|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 33, 23013-23019, August 13, 1999
From the Institute of Molecular Medicine and the
The multifunctional cytokine interleukin-6 (IL-6)
regulates growth and differentiation of many cell types and induces
production of acute-phase proteins in hepatocytes. Here we report that
IL-6 protects hepatoma cells from apoptosis induced by transforming growth factor- A balance between cell proliferation and apoptosis is critical for
tissue homeostasis. Maintenance of the size of liver is a notable
example of homeostasis which is heavily regulated by many growth
factors and cytokines (1). Among these factors, transforming growth
factor- TGF- Interleukin-6 (IL-6) is a mutilfunctional cytokine acting in the immune
system, hepatocytes, and neuronal cells (23, 24). In the liver, IL-6
induces synthesis of acute-phase proteins and plays central roles in
preventing acute hepatitis and initiating liver regeneration (25, 26).
Mice with targeted disruption of the IL-6 gene have impaired liver
regeneration characterized by liver necrosis and failure (26). The
signaling mechanism of IL-6 in hepatocytes is, however, not fully
understood. In hematopoietic cells, binding IL-6 to the In the liver, IL-6 acts as a hepatoprotecting and/or mitogenic factor,
whereas TGF- Cell Culture and Transfection--
Human hepatoma cell line
Hep3B was cultured as described previously (11). Hep3B cells stably
expressing the dominant-negative mutant of Akt were reported previously
(13). Transfection was performed using LipofectAMINE reagent (Life
Technologies) according to the manufacturer's instructions. For
transient transfection, cells were harvested at 48 h after
transfection. For selecting of stable clones, G418 (700 µg/ml) was
added into culture medium at 48 h after transfection. The
G418-resistant clones were individually picked, expanded, and assayed
for expression of the transfected cDNAs by Western blotting.
Antibodies and Reagents--
Mammalian expression plasmids for
the two dominant-negative mutants of STAT3, STAT3D and STAT3F (35),
were kindly provided by Dr. T. Hirano. Antibodies for p85 subunit of
the PI 3-kinase, Akt, and phosphotyrosine were purchased from Santa
Cruz Biotechnology. The anti-STAT3 antibody was from UBI. Wortmannin
and LY294002, the two PI 3-kinase inhibitors, were from Sigma.
Apoptosis Assays--
Apoptosis was quantitated with TUNEL and
Cell-Death Detection ELISA assays. For TUNEL assay (36), cells seeded
on chamber slides were serum starved for 24 h and then treated
with or without various cytokines and/or agents for 18 h. Cells
were fixed with 4% paraformaldehyde, and permeabilized with 0.1%
Triton X-100 and 0.1% sodium citrate. TUNEL assays were performed
using the In Situ Death Detection Kit, Fluorescein (Roche
Molecular Biochemicals) according to the manufacturer's instructions.
Apoptotic cells were visualized with fluorescence microscopy. The
Cell-Death Detection ELISA assay (Roche Molecular Biochemicals)
measures the presence of soluble histone-DNA complex as a result of DNA
fragmentation (37). For this assay, cells were seeded on 96-well plates
at a density of 0.5 × 104 cells/well and were
serum-starved and treated with various agents as for TUNEL assays.
Cell-Death ELISA assays were performed according to the manufacturer's instructions.
Caspase-3 Activity Assays--
Cells were seeded onto 6-well
plates, serum starved for 24 h, and treated with or without
TGF- Luciferase Assays--
Reporter plasmids p15P113-luc (38) and
p800luc (39), kindly provided by Drs. X.-F. Wang and R. Derynck,
contain the luciferase expression unit driven by the TGF- PI 3-Kinase Assay--
PI 3-kinase activities were assayed as
described previously (40). Briefly, 107 cells were washed
twice with ice-cold phosphate-buffered saline and lysed with 1 ml of
lysis buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM CaCl2,
1% Nonidet P-40, 10% glycerol, 1 mg/ml bovine serum albumin, 20 mM Tris, pH 8.0, 2 mM orthovanadate). Cell
extracts were incubated with 1 µg of anti-phosphotyrosine antibody
overnight at 4 °C. The immunocomplex was precipitated with 50 µl
of protein A-Sepharose for 1 h at 4 °C, washed three times with
lysis buffer, twice with LiCl buffer (0.5 M LiCl, 100 mM Tris, pH 7.6) and twice with TNE buffer (10 mM Tris, pH 7.6, 100 mM NaCl, 1 mM
EDTA). The immunocomplex was preincubated with 10 µl of 20 mM Hepes (pH 7.4), containing 2 mg/ml PI (Sigma) on ice for
10 min. Kinase reaction was performed by the addition of 40 µl of
reaction buffer (10 µCi of [ In Vitro Akt Kinase Assay--
Akt kinase assays were performed
essentially as described (41). Briefly, cells were lysed in lysis
buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM
sodium fluoride, 1 mM NaVO4, 1 mM
sodium pyrophosphate, 2 µM aprotinin, 2 µM
leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The
HA-tagged Akt was precipitated from cell lysates with anti-HA antibody
followed by protein A-Sepharose conjugated with rabbit anti-mouse
antibody. The immunocomplexes were washed once with Akt kinase buffer
containing 20 mM Hepes (pH 7.4), 1 mM
dithiothreitol, 10 mM MnCl2, 10 mM
MgCl2 and then incubated in 30 µl of kinase buffer
containing 5 µM ATP, 100 µg of histone 2B, and 10 µCi
of [ IL-6 Protects Hepatoma Cells from TGF- IL-6 Does Not Affect the Ability of TGF- IL-6 Signaling Prevents TGF- PI 3-Kinase Is Involved in the Antiapoptotic Signaling of
IL-6--
Our previous studies indicated not only that the PI
3-kinase/Akt pathway mediates the anti-apoptotic signal of insulin
against TGF- Akt Is Activated following IL-6 Stimulation and Is Involved in the
Antiapoptotic Signaling of IL-6--
Having demonstrated the
involvement of PI 3-kinase in the anti-apoptotic signaling of IL-6, we
next investigated whether the serine/threonine kinase Akt, a downstream
target of PI 3-kinase, is a critical component of this anti-apoptotic
signaling pathway. Attempts to evaluate the activity of endogenous Akt
were hindered by the lack of an antibody that could efficiently
immunoprecipitate Akt from the lysates of Hep3B cells. Therefore, an
HA-tagged Akt (19) was transiently transfected into Hep3B cells. The
transfected cells were treated with IL-6 for various time intervals and
cell lysates were used for immunoprecipitations with an anti-HA
antibody followed by in vitro kinase assays. As shown in
Fig. 5A, IL-6 substantially
increased Akt activity, and the kinetics of Akt activation resembled
that of the IL-6-induced tyrosine phosphorylation of p85 (Fig.
