MAPKK, MKK6, which lies directly upstream of p38; among
ERK, JNK, and p38, MKK6 activates only p38 (18, 20). Although MKK6
itself can be activated by several upstream kinases, a particularly
interesting finding is that in some cells, MKK6 can be activated by the
MAPKKK, TGF--activated protein kinase (Tak1) (18, 20). Tak1 was
originally named for its ability to be activated by TGF-. However,
Tak1 can also be activated by cytokines, such as interleukin-1 or
TNF- (20-22) (see Fig. 7 for reference).
In comparison with the p38 pathway, the NF-B pathway is activated
through a series of events that are also mediated by a cascade of
kinases, many of which are believed to be unique to that pathway.
NF-B, which is comprised of two subunits (p65 and p50), is retained
in the cytoplasm by virtue of its interaction with the inhibitor of
B (IB). IB is phosphorylated in response to NF-B-activating
signals; this phosphorylation leads to the ubiquitination and
subsequent degradation of IB. This then allows NF-B to
translocate to the nucleus, where it binds to critical elements in
cytokine genes and increases their transcription (23, 24). The kinases
responsible for the phosphorylation of IB belong to the IB kinase
family, or the IKKs (25-29). NF-B-inducing kinase phosphorylates
and activates IKK (26, 30, 31). Although it preferentially activates
IKK, NF-B-inducing kinase, which bears strong sequence homology to,
and is now considered a member of, the MAPKKK family, can also activate
the JNK MAPK pathway (32). Interestingly, in addition to being
activated by NF-B-inducing kinase, IKK can also be activated by
MEKK1 (27), a MAPKKK that is also known to activate JNK in some cell
types (33) and by Tak1 (refer to Fig. 5). Thus, at least at the MAPKKK
level, there exists the potential for cross-talk between the NF-B
and MAPK pathways.
In the present study, we examined the nature of the cross-talk between
the NF-B and p38 pathways. Because of the clear importance of these
pathways in promoting the recovery, or maintenance of, heart tissue
function following stress or injury, we used cardiac myocytes as the
model system. To this end, we explored whether NF-B and p38
collaboratively activate the IL-6 gene in cardiac myocytes and,
further, whether IL-6 itself has roles that are consistent with
functional recovery of the tissue following stress. We have found the
following: 1) agonists, such as TNF-, can activate IL-6
transcription in cardiac myocytes in a manner that requires both
NF-B and p38; 2) the coordinate activation of the NF-B and p38
pathways by TNF- takes place largely through the MAPKKK, Tak1; 3)
the p38 pathway can influence NF-B activation, at least partly,
through the physical association of MKK6 and IKK; and 4) IL-6 can
protect cardiac myocytes from undergoing apoptosis induced by
sphingosine, a signaling lipid known to increase in the heart following
ischemic stress.
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MATERIALS AND METHODS |
Cell Culture
Primary ventricular myocytes were prepared from 1-4 day old
Harlan Sprague-Dawley rats as described (34, 35). 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 were
plated in DMEM/F-12 (1:1) containing 10% fetal bovine serum for
approximately 16 h. After washing with DMEM/F-12 (1:1), the
cultures were incubated in DMEM/F-12 (1:1) with or without any test
agents for the indicated times.
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 combinations of
plasmids indicated in the figure legends in a total volume of 300 µl
of DMEM/F-12 (1:1). The total amount of plasmids used for each
electroporation was equalized using pCMV6. Dose-response experiments
were carried out to determine optimal quantities of plasmids for
transfection and to verify that the results obtained were consistent
over a range of plasmid levels. Generally, the dose-response
experiments led to using the following quantities of each plasmid type
in each electroporation: 1 µg each of pCMV6 (control) or
p382; 15 µg of MKK6(Glu), Tak1, or Tab1; 20 µg each
of luciferase and -galactosidase reporters; 45 µg of IKK-M,
MKK6-M1, MKK6-M2, and Tak1-M. 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 (34, 36). 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.
Plasmids
MKK6(Glu), MKK6-M1, and MKK6-M2--
pcDNA3 FLAG-MKK6 (Glu)
and pcDNA3 FLAG-MKK6(K82A), the latter of which we call MKK6-M1,
code for activated and kinase-inactive human MKK6, respectively (19)
and were obtained from R. J. Davis (University of Massachusetts,
Worcester, MA). In some cases, instead of MKK6-M1 we used MKK6-M2
(FLAG-MKK6(K82A; S207A; T211A)), which is a kinase-inactive form of
MKK6 that also has the sites normally phosphorylated by an upstream
MAPKKK, Ser207 and Thr211, mutated to Ala.
MKK6-M2 produced similar inhibitory effects on the p38 pathway as
MKK6-M1.
p38--
Sr3 HA-p38-2, which codes for wild type human
p38-2, was obtained from B. Stein (37) (Signal Pharmaceuticals, Inc.,
San Diego, CA). p38-2 is distinct from p38, -
, and -
; however, it is identical to human p382 (38). In the present work,
we have adopted the p382 nomenclature.
IKK and IKK-M--
pRK5 C-FLAG-IKK and pRK5
C-FLAG-IKK (K44A), which code for wild type human IKK and
kinase-dead human IKK, were obtained from M. Rothe (26) (Tularik,
San Francisco, CA).
NF-B-luc--
p2X NF-B, which codes for a luciferase
reporter driven by a minimal prolactin promoter with two nearby,
upstream NF-B consensus sites, was obtained from M. Karin (39)
(University of California at San Diego, La Jolla, CA).
