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B Activation1
Department of Biochemistry and Molecular Biology, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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B by TNF involves the
stimulation of a novel signaling cascade. In this paper we show that
phosphatidylinositol 3-kinase (PI 3-kinase) may play a pivotal role in
TNF-mediated activation of NF-B-dependent genes. Consistent with its
involvement in TNF signaling, PI 3-kinase activities in HepG2 and U937
cells can be stimulated by TNF in a rapid but transient manner through
a mechanism that may involve its association with the insulin receptor
substrate-1. A dominant-negative mutant of the p85 regulatory subunit
of PI 3-kinase, which is a potent inhibitor of PI 3-kinase signaling,
effectively blocked the TNF-induced expression of an NF-B-dependent
reporter gene. Although PI 3-kinase may be required for NF-B
activation, overexpression of its p110 catalytic subunit alone was
unable to induce an NF-B/chloramphenicol acetyltransferase (CAT)
reporter gene. However, when TNF was added to p110-overexpressing
cells, there was a synergistic activation of the NF-B/CAT reporter,
suggesting that other TNF-inducible signals may cooperate with PI
3-kinase to activate NF-B. Consistent with its role in NF-B
activation, inhibition of PI 3-kinase activity by wortmannin or
LY294002 greatly potentiated TNF-induced apoptosis. This
TNF/wortmannin-induced apoptosis was markedly prevented in cells
overexpressing Rel A. Taken together, our results indicate that a PI
3-kinase-regulated step in TNF-signaling is critical for the expression
of NF-B-dependent genes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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B. This has prompted
much investigation into the nature of the signaling mechanisms that are
triggered by TNF to activate NF-B.
The activation of NF-B is a tightly regulated process that involves
its translocation from the cytoplasm to the nucleus where it binds to
cognate DNA sequences (4). NF-B is composed of homo-
and heterodimers of members of the Rel family of transcription factors
and is normally sequestered in the cytoplasm through its interaction
with the I-B (inhibitory of
NF-B)3 family of
inhibitory proteins (4). In response to external stimuli,
I-B proteins undergo rapid phosphorylation on specific serine
residues. Phosphorylation of I-B
on serines 32 and 36 and of
I-Bß on serines 19 and 23 facilitates their ubiquitination on
neighboring lysine residues, thereby targeting these proteins for rapid
degradation by a proteosome (4). Following the degradation
of I-B, NF-B is released and is free to translocate to the
nucleus and to activate target genes. In addition to TNF, IL-1, phorbol
esters, viruses, LPSs, and UV light are potent inducers of NF-B
(4).
TNF-induced signals for the activation of NF-B are first transmitted
by the TNF receptors across the plasma membrane and then relayed
through specific cytoplasmic proteins. One of these cytoplasmic
proteins, TNFR-associated death domain-containing protein (TRADD), is a
protein adaptor that interacts specifically with TNFR type I and is
required for TNF-mediated induction of NF-B and apoptosis
(5). TRADD interacts, in turn, with another adaptor
protein, TRAF2 (TNFR-associated factor 2), which is dedicated to the
activation of NF-B and the c-Jun N-terminal kinase (JNK) pathway
(6). Because IL-1 also employs a member of the TRAF
family, TRAF6, to activate NF-B (7), these TRAFs may be
common mediators in cytokine-mediated NF-B activation.
The recent identification of two protein kinases that induce NF-B
upon overexpression has furthered our understanding of how TNF and IL-1
activate NF-B. One of these kinases, the NF-B-inducing kinase
(NIK), is closely related to the mitogen-activated protein kinase
kinase kinases and physically interacts with TRAF2 (8).
The second protein kinase, I-B kinase (IKK), physically
interacts with both NIK and I-B and phosphorylates I-B in
vitro on serines 32 and 36 (9, 10). Phosphorylation of
these residues is a prerequisite for the signal-induced ubiquitination
and degradation of I-B. IKK may exist as part of a large 500- to
900-kDa complex, which is composed of several other polypeptides
(11, 12). In addition to IKK, inducible phosphorylation
of I-B may, in fact, be the cooperative effort of two other
proteins, IKKß (11, 12) and IKK
(13),
which are also part of the 500- to 900-kDa protein complex. Although
they appear to have significant differences in their biochemical
properties, all three proteins may play an important role in NF-B
and IKK activation and may even form part of a kinase cascade within
the complex. The mechanisms by which IKKs and NIK are activated or
recruited to the TRAFs are not clear and remain to be elucidated.
Furthermore, the relationships of the TRAFs, NIK, and IKKs with other
proteins such as TANK/ITRAF and IL-1R-associated kinase (IRAK) are
important in the TNF (14) and IL-1 (15)
signaling pathways, respectively, and are under investigation. There is
also evidence that protein kinases other than the IKKs, such as
mitogen-activated /extracellular signal-regulated protein kinase kinase
1 (MEKK1), a regulator of the JNK pathway, are also involved
(16). This raises the possibility that the phosphorylation
of I-B in response to various inducers may be orchestrated by a
network of kinases. In addition to I-B, phosphorylation of NF-B
has also been reported (17). Because this phosphorylation
is critical for NF-B function, it would be of great interest to
identify the protein kinases that phosphorylate the relevant sites.
