Department of Medicine and Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine the
role of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear
factor-B (NF-
B) in protecting hepatocytes from tumor necrosis
factor-
(TNF-
)- and Fas-mediated apoptosis, we pretreated
primary cultures of mouse hepatocytes with pharmacological and
adenovirus-mediated inhibitors of the PI3K/Akt and NF-
B pathways followed by treatment with TNF-
or Jo2, an anti-Fas antibody. Jo2
and, to a lesser extent, TNF-
phosphorylate Akt. The PI3K inhibitor
LY-294002 blocks TNF-
- and Fas-mediated Akt phosphorylation. LY-294002 pretreatment reduces NF-
B binding activity and
transcriptional activity and NF-
B-responsive gene expression by
TNF-
or Jo2. LY-294002 promotes apoptosis after TNF-
or
Jo2. The expression of dominant-negative Akt blocks NF-
B activation
and sensitizes hepatocytes to TNF-
- and Fas-mediated
apoptosis. The expression of constitutively active Akt rescues
LY-294002-pretreated cells from TNF-
- and Fas-mediated
apoptosis. Active Akt induces NF-
B transcriptional activity
but not NF-
B binding activity or I
B degradation. Furthermore,
LY-294002 pretreatment blocks TNF-
- and Jo2-induced Bcl-xL levels in
hepatocytes, with no effect on the phosphorylation levels of Bad.
Bcl-xL overexpression protects hepatocytes from Fas- but not
TNF-
-induced apoptosis after sensitization by actinomycin D
or the I
B superrepressor. Together, the PI3K/Akt pathway has a
protective role in Fas-mediated apoptosis, which requires
NF-
B activation, partially through the subsequent induction of
Bcl-xL.
Bcl-xL; phosphatidylinositol 3-kinase; IB kinase; transcription; signaling
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DEATH FACTORS SUCH
AS tumor necrosis factor- (TNF-
) and Fas ligand (FasL)
induce apoptosis, or programmed cell death (33, 34). Overexpression of the TNF/TNF receptor (TNFR) or FasL/Fas system causes hepatic damage in animal models and is associated with
diverse diseases in patients. TNF-
injection with pretreatment of
protein synthesis inhibitor or RNA transcriptional inhibitor induces
massive apoptosis in the mouse liver (27).
Intraperitoneal injection of anti-Fas agonistic antibody (Jo2) in mice
results in fulminant hepatitis and death within hours
(35). On the other hand, TNF-
is a comitogen for
hepatocytes (17). Furthermore, a recent study
(12) showed that Fas engagement of hepatocytes in
regeneration promotes cell growth. This indicates that TNF-
and Fas
may mediate either liver damage or stimulate protective regenerative responses.
Nuclear factor-B (NF-
B) has been strongly implicated in the
regulation of apoptosis induced by death factors, such as
TNF-
(49, 50). The NF-
B superfamily consists of
transcriptional activators such as p65 and p50 that form homo- or
heterodimers as well as inhibitory subunits such as I
B
that
function to retain the transcription factor in the cytoplasm
(25). Various stimuli, such as TNF-
, interleukin-1
(IL-1), viral infection, and lipopolysaccharide, activate the I
B
kinase complex (IKK), which results in the phosphorylation, ubiquination, and degradation of I
B, followed by the translocation of NF-
B and activation of NF-
B-responsive genes. Additional mechanisms activate NF-
B transcriptional activity, such as the phosphorylation of the NF-
B transactivation domains, without affecting its DNA binding (31). NF-
B activation
suppresses TNF-
-induced apoptosis through NF-
B-responsive
protective genes (49). TNFR-associated factor (TRAF) 1, TRAF2, and the inhibitor-of-apoptosis (IAP) proteins c-IAP1 and
c-IAP2 are gene targets of NF-
B transcriptional activity
(51). We (19) have demonstrated that NF-
B
activation and nitric oxide (NO), synthesized by inducible NO synthase
(iNOS), protect primary mouse hepatocytes from TNF-
- and
Fas-mediated apoptosis. Therefore, iNOS is an NF-
B-inducible
gene that mediates resistance to TNF-
- and Fas-induced
hepatotoxicity. However, there must be additional NF-
B-responsive
genes whose expression in hepatocytes is required for protection from
TNF-
- and Fas-mediated apoptosis, because iNOS is not
sufficient for complete protection.
Akt/protein kinase B is a serine/threonine protein kinase that mediates
cell-survival signals from growth factors and cytokines (8). Several targets of the phosphatidylinositol 3-kinase
(PI3K)/Akt signaling pathway have been identified to promote survival
or inhibit apoptosis. These substrates include Bad, caspase-9,
transcription factors of the forkhead family, and IKK. TNF-
activates PI3K and its downstream target Akt in 293 cells
(36). Both Akt and NF-
B-inducing kinase (NIK) are
necessary for TNF-
-induced activation of NF-
B, and Akt mediates
IKK
phosphorylation in 293 cells (36). On the other
hand, in MCF7 breast carcinoma cells, whereas both TNFR1 and NIK are
partially involved in Akt-induced NF-
B stimulation, I
B
superrepressor completely blocked Akt-NF-
B cross-talk
(4). Also NF-
B activation by platelet-derived growth
factor (PDGF) is a target of the antiapoptotic Ras/PI3K/Akt pathway
(42). However, PDGF-induced Akt phosphorylation does not
activate NF-
B in human vascular smooth muscle cells and fibroblasts
(38). These results indicate NF-
B activation by Akt
might be a cell-specific event. Growth factors such as epidermal growth
factor (EGF) and interleukin (IL)-6 inhibit transforming growth
factor-
(TGF-
)-mediated apoptosis through PI3K/Akt
signaling pathway in rat hepatocytes and human hepatoma cell lines,
respectively (7, 41). PI3K/Akt mediates the
antiapoptotic effects of EGF and intestinal trefoil factor
(18, 48). However, the role of Akt in death
receptor-mediated apoptosis in hepatocytes remains unclear.
We hypothesized that TNF-/TNFR and FasL/Fas stimulate survival
signals to protect hepatocytes from apoptosis, because TNF-
and anti-Fas agonistic antibody alone induce minimal cytotoxicity in
cultured hepatocytes. In this study, we aimed to determine whether
1) TNF-
or Jo2 activates PI3K/Akt pathway in mouse
hepatocytes, 2) active Akt induces NF-
B activation, and
3) active Akt protects hepatocytes from TNF-
- or
Fas-mediated apoptosis.