IL-6-luc, IL-6 (NF-B mut)-luc, and
NF-B/IL-6-luc--
p1168hu.IL6-luc and pmut1168hu.IL6-luc, which
code for 1168 nt of the wild type or NF-B-mutated human IL-6
promoter driving luciferase in pGL3, respectively (15), were obtained
from G. Haegeman (University of Gent, Belgium). NF-B/IL-6-luc was
prepared by ligating three IL-6 NF-B elements upstream of the
noninducible IL-6 minimal promoter, which is composed of 50 nt of IL-6
5'-flanking sequence. This construct was also obtained from G. Haegeman.
Tak1, Tab1, and Tak1-M--
pFLAG-Tak1 and pFLAG-Tak1-M encode
human, full-length wild type Tak1 and human full-length Tak1 K63W,
respectively. pHA-Tab1 encodes human, full-length Tab1 (22). All Tak1
and Tab1 constructs used in this study were obtained from T. Sugita
(Tanabe Seiyaku Co., Ltd., Osaka, Japan).
Preparation of Recombinant Adenovirus
AdV-p38 and AdV-MKK6(Glu)--
The AdEasy system was used for
preparing recombinant adenoviral strains using previously described
methods (40). Briefly, human MKK6(Glu) and human p382
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 (GFP), and a CMV-flanked multiple
cloning site for the insertion of the gene of interest. Polymerase
chain reaction-amplified p382 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/p382 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-p382 and pAdTrack-CMV-MKK6(Glu).
pAdTrack-CMV-p382 or pAdTrack-CMV-MKK6(Glu) was
linearized and then co-transformed with the adenoviral vector,
pAdEasy-1, into Escherichia coli strain BM5183. 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 GFP fluorescence, as described previously (40).
The recombinant viruses were then harvested 7-10 days postinfection.
Viral titers were determined by observing GFP 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
dithiothreitol. 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 stopping each reaction by adding 100 µl of 1 M Na2CO3, the 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) was added to 100 µl of
cell lysate. Light emission of each sample was measured by a BioOrbit 1251 luminometer for 30 s. The relative luciferase activities were
determined by dividing the relative luciferase activity by the relative
-galactosidase activity.
Cytokine ELISAs
IL-6--
To measure IL-6 secretion, IL-6 ELISAs were carried
out using a kit according to the manufacturer's protocol
(BIOSOURCE International). After the myocardial
cells were preplated as described (17) to eliminate noncardiac
myocytes, the myocytes were plated at a density of 1300 cells/mm2 in 10% fetal calf serum for 24 h. Cells
were washed and cultured in media with or without recombinant TNF-
(Genzyme), with or without 5 µM SB203580, or the cells
were infected with AdV-MKK6(Glu), AdV-p382, or
AdV-Control. Following 48 h of incubation, media were collected
and assayed using the IL-6 ELISA kit.
Apoptosis
TUNEL--
Myocardial cells were cultured with or without
recombinant IL-6 (1 ng/ml) (BIOSOURCE
International) supplemented DMEM/F-12 with or without 5 µM SB203580 for 48 h prior to the addition of 10 µM sphingosine (Calbiochem) for 6 h. TUNEL analyses
of fragmented DNA were performed on cultured cardiac myocytes as
described previously (17) and according to the manufacturer's protocol
(Roche Molecular Biochemicals). Cells were scored for TUNEL-positive
nuclei in a researcher-blinded manner.
DNA Laddering--
Assessment of apoptosis was performed by DNA
laddering essentially as described previously (17). Approximately
106 cardiac myocytes were lysed in a digestion buffer
containing Proteinase K, scraped, pipetted several times, and incubated
at 55 °C for 5-14 h. Samples were then extracted with
phenol/chloroform/isoamyl alcohol, and then DNA was isopropyl
alcohol-precipitated and washed several times with ethanol and allowed
to air-dry. After dissolving the DNA and incubating with RNase A, DNA
fragments were fractionated on an agarose gel.
Immunocytofluorescence
p65 Immunocytofluorescence--
To study the cellular
localization of the p65 subunit of NF-B, myocardial cells were
co-transfected with test expression constructs (1 µg each of pCMV6,
p382 or 15 µg of MKK6(Glu)) and 5 µg of a plasmid
encoding GFP. Following 48 h of culture in minimal media, cells
were fixed as described (35), and immunocytofluorescence was carried
out using a polyclonal antibody raised against p65/NF-B (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) followed by Texas Red-conjugated
anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR). Transfected
cardiac myocytes were identified as GFP-positive cells, and p65
localization was visualized in only the transfected cells.
Kinase Assays
MAPKAP-K2 Assay--
Myocardial cells were treated with or
without TNF- (1 ng/ml) for 10 min and then 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 orthovanadate, 0.1%
2-mercaptoethanol, 0.1 mM PMSF, 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
submitted to PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) analysis.
IKK Assay--
IKK kinase activity was assessed in
myocardial cells by co-transfecting C-FLAG-IKK with or without test
expression constructs with or without SB203580 (5 µM).
After the appropriate times, cultures were extracted in a 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
orthovanadate, 10 µg/ml aprotinin, 2 mM DTT, 1 mM PMSF, 1 mM p-nitrophenyl
phosphate, and 10 µg/ml of leupeptin. After brief centrifugation,
extracts were incubated for 2 h at 4 °C with anti-IKK
antibody (Santa Cruz Biotechnology), followed by protein G-Sepharose
(Amersham Pharmacia Biotech) precipitation. Immunocomplex kinase assays
were carried out using 1 µg of recombinant IB--(1-317) (Santa
Cruz Biotechnology) per sample and 10 µM [-32P]ATP (5000 Ci/mmol) in a final volume of 30 µl
of kinase buffer (30 mM HEPES, pH 7.4, 10 mM
MgCl2, 1 mM DTT) at 25 °C for 15 min. The
reactions were terminated by the addition of Laemmli sample buffer, and
the phosphorylation level of IB- was evaluated by SDS-PAGE
followed by autoradiography and PhosphorImager analyses. The quantity
of IKK in each sample was determined by Western analysis; the
observed levels were used to normalize relative IB-
phosphorylation levels found in PhosphorImager analyses.