Our recent studies on IL-1 signaling pathways revealed that
phosphatidylinositol 3-kinase (PI 3-kinase) plays a major role in the
ability of IL-1 to activate NF-B and AP-1 (18). In
addition, Sontag et al. (19) have shown that activation of
NF-B by the SV40 small t Ag requires PI 3-kinase and the atypical
protein kinase C 
(PKC) isoform. PI 3-kinase is a lipid kinase
that is composed of two polypeptides, a p85 regulatory subunit, and a
p110 catalytic subunit and is activated by a large spectrum of
cytokines, hormones, and growth factors (20). It has been
implicated as a key signaling molecule in processes as diverse as
glucose transport (21), transcription factor activation
(22), and cell survival (23, 24, 25, 26, 27). Our studies
on IL-1 signal transduction implicating PI 3-kinase in the activation
of NF-B together with the report (28) on TNF-induced
physical interaction between the insulin receptor substrate-1 (IRS-1)
and PI 3-kinase suggest that this enzyme may also participate in
TNF-induced activation of NF-B. In this study, we present evidence
that further supports a role for PI 3-kinase in TNF signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained at 37°C in MEM containing 10% FBS and antibiotics. Wortmannin, insulin, and PI were from Sigma (St. Louis, MO). LY294002 was from Biomol (Plymouth Meeting, PA), IL-1 was obtained from the Biological Response Modifiers Program of the National Cancer Institute (Frederick, MD), and TNF was purchased from PeproTech (Rocky Hill, NJ). Anti-phosphotyrosine PY20 Abs were obtained from Transduction Labs (Lexington, KY). Anti-IRS-1 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Abs against the phosphorylated form of Akt kinase (serine 473) were from New England Biolabs (Beverly, MA).
PI 3-kinase and Akt kinase assays
HepG2 or U937 cells (5 x 105) were seeded in 60-mm culture dishes and cultured overnight before they were serum starved for 4 h and treated with or without 1.4 nM TNF for the indicated times. Whole-cell lysates were prepared in ice-cold lysis buffer containing 50 mM HEPES (pH 7.5), 10% glycerol, 150 mM NaCl, 1% Nonidet P-40, 1.5 mM MgCl2, 1 mM EGTA, 50 mM NaF, 1 mM sodium orthovanadate, 14 mM 2-ME, 0.2 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 3 mM benzamidine. After 20 min on ice, the lysates were clarified by centrifugation at 14,000 x g. The supernatant was then incubated at 4°C with either PY20, anti-IRS-1, or anti-phosphoAkt Abs for 1 h. Immune complexes from extracts immunoprecipitated with PY20 or anti-IRS-1 Abs were then utilized for PI 3-kinase assays (29), and the labeled [32P]PI 3-phosphate was extracted and resolved by thin-layer chromatography (30). Anti-phosphoAkt immune complexes were assayed for Akt kinase activities (25) using myelin basic protein (MBP) as substrate. The kinase incubation mixtures were then resolved on 10% SDS-PAGE gels and phosphorylated MBP was visualized by autoradiography.
EMSA
Cells were treated with TNF and various concentrations of wortmannin. Nuclear extracts were prepared from treated cells and were incubated with a radiolabeled probe (2 x 104 cpm) as described (18). Protein-DNA complexes were resolved on 5% polyacrylamide gels and visualized by autoradiography.
Transient transfection reporter assays
HepG2 (5 x 105) cells were plated in
100-mm dishes and transfected the next day with the
(NF-B)3/ chloramphenicol acetyltransferase
(CAT) reporter gene and various expression vectors (18).
Transfections were done using the Polybrene method (31).
Sixteen hours after transfection, cells were treated with cytokines or
left untreated for 20 h. All other procedures were performed as
described (31).
Apoptosis assays
U937 cells were treated for 90 min with 1.4 nM TNF or IL-1 in the presence of either 100 nM wortmannin or 10 µg/ml cycloheximide. Whereas cycloheximide was added at the same time as TNF or IL-1, wortmannin was added 45 min before the cytokines. In those experiments that required pretreatment, cells were incubated with 1.4 nM IL-1 or 1 µM insulin for 5 h with or without the inhibitors and then were treated with TNF plus either cycloheximide or wortmannin. After treatment for the indicated time periods, cells were immediately chilled on ice, harvested, and used for DNA (for DNA fragmentation analysis) or protein (for immunodetection of poly(ADP-ribose) polymerase (PARP)) extraction. For DNA extraction, cells were lysed in buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 25 mM EDTA, and 0.5% SDS and incubated at 50°C for 10 min. Lysates were then incubated with ribonuclease at 37°C for 1 h, and then freshly prepared proteinase K (3 mg/ml) was added to digest protein at 37°C for 16 h. Fragmented DNA was resolved on 1.8% agarose gels at 35 V for 8 h and visualized by ethidium bromide staining. To detect PARP, nuclear extracts were prepared as described for EMSA, boiled in Laemmli buffer, and resolved on 7% SDS-polyacrylamide gels. Protein was transferred overnight to nitrocellulose membranes at 4°C. These membranes were then blocked with 5% nonfat dry milk before being incubated with anti-PARP mAb (PharMingen, San Diego, CA) for 60 min and then with enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Immunofluorescence microscopy
Green fluorescent protein (GFP) expression vector was transfected (Fugene 6, Boehringer Mannheim, Indianapolis, IN) into U937 cells with expression vector for p65 Rel A or with empty vector control at a ratio of 1:3. After 20 h, cells were treated with various combinations of TNF, cycloheximide, or wortmannin. Samples were then coded and processed in a double-blinded fashion. After treatment, cells were harvested, stained with Hoechst 33342, and observed under a fluorescent microscope. Photographs of GFP and Hoechst-stained cells were taken of randomly selected fields in each treatment. For statistical analysis, the total number of GFP-expressing cells were counted along with the number of GFP-positive cells that were nonapoptotic as determined by Hoechst staining. Results from three independent experiments were used to estimate the cell viability and to calculate the SEs.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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In our previous studies on the signaling pathways triggered by
IL-1, we found that PI 3-kinase was a necessary component of
IL-1-induced NF-B activation (18). Because TNF is the
other major cytokine that can potently activate NF-B, we sought to
evaluate the possibility that PI 3-kinase might also be an integral
part of TNF signaling. We first examined whether PI
3-kinase activity and its downstream target, Akt kinase, could be
induced in response to TNF stimulation. HepG2 cells were treated with
TNF for various periods of time and used to prepare whole cell extracts
for immunoprecipitation of PI 3-kinase and phosphorylated Akt kinase
activities. Two different Abs were used to immunoprecipitate PI
3-kinase activity from these extracts: 1) anti-phosphotyrosine Abs
(PY20), which are routinely used to detect changes in PI
3-kinase activity after treatment with various agents (see references
in Ref. 20), and 2) anti-IRS-1 Abs, because IRS-1 has
been reported to interact with PI 3-kinase upon TNF stimulation
(28). Abs against phosphorylated Akt (serine 473) were
used to immunoprecipitate Akt kinase activity. As shown in Fig. 1
A, there was a rapid but
transient increase in PI 3-kinase activity after treatment with TNF.