![]() |
MATERIAL AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primary hepatocyte cultures.
C57Bl6 male mice (~8 wk old) were anesthetized with
ketamine-acepromazine maleate administered by intraperitoneal
injection. For some experiments, we used Bcl-xL transgenic mice for
hepatocyte cultures (a kind gift from Dr. A. Mignon), in which
expression of human Bcl-xL cDNA is directed by the regulatory sequences
of the rat L-type pyruvate kinase gene (10). Transgenic
mice were identified by Southern blot analysis using the coding 0.9-kb
Bcl-xL sequence as a probe (10). Hepatocytes were then
isolated by a retrograde, nonrecirculating in situ collagenase
perfusion of livers cannulating through the inferior vena cava by a
procedure modified from Moldeus et al. (32). Livers were
first perfused in situ with oxygenated 0.5 mM EGTA containing
calcium-free salt solution (10 ml/min at 37°C for 5 min), followed by
perfusion with solution containing 0.04% collagenase type I
(Worthington Biochemical, Lakewood, NJ) for 10 min. The liver was then
gently minced on a Petri dish and filtered with polyamide mesh (I 003 Y
Nitex 3-60/45, Tetko). Hepatocytes were washed two times and centrifuged at 50 g for 2 min. Cell viability was
consistently >90% as determined by trypan blue exclusion. Hepatocyte
cultures contained <1% Kupffer cells and hepatic stellate cells as
determined by FITC-labeled latex beads (1 µm, Polysciences,
Warrington, PA) and autofluorescence, respectively. Cells (4 × 105) were plated on six-well plates coated with mouse
collagen type I in Waymouth's medium containing 10% fetal bovine
serum, 0.1 µM insulin, and 0.1 µM dexamethazone. In preliminary
experiments, 0.1 µM insulin did not affect Akt phosphorylation. Next,
1.5 × 106 or 4 × 106 cells were
plated on 60- or 100-mm dishes, respectively. After 1.5-2 h, the
culture was washed with PBS and changed to hormonally defined medium
(HDM) containing 0.1 µM insulin, 2 mM L-glutamine, 5 µg/ml transferrin, 3 µM selenium, and 10 nM free fatty acids in
RPMI basal medium. Cells were pretreated with the PI3K inhibitor LY-294002 (Calbiochem-Novabiochem, San Diego, CA) or wortmannin (Sigma,
St. Louis, MO) for 1 h before the exposure of recombinant murine
TNF- ( R&D Systems, Minneapolis, MN) or Jo-2 (Pharmingen, San Diego,
CA). In some experiments, cells were infected with recombinant
adenoviruses in HDM containing 30 plaque-forming units/cell for 3 h at 37°C and then changed to HDM containing TNF-
or Jo2. All
animals received humane care in compliance with the guidelines of the
University of North Carolina.
Western blot analysis for Akt, IB
, Bad, and Bcl-xL.
Whole cell extracts were prepared by lysing the cells in lysis buffer
(10 mM HEPES, pH 7.9, 0.42 M NaCl, 1.5 mM MgCl2, 0.5 mM
dithiothreitol, 0.5% Nonidet P-40, and 25% glycerol) containing protease inhibitors (40 µg/ml bestatin, 0.5 mM Pefabloc, 700 ng/ml pepstatin A, 2 µg/ml aprotinin, and 0.5 µg/ml leupeptin; all from Roche, Indianapolis, IN). To detect the phosphorylation status of the
protein, we used cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerolphosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin) plus 1 mM phenylmethylsulfonyl fluoride according to the manufacturer's instructions. The protein concentration of the
extracts was determined by the Bradford method. Lysates containing 500 µg of protein were separated by electrophoresis on 10% acrylamide SDS gels. The proteins were transferred into nitocellulose membranes (Schleicher & Schuell, Keene, NH). Equal loading was confirmed by
Ponceau S staining. Phosphorylated Akt was detected using rabbit polyclonal phospho-Akt (serine-473) antibody (New England Biolabs, Beverly, MA) and secondary anti-rabbit horseradish
peroxidase-conjugated antibody (New England Biolabs). Anti-total Akt
antibody (New England Biolabs) was used for internal control. Antibody
was used at 1:1,000 dilution. Proteins were detected with enhanced
chemiluminescence detection reagents (Amersham, Arlington Heights, IL).
The protein levels of phosphorylated Akt were quantified using
AlphaImager 2000 (Alpha Innotech, San Leandro, CA). For I
B
,
Bcl-xL, and Bad Western blot analysis, lysates containing 50 µg
protein were separated by 12% acrylamide SDS gels. I
B
expression
was detected using rabbit polyclonal I
B
antibody (C-21, Santa
Cruz Biotechnology, Santa Cruz, CA) at 1:1,000 dilution. Phosphorylated
Bad was detected using rabbit polyclonal phospho-Bad (serine-136)
antibody and phospho-Bad (serine-112) (New England Biolabs) at 1:500
dilution. Anti-total Bad antibody (New England Biolabs) was used for
internal control. Bcl-xL was detected using rabbit polyclonal Bcl-xL
antibody (S-18, Santa Cruz Biotechnology) at 1:1,000 dilution.
Measurements of apoptosis.
For quantitation of cell viability (presented as means ± SE),
cells were infected and treated as described above. After 20 h of
TNF- or Jo2 treatment, cell viability was determined by exclusion of
trypan blue. Viable cells were counted in three different ×200 power
fields, and the percentage of treated viable cells to untreated viable
cells was expressed as a percentage of viability. For propidium iodide
nuclear staining, cells were fixed in methanol-acetic acid (3:1),
stained with 10 µg/ml propidium iodide, and viewed with an Olympus
fluorescence microscope using a rhodamine filter set. Hepatocyte cell
death was confirmed as apoptosis by terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling (TUNEL) (Boehringer
Mannheim, Mannheim, Germany). TUNEL staining was performed according to
the manufacturer's suggested protocol. Positive (apoptotic) cells
were counted in three different ×200 power fields. As described previously (19), we used the FluorAce kit (Bio-Rad
Laboratories, Hercules, CA) to perform
7-amino-4-triflouromethyl-coumarin (AFC) release assays for caspase-3.
Nuclear extract preparation and electrophoretic mobility shift
assay.
Four million cells were cultured overnight after adenoviral infection.