4A). Furthermore, both LY294002 and wortmannin efficiently suppressed the IL-6-induced Akt activity, indicating that the activation of Akt by IL-6 is mediated through PI 3-kinase. To assess
the role of Akt in the anti-apoptotic activity of IL-6 against TGF- STAT3 Elicits a Weak Anti-apoptotic Effect against TGF- PI 3-Kinase/Akt and STAT3 Pathways Act Cooperatively--
As
described earlier, both PI 3-kinase/Akt and STAT3 pathways were
involved in the IL-6-induced anti-apoptosis. We therefore determined
exactly how this anti-apoptosis could be affected by blocking both
pathways. For this purpose, parental Hep3B and the STAT3D transfectants
were treated with or without TGF- IL-6 is a pleiotropic cytokine capable not only of inducing growth
and differentiation in many cell types but also of stimulating acute-phase protein synthesis in liver cells (23, 24). However, the
anti-apoptotic effect of IL-6 has not been well documented. In this
study, we demonstrated that IL-6 suppressed TGF- This study provides several insights into the anti-apoptotic mechanism
of IL-6. The PI 3-kinase/Akt and the JAK/STAT3 pathways are both
activated by IL-6 and function cooperatively to achieve the maximal
anti-apoptotic effect of IL-6 against TGF- IL-6 was reported to protect multiple myeloma plasma cells from
anti-Fas- and dexamethasone-induced apoptosis (49). Inhibition of
JNK/SAPK pathway is involved in IL-6-induced protection against anti-Fas but not dexamethasone. These results support a hypothesis that
multiple mechanisms are involved in the IL-6-induced anti-apoptosis. However, blockage of JNK/SAPK pathway is unlikely to account for the
mechanism by which IL-6 suppresses TGF- Previous studies demonstrated that dominant-negative inhibition of
STAT3 activity completely abolishes gp130-mediated survival signal in a
pro-B cell line (33). In the case of Hep3B cells, however, the same
dominant-negative mutants only partially blocked the anti-apoptotic
effect of IL-6 against TGF- A recent investigation indicated that IL-6 treatment of prostate cancer
cell line LNCaP induces an increase in tyrosine phosphorylation of the
p85 subunit of PI 3-kinase and its kinase activity (34). Accordingly,
we observed a rapid induction of p85 tyrosine phosphorylation and PI
3-kinase activity by IL-6 in Hep3B cells. In addition, two specific
inhibitors of PI 3-kinase, wortmannin and LY294002, blocked the
anti-apoptotic effect of IL-6, implying the activation of PI 3-kinase
by IL-6. Furthermore, we demonstrated for the first time that the
serine/threonine kinase Akt is activated upon IL-6 treatment. A similar
induction of PI 3-kinase and Akt activities was found in cardiac
myocytes stimulated with leukemia inhibitory factor, a cytokine
transducing signal via gp130 (51). Thus, in addition to JAK/STAT and
Ras/MAP kinase pathways, the PI 3-kinase/Akt could be an important
signaling pathway activated by various cytokines. The mechanism of
IL-6-induced activation of PI 3-kinase remains unclear, although JAK
can bind PI 3-kinase upon activation of gp130 (51).
In the liver, IL-6 plays a crucial role in anti-inflammatory responses
to prevent liver injury (25, 52) and is a key growth factor to initiate
liver regeneration (1, 26). This study demonstrates another important
activity of IL-6 in the liver, namely, anti-apoptosis. We propose that
the ability of IL-6 to suppress apoptosis induced by TGF- We thank Dr. H.-F. Yang-Yen for helpful
instructions on PI 3-kinase assay, Drs. R. Derynck and X.-F. Wang for
providing reporter constructs, Dr. T. Hirano for the dominant-negative
STAT3, and Rachel L. Chuang for excellent technical assistance. We also
acknowledge Dr. C.-H. Wu for critical reading of the manuscript.
*
This work was supported by grants from the National Health
Research Institute, Department of Health (DOH87-HR-729) and National Science Council (NSC88-2312-B002-050) (to R.-H. C.) and National Science Council Grant NSC88-2314-B002-094 (to M.-L. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Laboratory of
Molecular and Cellular Toxicology, Institute of Toxicology,
College of Medicine, National Taiwan University, Taipei, Taiwan. Tel.: 886-2-23970800 (ext. 8607); Fax: 886-2-23410217; E-mail:
toxkml@ha.mc.ntu.edu.tw.
The abbreviations used are:
TGF-
Interleukin-6 Inhibits Transforming Growth Factor-
-induced
Apoptosis through the Phosphatidylinositol 3-Kinase/Akt and Signal
Transducers and Activators of Transcription 3 Pathways*
,§
Institute of Toxicology, College of Medicine, National
Taiwan University, Taipei, Taiwan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-), a well known apoptotic inducer in liver cells. Addition of IL-6 blocked TGF--induced activation of caspase-3 while showing no effect on the induction of plasminogen activator inhibitor-1 and p15INK4B genes, indicating that IL-6 interferes
with only a subset of TGF- activities. To further elucidate the
mechanism of this anti-apoptotic effect of IL-6, we investigated which
signaling pathway transduced by IL-6 is responsible for this effect.
IL-6 stimulation of hepatoma cells induced a rapid tyrosine
phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI
3-kinase) and its kinase activity followed by the activation of Akt.
Inhibition of PI 3-kinase by wortmannin or LY294002 abolished the
protection of IL-6 against TGF--induced apoptosis. A
dominant-negative Akt also abrogated this anti-apoptotic effect.
Dominant-negative inhibition of STAT3, however, only weakly attenuated
the IL-6-induced protection. Finally, inhibition of both STAT3 and PI
3-kinase by treating cells overexpressing the dominant-negative STAT3
with LY294002 completely blocked IL-6-induced survival signal. Thus,
concomitant activation of the PI 3-kinase/Akt and the STAT3 pathways
mediates the anti-apoptotic effect of IL-6 against TGF-, with the
former likely playing a major role in this anti-apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-)1 is a potent inducer of
apoptosis in hepatocytes and several
hepatoma cell lines, as well as in regressing liver in vivo
(2-6). TGF- also inhibits liver cell proliferation in
vitro (7) and plays a crucial role in terminating liver
regeneration after partial hepatectomy (1). TGF- exerts its
biological effects through the action of two types of transmembrane
serine/threonine kinase receptors. These receptors subsequently
propagate the signal by phosphorylating the intracellular targets,
Smads. Phosphorylated Smad2 or Smad3 can form a stable complex with
Smad4, which then translocates to the nucleus to regulate
transcriptional responses to TGF- (8-10). Although the signal
transduction pathway of TGF- has been well studied, mechanism of its
apoptotic effect is still not fully characterized. Nevertheless,
induction of oxidative stress (6), activation of caspase-3 (3, 11), and
inhibition of pRb expression (2) have been implicated in mediating
TGF--induced apoptosis.
-induced apoptosis in liver cells is blocked by growth factors
such as insulin and insulin-like growth factor-1 as well as by elevated
expression of insulin receptor substrate-1 (12). Our recent studies
have revealed that the phosphatidylinositol 3-kinase (PI 3-kinase) and
its downstream target, Akt, are responsible for the anti-apoptotic
activity of insulin against TGF- (13). PI 3-kinase was reported to
suppress apoptotic cell death induced by a variety of stimuli (14-19).
PI 3-kinase elicits this anti-apoptotic activity through the action of
the serine/threonine kinase, Akt. Recent studies have demonstrated that
activated Akt can phosphorylate the proapoptotic protein BAD (20, 21).
This phosphorylation allows for BAD association with 14-3-3 and
dissociation from BCL-XL, which is then free to resume its
function as a suppressor of apoptosis (22).
subunit of
its receptor triggers the recruitment of gp130, subsequently leading to
the activation of the gp130-associated Janus kinases (JAKs) (27-29).