Western Analysis
p38, JNK, and ERK--
Cultures (approximately 2 × 106 myocytes) were lysed in 100 µl of supplemented
Laemmli sample buffer supplemented with 0.1 mM sodium
orthovanadate, 10 µg/ml aprotinin, 2 mM DTT, 1 mM PMSF, 1 mM p-nitrophenyl
phosphate, and 10 µg/ml leupeptin; boiled for 5 min; and then
submitted to 10% 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. Western analyses were then
performed using 1:1000 dilutions of antisera specific for phospho-p38
(New England Biolabs, Beverly, MA; catalog no. 9211S), phospho-JNK
(Santa Cruz Biotechnology; catalog no. SC6254), or phospho-ERK (New
England Biolabs; catalog no. 9101S). 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 dilutions of antisera specific for
either p38 (Stressgen Biotechnologies Corp., Victoria, Canada; catalog
no. KAP-MAOO9E), JNK (Santa Cruz Biotechnology; catalog no. SC-474), or
ERK (Santa Cruz Biotechnology; catalog no. SC-093) for normalization purposes.
STAT3 and P-STAT3--
Myocytes were cultured as described above
and then treated with or without various test agents for 15 min. The
cells from 3-35-mm culture wells (4.5 × 106
myocytes) were collected by scraping into 450 µl of lysis buffer, which consisted of 20 mM Tris-HCl (pH 7.6), 20 µM sodium glycerophosphate, 250 mM NaCl, 3 mM EGTA, 3 mM EDTA, 0.5% Nonidet P-40, 0.25 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 50 mM NaF, and 2 mM
Na3VO4. Extracts were centrifuged for 15 s at 14,000 rpm in a microcentrifuge, and the supernatants were precleared by incubating with protein A-Sepharose beads for 30-60 min
at 4 °C. Extracts were then incubated for 12-18 h at 4 °C with
anti-STAT3 antibody (New England Biolabs; kit no. 9130) at 1:100.
Immunoprecipitates were collected on protein A-Sepharose beads, eluted
with Laemmli buffer, and analyzed by SDS-PAGE followed by blotting on
nitrocellular paper, as described above. Blots were then probed with
either anti-STAT3 antibody or with anti-Y-705-STAT3 antibody and washed
according to the manufacturer's protocol (New England Biolabs, Inc.,
Beverly, MA). Upon visualizing using chemiluminescence, blots were
digitized using a PhosphorImager, and the bands representing Y-705-STAT3 and total STAT3 were quantitated using ImageQuant software
(Molecular Dynamics).
IKK/MKK6 Association--
Myocytes were cultured for 72 h in DMEM/F-12 containing 1% fetal bovine serum. 9 × 106 myocytes were lysed in 500 ml of extraction 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
orthovanadate, 10 µg/ml aprotinin, 2 mM DTT, 1 mM PMSF, 1 mM p-nitrophenyl
phosphate, and 10 µg/ml leupeptin. The cell debris was removed by
brief centrifugation. Lysates were incubated at 4 °C with
anti-IKK polyclonal antibody (Santa Cruz Biotechnology) or with
anti-MEK-6 polyclonal antibody (Stressgen) at a dilution of 1:250 for
2 h. Thirty µl of a protein G-Sepharose slurry was then added to
each sample and incubated for 30 min at 4 °C. Following
centrifugation, Laemmli buffer was added to the pellet, which was then
boiled for 5 min. Proteins were resolved by a 12% SDS-PAGE and
transferred to a nitrocellulose membrane in methanol transfer buffer at
100 V for 2 h. Blots were blocked for 30 min in 5% nonfat milk
dissolved in TBS-Tween (0.01%) at room temperature and then probed
with anti-FLAG M2 monoclonal antibody (Sigma) at a dilution of 1:300 in
5% nonfat milk for 2 h at room temperature. After washing for
1 h in TBS-Tween (0.01%), the membrane was incubated in 1:2000
dilution of goat anti-mouse IgG horseradish peroxidase (Jackson
ImmunoResearch Laboratories). Visualization of immune complex was
carried out by an enhanced chemiluminescence (ECL) method using ECL
Western blotting detection reagents (NEN Life Science Products)
according to the manufacturer's instructions.
| |
RESULTS |
MKK6 and p38 Induce NF-B Translocation and
Transcription--
Previous studies have demonstrated that
constitutively active MKK6 (MKK6(Glu)) stimulates myocardial cell p38
but not ERK or JNK MAPK (35, 41). MKK6(Glu) was also shown to protect cardiac myocytes from apoptosis (17). Since NF-B is known to protect
several other cell types from apoptosis (e.g. see Ref. 12),
we explored whether p38 could activate NF-B in primary cardiac
myocytes. Accordingly, myocardial cell cultures were transfected with
constructs that code for the expression of wild type
p3822 and/or
MKK6(Glu). NF-B activation was estimated qualitatively by
visualizing the extent of nuclear translocation of one of the NF-B
subunits, p65/NF-B, by immunocytofluorescence and quantitatively by
examining the activation of NF-B-dependent transcription
of a co-transfected reporter gene. In cells transfected with a control plasmid, p65/NF-B cross-reactivity was distributed in a punctate pattern mostly in the cytoplasm of cardiac myocytes (Fig.
1A).3
In cells transfected with a plasmid encoding wild type
p382 only, there was a small increase in cross-reactive
p65/NF-B in the nucleus (Fig. 1B), compared with control
cells. However, in cells transfected with a plasmid encoding MKK6(Glu),
there were clear increases in nuclear p65/NF-B (Fig. 1C),
which was even more pronounced upon co-transfection with plasmids
encoding p382 and MKK6(Glu) (Fig. 1D).