Within 0.5 min there was an 
3-fold activation of the enzyme, and
maximum activation (4- to 7-fold) was observed within 1 min of
stimulation. Although the extent of PI 3-kinase activation detected
with anti-IRS-1 Abs was lower than that observed with PY20, the
kinetics of activation observed with either Ab were nearly identical.
To ensure that the induction of PI 3-kinase activity by TNF is not
unique to HepG2 cells, U937 cells were also stimulated with TNF and the
effect on PI 3-kinase activity was determined. Indeed, addition of TNF
also stimulated PI 3-kinase activity in U937 cells with induction
kinetics similar to those in HepG2 cells (Fig. 1A).
Consistent with its function in the PI 3-kinase signaling cascade, Akt
kinase activity was also increased (3-fold in 20 min; Fig. 1B) in response to TNF. The magnitude and kinetics of
induction of Akt kinase by TNF are comparable to those observed for
insulin (30). This further supports a functional role of
the PI 3-kinase pathway in TNF signal transduction.
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B. Therefore, it is likely that at least one of these signaling
proteins, such as the TNF receptors, interacts indirectly with PI
3-kinase through IRS-1.
PI 3-kinase is not involved in the activation of
DNA-binding activity of NF-B by TNF
To identify the role of PI 3-kinase in TNF signaling, we used
various inhibitors to see whether they would block the activation of
known transcription factors by TNF. First, we tested the effect of
wortmannin, a selective PI 3-kinase inhibitor, on the ability of TNF to
increase the DNA-binding activity of NF-B. This activity of TNF is
based on its ability to induce the degradation of the NF-B
inhibitor, I-B, and is recognized as a critical step before the
transcription factor can be translocated to the nucleus. Wortmannin had
little or no effect on TNF-induced NF-B-binding activity (Fig. 1
C) even at concentrations of 400 nM, suggesting that PI
3-kinase is not involved in the signaling pathway leading to the
degradation of I-B.
PI 3-kinase is required for the activation of NF-B-dependent
reporter gene
Because I-B degradation alone does not fully account for the
functional activation of NF-B-dependent genes (17), we
used transient cotransfection assays to examine whether PI 3-kinase was
necessary to transactivate NF-B-dependent reporter gene expression.
An (NF-B)3/CAT reporter gene that contained
three copies of the NF-B-binding site was cotransfected into HepG2
cells with an expression vector encoding either the dominant-negative
mutant of the p85 subunit of PI 3-kinase (p85DN) (21) or
the catalytic p110 subunit (20). The dominant-negative
mutant p85DN is identical with the wild-type p85 except that it lacks
the sequence essential for its association with the p110 subunit
(21). Consequently, p85DN is incapable of binding to p110,
though it still can compete with wild-type p85 for specific PI
3-kinase-binding sites. This means that, relative to wild-type p85,
p85DN would be competitively recruited by various PI 3-kinase-binding
proteins such as IRS-1 but would be defective in transmitting signals
downstream. As such, p85DN has been used frequently to demonstrate a
role for PI 3-kinase in various cellular processes (21, 32) including SV40 small t Ag-induced NF-B activation
(19).
Because wortmannin had not blocked the activation of NF-B-binding
activity, it was surprising when overexpression of p85DN in HepG2 cells
inhibited the TNF-mediated induction of the
(NF-B)3/CAT reporter gene by about 80% (Fig. 2A). These differential
effects of PI 3-kinase inhibitors suggest that NF-B is regulated at
different steps through parallel signaling pathways. One pathway leads
to the degradation of I-B and the other may lead to an increase in
the transactivation potential of NF-B. Therefore, the effect of
p85DN implicates PI 3-kinase in TNF-induced transactivation of
NF-B-dependent genes. The inhibition was dose-dependent and at a
level comparable to that observed with IL-1. These inhibitory effects
were deemed to be specific because in control experiments p85DN
had no effect on the induction of an estrogen-responsive reporter gene
by estradiol (Fig. 2B) or on the induction of a T1 kininogen
gene by the combination of IL-6 and dexamethasone
(18).
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B-dependent gene
expression by TNF and the p110 catalytic subunit of PI 3-kinase
Because PI 3-kinase appears to be necessary for TNF to induce
NF-B, we examined the possibility that overexpression of the
catalytic subunit of this enzyme would activate NF-B-dependent gene
expression. However, cotransfection of the
(NF-B)3/CAT reporter gene with p110 did not
result in its induction (Fig. 2C). This is consistent with
the fact that several inducers of PI 3-kinase, such as hepatocyte
growth factor and erythropoietin, are unable to activate NF-B.
We next investigated the effect of TNF on p110-overexpressing cells
because we had previously observed synergism between IL-1 and PI
3-kinase (18). When p110-overexpressing cells were
stimulated with TNF, there was a dramatic induction of the
(NF-B)3/CAT reporter gene (Fig. 2C), signifying synergism between PI 3-kinase and TNF. To
verify that this effect was mediated specifically through NF-B, we
cotransfected I-B-expression plasmids with the CAT reporter gene.
As shown in Fig. 2C, the induction of the CAT reporter by
TNF and by the combination of TNF and p110 were both inhibited by the
coexpression of I-B.