JAKs phosphorylate gp130 on several tyrosine residues and these
phosphotyrosines recruit various SH2 domain-containing proteins, such
as STAT3 and SHP-2 (30-32). SHP-2 links cytokine receptor to the
Ras/MAP kinase pathway and is essential for mitogenic activity, whereas STAT3 can induce BCL-2 and is involved in anti-apoptosis (33). In
addition to JAK/STAT and Ras/MAP kinase pathways, IL-6 was recently
shown to activate PI 3-kinase in prostate cancer cells (34). Whether
IL-6 in liver cells activates these pathways remains to be investigated.
elicits an apoptotic and/or growth-arresting effect. In
this study, we elucidate a cross-talk between signaling pathways
induced by these two factors in liver cells. Our results indicate that
IL-6 suppressed TGF--induced apoptotic death of hepatoma cells
in a dose-dependent manner. IL-6 inhibited TGF--induced activation of caspase-3 but did not affect its induction of an extracellular matrix protein and a cell cycle inhibitor, suggesting that IL-6 signaling blocks only the apoptotic signaling of TGF-. Furthermore, we demonstrate that the concomitant activation of the PI
3-kinase/Akt and the STAT3 signaling pathways mediates the
anti-apoptotic effect of IL-6 against TGF-.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(5 ng/ml) and/or IL-6 (60 ng/ml) for various times. Cells were
lysed in RIPA buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, and 10 µg/ml aprotinin and cell lysates were centrifuged
at 14,000 rpm. Cell lysate containing 100 µg of proteins in a 1-ml
assay buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 1 mM EGTA was incubated with a
fluorogenic peptide substrate, Ac-DEVD-AMC (5.6 µM) at
37 °C for 30 min. Fluorescence was quantified with a
spectrofluorometer (excitation 380 nm, emission 460 nm).
-responsive
elements in p15INK4B and plasminogen activator inhibitor-1
(PAI-1) promoters, respectively. These plasmids, together with a
plasmid for constitutive expression of -galactosidase, were
transfected to Hep3B. One day after transfection, the cells were
serum-starved for 10 h and then treated with TGF- (5 ng/ml)
and/or IL-6 (60 ng/ml) for 12 h. Luciferase and -galactosidase activities were quantitated by the Luciferase Assay System and the
-galactosidase Enzyme Assay System (Promega), respectively.
-32P] ATP, 20 mM Hepes, pH 7.4, 20 µM ATP, 5 mM
MgCl2) at room temperature for 15 min. The reaction was
stopped by the addition of 100 µl of 1 M HCl and
extracted with 200 µl of a 1:1 mixture of chloroform and methanol.
The radiolabeled lipids were separated by thin-layer chromatography and
visualized using a PhosphorImager.
-32P]ATP at 30 °C for 30 min. The kinase
reaction was terminated by addition of an equal volume of 2 times
SDS-PAGE sample buffer. The samples were boiled for 5 min prior to
electrophoretic separation by SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced
Apoptosis--
Previous studies revealed that human hepatoma cell line
Hep3B is highly sensitive to the apoptotic activity of TGF- (4). To
investigate whether IL-6 affects apoptosis induced by TGF-, Hep3B
cells were co-treated with 5 ng/ml TGF- and IL-6 at various concentrations. Using an ELISA assay measuring the presence of soluble
histone-DNA complex resulted from DNA fragmentation, we observed that
IL-6 suppressed TGF--induced apoptosis in a
dose-dependent manner (Fig.
1, A and B). The
anti-apoptotic effect of IL-6 was readily detected at a dose as low as
0.1 ng/ml (Fig. 1A). At a concentration of 60 ng/ml, IL-6
elicited a maximal protection. Notably, in the absence of TGF-, IL-6
did not promote nor suppress cell survival (Fig. 1B). These
findings suggest the existence of a cross-talk between the signaling
pathways transduced by TGF- and IL-6.
View larger version (16K):
[in a new window]
Fig. 1.
IL-6 suppresses
TGF- -induced apoptosis in hepatoma cells.
Hep3B cells were treated with TGF- (5 ng/ml) and IL-6 at low
(A) or high concentrations (B) as indicated.
Apoptotic cells at 18 h after treatment were determined by the
Cell-Death Detection ELISA. Data from three independent experiments are
presented as mean ± S.D.
to Induce PAI-1 and
p15INK4B Genes--
To explore the mechanism by which IL-6
signaling prevents TGF--induced apoptosis, we investigated whether
IL-6 could block all cellular responses to TGF- or affect only a
subset of the activities. The PAI-1 and p15INK4B genes are
highly induced by TGF- and are frequently used as indicators for the
effects of TGF- on production of extracellular matrix and cell cycle
arrest, respectively (42, 43). The reporter plasmid p800luc, containing
the TGF- responsive element in PAI-1 promoter (39), was used to
evaluate the induction of PAI-1 gene. When Hep3B cells were transiently
transfected with this reporter, TGF- induced a ~3.5-fold increase
in luciferase activity. This induction was not significantly affected
by co-treatment with IL-6 at 60 ng/ml (Fig.
2A), i.e. a dosage
that showed a maximal inhibition of TGF--induced apoptosis (Fig.
1B). Similar results were observed using the p15P113-luc
reporter (38) containing the p15INK4B promoter (Fig.
2B). Our data thus indicate that IL-6, although preventing
apoptosis induced by TGF-, did not affect the transcriptional up-regulation of PAI-1 and p15INK4B genes in response to
TGF-. IL-6 signaling is likely to block a step specific to the
apoptotic activity of TGF-.
View larger version (16K):
[in a new window]
Fig. 2.
IL-6 does not affect
TGF- -induced activation of PAI-1 and
p15INK4B promoters. Hep3B cells transiently transfected
with the reporter plasmid were treated with or without TGF- (5 ng/ml) and/or IL-6 (60 ng/ml) for 12 h. The reporter plasmid
p800luc containing the PAI-1 promoter was used in A, whereas
the p15P113-luc reporter was used in B. For each
transfection, luciferase activity was normalized to transfection
efficiency and cell survival by using -galactosidase activity as an
internal control. Values are mean ± S.D. of three independent
transfections.
-induced Activation of
Caspase-3--
Our previous studies demonstrated that caspase-3 is
activated upon TGF- treatment of Hep3B cells (13). To evaluate
whether IL-6 could block this activity of TGF-, cell extract derived from Hep3B treated with or without TGF- and/or IL-6 was analyzed for
caspase-3 activity by measuring cleavage of a fluorogenic substrate,
Ac-DEVD-AMC. As shown in Fig. 3,
TGF--induced caspase activity was abolished by co-treatment of IL-6.
This finding suggests that IL-6 signaling prevents the apoptotic
activity of TGF- by inhibiting the activation of caspase-3.
View larger version (19K):
[in a new window]
Fig. 3.
IL-6 inhibits
TGF- -induced caspase-3-like activity.
Caspase-3 activities in Hep3B cells treated with various agents were
measured by the amount of fluorescence generated from the cleavage of
Ac-DEVD-AMC.
but also that this signal leads to a blockage of
caspase-3 activation in response to TGF- (13). Therefore, we sought
to investigate whether IL-6 induces PI 3-kinase in Hep3B cells and whether the anti-apoptotic activity of IL-6 is mediated through the
function of PI 3-kinase. We observed that IL-6 treatment of Hep3B cells
stimulated tyrosine phosphorylation of the regulatory subunit of PI
3-kinase, p85, as determined by immunoprecipitations with an
anti-phosphotyrosine antibody followed by Western blotting using an
anti-p85 antibody (Fig. 4A).
Furthermore, IL-6 induced a substantial increase of PI 3-kinase
activity in immunoprecipitates with the anti-phosphotyrosine antibody
(Fig. 4B). Both tyrosine phosphorylation of the p85 and PI
3-kinase activity were readily evident within 5 min and declined at 60 min after exposure to IL-6, indicating a rapid activation of PI
3-kinase by IL-6. To determine the involvement of PI 3-kinase in the
anti-apoptotic effect of IL-6, two specific inhibitors of PI 3-kinase,
wortmannin and LY294002 (44, 45), were used. As shown in Fig.