Cell were harvested 30 min after TNF- or Jo2 treatment. Nuclear
protein extracts were prepared from primary mouse hepatocytes as
previously described (2, 22). Protein-DNA binding
reactions were carried out for 20 min on ice, using 5 µg of nuclear
extract and 32P-labeled DNA probes for the NF-
B
consensus binding site (3). Complexes were separated by
electrophoresis on nondenaturing 5% acrylamide gels and assayed by
autoradiography and PhosphorImager analysis (Molecular Dynamics,
Sunnyvale, CA). For supershift assays, 8 µg of antibody against the
p65 or p50 subunit of the NF-
B complex (Santa Cruz Biotechnology)
were added to the reaction mixture, and the incubation time was
extended for an additional 30 min.
Transfection and luciferase reporter assays.
Primary mouse hepatocytes plated on six-well plates were transiently
transfected using the Targefect F-1 (Targeting Systems, Santee, CA).
One microgram of 3× B luciferase [(
B)3-Luc]
plasmid (54) DNA was diluted in 1 ml of Opti-MEM and mixed
with 2.5 µl of F-1 reagent. Complexes were allowed to form for 25 min
at 37°C. Hepatocytes were washed twice with Opti-MEM, and then
F-1-DNA complexes were added to the cells. Twelve hours after
transfection, cells were washed with PBS and pretreated with LY-294002
for 1 h. Five hours after treatment of TNF-
or Jo2, cells were
lysed in cell lysis buffer, and luciferase assays were performed with enhanced luciferase assay systems (Analytical Luminescence Laboratory, San Diego, CA) according to the manufacturer's instructions. For some
experiments, cells were infected with adenovirus vector 12 h after
transfection and then lysed at 22 h after infection.
RT-PCR for iNOS and intercellular adhesion molecule-1.
Total RNA was extracted with RNeasy mini kit (Qiagen, Lalencia, CA)
according to the manufacturer's suggested protocol. First-strand cDNA
was synthesized using 1 µg of total RNA, 10 mM dNTPs (Pharmacia, Piscataway, NJ), and 200 U of Moloney murine leukemia virus RT (GIBCO,
Grand Island, NY) in a final volume of 25 µl. The reaction was
carried out for 60 min at 42°C. The synthesized cDNA was amplified using specific sets of primers for iNOS and -actin. The iNOS sense
primer sequence was 5'-TAGAAACAACAGGAACCTACCA-3', and the antisense
primer was 5'-ACAGGGGTGATGCTCCATGACA-3'. The primers for
-actin were
as described previously (3). The PCR reactions were cycled
as follows: after initial denaturation for 4 min at 99.9°C, 40 cycles
at 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s; final extension was carried out at 72°C for 5 min. The
intercellular adhesion molecule-1 (ICAM-1) sense primer sequence was
5'-TGGAACTGCACGTGCTGTAT-3', and the antisense primer was
5'-GACGAAAACTTGTCTTACCA-3'. The PCR reactions were cycled as follows:
after initial denaturation for 4 min at 99.9°C, 30 cycles at 95°C
for 60 s, 59°C for 90 s, and 72°C for 10 s; final
extension was carried out at 72°C for 5 min. The PCR products were
electrophoresed in a 2% agarose gel, stained with ethidium bromide,
and photographed.
Adenoviruses.
The adenovirus 5 variants Ad5IB and Ad5GFP, expressing hemagglutinin
(HA)-I
B
(S32A, S36A) and green fluorescent protein, respectively,
were as described previously (2, 22).
Dominant-negative (dn) Ad5IKK
expresses a catalytic mutant with
lysine changed to methionine (19). AddeltaNIK expressing
dnNIK was as described previously (20). An adenovirus
expressing constitutively active Akt encoding an amino-terminal
myristylation signal (AdmyrAkt) was a kind gift from Dr. J. A. Romashkova (42). An adenovirus expressing dnAkt (AddnAkt)
was a kind gift from Dr. W. Ogawa (26). Lysates were
prepared from 4 × 105 hepatocytes at 24 h after
adenoviral infection. The HA-tagged myrAkt and dnAkt were detected
using mouse anti-HA antibody (Boehringer Mannheim).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TNF- and anti-Fas antibody phosphorylate Akt through PI-3 K in
primary mouse hepatocytes.
To determine whether TNF-
or Jo2 phosphorylates Akt in hepatocytes,
we performed Western blot analysis using cell lysates from primary
mouse hepatocytes. Hepatocytes were incubated for 12 h without
serum before treatment to reduce the basal level of phosphorylated Akt.
Phospho-Akt antibody detects Akt only when phosphorylated at
serine-473. Cell lysate from hepatocytes treated with 10 ng/ml of EGF
was used as a positive control (41). TNF-
phosphorylated Akt at 5 min after treatment, and Jo2 phosphorylated Akt
15 min after treatment (Fig.
1A). The results from
densitometry showed that Jo2 is a more potent inducer for Akt
phosphorylation than TNF-
(Fig. 1B). Furthermore, to
determine whether PI3K is involved in TNF-
- and Jo2-induced
Akt phosphorylation, we pretreated cells with the PI3K-specific
inhibitor LY-294002 for 1 h at 25 µM, as performed previously
(41). Immunoblot analysis demonstrated that the PI3K
inhibitor LY-294002 blocks TNF-
- and Fas-mediated Akt
phosphorylation, indicating that PI3K is upstream in TNF-
- and
Jo2-induced Akt phosphorylation.
|
PI3K inhibitor sensitizes mouse hepatocytes to TNF-- and
Fas-mediated apoptosis.
To determine the role of PI3K/Akt pathway on survival for TNF-
- and
Fas-mediated apoptosis, primary mouse hepatocytes were pretreated with LY-294002 for 1 h and treated with TNF-
or Jo2. Percent viability was determined after 20 h by the trypan blue extraction test. TNF-
, Jo2, or LY-294002 alone induced minimal cytotoxicity. However, pretreatment with LY-294002 promotes cell death
after TNF-
or Jo2 (35% and 70%, respectively, Fig.
2B). The morphological changes
demonstrated by the phase-contrast microscopy included cellular
rounding, loss of attachment, and increased refractility (Fig.
2A). The hepatocytes treated with LY-294002 plus TNF-
or
LY-294002 plus Jo2 displayed nuclear condensation and fragmentation by
propidium iodide staining, characteristics of apoptosis (Fig.
2C, middle and right), whereas cells
treated with LY-294002 displayed normal nuclear morphology (Fig.