That PI 3-kinase was apparently required but insufficient to activate
NF-B suggests that PI 3-kinase may need to cooperate with another
TNF-inducible signal(s) to activate NF-B. This notion is
consistent with our observation that p110 synergized with TNF to
induce NF-B-dependent gene expression. This mechanism of NF-B
activation may be similar for both TNF and IL-1 because p110 synergizes
with IL-1 and may involve the cooperation ofp110 with IRAK
(18). It is presently unclear whether PI 3-kinase would
cooperate with any of the known transducers in the TNF signaling
pathway.
Wortmannin and LY294002 can potentiate TNF-induced apoptosis
Upon binding to its receptor, TNF initiates two very different
signals that diverge downstream of the TNF receptor-interacting protein
TRADD (5). One signal leads to the activation of NF-B,
whereas the other initiates apoptosis or programmed cell death.
TNF-induced apoptosis is potentiated in the presence of cycloheximide,
presumably due to inhibition of de novo synthesis of TNF-induced gene
products that function to protect cells from programmed cell death
(5). It has been demonstrated recently that these
antiapoptotic genes are downstream targets of NF-B; that is,
constitutive activation of NF-B can protect cells from apoptosis
while inhibition of its activation potentiates TNF-induced apoptosis
(5, 33, 34, 35).
Because we showed that TNF-mediated activation of NF-B-dependent
reporter gene requires PI 3-kinase, we sought to examine the effects of
wortmannin treatment on TNF-induced apoptosis through its inhibition of
NF-B transactivation activity. U937 cells were treated with TNF
alone or in combination with wortmannin or LY294002. To monitor the
extent of apoptosis, DNA fragmentation assays and immunodetection of
the apoptosis-induced cleavage of PARP were performed on treated cells.
As controls, U937 cells were treated with the combination of TNF and
cycloheximide. Consistent with earlier reports, neither TNF nor
cycloheximide alone could induce apoptosis (Fig. 3, A and B,
lanes 2 and 3), whereas treatment with TNF plus
cycloheximide resulted in extensive DNA fragmentation and PARP cleavage
(Fig. 3, A and B, lane 4). When
wortmannin was added together with TNF, it also strongly potentiated
TNF-induced apoptosis to a level comparable to that observed with
cycloheximide (Fig. 3, A and B, lanes
4 and 5). This is consistent with the ability of TNF to
activate PI 3-kinase in these cells (Fig. 1A). No apoptosis
could be detected in control cells that were treated with wortmannin
alone. LY294002, a more specific PI 3-kinase inhibitor than wortmannin,
also potentiated TNF-induced PARP degradation (Fig. 3B).
This data indicates that both inhibitors can potentiate TNF-mediated
apoptosis by specific inhibition of PI 3-kinase activities.
Furthermore, IL-1, which is not known to induce apoptosis, had no
effect either alone or in combination with cycloheximide or wortmannin
in these experiments. Therefore, these data are consistent with the
involvement of PI 3-kinase in TNF-induced activation of NF-B
function. It is noteworthy that although cycloheximide, wortmannin, and
LY294002 can potentiate the ability of TNF to induce apoptosis, their
sites of action differ: whereas cycloheximide blocks events downstream
of the activation of NF-B-dependent genes, wortmannin and LY294002
inhibit signaling events that are upstream to the activation of those
genes.
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B (35). Therefore, we examined whether
pretreatment with IL-1 could also protect cells from
TNF/wortmannin-induced apoptosis. Because both IL-1 and TNF can induce
PI 3-kinase activity (Ref. 18 and Fig. 1) and PI 3-kinase
activity has been shown to protect cells from serum withdrawal-, UV-,
and myc-induced apoptosis (23, 24, 25, 26, 27), we included
insulin pretreatment as a control to differentiate the NF-B effects
from those of PI 3-kinase. Cells pretreated for 5 h with IL-1 were
effectively protected from subsequent TNF/cycloheximide- or
TNF/wortmannin-induced apoptosis (Fig. 3C, lanes
2 and 5). The protection conferred by IL-1 could be
blocked by the simultaneous treatment with wortmannin to inhibit the
IL-1 signaling pathway (Fig. 3C, lanes 3 and
4). At the concentrations employed, wortmannin alone did not
induce apoptosis even after 7 h (see below). However, pretreatment
with insulin did not confer any protection (Fig. 3C,
lanes 6 and 7) despite its ability to activate PI
3-kinase in these cells (36) and to protect PC-12 cells
from serum withdrawal-induced apoptosis (23). This
suggests that PI 3-kinase-dependent survival signals, although
sufficient to protect cells from serum withdrawal- and UV-induced
apoptosis, may not be sufficient to protect cells from TNF-induced
apoptosis. Because both IL-1 and insulin activate PI 3-kinase but only
IL-1 can also activate NF-B, these data also suggest that apoptosis
induced in cells by treatment with TNF and wortmannin is due to
inhibition of NF-B activation. Furthermore, the involvement of PI
3-kinase in NF-B activation is also supported by the observation
that wortmannin can inhibit the protection conferred by IL-1 (Fig. 3C). To provide further evidence that potentiation of
TNF-induced apoptosis by wortmannin is due mainly to inhibition of
NF-B rather than to the inhibition of other PI 3-kinase-associated
survival pathways, we examined whether overexpression of NF-B could
protect cells from apoptosis induced by the combination of TNF and
wortmannin. U937 cells were transfected with an expression vector for
p65 Rel A or with an empty vector. To mark the transfected cells,
GFP-expression vector was cotransfected with Rel A vector or the empty
vector control at a ratio of 1:3. Approximately 20 h after
transfection, cells were treated with various combinations of TNF,
cycloheximide, and wortmannin, and the extent of apoptosis was analyzed
after Hoechst staining. Treatment of U937 cells with TNF,
cycloheximide, or wortmannin individually had minimal effects on cell
survival even after 5–7 h of treatment (Fig. 4A, panels 5–12
and panels 17–20, and B). In contrast, the
combination of TNF and cycloheximide reduced cell viability to almost
12% (Fig. 4B). However, overexpression of p65/Rel A in
these cells significantly increased the viability to 26%. These
results are consistent with earlier reports that cycloheximide
potentiates TNF-induced apoptosis by blocking NF-B-inducible gene
expression. In our case, we also observed protection from the combined
effects of TNF and cycloheximide in p65/Rel A-transfected cells, but
not in control cells.