4C, wortmannin markedly diminished the anti-apoptotic
activity of IL-6 at a dose of 10 nM. A similar
finding was observed with 25 µM LY294002. However, these
two inhibitors did not by themselves induce apoptosis in the absence of
TGF-. Taken together, our results demonstrated a critical role of PI
3-kinase in the anti-apoptotic signaling of IL-6 against TGF-.
View larger version (24K):
[in a new window]
Fig. 4.
PI 3-kinase is involved in the anti-apoptotic
effect of IL-6 against TGF- .
A, induction of tyrosine phosphorylation of p85 subunit of
the PI 3-kinase by IL-6. Hep3B cells were treated with IL-6 (60 ng/ml)
for various time intervals as indicated. Upper panel, cell
lysates with equal amount of proteins were immunoprecipitated with an
anti-phosphotyrosine (PY) antibody. The immunoprecipitates
were separated on SDS-PAGE and subjected to Western blot with antibody
against p85. Lower panel, cell lysates with equal amounts of
proteins were subjected to Western blot with the anti-p85 antibody to
verify the same amount of p85 in all samples. B, activation
of PI 3-kinase activity by IL-6. Cells were treated as indicated and
lysates with equal amounts of protein were subjected to
immunoprecipitations with anti-phosphotyrosine antibody. The
immunocomplex was used for PI 3-kinase assays. C, wortmannin
and LY294002, two specific inhibitors of PI 3-kinase, block the
protection of IL-6 against TGF--induced apoptosis. Hep3B cells were
treated with various agents as indicated. The concentrations of
wortmannin and LY294002 used were 10 nM and 25 µM, respectively. Apoptotic cells were stained with the
TUNEL assay and identified under a fluorescent microscope. Data are
presented as means of three experiments.
,
we evaluated whether a dominant-negative mutant of Akt can block
IL-6-induced protection. Our previous study generated Hep3B cells
stably expressing a kinase-defective mutant of Akt (Akt 179A) (13),
which can inhibit wild type Akt in a dominant-negative fashion (19).
Akt(K
)-8, a stable transfectant of Hep3B expressing a
high level of such mutated Akt, exhibited a markedly reduced protection
of IL-6 against TGF- (Fig. 5B). A similar but less
prominent reduction of IL-6-induced anti-apoptosis was observed using a
mixture of eight stable clones, Akt(K)-M (13), expressing
a lower level of the mutated Akt (Fig. 5B). Taken together,
our findings indicate that the PI 3-kinase/Akt pathway is activated by
IL-6 and mediates the anti-apoptotic effect of IL-6.
View larger version (28K):
[in a new window]
Fig. 5.
Akt, a downstream effector of PI 3-kinase,
mediates the survival signal transduced by IL-6. A,
IL-6 stimulates Akt activity. Upper panel, Hep3B cells
transiently transfected with an HA-tagged Akt were treated with 60 ng/ml IL-6 in the presence or absence of 10 nM wortmannin
(W) or 25 µM LY294002 (LY) for
various time intervals as indicated. Cell lysates were subjected to
immunoprecipitations with an anti-HA antibody followed by in
vitro kinase assays using histone 2B as a substrate. Lower
panel, the same immunoprecipitates were used for Western blotting
with an anti-Akt antibody to verify equal amount of proteins were
loaded. Akt kinase activity in each lane was quantitated and normalized
to the equal amount of proteins and was shown under the upper
panel. B, dominant-negative Akt inhibits the anti-apoptotic effect
of IL-6. Parental Hep3B and stable transfectants expressing the
dominant-negative Akt, Akt(K ), were treated with various
agents as indicated and apoptosis was assayed as for Fig.
4C.
--
In
hematopoietic cells, STAT3 mediates the survival signal of cytokines
through its ability to induce BCL-2 (33). To investigate whether STAT3
is involved in suppressing TGF--induced apoptosis by IL-6, a STAT3
dominant-negative mutant (STAT3D) carrying EE to AA substitutions at
positions 434 and 435 (35) was introduced to Hep3B cells. Seven stable
transfectants were generated and Fig.
6A displayed the expression
levels of the endogenous, wild type STAT3 and the mutant proteins in
these cells. A high expressor (STAT3D-1) and a mixture of all seven
clones (STAT3D-M) were analyzed in terms of their responsiveness to
IL-6-induced anti-apoptosis. As shown in Fig. 6B, IL-6
induced a slightly reduced protection against TGF--induced apoptosis
in both STAT3D-1 and STAT3D-M cells, comparing to the parental Hep3B
cells. This reduction was consistently observed in three independent
experiments (Table I). In addition, this
reduced protection was also observed from Hep3B cells stably expressing
a second STAT3 dominant-negative mutant, STAT3F (35) (data not shown).
Thus, our results suggest that STAT3 is involved in the anti-apoptotic
signaling of IL-6.
View larger version (30K):
[in a new window]
Fig. 6.
The anti-apoptotic activity of IL-6 can be
partially inhibited by a dominant-negative mutant of STAT3.
A, expression of the dominant-negative STAT3 (STAT3D) in 7 stable transfectants assayed by Western blot using an anti-STAT3
antibody. An equal amount of proteins was loaded in each lane, and
therefore the first lane represented the level of endogenous, wild type
STAT3. B, expression of STAT3D attenuates the survival
signal transduced by IL-6. STAT3D-M is a mixture of all stable
transfectants shown in A. Cells were treated with various
agents as indicated and apoptosis was assayed by the Cell-Death
Detection ELISA.
Apoptotic death of Hep3B and STAT3D stable transfectants induced by
TGF- and/or IL-6
, IL-6, and/or LY294002. As shown
in Fig. 7, the anti-apoptotic activity of
IL-6 against TGF- was completely abolished in the STAT3D
transfectants treated with LY294002. This complete inhibition of
IL-6-induced anti-apoptosis was not observed in parental Hep3B
cells treated with LY294002. In three independent experiments (Table
II), we consistently observed a more
complete abrogation of the anti-apoptotic activity of IL-6 in STAT3D
transfectants than in parental Hep3B cells. These findings allow us to
infer that PI 3-kinase/Akt and STAT3 pathways act cooperatively to
mediate the anti-apoptotic effect of IL-6.
View larger version (25K):
[in a new window]
Fig. 7.
The anti-apoptotic effect of IL-6 is
completely abolished by blocking both PI 3-kinase and STAT3 signaling
pathways. Hep3B, STAT3D-1, and STAT3D-M were treated with or
without TGF- (5 ng/ml), IL-6 (60 ng/ml), and LY294002 (25 µM) and apoptosis was assayed as for Fig.
6B.
Apoptotic death of Hep3B and STAT3D stable transfectants induced by
various agents
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced apoptotic
death of hepatoma cells in a dose-dependent manner. The
anti-apoptotic effect of IL-6 correlated with its inhibition of
TGF--induced activation of caspase-3. IL-6 stimulation, however, did
not alter the effect of TGF- on the induction of PAI-1 and p15INK4B genes. Our results suggest that IL-6 interferes with
only a subset of TGF- signaling without affecting general TGF- signaling.