2C, left). To confirm death by apoptosis,
a TUNEL assay was performed. Although TUNEL-positive cells were minimal
after TNF-
or Jo2 treatment in hepatocytes (2 ± 1, 11.7 ± 1.5, means ± SE cells/×200, respectively), positive
hepatocytes were observed after TNF-
or Jo2 treatment in hepatocytes
pretreated with LY-294002 (39.6 ± 4.7 and 94 ± 10, respectively). Furthermore, AFC release assay showed that TNF-
or
Jo2 induced 2.5- or 6-fold caspase-3 activation in hepatocytes pretreated with LY-294002, respectively, compared with that in hepatocytes without treatment. To confirm these results, hepatocytes were pretreated with wortmannin, another PI3K inhibitor. Wortmannin also promotes cell killing after treatment with TNF-
or Jo2
(21.4 ± 5.2% or 11.6 ± 4.3%, respectively, as %control
viability). Both PI3K inhibitors induce more apoptosis in cells
treated with Jo2 than TNF-
, which may reflect the more potent
phosphorylation activity of Akt by Jo2.
|
PI3K inhibition attenuates NF-B binding activity and
transcriptional activity and NF-
B-mediated gene expression by
TNF-
or anti-Fas antibody.
NF-
B is an antiapoptotic mediator in the liver in vivo
(14, 28, 43) and in cultured hepatocytes (2,
20). Inhibition of NF-
B activation by the I
B
superrepressor, a proteasome inhibitor, dnNIK, or dnIKK
sensitizes
mouse hepatocytes to TNF-
- and Fas-mediated apoptosis
(19, 20, 45). To explore the mechanism by which LY-294002
sensitizes hepatocytes to TNF-
- and Fas-mediated apoptosis, we examined NF-
B DNA-binding activity by electrophoretic mobility shift assay (EMSA) (Fig. 3A),
NF-
B transcriptional activity by reporter gene assay (Fig.
3B), and NF-
B-responsive gene expression by RT-PCR (Fig.
3C). EMSA showed that TNF-
or Jo2 induced NF-
B binding
activities (Fig. 3A, middle band in lanes 2 and
4), which is composed of p65/p50 heterodimers, as
demonstrated by the supershift analysis (data not shown). Pretreatment
with LY-294002 reduced NF-
B binding activities by TNF-
or Jo2
(59% or 33% reduction, respectively, demonstrated with PhosphorImager
analysis). Pretreatment with LY-294002 also reduced NF-
B
transcriptional activities by TNF-
or Jo2, as demonstrated by the
(
B)3Luc luciferase activities.
|
dnAkt blocks NF-B binding activity and I
B degradation and
sensitizes hepatocytes to TNF-
- and Fas-mediated apoptosis.
To extend the results obtained with the pharmacological PI3K inhibitor
LY-294002, we used AddnAkt to block activation of Akt. HA-tagged mutant
Akt (dnAkt) has a lysine-179 in the kinase domain replacing Asp, as
previously described (26). The adenovirus-mediated expression of dnAkt was confirmed by detecting HA (Fig.
4A). dnAkt abolished TNF-
-
and Jo2-induced NF-
B binding activity, whereas GFP expression had no
effect on NF-
B binding activity (Fig. 4B). TNF-
induced I
B degradation in hepatocytes from 15 to 30 min after
treatment (Fig. 4C, top), whereas I
B
degradation was not observed in Jo2-treated cells (data not shown).
Consistent with this result, the Fas-interacting proteins FADD, Casper,
and caspase-8 also activate NF-
B by an alternative pathway in other
cell types (21). dnAkt expression (Fig. 4C,
bottom) blocks TNF-
-induced I
B degradation (Fig.
4C, middle). To confirm the effect of Akt on
survival after treatment with TNF-
or Jo2, hepatocytes were infected
with AddnAkt or control adenovirus AdGFP and treated with TNF-
or
Jo2. Measurements of cell viability demonstrate that dnAkt sensitizes
hepatocytes to killing by TNF-
or Fas (Fig. 4D). The
hepatocytes expressing dnAkt treated with TNF-
or Jo2 displayed
nuclear condensation and fragmentation by propidium iodide staining,
characteristics of apoptosis (Fig. 4E,
middle and right), whereas cells expressing dnAkt
without the treatment displayed normal nuclear morphology (Fig.
4E, left). These results demonstrate that either
dnAkt or LY-294002 sensitize hepatocytes to TNF-
- and Fas-mediated
apoptosis through inhibition of NF-
B activation.
|
Constitutively active Akt rescues hepatocytes from sensitization by
LY-294002 to TNF-- and Fas-mediated apoptosis.
To confirm that Akt is involved in the antiapoptotic pathway in
TNF-
- and Fas-mediated apoptosis, we used AdmyrAkt
(42). After infection with AdmyrAkt, cells were pretreated
with LY-294002, followed by treatment with TNF-
or Jo2. Myristylated
Akt expression was confirmed by HA Western blotting (Fig.
5A). Constitutively active Akt
rescues LY-294002-pretreated cells from TNF-
- and Fas-mediated
apoptosis (Fig. 5B). Thus Akt is the downstream
effector of PI3K that mediates protection from TNF-
- or Jo2-induced
apoptosis.
|
|
NF-B is a downstream target of Akt.
To explore the signaling pathways of Akt/NF-
B in primary
hepatocytes, we tested whether dnNIK, dnIKK
, dnIKK
, or I
Bsr
would influence TNF-
-induced NF-
B activation in hepatocytes
expressing active myrAkt, using EMSA and I
B
Western blot
analysis. Similar experiments could not be performed with Jo2, since
Jo2 appears to activate NF-
B independent of I
B. As shown in Figs.
3A and 4C, TNF-
induced NF-
B binding
activity and I
B degradation (Fig. 7,
A, lane 2, and B, top
left). Active myrAkt alone did not induce NF-
B binding activity
but accelerated I
B degradation by TNF-
(Fig. 7, A,
lanes 3 and 4, and B, middle
left). As expected, I
Bsr completely blocked NF-
B activation
and I
B
degradation by TNF-
in hepatocytes expressing active
myrAkt (Fig. 7, A, lane 5, and B,
bottom left). Furthermore, expression of either dnIKK
or
dnNIK, but not dnIKK
, blocked NF-
B activation and I
B
degradation by TNF-
in hepatocytes expressing active myrAkt (Fig.
7). These results are similar to those obtained from hepatocytes
without adenovirus-mediated active myrAkt (19, 20).
|
|
TNF- and Jo2 induce antiapoptotic Bcl-xL.