Consistent with the nuclear accumulation of p65/NF-B were the
abilities of overexpressed p382 and/or MKK6(Glu) to
increase luciferase expression from an NF-B-dependent luciferase reporter plasmid (Fig. 1E). Overexpression of
either p382 or MKK6(Glu) resulted in approximately 2- and 3-fold induction of NF-B/luciferase, respectively. However,
co-expression of p382 and MKK6(Glu) conferred a robust,
16-fold induction of reporter expression. The p38-specific
inhibitor,4 SB203580,
diminished this induction by about 70%. Thus, upon overexpression of
wild type p382 and/or MKK6(Glu), the extent of
NFB-dependent luciferase induction correlated well with
the nuclear accumulation of p65/NF-B. These results are consistent with the hypothesis that in cardiac myocytes, activation of the p38
pathway can lead to the nuclear translocation of NF-B and to the
induction of NF-B-sensitive genes.
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Fig. 1.
Effects of p38 and MKK6(Glu) on
NF-B translocation and
NF-B-mediated transcription.
A-D, myocardial cells were transfected with 1 µg of
plasmids encoding either no protein (pCMV6 (Control; 1 µg))3 or p382 (1 µg) and/or MKK6(Glu)
(15 µg), as shown. Cultures were plated onto glass slides and
incubated for 48 h in serum-free media, after which they were
fixed and stained for p65/NF-B and visualized using a Texas
Red-conjugated second antibody. Shown are single cells that are
representative of the population of cells observed following the
treatments indicated. E, myocardial cells were
co-transfected with p2X NF-B (20 µg)3 and pCH110
(-gal; 20 µg) with or without plasmids encoding
p382 (1 µg) and with MKK6(Glu) (15 µg) and treated
with or without SB203580 (5 µM), as indicated. After
48 h in serum-free medium, culture extracts were assayed for
luciferase and -galactosidase activities. Relative luciferase
(Rel Luc, luciferase/-gal) was normalized to the maximum
relative luciferase value in each experiment and then displayed as the
percentage of that value. Each bar represents the mean of
the results obtained from three identically treated cultures with or
without S.E. Each experiment was replicated at least three times; the
results shown are from one representative experiment.
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MKK6 and p38 Induce NF-B-dependent IL-6
Transcription--
Since p38 is activated during the stress response
in many cells types and since NF-B is known to facilitate cytokine
gene induction during cell stress, we explored the hypothesis that, together, p38 and NF-B could mediate cytokine induction in cardiac myocytes. Accordingly, myocardial cells were transfected with a
construct that possesses 1186 nt of the IL-6 gene-regulatory region and
promoter-driving luciferase, wild type IL-6 (IL-6 (wt)) (Fig. 2A). Overexpressing wild
type p382 or MKK6(Glu) resulted in approximately 2- or
3-fold reporter induction, respectively (Fig. 2B). However,
co-expression of p382 and MKK6(Glu) together conferred
an approximately 7-fold induction of reporter expression (Fig.
2B). These results indicate that the IL-6 promoter is
transcriptionally active in cardiac
myocytes5 and that myocardial
cell IL-6 transcription can be stimulated by p382 and
MKK6. IL-6 transcriptional induction in response to p382
and/or MKK6(Glu) was decreased by 70-100% upon the addition of
SB203580 (Fig. 2B). Notably, IL-6 transcriptional induction was completely dependent upon NF-B, since a point mutation that abolishes the only NF-B binding site in the IL-6 gene
(IL-6-M in Fig. 2A; Ref. 15) resulted in a nearly
complete loss of p382- and/or MKK6(Glu)-inducible IL-6
reporter activity. Interestingly, the abilities of p38 and/or MKK6(Glu)
to induce the IL-6 promoter were also inhibited by overexpression of a
kinase-inactive, dominant-interfering form of IKK, IKK-M (Fig.
2B). Taken together, these results indicate that IL-6
transcriptional induction in cardiac myocytes depends on the activation
of NF-B and is increased upon stimulation of the p38 pathway in a
manner that is dependent upon IKK.
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Fig. 2.
Effects of p38, MKK6(Glu),
IKKM, Tak1, Tak1-M, and
TNF- on IL-6 promoter activity. A,
reporter diagram. The three IL-6 reporter constructs used in this study
are diagrammed. Wild type IL-6 (IL-6 (wt)) is composed of
1168 nt of the native human IL-6 5'-flanking sequence-driving
luciferase; IL-6-M is composed of the same 1168 nt of the human IL-6
5'-flanking sequence except that the sole promoter-proximal NF-B
binding site was mutated so that it no longer binds NF-B;
NF-B/IL-6 is comprised of a concatamer of three IL-6 NF-B sites
ligated directly to 50 nt of the human IL-6 5'-flanking sequence, a
noninducible, minimal promoter-driving luciferase. Each of these IL-6
promoter/reporter constructs is described in more detail under
"Materials and Methods" (see Ref. 15). B-E, IL-6
promoter induction. Myocardial cells were co-transfected with 20 µg3 of either wild type IL-6, IL-6-M (B) or
with NF-B/IL-6 (C-E) and pCH110 (-galactosidase).
Some cultures were also co-transfected with combinations of plasmids
that encode the following proteins: p382 (1 µg),
MKK6(Glu) (15 µg), IKK-M (45 µg), MKK6-M (45 µg), Tak1 (15 µg), Tab1 (15 µg), and Tak1-M (45 µg), as indicated in the
figure (see "Materials and Methods" for plasmid
details). Cultures were treated with or without SB203580 (5 µM), as indicated. After 48 h, culture extracts were
assayed for luciferase and -galactosidase. Relative luciferase
values are expressed as percentage of the maximum, as described in the
legend to Fig. 1. Each bar represents the mean of three
cultures ± S.E. Each experiment was replicated at least three
times; the results shown are from one representative experiment.