. Previous investigations
have indicated that the PI 3-kinase/Akt is involved in preventing
apoptosis induced by various growth factors in many different cell
types (14-21), whereas STAT3 is a critical mediator of the
anti-apoptotic effect of oncostatin M in osteosarcoma cells (46) and
the survival signals transduced by gp130 in B cells (33). The
anti-apoptotic mechanism of PI 3-kinase/Akt is at least partially
attributed to phosphorylation of the BCL-2 family member BAD by Akt
(20, 21). The phosphorylated BAD is then associated with 14-3-3, which
sequesters BAD from BCL-XL, thereby promoting cell survival
(22). Regulating the BCL-2 family member is also considered as one of
the anti-apoptotic mechanisms of STAT3, which was reported to be
capable of inducing BCL-2 in pro-B cells (33). Thus, both
anti-apoptotic signaling pathways transduced by IL-6 are likely to
converge to BCL-2 family members, which could act upstream of caspase-3
(47, 48). This is consistent with our finding that IL-6 blocked the
TGF--induced activation of caspase-3. In addition to induction of
BCL-2, STAT3 can directly up-regulate the transcription of p21, which
is implicated in the anti-apoptosis (46). In our system, an increased
expression of p21 upon IL-6 stimulation was also observed (data not
shown). Whether p21 and BCL-2 are induced independently by STAT3 and
exactly how p21 promotes survival remain to be investigated.
-induced apoptosis, since
TGF- failed to induce JNK/SAPK activity in Hep3B cells (data not
shown). Thus, distinct signaling pathways could mediate IL-6-induced
protection from apoptosis induced by different stimuli.
. Interestingly, in mouse leukemia M1
cells, STAT3 is involved in the differentiation and growth arrest but
not required for the anti-apoptotic signal (35, 50). These findings
highlight the significance of cellular context in determining the
biological functions of STAT3. STAT3 may induce the expression of a
distinct set of genes depending on cell type involved. Another
determinant of the biological consequences of STAT3 activation is
likely to be the concomitant activation of other STAT family members
and/or other signaling pathways, such as the PI 3-kinase/Akt pathway,
upon ligand binding to the cytokine receptors.
could be
physiologically important. TGF- is thought to be a terminator of
liver regeneration through its growth-inhibitory and apoptotic effects
(1). However, the mRNA of TGF- is induced at an initiation stage
of liver regeneration (53), indicating that hepatocytes can proceed
with regeneration despite the increase in the concentrations of
TGF-. Accordingly, hepatocytes isolated from actively regenerating
liver are resistant to TGF- (54). This raises the possibility that
the effect of TGF- is blocked by other growth factors and/or
cytokines as part of their regeneration-promoting effects. IL-6 is a
logical candidate of these factors, because the timing of its induction
correlates with the resistance of hepatocytes to TGF- (55).
Additional studies would be required to further define the
physiological roles of the anti-apoptotic activity of IL-6 against
TGF- in liver cells.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
, transforming growth factor-;
PI 3-kinase, phosphatidylinositol
3-kinase;
IL-6, interleukin-6;
JAK, Janus kinase;
STAT, signal
transducer and activator of transcription;
PAI-1, plasminogen activator
inhibitor-1;
SH2, Src homology domain 2;
MAP, mitogen-activated
protein;
PAGE, polyacrylamide gel electrophoresis;
ELISA, enzyme-linked
immunosorbent assay.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Michalopoulos, G. K.,
and DeFrances, M. C.
(1997)
Science
276,
60-66 2.
Fan, G.,
Ma, X.,
Kren, B.,
and Steer, C. J.
(1996)
Oncogene
12,
1909-1919[Medline]
[Order article via Infotrieve]
3.
Fukuda, K.,
Kojiro, M.,
and Chiu, J. F.
(1993)
Hepatology
18,
945-952[CrossRef][Medline]
[Order article via Infotrieve]
4.
Lin, J. K.,
and Chou, C. K.
(1992)
Cancer Res.
52,
385-388 5.
Oberhammer, F. A.,
Pavelka, M.,
Sharma, S.,
Tiefenbacher, R.,
Purchio, A. F.,
Bursch, W.,
and Schulte-Hermann, R.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
5408-5412 6.
S 
nchez, A.,
Çlvarez, A. M.,
Benito, M.,
and Fabregat, I.
(1996)
J. Biol. Chem.
271,
7416-74227.
Inagaki, M.,
Moustakas, A.,
Lin, H. Y.,
and Carr, B. I.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
5359-5363 8.
Heldin, C.-H.,
Miyazono, K.,
and ten Dijke, P.
(1997)
Nature
390,
465-471[CrossRef][Medline]
[Order article via Infotrieve]
9.
Massague, J.,
Hata, A.,
and Liu, F.
(1997)
Trends Cell Biol.
7,
187-192
10.
Zhang, Y.,
Musci, T.,
and Derynck, R.
(1997)
Curr. Biol.
7,
270-276[CrossRef][Medline]
[Order article via Infotrieve]
11.
Chen, R.-H.,
and Chang, T. Y.
(1997)
Cell Growth Diff.
8,
821-827[Abstract]
12.
Tanaka, S.,
and Wands, J. R.
(1996)
Cancer Res.
56,
391-394
13.
Chen, R.-H.,
Su, Y.-H.,
Chuang, R. L. C.,
and Chang, T.-Y.
(1998)
Oncogene
17,
1959-1968[CrossRef][Medline]
[Order article via Infotrieve]
14.
Ahmed, N. N.,
Grimes, H. L.,
Bellacosa, A.,
Chan, T. O.,
and Tsichlis, P. N.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3627-3632 15.
Dudek, H.,
Datta, S. R.,
Franke, T. F.,
Bimbaum, M. J.,
Yao, R.,
Cooper, G. M.,
Segel, R. S.,
Kaplan, D. R.,
and Greenberg, M. E.
(1997)
Science
275,
661-665 16.
Kauffmann-Zeh, A.,
Rodriguez-Vicana, P.,
Ulrich, E.,
Gilbert, C.,
Coffer, P.,
Downward, J.,
and Evan, G.
(1997)
Nature
385,
544-548[CrossRef][Medline]
[Order article via Infotrieve]
17.
Kennedy, S. G.,
Wagner, A. J.,
Conzen, S. D.,
Jordan, J.,
Bellacosa, A.,
Tsichlis, P. N.,
and Hay, N.
(1997)
Genes Dev.
11,
701-713 18.
Khwaja, A.,
Rodriguez-Vicana, P.,
Wennstrom, S.,
Warne, P. H.,
and Downward, J.
(1997)
EMBO J.
16,
2783-2793[CrossRef][Medline]
[Order article via Infotrieve]
19.
Kulik, G.,
Klippel, A.,
and Weber, M. J.
(1997)
Mol. Cell. Biol.
17,
1595-1606[Abstract]
20.
Datta, S. R.,
Dudek, H.,
Tao, X.,
Masters, S.,
Fu, H.,
Gotoh, Y.,
and Greenberg, M. E.
(1997)
Cell
91,
231-241[CrossRef][Medline]
[Order article via Infotrieve]
21.
del Peso, L.,
Gonzáles-Garcia, M.,
Page, C.,
Herrera, R.,
and Nunez, G.
(1997)
Science
278,
687-689 22.
Zha, J.,
Harada, H.,
Yang, E.,
Jockel, J.,
and Korsmeyer, S. J.
(1996)
Cell
87,
619-628[CrossRef][Medline]
[Order article via Infotrieve]
23.
Van Snick, J.
(1990)
Annu. Rev. Immunol.
8,
253-278[Medline]
[Order article via Infotrieve]
24.
Hirano, T.
(1994)
in
The Cytokine Handbook
(Thomson, A. W., ed), 2nd Ed.