Several putative antiapoptotic targets of PI3K/Akt pathway have
been proposed, including Bad, caspase-9, and the Forkhead transcriptional factors (8). To determine the involvement
of Bad in sensitization by LY-294002 to TNF-
- and Fas-mediated
apoptosis, we performed Western blot analysis with phospho-Bad
(serine-136), phospho-Bad (serine-112), and total Bad antibodies.
Phosphorylated Bad was not detected (data not shown), and total Bad
expression was unchanged during TNF-
- and Fas-mediated
apoptosis (Fig. 9A). Furthermore, active myrAkt had no influence on Bad levels. In addition,
the long form of Fas-associated death domain-like IL-1
-converting enzyme-inhibitory protein (FLIP) is downregulated in T cells and endothelial cells when they become susceptible to apoptosis
(23, 40). Partial hepatectomy exerts its protective effect
by maintaining levels of FLIP sufficient to inhibit Fas-induced
apoptosis (12). However, pretreatment with
LY-294002 or the expression of I
Bsr did not affect intracellular
FLIP, as demonstrated by Western blotting (data not shown).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we demonstrate that 1) TNF- and
anti-Fas agonistic antibody Jo2 phosphorylate Akt through PI3K,
2) pretreatment with a PI3K inhibitor or by
adenovirus-mediated expression of dnAkt reduces NF-
B activation and
sensitizes hepatocytes to TNF-
- and Fas-mediated apoptosis,
3) the expression of constitutively active Akt rescues PI3K
inhibitor-pretreated cells from TNF-
- and Fas-mediated
apoptosis, 4) active Akt induces NF-
B
transcriptional activity, but not NF-
B binding activity or I
B
degradation, 5) active Akt had no effect on dnNIK, dnIKK
,
or dnIKK
, or the I
Bsr in TNF-
-induced NF-
B binding
activity, I
B degradation, or apoptosis in hepatocytes, and
6) TNF-
and Jo2 induce Bcl-xL through NF-
B activation,
which is blocked by PI3K inhibition. These results indicate that the
PI3K/Akt pathway has a protective role in death receptor-induced
apoptosis mediated in part by NF-
B via Bcl-xL (Fig.
11).
|
Ozes et al. (36) suggested that Akt is involved in the
TNF--mediated activation of NF-
B in HeLa cells, implying that the antiapoptotic activity of Akt may be mediated in part through NF-
B. Furthermore, in MCF7 breast carcinoma cells, PI3K and Akt suppressed a dose-dependent induction of apoptosis by TNF-
and stimulated NF-
B activation in a dose-dependent manner,
suggesting a common link between these two pathways (4).
However, Delhase et al. (11) failed to detect any
involvement of Akt in the signaling pathway through which TNF-
leads
to NF-
B activation in HeLa cells. In human endothelial cells,
TNF-
and IL-1 activate the PI3K/Akt antiapoptotic pathway, but
the antiapoptotic effects of Akt are independent of NF-
B
(29). Together, the role of Akt and the extent to which it
is activated by TNF-
must be cell-type specific.
In our study, Jo2 and, to a lesser extent, TNF- phosphorylated Akt
(Fig. 1A). The PI3K inhibitor LY-294002 blocks TNF-
- and
Fas-mediated Akt phosphorylation (Fig. 1C). Pretreatment
with LY-294002 promotes cell death after TNF-
or Jo2 (Fig.
2B). PI3K inhibitors induce more apoptosis in cells
treated with Jo2 than TNF-
. This observation might result from the
more potent phosphorylation activity of Akt by Jo2. These results are
consistent with the previous reports (41) suggesting
TNF-
-mediated PI3K/Akt activation in isolated rat hepatocytes
treated with TGF-
is minimal compared with EGF-mediated PI3K/Akt
activation. Furthermore, our study revealed the novel finding that
PI3K/Akt protects cultured hepatocytes from Fas-mediated
apoptosis. Although the mechanism by which Fas stimulates the
PI3K/Akt pathway is not clear, there are several studies supporting
PI3K/Akt activation as a protective mechanism in Fas-mediated
apoptosis. In a rat hepatoma cell line, bile salts mediate
hepatocyte apoptosis by increasing cell surface trafficking of
Fas (47) and some hydrophobic bile acids activate
PI3K-dependent survival pathways, which prevents their toxicity
(44). The protective effect of hepatocyte growth factor in
bile acid-induced apoptosis requires Akt activation
(53). PTEN, a phosphatidylinositol phosphatase, acts as a
tumor suppressor, at least in part, by antagonizing PI3K/Akt signaling
(5). Fas-mediated apoptosis was impaired in PTEN
+/
mice, and T lymphocytes from these mice show reduced activation-induced cell death (13). PI3K
inhibitors restored Fas responsiveness in PTEN +/
cells
(13). These results indicate that PTEN is an essential
mediator of the Fas response and a repressor of autoimmunity and thus
implicate the PI3K/Akt pathway in Fas-mediated apoptosis.
Pharmacological inhibition of PI3K attenuates NF-B binding activity,
NF-
B transcriptional activity, and NF-
B-mediated gene expression
by TNF-
or anti-Fas antibody (Fig. 3). Adenovirus-mediated expression of dnAkt reduces NF-
B activation and sensitizes
hepatocytes to TNF-
- and Fas-mediated apoptosis (Fig. 4).
The expression of constitutively active Akt rescues PI3K
inhibitor-pretreated cells from TNF-
- and Fas-mediated
apoptosis (Fig. 5B) and active Akt induces NF-
B
transcriptional activity, but not NF-
B binding activity and I
B
degradation (Fig. 6). Taken together, active Akt is necessary but not
sufficient for optimal death receptor-mediated NF-
B activation.
In HeLa cells, IB is a target for Akt (36). However,
other studies (30, 31, 39, 46) have found no evidence for the involvement of Akt in I
B degradation but rather propose an I
B-independent mechanism in which Akt affects the transcriptional activity of NF-
B. IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-
B, a distinct process from the liberation of NF-
B from its cytoplasmic inhibitor I
B (30, 46).
Akt stimulates NF-
B transcriptional activity by phosphorylating the transactivation domain of NF-
B p65 (30, 31). These
recent studies (30, 31) are consistent with our
observation that active Akt induces NF-
B transcriptional activity
but not NF-
B binding activity and I
B degradation in primary hepatocytes.
NF-B reporter gene activity in HeLa cells is induced by
constitutively active Akt, and this is inhibited by dnIKK
(55). Similarly, in our study, expression of either
dnIKK
or dnNIK, but not dnIKK
, blocked NF-
B activation and
I
B degradation by TNF-
in hepatocytes expressing active myrAkt.