F, assessment of transgene expression. Duplicate myocardial
cell cultures were transfected as described above with plasmids
encoding the proteins shown. Cultures were then extracted, submitted to
immunoprecipitation with either anti-FLAG (MKK6, IKK-, IKK--M,
Tak, and Tak-M) or with anti-HA (p38) antisera, and then Western
blotting to assess the approximate levels of expression of each of the
test proteins. As can be seen, each of the transgenes is expressed at
approximately the same level.
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TNF- stimulates p38 in other cell types (42, 43). Consistent with
those results, we found that in cardiac myocytes, among the MAPKs,
TNF- preferentially stimulated p38 by about 6-fold, compared with
approximately 2-fold for either JNK or ERK (Fig. 3, A and B).
Accordingly, we evaluated whether TNF- could augment NF-B-dependent transcription and, if so, whether this
enhancement was sensitive to the inhibition of p38. For these
experiments, we employed a reporter gene composed of the minimal IL-6
promoter flanked only by NF-B binding sites
(NF-B/IL-6 in Fig. 2A). Indeed, TNF- conferred a robust, 10-fold induction of
NF-B-dependent IL-6 transcription in cardiac myocytes
(Fig. 2C), which was diminished by about 30-40% by
SB203580 or by a dominant-negative, kinase-inactive MKK6 (MKK6-M1),
indicating a partial requirement for p38. TNF--mediated induction of
IL-6 transcription was not inhibited by treating cells with 5 µM of the MEK inhibitor, PD098059, or by co-transfecting cells with a dominant-negative form of MEK (not shown), indicating that
ERK is probably not involved. Transfection with IKK-M resulted in an
approximately 40-50% reduction in TNF--mediated induction of IL-6
transcription, and SB203580 and IKK-M together completely blocked
the effects of TNF-. The effectiveness of SB203580 as an inhibitor
of p38 was assessed by assaying MAPKAP-K2, a kinase that lies
downstream of p38. When cultures were treated with 5 µM,
SB203580, there was a complete blockade of the ability of TNF- to
stimulated MAPKAP-K2, supporting the contention that at this
concentration, SB203580 is an effective inhibitor of myocardial cell
p38 (Fig. 3C). These results are consistent with a signaling process whereby TNF--activated p38 and NF-B constitute
the major pathways responsible for TNF--mediated
NF-B-dependent IL-6 transcriptional induction.
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Fig. 3.
Effects of TNF- on
the MAPKs and MAPKAP-K2. A and B, MAPK
activity. Triplicate cultures were treated with or without TNF- (1 ng/ml) for 5 min and then extracted and submitted to Western analyses
to determine P-p38, P-JNK, P-ERK, total p38, total JNK, or total ERK,
as described under "Materials and Methods." The phosphor image from
each triplicate culture set is shown in A, and the results
of the densitometric analysis of each image, carried out using
Molecular Dynamics ImageQuant software, are shown in B. Each
bar represents the mean of three cultures ± S.E. C,
MAPKAP-K2 activity. Triplicate cultures were treated with or without
TNF- (1 ng/ml), with or without SB203580 (5 µM) for 10 min and then extracted, and the activity of MAPKAP-K2 was determined as
described under "Materials and Methods." The phosphor image from
each triplicate culture set is shown at the top of
C, and the results of the densitometric analysis of each
image, carried out using Molecular Dynamics ImageQuant software, are
shown at the bottom of C. Each bar
represents the mean of three cultures ± S.E.
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Recently, it has been shown that in HeLa cells, TNF- stimulates
TGF--activated protein kinase 1 (Tak1), a newly discovered, multifunctional MAPKKK capable of binding to and activating IKK and
MKK6 (Fig. 7; Ref. 22). To determine whether this MAPKKK can mediate
NF-B- and p38-dependent IL-6 induction in myocardial cells, Tak1 and its required activator protein, Tak1-binding protein-1 (Tab1) (44), were both expressed in cardiac myocytes, along with the
NF-B/IL-6 luciferase reporter. This combination of Tak1 and Tab1
resulted in an approximate 5-fold activation of
NF-B-dependent IL-6 reporter gene
expression,6 which was
inhibited by about 50-60% by SB203580, by kinase-inactive MKK6
(MKK6-M2), or by kinase-inactive IKK (IKK-M) (Fig.
2D). Together, SB203580 and IKK-M completely blocked
Tak1-stimulated NF-B-dependent IL-6 reporter gene
expression. These results are consistent with the capacity of Tak1 to
stimulate both the IKK/NF-B and the MKK6/p38 pathways in cardiac
myocytes, the latter of which we have
observed.7 Moreover, in
conjunction with results shown in Fig. 2C, these findings
indicate that the p38 pathway is required for optimal TNF-- and
Tak1-mediated NF-B activation in cardiac myocytes.
To evaluate whether TNF--mediated IL-6 induction involves the
endogenous myocardial cell Tak1, which is present in neonatal rat heart
(45), a kinase-inactive Tak-1 mutant (Tak1-M) was expressed. Tak1-M
strongly inhibited TNF--induced IL-6 transcription by about 70%
(Fig. 2E). As expected from the results in Fig.
2C, the p38 inhibitor, either SB203580 or MKK6-M1, blocked
TNF--inducible IL-6 transcription by about 50%. Further, SB203580
and the Tak1-M together nearly completely inhibited the effects of
TNF- (Fig. 2E). These results indicate that in cardiac
myocytes, TNF- signals through Tak1 to activate IL-6 transcription
in a partially p38-dependent manner. This is consistent
with the ability of Tak1 to activate MKK6 and p38.