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), it potentiated TNF-induced apoptosis resulting in
reduced cell viability to about 35% (Fig. 4A,
panels 21 and 22, and B). However,
when these cells were transfected with p65/Rel A, they showed
considerable resistance to apoptosis induced by this combination of TNF
and wortmannin (Fig. 4A, panels 23 and
24, and B). The protection from
TNF/cycloheximide- and TNF/wortmannin-mediated apoptosis by p65/Rel A
are highly significant, as indicated by their p values. If
NF-B activation in the TNF signaling pathway had not been interfered
with, overexpression of p65/Rel A would not have been sufficient to
protect these cells from apoptosis (33, 34, 35). Therefore,
the fact that p65/Rel A overexpression can protect cells from
TNF/wortmannin-induced apoptosis suggested that wortmannin potentiates
TNF-induced apoptosis largely by inhibiting NF-B activity.
Therefore, taken together, our data are consistent with the involvement
of PI 3-kinase in TNF-induced NF-B activation. However, we cannot
completely rule out a significant role for PI 3-kinase-mediated,
NF-B-independent survival pathways under our experimental
conditions.
We have used a dominant-negative mutant of p85 PI 3-kinase to assess
the involvement of PI 3-kinase in TNF-mediated activation of NF-B.
In this paper and in a previous report (18), we have
provided evidence that this enzyme may be common to the IL-1 and TNF
pathways leading to the activation of NF-B, albeit may be through
different mechanisms. We have also shown here that PI 3-kinase alone is
insufficient to activate NF-B but that it may interact with other
mediators of TNF signal transduction because it greatly synergized with
TNF. The other mediators of TNF signal transduction that PI 3-kinase
interacts with to activate NF-B are uncertain, but such interactions
appear to be essential. Therefore, this is reminiscent of the IL-1
signal-transduction pathway in which NF-B activation may be
dependent, at least in part, on the interaction between PI 3-kinase and
the serine/threonine protein kinase IRAK (18). Whether the
synergism between PI 3-kinase and IRAK is mediated by common mediators
of the IL-1 and TNF signaling pathways, such as NIK, or by the IKK
kinase complex remains to be determined. Functional interaction of PI
3-kinase with specific TNF-inducible signals may contribute to
superactivate downstream signaling proteins, to target them
appropriately to subcellular compartments, or to relieve tight negative
regulation.
PI 3-kinase may not, in fact, participate in any linear pathway that
leads to NF-B activation in as much as several known PI 3-kinase
activators such as HGF cannot induce NF-B. Given that overexpression
of proteins such as TRAF2 and TRAF6 can activate NF-B, it would
appear possible that a linear pathway for NF-B activation may be
functional in the absence of PI 3-kinase. However, without PI 3-kinase,
the kinetics and/or the extent of NF-B activation or transactivation
potential may be severely compromised. We favor the last
possibility, because wortmannin only minimally inhibited (if at
all) the DNA-binding activity of TNF-induced NFB in gel-mobility
shift assays, whereas expression of the dominant-negative inhibitor
p85DN blocked TNF-induced NF-B-dependent gene expression by about
80%. The inhibitory effects of p85DN on the induction of
NF-B-dependent genes by TNF suggest that PI 3-kinase may be involved
in a step or a series of steps that regulate the transactivation
potential of NF-B but not its DNA-binding. Our data suggest that
while PI 3-kinase may participate in modulating increases in the
transactivation potential of NF-B, it appears not to be involved in
TNF-induced I-B degradation. A similar role for PI 3-kinase has been
postulated in the activation of STAT3 (22). Tyrosine
phosphorylation of STAT3 is sufficient for a profound activation of
this transcription factor; however, phosphorylation of STAT3 on a
specific serine residue mediated by PI 3-kinase is necessary for it to
achieve full activity.
Two recent reports by Ozes et al. (37) and Romashkova and
Makarov (38) have implicated PI 3-kinase/Akt kinase
signaling pathway in NF-B activation. These studies showed that both
platelet-derived growth factor and TNF-mediated NF-B activation
involve the activation of Akt kinase and its interaction with the IKKs.
However, they disagree on whether TNF-induced NF-B activation
involves the activation of PI 3-kinase. Although Romashkova and Makarov
(38) failed to observe any inhibitory effects of
wortmannin on the induction of a NF-B reporter gene by TNF, Ozes et
al. (37) reported that wortmannin, a dominant-negative p85
PI 3-kinase mutant, and a kinase deficient Akt kinase are all able to
abrogate TNF-induced activation of NF-B. Although our data indicate
a role for the PI 3-kinase pathway in TNF-mediated NF-B activation,
there are mechanistic differences between the model we propose and that
suggested by the observation of Ozes et al. (37). Indeed,
because we did not observe any inhibitory effects of wortmannin on the
ability of TNF to increase the DNA-binding activities of NF-B, our
data would suggest that PI 3-kinase may be dispensable for I-B
degradation. These differences, while they cannot be fully explained,
may suggest that PI 3-kinase affects NF-B activation in more than
one way and that the nature of its effects may vary in a cell
type-specific manner.