Active myrAkt fails to rescue hepatocytes expressing the I
B
superrepressor, dnNIK, and dnIKK
from TNF-
- and Fas-mediated
apoptosis. Together, these observations show that an intact
mitogen-activated protein kinase kinase kinase (MAPKKK)/IKK
/NF-
B
is required for the protective effect of activated Akt in cultured
hepatocytes. What is the relationship between the classic IKK signaling
pathway with nuclear translocation of NF-
B and the increased NF-
B
transcriptional activity mediated by Akt? Based on our present study
and the recent literature (30, 31, 45), we propose a
working model in Fig. 11. Here the death receptors activate an upstream
MAPKKK such as NIK (56) and then the IKK complex,
resulting in I
B phosphorylation and degradation, with subsequent
release and nuclear translocation of NF-
B. Through a distinct second
pathway, Akt is activated by the same death receptors and in turn
activates NF-
B transcriptional activity, probably through the
phosphorylation of the NF-
B transactivation domain (30, 31,
45). p65 is not a direct substrate for Akt, so unknown
intermediate steps are required (30, 31). Furthermore, Akt
potentiates the activation of IKK, demonstrating cross talk between
these two pathways. Finally, the death receptors activate p38 MAPK in
primary hepatocytes, which is required for optimal induction of NF-
B
(Hatano, unpublished observations). Recently (30), p38 was
shown to increase the transcription of NF-
B-responsive genes by
interacting with the coactivator cAMP-response element binding protein
binding protein. This model explains why a low level of the NF-
B
translocation pathway is required to reveal the Akt-NF-
B
transcriptional activation, since NF-
B is required to act as a
substrate for activation by the Akt pathway.
Several targets of the PI3K/Akt signaling pathway have been recently
identified (8) that may underlie the ability of this regulatory cascade to promote survival. These substrates include components of the intrinsic cell death machinery (Bad, caspase-9, and
c-FLIP), transcription factors of the forkhead family, and a kinase
(IKK) that regulates the NF-B transcription factor. Growth factor
activation of the PI3K/Akt signaling pathway culminates in the
phosphorylation of the Bcl-2 family member Bad, thereby suppressing
apoptosis and promoting cell survival (9). Akt phosphorylates Bad in vitro and in vivo and blocks the Bad-induced death of primary neurons in a site-specific manner (9).
However, we could not detect phosphorylation of Bad in mouse
hepatocytes stimulated by TNF-
, Jo2, or active Akt. Similarly,
phosphorylated Bad was not detected in EGF-mediated protection from
TGF-
-induced apoptosis in rat fetal hepatocytes
(15) nor did PI3K inhibition affect Bad phosphorylation by
hepatocyte growth factor (53). Furthermore, caspase-9 is
one caspase upstream of caspase-3 and its activation is stimulated by
apoptotic protease activating factor-1/cytochrome c and
inhibited by Akt signals (8). However, the Akt
phosphorylation site found in human caspase-9 is absent in mouse
caspase-9 (16).
c-FLIP has been suggested (23) to play a role in
protecting activated peripheral T cells from CD95-mediated
apoptosis. Partial hepatectomy protected mice against the
lethal effects of Jo2 through upregulation of FLIP (12).
FLIP is an apoptosis inhibitor whose expression or function is
regulated by the PI3K/Akt pathway in some tumor cell lines
(37). However, we could not detect any change in c-FLIP
levels by Western blot analysis after TNF- or Jo2 stimulation in
hepatocytes pretreated with LY-294002 or expressing I
B
superrepressor (data not shown).
Finally, we focus on the role of Bcl-xL, a member of the
antiapoptotic Bcl family in the protection from TNF-- and
Fas-mediated apoptosis. TNF-
and Jo2 induced Bcl-xL, and
Fas-mediated Bcl-xL induction was especially reduced by the PI3K
inhibitor. These results are consistent with a study (24)
using Akt transgenic mice in which both thymocytes and T cells
overexpressing active Akt displayed elevated levels of Bcl-xL.
Furthermore, Bcl-xL induction was blocked by the I
B superrepressor
in hepatocytes. These results indicate that Bcl-xL is an
antiapoptotic NF-
B-responsive gene in hepatocytes as previously
described (6, 24) in other cell types. Our proposed
Bcl-xL-mediated protection by Akt was confirmed by the study using
Bcl-xL transgenic mice, demonstrating that induced levels of Bcl-xL
protect hepatocytes from Fas- but not TNF-
-mediated
apoptosis. Together, Bcl-xL might be a key
antiapoptotic-NF-
B-responsive gene, mediated by PI3/Akt in
Fas-mediated apoptosis in hepatocytes.
In conclusion, we demonstrated that the PI3K/Akt pathway has a
protective role in death factor-mediated apoptosis, especially by Fas, including the activation of NF-B and induction of Bcl-xL. Activation of PI3K/Akt might be a therapeutic target for several types
of death factor-related liver diseases.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. W. Ogawa (Kobe University School of Medicine, Kobe, Japan) for kindly providing AddnAkt and Dr. J. A. Romashkova (University of North Carolina, Chapel Hill, NC) for kindly providing AdmyrAkt.
![]() |
FOOTNOTES |
---|
This work was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (E. Hatano) and National Institutes of Health Grants GM-41804, DK-34987, and AA-11605 (D. A. Brenner).
Address for reprint requests and other correspondence: D. A. Brenner, CB 7038, 156 Glaxo, Univ. of North Carolina, Chapel Hill, NC 27599 (E-mail: dab{at}med.unc.edu).
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.
Received 5 June 2001; accepted in final form 27 August 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bonnard, M,
Mirtsos C,
Suzuki S,
Graham K,
Huang J,
Ng M,
Itie A,
Wakeham A,
Shahinian A,
Henzel WJ,
Elia AJ,
Shillinglaw W,
Mak TW,
Cao Z,
and
Yeh WC.
Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-B-dependent gene transcription.
EMBO J
19:
4976-4985,
2000
2.
Bradham, CA,
Qian T,
Streetz K,
Trautwein C,
Brenner DA,
and
Lemasters JJ.
The mitochondrial permeability transition is required for tumor necrosis factor -mediated apoptosis and cytochrome c release.
Mol Cell Biol
18:
6353-6364,
1998
3.
Bradham, CA,
Stachlewitz RF,
Gao W,
Qian T,
Jayadev S,
Jenkins G,
Hannun Y,
Lemasters JJ,
Thurman RG,
and
Brenner DA.
Reperfusion after liver transplantation in rats differentially activates the mitogen-activated protein kinases.