MKK6 and p38 MAPK Activate IKK--
Since MKK6 and p38 can
activate NF-B and, thus, lead to the induction of IL-6, a cytokine
with potentially important functions in the heart, we carried out
experiments to begin elucidating the mechanism of cross-talk between
the p38 and NF-B pathways. Initially, the effects of
p382 and/or MKK6(Glu) on the activity of IKK in
cultured myocardial cells were assessed. While expression of wild type
p382 alone had little effect on the kinase activity of
IKK, expression of MKK6(Glu) or MKK6(Glu) together with
p382 increased IKK kinase activity by about 2- and
5-fold, respectively (Fig.
4A). The IKK activation
mediated by p382 and MKK6(Glu) was decreased by about
50% upon the addition of SB203580, indicating a requirement for
p38.
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Fig. 4.
Interaction of IKK
and MKK6. A, IKK kinase activity. Duplicate
myocardial cultures were transfected with IKK and the combinations
of plasmids encoding p382 and/or MKK6(Glu) with or
without SB203580, as shown. After 72 h, extracts were submitted to
immunoprecipitation using a IKK-specific antibody. IKK kinase
assays were then carried out using IB- as the substrate (see
"Materials and Methods"). The phosphorylation of IB- was
assessed by SDS-PAGE followed by autoradiography. The relative levels
of IB- phosphorylation following each treatment were assessed
using Molecular Dynamics ImageQuant software. Shown above
each autoradiogram is the -fold stimulation normalized to total IKK
protein levels as determined by subsequent Western analysis of each
kinase assay. B, IKK MKK6 association. Myocardial cells
were transfected with plasmids encoding IKK and/or MKK6(Glu). After
72 h, culture extracts were submitted to immunoprecipitation using
IKK- or MKK6-specific antibody. Immunoprecipitates were then
submitted to SDS-PAGE and electroelution onto a nitrocellulose
membrane. Western blotting was then carried out using a FLAG-specific
antibody. This experiment was repeated at least three times; the
results shown are from one representative experiment.
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MKK6 Associates with IKK--
To pursue possible mechanisms by
which MKK6(Glu) and p382 can influence IKK activity,
experiments were carried out to evaluate whether p382
and/or MKK6 can interact directly with IKK. Myocardial cells were
transfected with constructs encoding FLAG-IKK and FLAG-MKK6(Glu)
and/or FLAG-p38 and the abilities of these proteins to interact with
each other were assessed using immunoprecipitation followed by Western
blotting. While transfection of FLAG-IKK together with
FLAG-p382 did not result in any apparent complex formation (not shown), co-expression of FLAG-IKK and FLAG-MKK6 did
result in complex formation. Immunoprecipitating with either IKK- or
MKK6-specific antisera resulted in the pull-down of both IKK and
MKK6 (Fig. 4B), consistent with the abilities of the two
proteins to interact in vivo. Taken together, the results of
these kinase and association experiments indicate that MKK6 can
directly interact with IKK and that this interaction may position
p38 in the NF-B pathway, resulting in an enhancement in the activity
of IKK.
MKK6 and p38 MAPK Induce IL-6 Release from Cardiac
Myocytes--
To evaluate whether IL-6 promoter induction correlates
with increased expression of IL-6 protein itself, cardiac myocytes were
treated with various doses of TNF-, and the quantity of IL-6
released into the culture medium was assessed by ELISA. TNF- increased the quantity of IL-6 cross-reactive material in the medium in
a dose-dependent manner by up to about 3-fold (Fig. 5A). The quantity of IL-6 in
the medium was reduced significantly when SB203580 was added,
consistent with a role for p38 in TNF--simulated IL-6 release from
myocardial cells.
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Fig. 5.
Effect of TNF-,
p382, and
MKK6(Glu) on IL-6 cytokine secretion. A, TNF-
induction of IL-6 secretion. Myocardial cells were incubated with
various levels of recombinant rat TNF- with or without SB203580, as
shown. After 24 h, the levels of IL-6 in medium samples were determined by ELISA. B, characterization of
adenoviral MKK6(Glu). Myocardial cells that had been in culture for 24 h in serum-free medium were infected with AdV-MKK6(Glu) (see
"Materials and Methods"); this virus strain expresses both GFP and
MKK6(Glu) under the control of separate CMV promoters (see "Materials
and Methods" and Ref. 40). After 48 h, cultures were visualized under
low magnification (×20) phase contrast (Phase-Low), low
magnification green fluorescence (GFP-Low) (note that the
reference arrow in Phase-Low and GFP-Low
panels points to the same cell in each field), high magnification
(×100) green fluorescence (GFP-High), or high magnification
red fluorescence (Phalloidin-High). C, effects of
adenoviral MKK6(Glu) on IL-6 secretion. Myocardial cells were infected
with AdV-(Con), AdV-p382, and/or AdV-MKK6(Glu) such that
100% of the cells were transfected, and they were treated with or
without SB203580, as shown and as described for B and under
"Materials and Methods." After 48 h, medium samples were assayed
for the presence of IL-6 by ELISA. Each value represents the mean of
three identically cultures ± S.E.
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To further determine the role of the p38 pathway in myocardial cell
IL-6 induction and release, cardiac myocytes were treated with
recombinant adenoviral strains harboring the genes for
p382 or MKK6(Glu). The levels of virus were adjusted so
that all of the myocytes expressed the transgenes. Shown in Fig.