The mechanism of action of PI 3-kinase in NF-B activation may also
involve its phosphorylated lipid products. These lipid mediators may
regulate the activity of protein kinases such as PKC
(39), an atypical PKC isoform that has been implicated in
NF-B activation (40). These protein kinases may then
regulate signaling proteins involved in TNF signal transduction. This
possibility is also supported by recent observations that SV40 small t
Ag-mediated NF-B activation is dependent on both PKC and PI
3-kinase (19). These studies also indicated that PKC is
a downstream target of PI 3-kinase in vivo. The small G protein Cdc42
(41) and the protein kinase MEKK1 (16) are
some of the other proteins implicated in TNF-induced activation of
NF-B. Because physical interaction between Cdc42 and PI 3-kinase
(42) may result in the stimulation of PI 3-kinase activity
(43), it remains to be seen whether these interactions are
important for the activation of NF-B. MEKK1, a key participant in
the activation of JNK, has been implicated in the activation of the
700-kDa I-B kinase complex (16). A signaling link
between MEKK1 and PI 3-kinase seems possible because overexpression of
PI 3-kinase results in JNK activation (44). To understand
the nature of the involvement of PI 3-kinase in NF-B activation, it
is important to identify its relationship with the other signaling
molecules recruited by TNF.
|
Acknowledgments |
|---|
expression vector; C. Reynolds and the Biological Response Modifiers
Program, National Cancer Institute, for IL-1ß; and P. J. Chiao
for the I-B expression vector. We are grateful to Karen Hensley
for help with the figures and to Zhanyong Bing for help with
statistical analysis. We are also very thankful to Drs. Athula
Wikramanayake and Masahiro Higuchi for help with the immunofluorescence
experiments. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Warren Liao, Department of Biochemistry and Molecular Biology, Box 117, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030. E-mail address:
3 Abbreviations used in this paper: I-B, inhibitor of NF-B; TRAF2, TNFR-associated factor 2; JNK, c-Jun N-terminal kinase; NIK, NF-B-inducing kinase; MEKK1, mitogen-activated/extracellular signal-regulated protein kinase kinase 1; PI 3-kinase, phosphatidylinositol 3-kinase; IRS-1, insulin receptor substrate-1; MBP, myelin basic protein; CAT, chloramphenicol acetyltransferase; PARP, poly(ADP-ribose) polymerase; GFP, green fluorescent protein; IRAK, IL-1R-associated kinase; PKC, protein kinase C ; IKK, I-B kinase .
Received for publication April 22, 1999. Accepted for publication November 10, 1999.
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References |
|---|
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
B and IB proteins:
new discoveries and insights. Annu. Rev. Immunol. 14:649.
B activation prevents cell death. Cell 87:565.[Medline]
B induction by TNF, CD95 and IL-1. Nature 385:540.[Medline]
B kinase. Cell 90:373.[Medline]
B kinase that activates the transcription factor NFB. Nature 388:548.[Medline]
B kinases essential for NFB activation. Science 278:860.B kinase complex (IKK) contains two kinase subunits, IKK and IKKß, necessary for IB phosphorylation and NFB activation. Cell 91:243.[Medline]
is an essential regulatory subunit of the IB kinase complex. Nature 395:297.[Medline]
B activation. Genes Dev. 10:963.B kinase complex by MEKK1, a kinase of the JNK pathway. Cell 88:213.[Medline]
B-dependent transcription by tumor necrosis factor- is mediated through phosphorylation of RelA/p65 on serine 529. J. Biol. Chem. 273:29411.B and AP-1 activation. J. Biol. Chem. 272:29167. signaling targeted by SV40 small t to promote cell growth and NFB activation. EMBO J. 16:5662.[Medline]
B in preventing TNF--induced cell death. Science 274:782.-induced apoptosis by NFB. Science 274:787.B. Science 274:784.B activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401:82.[Medline]
B is a target of AKT in anti-apoptotic PDGF signalling. Nature 401:86.[Medline]
isozyme of protein kinase C by phosphatidylinositol 3,4,5,-triphosphate. J. Biol. Chem. 268:13. subspecies blocks NFB activation. Mol. Cell. Biol. 13:4770.B by Rho, CDC42 and Rac-1 proteins. Genes Dev. 11:463.This article has been cited by other articles:
|
|
 |
|
  R. P. Cherla, S.-Y. Lee, R. A. Mulder, M.-S. Lee, and V. L. Tesh Shiga Toxin 1-Induced Proinflammatory Cytokine Production Is Regulated by the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin Signaling Pathway Infect. Immun., September 1, 2009; 77(9): 3919 - 3931. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Lasbury, P. J. Durant, C.-P. Liao, and C.-H. Lee Effects of Decreased Calmodulin Protein on the Survival Mechanisms of Alveolar Macrophages during Pneumocystis Pneumonia Infect. Immun., August 1, 2009; 77(8): 3344 - 3354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Pincheira, A. F. Castro, O. N. Ozes, P. S. Idumalla, and D. B. Donner Type 1 TNF Receptor Forms a Complex with and Uses Jak2 and c-Src to Selectively Engage Signaling Pathways That Regulate Transcription Factor Activity J. Immunol., July 15, 2008; 181(2): 1288 - 1298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Cheung, V. Ravyn, L. Wang, A. Ptasznik, and R. G. Collman Signaling Mechanism of HIV-1 gp120 and Virion-Induced IL-1{beta} Release in Primary Human Macrophages J. Immunol., May 15, 2008; 180(10): 6675 - 6684. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Scortegagna, C. Cataisson, R. J. Martin, D. J. Hicklin, R. D. Schreiber, S. H. Yuspa, and J. M. Arbeit HIF-1{alpha} regulates epithelial inflammation by cell autonomous NF{kappa}B activation and paracrine stromal remodeling Blood, April 1, 2008; 111(7): 3343 - 3354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kawamata, T. Imamura, J. L. Babendure, J.-C. Lu, T. Yoshizaki, and J. M. Olefsky Tumor Necrosis Factor Receptor-1 Can Function through a G{alpha}q/11-beta-Arrestin-1 Signaling Complex J. Biol. Chem., September 28, 2007; 282(39): 28549 - 28556. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Chen, S. Xiong, H. She, S. W. Lin, J. Wang, and H. Tsukamoto Iron Causes Interactions of TAK1, p21ras, and Phosphatidylinositol 3-Kinase in Caveolae to Activate I{kappa}B Kinase in Hepatic Macrophages J. Biol. Chem., February 23, 2007; 282(8): 5582 - 5588. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Nanua, S. M. Zick, J. E. Andrade, U. S. Sajjan, J. R. Burgess, N. W. Lukacs, and M. B. Hershenson Quercetin Blocks Airway Epithelial Cell Chemokine Expression Am. J. Respir. Cell Mol. Biol., November 1, 2006; 35(5): 602 - 610. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bernier, Y.-K. Kwon, S. K. Pandey, T.-N. Zhu, R.-J. Zhao, A. Maciuk, H.-J. He, R. DeCabo, and S. Kole Binding of Manumycin A Inhibits I{kappa}B Kinase beta Activity J. Biol. Chem., February 3, 2006; 281(5): 2551 - 2561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. B. Kerbauy, V. Lesnikov, N. Abbasi, S. Seal, B. Scott, and H. J. Deeg NF-{kappa}B and FLIP in arsenic trioxide (ATO)-induced apoptosis in myelodysplastic syndromes (MDSs) Blood, December 1, 2005; 106(12): 3917 - 3925. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-C. Huang and C.-C. Chen Akt Phosphorylation of p300 at Ser-1834 Is Essential for Its Histone Acetyltransferase and Transcriptional Activity Mol. Cell. Biol., August 1, 2005; 25(15): 6592 - 6602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Puri, T. A. Doggett, C.-Y. Huang, J. Douangpanya, J. S. Hayflick, M. Turner, J. Penninger, and T. G. Diacovo The role of endothelial PI3K{gamma} activity in neutrophil trafficking Blood, July 1, 2005; 106(1): 150 - 157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-M. Bian, S. G. Elner, A. Yoshida, and V. M. Elner Differential Involvement of Phosphoinositide 3-Kinase/Akt in Human RPE MCP-1 and IL-8 Expression Invest. Ophthalmol. Vis. Sci., June 1, 2004; 45(6): 1887 - 1896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Strassheim, K. Asehnoune, J.-S. Park, J.-Y. Kim, Q. He, D. Richter, K. Kuhn, S. Mitra, and E. Abraham Phosphoinositide 3-Kinase and Akt Occupy Central Roles in Inflammatory Responses of Toll-Like Receptor 2-Stimulated Neutrophils J. Immunol., May 1, 2004; 172(9): 5727 - 5733. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Puri, T. A. Doggett, J. Douangpanya, Y. Hou, W. T. Tino, T. Wilson, T. Graf, E. Clayton, M. Turner, J. S. Hayflick, et al. Mechanisms and implications of phosphoinositide 3-kinase {delta} in promoting neutrophil trafficking into inflamed tissue Blood, May 1, 2004; 103(9): 3448 - 3456. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Kasper, E. Brandt, S. Bulfone-Paus, and F. Petersen Platelet factor 4 (PF-4)-induced neutrophil adhesion is controlled by src-kinases, whereas PF-4-mediated exocytosis requires the additional activation of p38 MAP kinase and phosphatidylinositol 3-kinase Blood, March 1, 2004; 103(5): 1602 - 1610. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Y. Lee, J. Ye, Z. Gao, H. S. Youn, W. H. Lee, L. Zhao, N. Sizemore, and D. H. Hwang Reciprocal Modulation of Toll-like Receptor-4 Signaling Pathways Involving MyD88 and Phosphatidylinositol 3-Kinase/AKT by Saturated and Polyunsaturated Fatty Acids J. Biol. Chem., September 26, 2003; 278(39): 37041 - 37051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Okugawa, Y. Ota, T. Kitazawa, K. Nakayama, S. Yanagimoto, K. Tsukada, M. Kawada, and S. Kimura Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages Am J Physiol Cell Physiol, August 1, 2003; 285(2): C399 - C408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Mayo, C. E. Denlinger, R. M. Broad, F. Yeung, E. T. Reilly, Y. Shi, and D. R. Jones Ineffectiveness of Histone Deacetylase Inhibitors to Induce Apoptosis Involves the Transcriptional Activation of NF-{kappa}B through the Akt Pathway J. Biol. Chem., May 23, 2003; 278(21): 18980 - 18989. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Osawa, M. Nagaki, Y. Banno, D. A. Brenner, Y. Nozawa, H. Moriwaki, and S. Nakashima Expression of the NF-{kappa}B Target Gene X-Ray-Inducible Immediate Early Response Factor-1 Short Enhances TNF-{alpha}-Induced Hepatocyte Apoptosis by Inhibiting Akt Activation J. Immunol., April 15, 2003; 170(8): 4053 - 4060. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Albanese, K. Wu, M. D'Amico, C. Jarrett, D. Joyce, J. Hughes, J. Hulit, T. Sakamaki, M. Fu, A. Ben-Ze'ev, et al. IKKalpha Regulates Mitogenic Signaling through Transcriptional Induction of Cyclin D1 via Tcf Mol. Biol. Cell, February 1, 2003; 14(2): 585 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. W. Dawson, G. Tramountanis, A. G. Eliopoulos, and L. S. Young Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) Activates the Phosphatidylinositol 3-Kinase/Akt Pathway to Promote Cell Survival and Induce Actin Filament Remodeling J. Biol. Chem., January 31, 2003; 278(6): 3694 - 3704. [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]
|
|
|
|
|
|