Hepatology
25:
1128-1135,
1997[ISI][Medline].
4.
Burow, ME,
Weldon CB,
Melnik LI,
Duong BN,
Collins-Burow BM,
Beckman BS,
and
McLachlan JA.
PI3-K/AKT regulation of NF-B signaling events in suppression of TNF-induced apoptosis.
Biochem Biophys Res Commun
271:
342-345,
2000[ISI][Medline].
5.
Cantley, LC,
and
Neel BG.
New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway.
Proc Natl Acad Sci USA
96:
4240-4245,
1999
6.
Chen, C,
Edelstein LC,
and
Gelinas C.
The Rel/NF-B family directly activates expression of the apoptosis inhibitor Bcl-x(L).
Mol Cell Biol
20:
2687-2695,
2000
7.
Chen, RH,
Chang MC,
Su YH,
Tsai YT,
and
Kuo ML.
Interleukin-6 inhibits transforming growth factor--induced apoptosis through the phosphatidylinositol 3-kinase/Akt and signal transducers and activators of transcription 3 pathways.
J Biol Chem
274:
23013-23019,
1999
8.
Datta, SR,
Brunet A,
and
Greenberg ME.
Cellular survival: a play in three Akts.
Genes Dev
13:
2905-2927,
1999
9.
Datta, SR,
Dudek H,
Tao X,
Masters S,
Fu H,
Gotoh Y,
and
Greenberg ME.
Akt phosphorylation of Bad couples survival signals to the cell-intrinsic death machinery.
Cell
91:
231-241,
1997[ISI][Medline].
10.
De la Coste, A,
Fabre M,
McDonell N,
Porteu A,
Gilgenkrantz H,
Perret C,
Kahn A,
and
Mignon A.
Differential protective effects of Bcl-xL and Bcl-2 on apoptotic liver injury in transgenic mice.
Am J Physiol Gastrointest Liver Physiol
277:
G702-G708,
1999
11.
Delhase, M,
Li N,
and
Karin M.
Kinase regulation in inflammatory response.
Nature
406:
367-368,
2000[ISI][Medline].
12.
Desbarats, J,
and
Newell MK.
Fas engagement accelerates liver regeneration after partial hepatectomy.
Nat Med
6:
920-923,
2000[ISI][Medline].
13.
Di Cristofano, A,
Kotsi P,
Peng YF,
Cordon-Cardo C,
Elkon KB,
and
Pandolfi PP.
Impaired Fas response and autoimmunity in PTEN +/ mice.
Science
285:
2122-2125,
1999
14.
Doi, TS,
Marino MW,
Takahashi T,
Yoshida T,
Sakakura T,
Old LJ,
and
Obata Y.
Absence of tumor necrosis factor rescues RelA-deficient mice from embryonic lethality.
Proc Natl Acad Sci USA
96:
2994-2999,
1999
15.
Fabregat, I,
Herrera B,
Fernandez M,
Alvarez AM,
Sanchez A,
Roncero C,
Ventura JJ,
Valverde AM,
and
Benito M.
Epidermal growth factor impairs the cytochrome c/caspase-3 apoptotic pathway induced by transforming growth factor in rat fetal hepatocytes via a phosphoinositide 3-kinase-dependent pathway.
Hepatology
32:
528-535,
2000[ISI][Medline].
16.
Fujita, E,
Jinbo A,
Matuzaki H,
Konishi H,
Kikkawa U,
and
Momoi T.
Akt phosphorylation site found in human caspase-9 is absent in mouse caspase-9.
Biochem Biophys Res Commun
264:
550-555,
1999[ISI][Medline].
17.
Gallucci, RM,
Simeonova PP,
Toriumi W,
and
Luster MI.
TNF- regulates transforming growth factor-
expression in regenerating murine liver and isolated hepatocytes.
J Immunol
164:
872-878,
2000
18.
Gibson, S,
Tu S,
Oyer R,
Anderson SM,
and
Johnson GL.
Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation.
J Biol Chem
274:
17612-17618,
1999
19.
Hatano, E,
Bennett BL,
Manning AM,
Qian T,
Lemasters JJ,
and
Brenner DA.
NF-B stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-
- and Fas-mediated apoptosis.
Gastroenterology
120:
1251-1262,
2001[ISI][Medline].
20.
Hatano, E,
Bradham CA,
Stark A,
Iimuro Y,
Lemasters JJ,
and
Brenner DA.
The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes.
J Biol Chem
275:
11814-11823,
2000
21.
Hu, WH,
Johnson H,
and
Shu HB.
Activation of NF-B by FADD, Casper, and caspase-8.
J Biol Chem
275:
10838-10844,
2000
22.
Iimuro, Y,
Nishiura T,
Hellerbrand C,
Behrns KE,
Schoonhoven R,
Grisham JW,
and
Brenner DA.
NF-B prevents apoptosis and liver dysfunction during liver regeneration.
J Clin Invest
101:
802-811,
1998
23.
Irmler, M,
Thome M,
Hahne M,
Schneider P,
Hofmann K,
Steiner V,
Bodmer JL,
Schroter M,
Burns K,
Mattmann C,
Rimoldi D,
French LE,
and
Tschopp J.
Inhibition of death receptor signals by cellular FLIP.
Nature
388:
190-195,
1997[ISI][Medline].
24.
Jones, RG,
Parsons M,
Bonnard M,
Chan VS,
Yeh WC,
Woodgett JR,
and
Ohashi PS.
Protein kinase B regulates T lymphocyte survival, nuclear factor B activation, and Bcl-X(L) levels in vivo.
J Exp Med
191:
1721-1734,
2000
25.
Karin, M,
and
Ben-Neriah Y.
Phosphorylation meets ubiquitination: the control of NF-B activity.
Annu Rev Immunol
18:
621-663,
2000[ISI][Medline].
26.
Kotani, K,
Ogawa W,
Hino Y,
Kitamura T,
Ueno H,
Sano W,
Sutherland C,
Granner DK,
and
Kasuga M.
Dominant negative forms of Akt (protein kinase B) and atypical protein kinase C do not prevent insulin inhibition of phosphoenolpyruvate carboxykinase gene transcription.
J Biol Chem
274:
21305-21312,
1999
27.
Leist, M,
Gantner F,
Jilg S,
and
Wendel A.
Activation of the 55 kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release.
J Immunol
154:
1307-1316,
1995
28.
Li, Q,
Van Antwerp D,
Mercurio F,
Lee KF,
and
Verma IM.