, Academic Press, London
25.
Mizuhara, H.,
O'Neill, E.,
Seki, N.,
Ogawa, T.,
Kusunoki, C.,
Otsuka, K.,
Satoh, S.,
Niwa, M.,
Senoh, H.,
and Fujiwara, H.
(1994)
J. Exp. Med.
179,
1529-1537 26.
Cressman, D.,
Greenbaum, L.,
DeAngelis, R.,
Ciliberto, G.,
Furth, E.,
Poli, V.,
and Taub, R.
(1996)
Science
274,
1379-1383 27.
Murakami, M.,
Hibi, M.,
Nakagawa, N.,
Yasukawa, K.,
Yamanishi, K.,
Taga, T.,
and Kishimoto, T.
(1993)
Science
260,
1808-1810 28.
Lutticken, C.,
Wegenka, U. M.,
Yuan, J.,
Buschmann, J.,
Schindler, C.,
Ziemiecki, A.,
Harpur, A. G.,
Wilks, A. F.,
Yasukawa, K.,
Taga, T.,
Kishimoto, T.,
Barbirri, G.,
Pellegrini, S.,
Sendtner, M.,
Heinrich, P. C.,
and Horn, F.
(1994)
Science
263,
89-92 29.
Narazaki, M.,
Witthuhn, B. A.,
Yoshida, K.,
Silvennoinen, O.,
Yasukawa, K.,
Ihle, J. N.,
Kishimoto, T.,
and Taga, T.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
2285-2289 30.
Akira, S.,
Nishio, Y.,
Inoue, M.,
Wang, X. J.,
Wei, S.,
Matsusaka, T.,
Yoshida, K.,
Sudo, T.,
Naruto, M.,
and Kishimoto, T.
(1994)
Cell
77,
63-71[CrossRef][Medline]
[Order article via Infotrieve]
31.
Boulton, T. G.,
Stahl, N.,
and Yancopoulos, G. D.
(1994)
J. Biol. Chem.
269,
11648-11655 32.
Sadowski, H. B.,
Shuai, K.,
Darnell, J. E., Jr.,
and Gilman, M. Z.
(1993)
Science
261,
1739-1744 33.
Fukada, T.,
Hibi, M.,
Yamanaka, Y.,
Takahashi-Tezuka, M.,
Fujitani, Y.,
Yamaguchi, T.,
Nakajima, K.,
and Hirano, T.
(1996)
Immunity
5,
449-460[CrossRef][Medline]
[Order article via Infotrieve]
34.
Qiu, Y.,
Robinson, D.,
Pretlow, T. G.,
and Kung, H.-J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3644-3649 35.
Nakajima, K.,
Yamanaka, Y.,
Nakae, K.,
Kojima, H.,
Ichiba, M.,
Kiuchi, N.,
Kitaoka, T.,
Fukada, T.,
Hibi, M.,
and Hirano, T.
(1996)
EMBO J.
15,
3651-3658[Medline]
[Order article via Infotrieve]
36.
Gavrieli, Y.,
Sheman, Y.,
and Ben-Sasson, S. A.
(1992)
J. Cell Biol.
119,
493-501 37.
Thome, M.,
Schneider, P.,
Hofmann, K.,
Fickenscher, H.,
Meinl, E.,
Neipel, F.,
Mattmann, C.,
Burns, K.,
Bodmer, J. L.,
Schröter, M.,
Scaffidi, C.,
Krammer, P. H.,
Peter, M. E.,
and Tschopp, J.
(1997)
Nature
386,
517-521[CrossRef][Medline]
[Order article via Infotrieve]
38.
Li, J.-M.,
Nichols, M. A.,
Chandrasekharan, S.,
Xiong, Y.,
and Wang, X.-F.
(1995)
J. Biol. Chem.
270,
26750-26753 39.
Feng, X.-H.,
Filvaroff, E. H.,
and Derynck, R.
(1995)
J. Biol. Chem.
270,
24237-24245 40.
Whitman, M.,
Kaplan, D. R.,
Schaffhausen, B.,
Cantley, L.,
and Roberts, T. M.
(1985)
Nature
315,
239-242[CrossRef][Medline]
[Order article via Infotrieve]
41.
King, W. G.,
Mattaliano, M. D.,
Chan, T. O.,
Tsichlis, P. N.,
and Brugge, J. S.
(1997)
Mol. Cell. Biol.
17,
4406-4418[Abstract]
42.
Yingling, J. M.,
Datto, M. B.,
Wong, C.,
Frederick, J. P.,
Liberati, N. T.,
and Wang, X.-F.
(1997)
Mol. Cell. Biol.
17,
7019-7028[Abstract]
43.
Hu, P. P.,
Datto, M. B.,
and Wang, X.-F.
(1998)
Endocr. Rev.
19,
349-363 44.
Powis, G.,
Bonjouklian, R.,
Berggern, M.,
and Vlahos, C.
(1994)
Cancer Res.
54,
2419-2423 45.
Vlahos, C. J.,
Matter, W. F.,
Hui, K. Y.,
and Brown, R. F.
(1994)
J. Biol. Chem.
269,
5241-5248 46.
Bellido, T.,
O'Brien, C. A.,
Roberson, P. K.,
and Manolagas, S. C.
(1998)
J. Biol. Chem.
273,
21137-21144 47.
Cryns, V.,
and Yuan, J.
(1998)
Genes Dev.
12,
1551-1570 48.
Rao, L.,
and White, E.
(1997)
Curr. Opin. Gen. Dev.
7,
52-58[CrossRef][Medline]
[Order article via Infotrieve]
49.
Xu, F.-h.,
Sharma, S.,
Garner, A.,
Tu, Y.,
Raitano, A.,
Sawyers, C.,
and Lichtenstein, A.
(1998)
Blood
92,
241-251 50.
Yamanaka, Y.,
Nakajima, K.,
Fukada, T.,
Hibi, M.,
and Hirano, T.
(1996)
EMBO J.
15,
1557-1565[Medline]
[Order article via Infotrieve]
51.
Oh, H.,
Fujio, Y.,
Kunisada, K.,
Hirota, H.,
Matsui, H.,
Kishimoto, T.,
and Yamauchi-takihara, K.
(1998)
J. Biol. Chem.
273,
9703-9710 52.
Camargo, C. A., Jr.,
Madden, J. F.,
Gao, W.,
Selvan, R. S.,
and Clavien, P.-A.
(1997)
Hepatology
26,
1513-1520[CrossRef][Medline]
[Order article via Infotrieve]
53.
Braun, L.,
Mead, J. E.,
Panzica, M.,
Mikumo, R.,
and Bell, G. I.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
1539-1543 54.
Houck, K. A.,
and Michalopoulos, G. K.
(1989)
J. Cell. Physiol.
141,
503-509[CrossRef][Medline]
[Order article via Infotrieve]
55.
Rai, R. M.,
Yang, S. Q.,
McClain, C.,
Karp, C. L.,
Klein, S.,
and Diehl, A. M.
(1996)
Am. J. Physiol.