5B is an example using AdV-MKK6(Glu), a strain of adenovirus
that possesses sequences encoding both MKK6(Glu) and GFP under the
control of separate CMV promoters. Cultured myocytes were infected with
virus and then photographed under phase and fluorescence microscopy 48 h later (see "Materials and Methods"). Upon inspection of
low power micrographs by phase and fluorescence microscopy, it was apparent that essentially all of the cells visible in the phase field
expressed GFP (compare Phase-Low with GFP-Low in
Fig. 5B). Upon inspection of higher power micrographs, it
was apparent that AdV-MKK6(Glu)-infected myocytes displayed the visual
characteristics typical of hypertrophic cells, such as enlarged cell
area and extensive sarcomeric organization (Fig. 5B,
GFP-High and Phalloidin-High). This is consistent
with previous studies of the effects of MKK6(Glu) on cardiac myocyte
morphology (35, 41). Cells infected with AdV-MKK6(Glu) and
AdV-p382 display similar morphology (not shown).
Infecting myocytes with AdV-p382 alone did not
significantly increase medium levels of IL-6 compared with control
(Fig. 5C), consistent with the relatively weak activation of
IL-6 transcription afforded by p38 alone (see Fig. 2A).
However, infecting myocytes with AdV-MKK6(Glu) alone or with both
AdV-p382 and AdV-MKK6(Glu) together increased the
release of IL-6 by about 2- and 8-fold, respectively (Fig.
5C), levels that are very similar to their abilities to
induce IL-6 transcription (see Fig. 2A). Moreover, MKK6(Glu)- and p38/MKK6(Glu)-dependent IL-6 release were
both effectively inhibited by SB203580 (Fig. 5C), indicating
that the selective activation of the p38 pathway confers significant
IL-6 induction and release from cultured cardiac myocytes.
IL-6 Protects Cardiac Myocytes from Apoptosis--
To explore
whether IL-6 might exert functions consistent with myocardial recovery
following stress, the effect of IL-6 on myocardial cell apoptosis was
determined. When cardiac myocytes were treated for a limited time with
sphingosine, a lipid second messenger known to mediate apoptosis in
many cell types (46-48), there was an approximately 3-fold increase in
the number of apoptotic cells, as determined using TUNEL analysis (17).
Strikingly, IL-6 exerted potent, dose-dependent,
antiapoptotic effects, affording complete protection from
sphingosine-induced apoptosis at doses above 0.1 ng/ml (Fig.
6A). TNF- also conferred
protection against sphingosine-induced apoptosis, again with nearly
maximal effects being observed at 0.1 ng/ml cytokine. These results
were confirmed using DNA laddering, where the fragmentation of DNA to
create discrete increments of approximately 180 base pairs is
diagnostic of apoptosis. Treating cells with spingosine resulted in an
approximate 6-fold increase in the intensity of the DNA bands observed,
and incubation with either IL-6 or TNF- at 1 ng/ml resulted in
complete protection from apoptosis, as determined by DNA laddering
(Fig. 6, B and C). The mechanisms by which
TNF- and Il-6 confer protection from apoptosis remain to be
determined. However, several recent studies have suggested that certain
cytokines that signal through the gp130 receptor can activate the Janus
kinase/STAT and protect against apoptosis in cardiac myocytes (49,
50). Moreover, one recent study has shown that, although it does not
couple through gp130, TNF- can activate STAT3 in 3T3-L1 adipocytes
(51). Accordingly, we tested the abilities of TNF- and IL-6 to
activate myocardial cell STAT3 by evaluating the level of Janus
kinase-mediated phosphorylation of Y-705 on immunoprecipitated STAT3.
Treatment with TNF- resulted in a small, 2-fold increase in STAT3
activation, while treatment with IL-6 induced a robust 8.2-fold
increase in STAT3 activation (Fig. 6D). Thus, while it is
conceivable that TNF- could induce STAT3-sensitive genes involved in
protection from apoptosis, it seems more likely that TNF- exerts its
cardioprotective effects in this cultured cell model via the activation
of NF-B and the subsequent induction of cytokines, such as IL-6,
which may signal to cardiac myocytes in an autocrine manner.
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Fig. 6.
Effects of TNF- and
IL-6 on apoptosis and STAT3 activation. A, TUNEL
analyses. Myocardial cells were plated onto glass slides and cultured
for 48 h in serum-free medium with or without IL-6 (1 ng/ml) or
TNF- (1 ng/ml), as indicated. Cultures were then treated with or
without sphingosine (10 µM), as indicated, and after
4 h they were fixed for further TUNEL analysis. The number of
TUNEL-positive cells observed in each field was determined as described
under "Mate- rials and Methods" (see Ref. 17) and then normalized to the
total number of cells in each field on each slide. Each value
represents the mean of 10 separate fields ± S.E. B,
ladder analyses. Myocardial cells were treated for 24 h with media
lacking cytokine (lanes 2-4), with TNF- (1 ng/ml)
(lanes 6 and 7), or with IL-6 (1 ng/ml) (lanes 8 and 9). Sphingosine
(10 µM) was then added to some cultures (lanes
4-9), and after 4 h they were assessed for apoptosis
by DNA ladder analysis, as described under "Materials and Methods."
Standard DNA ladders consisting of fragments composed of 100-base pair
increments are shown in lanes 1 and
10. The 400-base pair standard is shown. C,
ladder quantitation. Densitometric analysis of the 360-base pair bands
from E was carried out, and the average ± S.D. of the
results of each treatment are shown relative to the control (no
cytokine, no sphingosine), which was set at 1.0. D, STAT3
phosphorylation. Duplicate cultures were treated for 15 min with or
without 10 ng/ml of each test compound shown, and then extracts were
assessed for the phosphorylation of STAT3 on Y-705 (P-STAT3) or for
total STAT3 levels as described under "Materials and Methods." The
controls are extracts of A431 cells that have been treated with (+) or
without ( ) epidermal growth factor, which is known to induce STAT3
phosphorylation on Y-705. The -fold increase in STAT3 phosphorylation
was determined by densitometry followed by normalizing each P-STAT3
value to the total STAT3 in that sample. Values shown are mean ± S.D.; n = 2 cultures/treatment. This blot is
representative of three similar experiments.