Severe liver degeneration in mice lacking the IB kinase 2 gene.
Science
284:
321-325,
1999
29.
Madge, LA,
and
Pober JS.
A phosphatidylinositol 3-kinase/Akt pathway, activated by tumor necrosis factor or interleukin-1, inhibits apoptosis but does not activate NFB in human endothelial cells.
J Biol Chem
275:
15458-15465,
2000
30.
Madrid, LV,
Mayo MW,
Reuther JY,
and
Baldwin AS, Jr.
Akt stimulates the transactivation potential of the RelA/p65 subunit of NF-B through utilization of the I
B kinase and activation of the mitogen activated protein kinase p38.
J Biol Chem
276:
18934-18940,
2001
31.
Madrid, LV,
Wang CY,
Guttridge DC,
Schottelius AJ,
Baldwin AS, Jr,
and
Mayo MW.
Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-B.
Mol Cell Biol
20:
1626-1638,
2000
32.
Moldeus, P,
Hogberg J,
and
Orrenius S.
Isolation and use of liver cells.
Methods Enzymol
52:
60-71,
1978[Medline].
33.
Nagata, S.
Apoptosis by death factor.
Cell
88:
355-365,
1997[ISI][Medline].
34.
Nagata, S,
and
Golstein P.
The Fas death factor.
Science
267:
1449-1456,
1995[ISI][Medline].
35.
Ogasawara, J,
Watanabe-Fukunaga R,
Adachi M,
Matsuzawa A,
Kasugai T,
Kitamura Y,
Itoh N,
Suda T,
and
Nagata S.
Lethal effect of the anti-Fas antibody in mice.
Nature
364:
806-809,
1993[ISI][Medline].
36.
Ozes, ON,
Mayo LD,
Gustin JA,
Pfeffer SR,
Pfeffer LM,
and
Donner DB.
NF-B activation by tumour necrosis factor requires the Akt serine-threonine kinase.
Nature
401:
82-85,
1999[ISI][Medline].
37.
Panka, DJ,
Mano T,
Suhara T,
Walsh K,
and
Mier JW.
Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells.
J Biol Chem
276:
6893-6896,
2001
38.
Rauch, BH,
Weber A,
Braun M,
Zimmermann N,
and
Schror K.
PDGF-induced Akt phosphorylation does not activate NF-B in human vascular smooth muscle cells and fibroblasts.
FEBS Lett
481:
3-7,
2000[ISI][Medline].
39.
Reddy, SA,
Huang JH,
and
Liao WS.
Phosphatidylinositol 3-kinase as a mediator of TNF-induced NF-B activation.
J Immunol
164:
1355-1363,
2000
40.
Refaeli, Y,
Van Parijs L,
London CA,
Tschopp J,
and
Abbas AK.
Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis.
Immunity
8:
615-623,
1998[ISI][Medline].
41.
Roberts, RA,
James NH,
and
Cosulich SC.
The role of protein kinase B and mitogen-activated protein kinase in epidermal growth factor and tumor necrosis factor -mediated rat hepatocyte survival and apoptosis.
Hepatology
31:
420-427,
2000[ISI][Medline].
42.
Romashkova, JA,
and
Makarov SS.
NF-B is a target of AKT in anti-apoptotic PDGF signalling.
Nature
401:
86-90,
1999[ISI][Medline].
43.
Rudolph, D,
Yeh WC,
Wakeham A,
Rudolph B,
Nallainathan D,
Potter J,
Elia AJ,
and
Mak TW.
Severe liver degeneration and lack of NF-B activation in NEMO/IKK
-deficient mice.
Genes Dev
14:
854-862,
2000
44.
Rust, C,
Karnitz LM,
Paya CV,
Moscat J,
Simari RD,
and
Gores GJ.
The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade.
J Biol Chem
275:
20210-20216,
2000
45.
Schwabe, RF,
Bennett BL,
Manning AM,
and
Brenner DA.
Differential role of IB kinase 1 and 2 in primary rat hepatocytes.
Hepatology
33:
81-90,
2001[ISI][Medline].
46.
Sizemore, N,
Leung S,
and
Stark GR.
Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-B p65/RelA subunit.
Mol Cell Biol
19:
4798-4805,
1999
47.
Sodeman, T,
Bronk SF,
Roberts PJ,
Miyoshi H,
and
Gores GJ.
Bile salts mediate hepatocyte apoptosis by increasing cell surface trafficking of Fas.
Am J Physiol Gastrointest Liver Physiol
278:
G992-G999,
2000
48.
Taupin, DR,
Kinoshita K,
and
Podolsky DK.
Intestinal trefoil factor confers colonic epithelial resistance to apoptosis.
Proc Natl Acad Sci USA
97:
799-804,
2000
49.
Van Antwerp, DJ,
Martin SJ,
Kafri T,
Green DR,
and
Verma IM.
Suppression of TNF--induced apoptosis by NF-
B.
Science
274:
787-789,
1996
50.
Wang, CY,
Mayo MW,
and
Baldwin AS, Jr.
TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-B.
Science
274:
784-787,
1996
51.
Wang, CY,
Mayo MW,
Korneluk RG,
Goeddel DV,
and
Baldwin AS, Jr.
NF-B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation.
Science
281:
1680-1683,
1998
52.
Wang, D,
and
Baldwin AS, Jr.
Activation of nuclear factor-B-dependent transcription by tumor necrosis factor-
is mediated through phosphorylation of RelA/p65 on serine 529.
J Biol Chem
273:
29411-29416,
1998
53.
Webster, CR,
and
Anwer MS.
Phosphoinositide 3-kinase, but not mitogen-activated protein kinase, pathway is involved in hepatocyte growth factor-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes.
Hepatology
33:
608-615,
2001[ISI][Medline].
54.
Westwick, JK,
Bielawska AE,
Dbaibo G,
Hannun YA,
and
Brenner DA.
Ceramide activates the stress-activated protein kinases.
J Biol Chem
270:
22689-22692,
1995
55.
Xie, P,
Browning DD,
Hay N,
Mackman N,
and
Ye RD.
Activation of NF-B by bradykinin through a G
(q)- and G
-dependent pathway that involves phosphoinositide 3-kinase and Akt.
J Biol Chem
275:
24907-24914,
2000
56.
Yin, L,
Wu L,
Wesche H,
Arthur CD,
White JM,
Goeddel DV,
and
Schreiber RD.
Defective lymphotoxin- receptor-induced NF-
B transcriptional activity in NIK-deficient mice.
Science
291:
2162-2165,
2001