270,
G909-918
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  T. H. Sakuma, G. Hans-Filho, K. Arita, M. Odashiro, D. N. Odashiro, N. R. Hans, G. Hans-Neto, and J. A. McGrath Familial Primary Localized Cutaneous Amyloidosis in Brazil Arch Dermatol, June 1, 2009; 145(6): 695 - 699. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Malinowska, H. Neuwirt, I. T Cavarretta, J. Bektic, H. Steiner, H. Dietrich, P. L Moser, D. Fuchs, A. Hobisch, and Z. Culig Interleukin-6 stimulation of growth of prostate cancer in vitro and in vivo through activation of the androgen receptor Endocr. Relat. Cancer, March 1, 2009; 16(1): 155 - 169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. Chuang, R. S. Yang, K. S. Tsai, F. M. Ho, and S. H. Liu Hyperglycemia Enhances Adipogenic Induction of Lipid Accumulation: Involvement of Extracellular Signal-Regulated Protein Kinase 1/2, Phosphoinositide 3-Kinase/Akt, and Peroxisome Proliferator-Activated Receptor {gamma} Signaling Endocrinology, September 1, 2007; 148(9): 4267 - 4275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-A Seong, H. Jung, K.-T. Kim, and H. Ha 3-Phosphoinositide-dependent PDK1 Negatively Regulates Transforming Growth Factor-beta-induced Signaling in a Kinase-dependent Manner through Physical Interaction with Smad Proteins J. Biol. Chem., April 20, 2007; 282(16): 12272 - 12289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Castellino and R. N. Germain Chemokine-Guided CD4+ T Cell Help Enhances Generation of IL-6R{alpha}highIL-7R{alpha}high Prememory CD8+ T Cells J. Immunol., January 15, 2007; 178(2): 778 - 787. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-C. Li, S.-L. Ye, R.-X. Sun, Y.-K. Liu, Z.-Y. Tang, Y. Kim, J. G. Karras, and H. Zhang Inhibition of Growth and Metastasis of Human Hepatocellular Carcinoma by Antisense Oligonucleotide Targeting Signal Transducer and Activator of Transcription 3 Clin. Cancer Res., December 1, 2006; 12(23): 7140 - 7148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. Ho, Z. F. Yang, C. K. Lau, K. H. Tam, J. Y. To, R. T. Poon, and S. T. Fan Therapeutic Potential of Cardiotrophin 1 in Fulminant Hepatic Failure: Dual Roles in Antiapoptosis and Cell Repair Arch Surg, November 1, 2006; 141(11): 1077 - 1084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Weigert, A. M. Hennige, R. Lehmann, K. Brodbeck, F. Baumgartner, M. Schauble, H. U. Haring, and E. D. Schleicher Direct Cross-talk of Interleukin-6 and Insulin Signal Transduction via Insulin Receptor Substrate-1 in Skeletal Muscle Cells J. Biol. Chem., March 17, 2006; 281(11): 7060 - 7067. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kwon, Y. Ling, L. A. Maile, J. Badley-Clark, and D. R. Clemmons Recruitment of the Tyrosine Phosphatase Src Homology 2 Domain Tyrosine Phosphatase-2 to the p85 Subunit of Phosphatidylinositol-3 (PI-3) Kinase Is Required for Insulin-Like Growth Factor-I-Dependent PI-3 Kinase Activation in Smooth Muscle Cells Endocrinology, March 1, 2006; 147(3): 1458 - 1465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-J. Qi, G. M. Wildey, and P. H. Howe Evidence That Ser87 of BimEL Is Phosphorylated by Akt and Regulates BimEL Apoptotic Function J. Biol. Chem., January 13, 2006; 281(2): 813 - 823. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kosakowska-Cholody, W. M. Cholody, A. Monks, B. A. Woynarowska, and C. J. Michejda WMC-79, a potent agent against colon cancers, induces apoptosis through a p53-dependent pathway Mol. Cancer Ther., October 1, 2005; 4(10): 1617 - 1627. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Weigert, A. M. Hennige, K. Brodbeck, H. U. Haring, and E. D. Schleicher Interleukin-6 acts as insulin sensitizer on glycogen synthesis in human skeletal muscle cells by phosphorylation of Ser473 of Akt Am J Physiol Endocrinol Metab, August 1, 2005; 289(2): E251 - E257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Gealy, M. Denson, C. Humphreys, B. McSharry, G. Wilkinson, and R. Caswell Posttranscriptional Suppression of Interleukin-6 Production by Human Cytomegalovirus J. Virol., January 1, 2005; 79(1): 472 - 485. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Bailey, K. M. Nieport, M. P. Herbst, S. Srivastava, R. A. Serra, and N. D. Horseman Prolactin and Transforming Growth Factor-{beta} Signaling Exert Opposing Effects on Mammary Gland Morphogenesis, Involution, and the Akt-Forkhead Pathway Mol. Endocrinol., May 1, 2004; 18(5): 1171 - 1184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sun, Z. Tian, S. Kulkarni, and B. Gao IL-6 Prevents T Cell-Mediated Hepatitis via Inhibition of NKT Cells in CD4+ T Cell- and STAT3-Dependent Manners J. Immunol., May 1, 2004; 172(9): 5648 - 5655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kiessling, G. Muller-Newen, S. N. Leeb, M. Hausmann, H. C. Rath, J. Strater, T. Spottl, K. Schlottmann, J. Grossmann, F. A. Montero-Julian, et al. Functional Expression of the Interleukin-11 Receptor {alpha}-Chain and Evidence of Antiapoptotic Effects in Human Colonic Epithelial Cells J. Biol. Chem., March 12, 2004; 279(11): 10304 - 10315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Land and F. Darakhshan Thymulin evokes IL-6-C/EBP{beta} regenerative repair and TNF-{alpha} silencing during endotoxin exposure in fetal lung explants Am J Physiol Lung Cell Mol Physiol, March 1, 2004; 286(3): L473 - L487. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Fukuda and R. Longnecker Latent Membrane Protein 2A Inhibits Transforming Growth Factor-{beta}1-Induced Apoptosis through the Phosphatidylinositol 3-Kinase/Akt Pathway J. Virol., February 15, 2004; 78(4): 1697 - 1705. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Oskeritzian, W. Zhao, A. L. Pozez, N. M. Cohen, M. Grimes, and L. B. Schwartz Neutralizing Endogenous IL-6 Renders Mast Cells of the MCT Type from Lung, but Not the MCTC Type from Skin and Lung, Susceptible to Human Recombinant IL-4-Induced Apoptosis J. Immunol., January 1, 2004; 172(1): 593 - 600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Brocke-Heidrich, A. K. Kretzschmar, G. Pfeifer, C. Henze, D. Loffler, D. Koczan, H.-J. Thiesen, R. Burger, M. Gramatzki, and F. Horn Interleukin-6-dependent gene expression profiles in multiple myeloma INA-6 cells reveal a Bcl-2 family-independent survival pathway closely associated with Stat3 activation Blood, January 1, 2004; 103(1): 242 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-L. Lin, R.-H. Chen, Y.-M. Chen, W.-C. Chiang, T.-J. Tsai, and B.-S. Hsieh Pentoxifylline Inhibits Platelet-Derived Growth Factor-Stimulated Cyclin D1 Expression in Mesangial Cells by Blocking Akt Membrane Translocation Mol. Pharmacol., October 1, 2003; 64(4): 811 - 822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Wang, B. Walia, J. Evans, A. T. Gewirtz, D. Merlin, and S. V. Sitaraman IL-6 Induces NF-{kappa}B Activation in the Intestinal Epithelia J. Immunol., September 15, 2003; 171(6): 3194 - 3201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-H. Li, Z.-X. Yu, C.