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DISCUSSION |
The way the heart responds to stress is critical for maintaining
proper cardiac function. One of the stress responses that has attracted
recent attention is the release of cardiac-derived cytokines. Although
it is widely believed that these cytokines serve important functions in
the heart, the precise nature of those functions remains unclear. Our
findings indicate that the myocardial IL-6 system serves an important
autocrine signaling role that contributes to myocyte survival following
certain cardiac stresses. Moreover, it appears that the optimal
induction of IL-6 in cardiac myocytes involves both the NF-B and p38
pathways. Our results indicate further that a potential point of
collaboration between these signaling pathways resides in the ability
of MKK6 to augment the activity of IKK in a
p38-dependent manner (Fig. 7).
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Fig. 7.
Diagram of
IKK/NF-B and the
MKK6/p38 pathways relevant to this study. Shown is a simplified
diagram depicting the signaling pathways under study in the present
report. Tak1 is a MAPKKK that requires a co-activator, Tab, for optimal
activity. IKK and MKK6 are MAPKK family members that can be
activated by Tak1. The shaded arrows indicate
possible cross-talk between the p38 and the NF-B pathways that is
supported by the present study.
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The possibility that p38 plays a role in NF-B activation has been
explored in cardiac myocytes (8, 17) and in other cell types
(e.g. see Ref. 42); however, the mechanism of interaction between the two pathways has not been fully resolved and has been addressed in only a few studies. To address whether p38/NF-B cross-talk could occur at the transcriptional level, Vanden Berghe et al. (15) used a Gal4-based one-hybrid assay and presented data indicating that p38 increased the transcriptional activation potential p65/NF-B. In contrast to that study, in preliminary experiments, we were unable to demonstrate such
trans-activation of p65/NF-B in cardiac
myocytes.8 Moreover, while
phosphorylation of p65/NF-B augments its ability to bind to DNA (52)
and is apparently required for its transcriptional activity (53),
recent data indicate that p38 itself does not phosphorylate p65/NF-B
(54). To our knowledge, the results in the present study constitute the
first demonstration that p38/NF-B cross-talk may exist at the level
of MKK6 and IKK.
Although the mechanism by which MKK6 augments IKK kinase activity is
yet to be determined, our results showing that MKK6 and IKK can
physically interact provide some clues. For example, it is formally
possible that MKK6, which has never been shown to phosphorylate any
protein other than p38 (19, 38), might phosphorylate IKK and, in so
doing, stimulate its kinase activity. However, we were unable to
observe any change in the phosphorylation status of IKK upon
incubation with MKK6(Glu) in vitro (not shown). Alternatively, in addition to activating p38, MKK6 might serve to
position p38 near IKK and other members of the NF-B signaling pathway. This positioning might then lead to the observed
p38-dependent increases in NF-B activity. Interestingly,
we have been unable to observe the binding of p38 itself to IKK,
which further supports an anchoring role for MKK6. IKK and MKK6 are
both members of the MAPKK family, and IKK is known to associate with
other MAPKKs in the NF-B signalsome (28, 29). Thus, while it has not
previously been shown that MKK6 can bind to other MAPKKs, it seems
probable that IKK could form dimers with MKK6 via the same binding
domains involved in the formation of IKK homodimers. Future studies
directed toward mapping such association domains in the IKKs, MKK6, and other MAPKKs will be required to address this provocative yet very
feasible hypothesis.
To the best of our knowledge, the present study is also the first to
demonstrate that a functional consequence of p38-mediated NF-B
activation by MKK6(Glu) is the induction of the IL-6 gene and release
of IL-6 from cardiac myocytes. Consistent with roles for both pathways
in IL-6 induction were our findings that either TNF- or Tak1, both
of which activate p38 and NF-B in other cell types (22) and in
cardiac myocytes,6 induced the IL-6 promoter. In agreement
with results showing that the activation of p38 and NF-B by
MKK6(Glu) leads to protection from apoptosis in cardiac myocytes (17)
was our finding that IL-6 is antiapoptotic.
Although many studies have been carried out using IL-6-related
cytokines, clear physiological functions for IL-6 in the stressed heart
remain elusive. For example, the expression of IL-6 in the heart has
been associated with acute ischemia and cardiac failure, findings that
have been interpreted as indicative of either a productive or a
deleterious role for the cytokine (2-5). Interestingly, in addition to
the findings reported in this study, there is considerable evidence,
mostly in noncardiac myocytes, that supports a cytoprotective and even
growth-promoting role for IL-6. For example, IL-6 has been shown to
protect hepatocytes and myeloma cells from apoptosis (55, 56).
Additionally, ET-1, which is a well known myocardial cell growth factor
(57), is believed to mediate some of its cardiac growth-promoting
effects through IL-6 (58). Interestingly, IL-6 was found to improve
survival in an animal model of viral myocarditis, in part by reducing
myocardial cell death (59). Moreover, the induction of IL-6 upon
myocardial ischemia (6, 8) and the finding that p38 can activate
NF-B during cardiac ischemia (16) suggest that upon such stresses
the heart responds by producing cytokines that may serve important
roles as barriers against apoptosis.
Consistent with possible cytoprotective roles for cytokines in the
heart was our finding that IL-6 afforded significant protection from
apoptosis. The mechanism by which IL-6 might confer such protection
remains to be elucidated. However, IL-6 belongs to the superfamily of
trophic factors that includes cardiotrophin (CT-1), ciliary
neurotrophic factor, leukemia inhibitory factor, and oncostatin M (60),
most of which have been shown to activate ERK, STAT3, and Akt (61, 62)
and foster the growth and survival of cardiac myocytes, neurons, and
other cells ty
TOP
ABSTRACT
INTRODUCTION