M. Guha and N. Mackman The Phosphatidylinositol 3-Kinase-Akt Pathway Limits Lipopolysaccharide Activation of Signaling Pathways and Expression of Inflammatory Mediators in Human Monocytic Cells J. Biol. Chem., August 23, 2002; 277(35): 32124 - 32132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Meng, L. Liu, P. C. Chin, and S. R. D'Mello Akt Is a Downstream Target of NF-kappa B J. Biol. Chem., August 9, 2002; 277(33): 29674 - 29680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-C. Chen, W.-T. Wu, F.-M. Ho, and W.-W. Lin Inhibition of Interleukin-1beta -induced NF-kappa B Activation by Calcium/Calmodulin-dependent Protein Kinase Kinase Occurs through Akt Activation Associated with Interleukin-1 Receptor-associated Kinase Phosphorylation and Uncoupling of MyD88 J. Biol. Chem., June 28, 2002; 277(27): 24169 - 24179. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Gronning, A. Cederberg, N. Miura, S. Enerback, and K. Tasken Insulin and TNF{alpha} Induce Expression of the Forkhead Transcription Factor Gene Foxc2 in 3T3-L1 Adipocytes via PI3K and ERK 1/2-Dependent Pathways Mol. Endocrinol., April 1, 2002; 16(4): 873 - 883. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Mayo, L. V. Madrid, S. D. Westerheide, D. R. Jones, X.-J. Yuan, A. S. Baldwin Jr., and Y. E. Whang PTEN Blocks Tumor Necrosis Factor-induced NF-kappa B-dependent Transcription by Inhibiting the Transactivation Potential of the p65 Subunit J. Biol. Chem., March 22, 2002; 277(13): 11116 - 11125. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Pandey, H.-J. He, A. Chesley, M. Juhaszova, M. T. Crow, and M. Bernier Wortmannin-Sensitive Pathway Is Required for Insulin-Stimulated Phosphorylation of Inhibitor {kappa}B{alpha} Endocrinology, February 1, 2002; 143(2): 375 - 385. [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]
|
|
|
|
|
|
H.-K. Yum, J. Arcaroli, J. Kupfner, R. Shenkar, J. M. Penninger, T. Sasaki, K.-Y. Yang, J. S. Park, and E. Abraham Involvement of Phosphoinositide 3-Kinases in Neutrophil Activation and the Development of Acute Lung Injury J. Immunol., December 1, 2001; 167(11): 6601 - 6608. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Garcia-Garcia, G. Sanchez-Mejorada, and C. Rosales Phosphatidylinositol 3-kinase and ERK are required for NF-{kappa}B activation but not for phagocytosis J. Leukoc. Biol., October 1, 2001; 70(4): 649 - 658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Liu, H. Perlman, L. J. Pagliari, and R. M. Pope Constitutively Activated Akt-1 Is Vital for the Survival of Human Monocyte-Differentiated Macrophages: Role of Mcl-1, Independent of Nuclear Factor (Nf)-{kappa}b, Bad, or Caspase Activation J. Exp. Med., July 16, 2001; 194(2): 113 - 126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Johnson, X. Wang, X.-L. Ma, S.-M. Huong, and E.-S. Huang Human Cytomegalovirus Up-Regulates the Phosphatidylinositol 3-Kinase (PI3-K) Pathway: Inhibition of PI3-K Activity Inhibits Viral Replication and Virus-Induced Signaling J. Virol., July 1, 2001; 75(13): 6022 - 6032. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Osawa, Y. Banno, M. Nagaki, D. A. Brenner, T. Naiki, Y. Nozawa, S. Nakashima, and H. Moriwaki TNF-{{alpha}}-Induced Sphingosine 1-Phosphate Inhibits Apoptosis Through a Phosphatidylinositol 3-Kinase/Akt Pathway in Human Hepatocytes J. Immunol., July 1, 2001; 167(1): 173 - 180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Bone and N. A. Williams Antigen-receptor cross-linking and lipopolysaccharide trigger distinct phosphoinositide 3-kinase-dependent pathways to NF-{{kappa}}B activation in primary B cells Int. Immunol., June 1, 2001; 13(6): 807 - 816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Diem, R. Meyer, J. H. Weishaupt, and M. Bahr Reduction of Potassium Currents and Phosphatidylinositol 3-Kinase-Dependent Akt Phosphorylation by Tumor Necrosis Factor-{alpha} Rescues Axotomized Retinal Ganglion Cells from Retrograde Cell Death In Vivo J. Neurosci., March 15, 2001; 21(6): 2058 - 2066. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Equils, E. Faure, L. Thomas, Y. Bulut, S. Trushin, and M. Arditi Bacterial Lipopolysaccharide Activates HIV Long Terminal Repeat Through Toll-Like Receptor 4 J. Immunol., February 15, 2001; 166(4): 2342 - 2347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. E. Kilpatrick, Y.-H. Song, M. W. Rossi, and H. M. Korchak Serine phosphorylation of p60 tumor necrosis factor receptor by PKC-delta in TNF-alpha -activated neutrophils Am J Physiol Cell Physiol, December 1, 2000; 279(6): C2011 - C2018. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Koul, Y. Yao, J. L. Abbruzzese, W. K. A. Yung, and S. A. G. Reddy Tumor Suppressor MMAC/PTEN Inhibits Cytokine-induced NFkappa B Activation without Interfering with the Ikappa B Degradation Pathway J. Biol. Chem., March 30, 2001; 276(14): 11402 - 11408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Martin, B. San-Antonio, and M. Fresno Regulation of Nuclear Factor kappa B Transactivation. IMPLICATION OF PHOSPHATIDYLINOSITOL 3-KINASE AND PROTEIN KINASE C zeta IN c-Rel ACTIVATION BY TUMOR NECROSIS FACTOR alpha J. Biol. Chem., May 4, 2001; 276(19): 15840 - 15849. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Learn, M. S. Boger, L. Li, and C. E. McCall The Phosphatidylinositol 3-Kinase Pathway Selectively Controls sIL-1RA Not Interleukin-1beta Production in the Septic Leukocytes J. Biol. Chem., June 1, 2001; 276(23): 20234 - 20239. [Abstract] [Full Text] [PDF]
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