-L. Li, H.-P. Nguyen, Y.-X. Zhou, C. Deng, and X.-J. Li Lack of Huntingtin-Associated Protein-1 Causes Neuronal Death Resembling Hypothalamic Degeneration in Huntington's Disease J. Neurosci., July 30, 2003; 23(17): 6956 - 6964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kortylewski, F. Feld, K.-D. Kruger, G. Bahrenberg, R. A. Roth, H.-G. Joost, P. C. Heinrich, I. Behrmann, and A. Barthel Akt Modulates STAT3-mediated Gene Expression through a FKHR (FOXO1a)-dependent Mechanism J. Biol. Chem., February 7, 2003; 278(7): 5242 - 5249. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Longo, J. A. Kennell, M. J. Ochocinska, S. E. Ross, W. S. Wright, and O. A. MacDougald Wnt Signaling Protects 3T3-L1 Preadipocytes from Apoptosis through Induction of Insulin-like Growth Factors J. Biol. Chem., October 4, 2002; 277(41): 38239 - 38244. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. M. Morel, C. C. Park, K. Zhu, P. Kumar, J. H. Ruth, and A. E. Koch Signal Transduction Pathways Involved in Rheumatoid Arthritis Synovial Fibroblast Interleukin-18-induced Vascular Cell Adhesion Molecule-1 Expression J. Biol. Chem., September 13, 2002; 277(38): 34679 - 34691. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. L. Webster, P. Usechak, and M. S. Anwer cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochrome c release Am J Physiol Gastrointest Liver Physiol, September 1, 2002; 283(3): G727 - G738. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Barragan, B. Bellosillo, C. Campas, D. Colomer, G. Pons, and J. Gil Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells Blood, April 15, 2002; 99(8): 2969 - 2976. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. McNally and J. M. Anderson {beta}1 and {beta}2 Integrins Mediate Adhesion during Macrophage Fusion and Multinucleated Foreign Body Giant Cell Formation Am. J. Pathol., February 1, 2002; 160(2): 621 - 630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Hatano and D. A. Brenner Akt protects mouse hepatocytes from TNF-alpha - and Fas-mediated apoptosis through NK-kappa B activation Am J Physiol Gastrointest Liver Physiol, December 1, 2001; 281(6): G1357 - G1368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-T. Lin, C.-Y. Juan, K.-J. Chang, W.-J. Chen, and M.-L. Kuo IL-6 inhibits apoptosis and retains oxidative DNA lesions in human gastric cancer AGS cells through up-regulation of anti-apoptotic gene mcl-1 Carcinogenesis, December 1, 2001; 22(12): 1947 - 1953. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tu, A. Gardner, and A. Lichtenstein The Phosphatidylinositol 3-Kinase/AKT Kinase Pathway in Multiple Myeloma Plasma Cells: Roles in Cytokine-dependent Survival and Proliferative Responses Cancer Res., December 1, 2000; 60(23): 6763 - 6770. [Abstract] [Full Text]
|
|
|
|
|
|
J. Wang, R. J. Homer, Q. Chen, and J. A. Elias Endogenous and Exogenous IL-6 Inhibit Aeroallergen-Induced Th2 Inflammation J. Immunol., October 1, 2000; 165(7): 4051 - 4061. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K L STREETZ, T LUEDDE, M P MANNS, and C TRAUTWEIN Interleukin 6 and liver regeneration Gut, August 1, 2000; 47(2): 309 - 312. [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-T. Tsai, Y.-H. Su, S.-S. Fang, T.-N. Huang, Y. Qiu, Y.-S. Jou, H.-m. Shih, H.-J. Kung, and R.-H. Chen Etk, a Btk Family Tyrosine Kinase, Mediates Cellular Transformation by Linking Src to STAT3 Activation Mol. Cell. Biol., March 15, 2000; 20(6): 2043 - 2054. [Abstract] [Full Text]
|
|
|
|
 |
|
 P. Ernfors Nuclear Factor-{kappa}B to the Rescue of Cytokine-induced Neuronal Survival J. Cell Biol., January 24, 2000; 148(2): 223 - 226. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Miyakawa, P. Rojnuckarin, T. Habib, and K. Kaushansky Thrombopoietin Induces Phosphoinositol 3-Kinase Activation through SHP2, Gab, and Insulin Receptor Substrate Proteins in BAF3 Cells and Primary Murine Megakaryocytes J. Biol. Chem., January 19, 2001; 276(4): 2494 - 2502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. M. Ceponis, F. Botelho, C. D. Richards, and D. M. McKay Interleukins 4 and 13 Increase Intestinal Epithelial Permeability by a Phosphatidylinositol 3-Kinase Pathway. LACK OF EVIDENCE FOR STAT 6 INVOLVEMENT J. Biol. Chem., September 8, 2000; 275(37): 29132 - 29137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-L. Shih, M.-L. Kuo, S.-E. Chuang, A.-L. Cheng, and S.-L. Doong Hepatitis B Virus X Protein Inhibits Transforming Growth Factor-beta -induced Apoptosis through the Activation of Phosphatidylinositol 3-Kinase Pathway J. Biol. Chem., August 11, 2000; 275(33): 25858 - 25864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kusch, S. Tkachuk, H. Haller, R. Dietz, D. C. Gulba, M. Lipp, and I. Dumler Urokinase Stimulates Human Vascular Smooth Muscle Cell Migration via a Phosphatidylinositol 3-Kinase-Tyk2 Interaction J. Biol. Chem., December 8, 2000; 275(50): 39466 - 39473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Peron, M. Rahmani, Y. Zagar, A.-M. Durand-Schneider, B. Lardeux, and D. Bernuau Potentiation of Smad Transactivation by Jun Proteins during a Combined Treatment with Epidermal Growth Factor and Transforming Growth Factor-beta in Rat Hepatocytes. ROLE OF PHOSPHATIDYLINOSITOL 3-KINASE-INDUCED AP-1 ACTIVATION J. Biol. Chem., March 23, 2001; 276(13): 10524 - 10531. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Jones, J. Conover, and A. J. Symes Identification of a Novel gp130-responsive Site in the Vasoactive Intestinal Peptide Cytokine Response Element J. Biol. Chem., November 10, 2000; 275(46): 36013 - 36020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Yang, A. Murti, S. R. Pfeffer, J. G. Kim, D. B. Donner, and L. M. Pfeffer Interferon alpha /beta Promotes Cell Survival by Activating Nuclear Factor kappa B through Phosphatidylinositol 3-Kinase and Akt J. Biol. Chem., April 20, 2001; 276(17): 13756 - 13761. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Pitts, S. Wang, E. A. Jones, and A. J. Symes Transforming Growth Factor-beta and Ciliary Neurotrophic Factor Synergistically Induce Vasoactive Intestinal Peptide Gene Expression through the Cooperation of Smad, STAT, and AP-1 Sites J. Biol. Chem., June 1, 2001; 276(23): 19966 - 19973. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-T. Lin, R.-C. Lee, P.-C. Yang, F.-M. Ho, and M.-L. Kuo Cyclooxygenase-2 Inducing Mcl-1-dependent Survival Mechanism in Human Lung Adenocarcinoma CL1.0 Cells. INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE/Akt PATHWAY J. Biol. Chem., December 21, 2001; 276(52): 48997 - 49002. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Craig, A. Larkin, A. M. Mingo, D. J. Thuerauf, C. Andrews, P. M. McDonough, and C. C. Glembotski p38 MAPK and NF-kappa B Collaborate to Induce Interleukin-6 Gene Expression and Release. EVIDENCE FOR A CYTOPROTECTIVE AUTOCRINE SIGNALING PATHWAY IN A CARDIAC MYOCYTE MODEL SYSTEM J. Biol. Chem., July 28, 2000; 275(31): 23814 - 23824. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |