the pi3k-akt pathway limits lps activation of signaling ... · the pi3k-akt pathway has been shown...
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The PI3K-Akt Pathway Limits LPS Activation of Signaling Pathways
and Expression of Inflammatory Mediators in Human Monocytic Cells
Mausumee Guha and Nigel Mackman1
Departments of Immunology and Cell Biology
The Scripps Research Institute
La Jolla, California
1Corresponding author: Departments of Immunology and Cell Biology, C-204,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.
Tel: 858-784-8594; Fax: 858-784-8480; E-mail: [email protected]
Running title: Regulation of LPS Signaling by PI3K-Akt
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Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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Summary
Monocytes and macrophages express cytokines and procoagulant molecules in
various inflammatory diseases. In sepsis, lipopolysaccharide (LPS) from Gram-
negative bacteria induces tumor necrosis factor-alpha (TNFα) and tissue factor
(TF) in monocytic cells via the activation of the transcription factors Egr-1, AP-1
and NF-κB. However, the signaling pathways that negatively regulate LPS-
induced TNFα and TF expression in monocytic cells are currently unknown. We
report that inhibition of the PI3K-Akt pathway enhances LPS-induced activation
of the MAP kinase pathways (ERK1/2, p38 and JNK) and the downstream targets
AP-1 and Egr-1. In addition, inhibition of PI3K-Akt enhanced LPS-induced
nuclear translocation of NF-κB and prevented Akt-dependent inactivation of
glycogen synthase kinase-β (GSK-3β), which increased the transactivational
activity of p65. We propose that the activation of the PI3K-Akt pathway in
human monocytes limits the LPS induction of TNFα and TF expression. Our
study provides new insight into the inhibitory mechanism by which the PI3K-Akt
pathway ensures transient expression of these potent inflammatory mediators.
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Introduction
Regulation of pro-inflammatory gene expression in a biological system is a
balance between positive and negative signal transduction pathways.
Lipopolysaccharide (LPS), the outer membrane component of Gram-negative
bacteria induces expression of many pro-inflammatory mediators in
monocyte/macrophages, one of the key cell types involved in sepsis (1-7). LPS
activation of the CD14-TLR4-MD2 complex results in the expression of TNFα
and TF (1;8-10). The signaling pathways that positively regulate TNFα and TF
gene expression in LPS-stimulated monocytes/macrophages are well
characterized (2;3;5;11-15). However, mechanisms and signaling pathways
that limit the magnitude of the induction of these genes are poorly understood.
Recent evidence suggests that activation of phosphatidylinositol 3-kinase (PI3K),
a ubiquitous lipid-modifying enzyme, may modulate positively acting signaling
pathways. PI3K is a heterodimeric protein consisting of a p85 regulatory subunit
and a p110 catalytic subunit. LPS stimulation of monocytes/macrophages
activates the PI3K pathway (16-18), although the steps between the CD14-
TLR4-MD2 complex and activation of PI3K have not been characterized.
Activation of PI3K appears to occur via phosphorylation of tyrosine residues in
the Src homology 2 (SH2) domain of p85, which permits docking of PI3K to the
plasma membrane and allows allosteric modifications that increase its catalytic
activity (19-21). Activated PI3K catalyzes the phosphorylation of membrane
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inositol lipids and the accumulation of phosphatidyl inositol 3,4,5-trisphosphate
[PI(3,4,5)P3] and its phospholipid phosphatase product phosphatidyl inositol 3,4-
bisphosphate [PI(3,4)P2] in the membrane. These membrane changes allow
docking of the lipid kinases phosphatidylinositol dependent kinase 1 (PDK-1)
and protein kinase B/Akt. Following membrane recruitment Akt is activated by
dual phosphorylation of Ser473 and Thr308 by PDK-1 and possibly PDK-1-
related kinase-2 (PRK-2) (22;23).
The PI3K-Akt pathway has been shown to negatively regulate NF-κB and the
expression of inflammatory genes. Wortmannin, a specific inhibitor of PI3K,
enhanced LPS-induced nitric oxide synthesis (iNOS) in murine peritoneal
macrophages (18) and activation of PI3K-Akt suppressed LPS-induced
lipoprotein lipase expression in J774-macrophages (24). Induction of iNOS in
C6 glial cells and rat primary astrocytes was also negatively regulated by
activation of PI3K (17) and a constitutively active PI3K inhibited induction of iNOS
gene expression in human astrocytes (25). Angiopoeitin-1, a potent activator of
PI3K, negatively regulated VEGF- and TNFα-induced TF expression in
endothelial cells (26). Finally, in endothelial cells the PI3K-Akt pathway limited
VEGF activation of the p38 MAPK pathway and TF gene expression (27).
In contrast to studies showing that the PI3K-Akt pathway negatively regulates
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expression of inflammatory genes in macrophages, a recent study demonstrated
that the PI3K-Akt pathway positively regulated NF-κB-dependent gene
expression in HepG2 cells via phosphorylation and increased transactivation
activity of p65 (28). Overexpression of a constitutively active form of Akt also
increased NF-κB-dependent gene expression in 3T3 fibroblasts via the
activation of IKK and the p38 MAPK (29). Activation of PI3K-Akt has been
implicated in playing a pivotal role in cytokine-induced transcriptional activation
of NF-κB- and AP-1-dependent gene expression and in inhibiting apoptosis
(30-34). Finally, activation of the TLR2 receptor in human monocytic cells by
Gram-positive bacteria activated the PI3K-Akt pathway and increased the
transactivation activity of p65 (35).
The PI3K-Akt pathway has been shown to negatively regulate many kinases
including Raf-1 and GSK-3β, which mediate induction of inflammatory genes.
We and others have shown that LPS-induced TNFα and TF expression in
monocytic cells is mediated, in part, via the activation of the Raf-MEK-ERK1/2
pathway (2;6;13;15). Activation of Akt has been shown to negatively regulate the
serine/threonine kinase Raf-1 and the downstream MEK-ERK1/2 signaling
pathway (36;37). Akt induces an inhibitory phosphorylation of Ser259 in the N-
terminal CR2 domain of Raf-1 which increases its association with 14-3-3
protein and masks the accessibility of residues in the kinase domain of Raf-1
necessary for its activation (38). Dephosphorylation of Ser259 and
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phosphorylation of Ser338 and Tyr341 in the C-terminal kinase domain is
required for Raf-1 activation and its interaction with downstream substrates
(39;40).
Glycogen synthase kinase-3β (GSK-3β) is another serine/threonine kinase that
is inhibited by Akt-dependent phosphorylation. Akt phosphorylates Ser9 in the
N-terminus of GSK-3β and inactivates the kinase (41). The phenotype of GSK-
3β-/- embryos is similar to that of RelA-/- embryos, suggesting that GSK-3β
may regulate the transactivational activity of p65 (42). Lithium chloride (LiCl) is a
potent inhibitor of GSK-3β and inactivates GSK-3β by inducing its
phosphorylation on Ser9 in its N-terminus (43). Indeed, the effect of LiCl on Wnt
signaling in wild-type cells mimicked the phenotype observed in GSK-3β null
cells (41). The role of GSK-3β in the regulation of inflammatory genes in
monocytes is currently undefined.
Our study demonstrates that inhibition of the PI3K-Akt pathway enhances LPS-
induced TNFα and TF gene expression via increased activation of Egr-1-, AP-1
and NF-κB. Inhibition of PI3K also enhanced TNFα and TF gene expression, in
part, by increasing the transactivational activity of p65 by inhibiting Akt-
dependent inactivation of GSK-3β. Therefore, activation of the PI3K-Akt
pathway in LPS-treated human monocytes and THP-1 cells limits the induction
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of TNFα and TF gene expression.
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Materials and Methods
LPS (Escherichia coli serotype 0111:B4) and the PI3K inhibitors Wortmannin and
LY294002 were obtained from Calbiochem (Carlsbad, CA). Lithium chloride
(LiCl) and sodium chloride (NaCl) were obtained from Sigma Chemical (St. Louis,
MO).
Cell culture
The human monocytic cell line THP-1 was obtained from American Type Culture
Collection (Manassas, VA). THP-1 cells were cultured in RPMI 1640 (Gibco
BRL Life Technologies, Gaithersburg, MD) with 8% fetal calf serum (Omega
Scientific, Tarzana, CA), L-glutamine (Gibco BRL, Gaithersburg, MD) and 2-
mercaptoethanol (Sigma). Human peripheral blood mononuclear cells (PBMCs)
were isolated from heparinized blood from healthy volunteers by buoyant density
gradient centrifugation on low endotoxin Ficoll-Hypaque (44).
TNFα ELISA
To study the effect of the PI3K inhibitors on TNFα production, THP-1 cells or
PBMCs (1 X 106) were preincubated with 100 nM wortmannin or 10µM
LY294002 for 1 hour at 37°C before the addition of LPS for 5 hours at 37°C. The
effect of inhibition of GSK-3β on TNFα release was studied by pre-incubating
THP-1 cells with LiCl (20 or 50 mM) for 1 hour at 37°C prior to stimulating with
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LPS for 5 hours at 37°C. NaCl (20 or 50 mM) served as an osmolarity control for
LiCl. TNFα protein levels were measured using a commercial ELISA kit (R&D
Systems, MN).
TF activity
THP-1 or PBMC cell pellets (1 X106) were solubilized at 37°C for 15 minutes
using 15 mM octyl-βD-glucopyranoside. TF activity in cell lysates was
measured using a one-stage clotting assay as described (45) with the PT
program on the Start 4 clotting machine (Diagnostica Stago, Asnieres, France).
Clotting times were converted to milliunits of TF activity by comparison with a
standard curve established with purified human brain TF.
Western blotting
Whole cell lysates and cytosolic and nuclear extracts were prepared from THP-1
cells (5 X106) (16;45). Protein concentrations were measured using a Bio-Rad
protein assay kit. Proteins were separated by SDS-PAGE and transferred to
Hybond-enhanced chemiluminescence membrane (Amersham Pharmacia
Biotech, Alameda, CA). Activation of Akt, ERK1/2, p38 and JNK was assessed
using 1:1000 dilution of anti-phosphospecific antibodies (New England Biolabs).
Inactivated GSK-3β was detected using a 1:1000 dilution of an antibody that
recognizes GSK-3β phosphorylated at Ser9. Activation of Raf-1 was monitored
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using 1:2000 dilution of an antibody that recognizes Raf-1 phosphorylated at
Ser338 (39;40) (United Biotechnology Incorporated, PA). When whole cell or
cytosolic extracts were used, blots were stripped and reprobed using a 1:1000
dilution of antibodies against the non-phosphorylated forms of each protein to
monitor protein loading. Levels of p65 were monitored in the nuclear extracts
using 1:1000 dilution of an anti-N-terminal RelA antibody (Santa Cruz
Biotechnology, SF). Egr-1 was visualized in nuclear extracts using 1:1000
dilution of an anti-Egr-1 antibody (Santa Cruz Biotechnology). To ensure equal
protein loading, blots with nuclear extracts were stripped and reprobed with
1:1000 dilution of an anti-histone antibody (Santa Cruz Biotechnology).
Northern Blotting
Total cellular RNA was isolated from THP-1 cells (5 x 106) stimulated with LPS
(10 µg/mL) using Trizol Reagent (Gibco). RNA (10 µg) was analyzed by
Northern blotting (41). A 641 base pair (bp) human TF cDNA fragment, a 800 bp
human TNFα cDNA fragment or a 1500 bp Egr-1 cDNA fragment was labeled
with [α32P], deoxycytidine triphosphate (ICN, Costa Mesa, CA) using a Prime-It
Kit (Strategene Cloning Systems, San Diego, CA). Blots were re-hybridized with
the house keeping gene glyceraldehyde 3-phosphate dehydrogenase (G3PDH;
Clonetech Laboratories, Palo Alto, CA). Bands were visualized by
autoradiography.
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Electrophoretic mobility shift assays
Nuclear extracts were prepared from THP-1 cells (5 X 106) as described
previously (46). Nuclear extracts were incubated with radiolabeled double-
stranded oligonucleotide probes (Operon Technologies, Alameda, CA) containing
the immunoglobulin IgκB site (underlined), 5’-CAGAGGGGACTTTCCGAGA-3’;
an AP-1 site (underlined), 5’-CTGGGGTGAGTCATCCCTT-3’ or a Sp1 site
(underlined), 5’-ATTCGATCGGGGCGGGGCGAGC-3’. Protein-DNA
complexes were separated from free DNA probe by electrophoresis through 6%
non-denaturing acrylamide gels (InVitrogen, Carlsbad, CA) in 0.5 X Tris borate
ethylenediaminetetraacetic acid (TBE) buffer. Gels were dried, and protein-DNA
complexes were visualized by autoradiography.
Plasmids
pTF-LUC contains 2106 bp of the human TF promoter. pTNFα-LUC contains
615 bp of the human TNFα promoter and pEgr-1-LUC contains 1200 bp of the
murine Egr-1 promoter. p(κB)5-LUC contains 5 copies of a NF-κB site, and
p(AP-1)4-LUC contains 4 copies of an AP-1 site. These sites were cloned
upstream of the minimal simian virus 40 (SV40) promoter expressing the firefly
luciferase (LUC) reporter gene in pGL2-Promoter (Promega, Madison, WI) (47).
A plasmid expressing dominant-negative (dnAkt) (S308A/S473A) was kindly
provided by G. Bokoch (The Scripps Research Institute, La Jolla, CA). The
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control plasmid pFA-CMV expresses the GAL4 DNA-binding domain alone and
was obtained from Stratagene. pFR-Luc (pGAL4-LUC) contains 5XGAL4
binding sites upstream of a minimal promoter. pGAL4-p65 contains the
transactivation domain (aa 386-551) of p65 fused to the DNA binding portion of
GAL4 (35). The pcDNA3 (InVitrogen, San Diego, CA) was used as a control
plasmid for transfections when expression plasmids were used.
Transfections
THP-1 cells were transfected using DEAE-dextran (14). After transfection, cells
were incubated in complete media for 46 hours at 37°C before stimulating with
LPS (10 µg/mL) for 5 hours at 37°C. In some experiments cells were incubated
with wortmannin (100 nM) or LY294002 (10µM) for 1 hour at 37°C before
stimulation with LPS. In other experiments cells were incubated with LiCl
(50mM) or NaCl (50 mM) for 1 hour at 37°C before stimulation with LPS. Cell
lysates were assayed for luciferase activity as described in the manufacturer’s
protocol (Promega) using a Monolight 2010 luminometer (Analytical
Luminescence Laboratory, San Diego, CA). Cells were co-transfected with
pRLTK, which expresses Renilla luciferase (Promega). Renilla luciferase was
measured according to the manufacturer’s protocol (Promega) and used to
normalize the activity of the firefly luciferase.
Data analysis
The number of experiments analyzed is indicated in each figure. Band intensity
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was quantified by densitometric analyses using a Personal Densitometer
(Molecular Dynamics, Sunnyvale, CA) and ImageQuant software. Data was
collected using a minimum of three experiments and used to calculate mean ±
standard deviation (SD). Statistical significance was calculated using an
unpaired Student’s t test and was considered significant at P values ≤ 0.05.
Results
The PI3K-Akt pathway inhibits LPS-induced TNF α and TF protein expression in
human monocytic cells.
We examined the role of the PI3K-Akt pathway in LPS induction of TNFα and TF
in human monocytic cells using two different pharmacological inhibitors
(LY294002 and wortmannin) that block the activation of PI3K by different
mechanisms. LPS induction of TNFα and TF in PBMCs was measured in
presence and absence of LY294002 (Fig. 1A). LY294002 enhanced LPS
induction of TNFα and TF expression 2.0 and 3.3 fold, respectively. THP-1 cells
represent a well-established model of human monocytes. LY294002 also
enhanced LPS induction of TNFα and TF expression in THP-1 cells 2.8 and 2.0
fold, respectively (Fig. 1B). Similar results were observed in THP-1 cells with
wortmannin (Fig. 1B). These results indicate that the PI3K-Akt pathway is a
negative regulator of LPS-induced TNFα and TF expression in human monocytic
cells. Since the response to LY294002 was similar in PBMCs and THP-1 cells,
we used THP-1 cells to further determine the mechanism by which activation of
PI3K-Akt negatively regulates LPS-induced TNFα and TF expression.
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GSK-3β positively regulates TNF α and TF protein expression in LPS stimulated
THP-1 cells.
GSK-3β is a kinase that is negatively regulated by Akt-dependent
phosphorylation. However, the role of GSK-3β in the regulation of inflammatory
genes is currently unknown. We wished to determine if GSK-3β played a role in
LPS induction of TNFα and TF gene expression. We used LiCl to inhibit GSK-
3β kinase activity (41;42). LiCl reduced LPS-induced TNFα and TF expression in
a dose-dependent manner (Fig. 1C). NaCl was used as an osmolarity control.
These results indicate that GSK-3β positively regulates TNFα and TF expression
in LPS-stimulated monocytic cells and may be a potential target for negative
regulation by the PI3K-Akt signaling pathway.
Activation of the PI3K-Akt pathway negatively regulates LPS induction of TNF α
and TF gene expression.
The role of the PI3K-Akt pathway in the LPS induction of TNFα and TF mRNA
expression was determined by Northern blot analysis (Fig. 2). LPS induced
maximal levels of TNFα and TF mRNAs at 1 hour. LY294002 enhanced the LPS
induction of TNFα at 1 hour by 4.2 fold. However, LY294002 had only a minor
affect on LPS-induced levels of TF mRNA at 1 hour but increased TF mRNA
expression at 2 hours by 5.8 fold. The slower migrating band represents a
differentially-spliced, non-functional TF transcript (48). These results indicate
that activation of PI3K limits the LPS induction of TNFα and TF mRNA
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expression in monocytic cells.
Next, we evaluated the role of PI3K-Akt signaling in the LPS-induced TNFα and
TF promoter activity. Wortmannin significantly enhanced LPS-induced TNFα
(p=0.0008) and TF (p=0.037) promoter activity (Fig. 3A). Importantly, co-
transfection of cells with a plasmid expressing a dominant-negative version of
Akt (dnAkt) also enhanced LPS induction of TNFα and TF promoter activity (Fig.
3B). The results using a pharmacological inhibitor and dnAkt indicate that LPS-
induced TNFα and TF gene expression is negatively regulated by activation of
Akt at the level of transcription.
LPS activates Akt.
Activation of PI3K-Akt differentially regulates downstream effectors in different
cell types (21;24-30). LPS stimulation of THP-1 cells resulted in a time-
dependent phosphorylation of Akt that was maximal at 1 hour (Fig. 4A). Pre-
incubation with either LY294002 (Fig. 4A) or wortmannin (not shown) completely
abolished LPS-induced activation of Akt.
Inhibition of PI3K enhances the LPS activation of MAPK pathways.
LPS stimulation of monocytic cells activates all three MAPK pathways, ERK1/2,
p38 and JNK (3;5;9;11;15). To further understand the mechanism by which PI3K
negatively regulates LPS-induced TNFα and TF expression, we evaluated the
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effect of LY294002 on activation of Raf-1 and its downstream target ERK1/2.
Raf-1 is activated by phosphorylation at multiple serine and tyrosine motifs of
which Ser338 is a key site that correlates with its activation (39;40). LPS
stimulated THP-1 cells showed an increase in Raf-1 phosphorylation that was
maximal at 30 minutes (Fig. 4B). Incubation of cells with LY294002 alone
resulted in an increase in basal Raf-1 phosphorylation. Additionally, LY294002
enhanced LPS-induced Ser338 phosphorylation of Raf-1 (Fig. 4B). LPS-
induced activation of p38 MAPK was analyzed using anti-phospho-specific p38
antibody. LY294002 enhanced LPS-induced phosphorylation of p38 at 1 hour
(Fig. 4C). LY294002 stimulated basal activation of ERK1/2 (Fig. 4E).
Furthermore, LY294002 enhanced LPS-induced phosphorylation of ERK1/2 and
JNK (Fig. 4E, F).
Inhibition of PI3K blocks GSK-3β inactivation.
Since inactivation of GSK-3β by LiCl inhibited TNFα and TF gene expression,
we monitored activation of GSK-3β in the same extracts used to study the
activation of the MAPKs. LPS induced a time-dependent phosphorylation of
GSK-3β on Ser9 (inactivation) that correlated with the kinetics of Akt activation
by LPS (Fig. 4D). Pre-incubation of cells with LY294002 completely abrogated
LPS-induced inactivation of GSK-3β, thereby retaining the kinase in its active
(dephosphorylated) state (Fig. 4D). These results demonstrated that GSK-3β is
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inactivated in a PI3K-dependent manner in LPS stimulated THP-1 cells.
Inactivation of GSK-3β may limit the LPS induction of TNFα and TF expression.
To further ascertain the role of GSK-3β in LPS-stimulated TNFα and TF
expression in THP-1 cells, we used LiCl to inhibit GSK-3β. Pretreatment of cells
with 50 mM LiCl, a dose at which TNFα and TF expression were significantly
inhibited, had no effect on LPS-induced activation of ERK1/2, p38 or JNK
MAPKs (not shown). In contrast, LiCl enhanced LPS-induced phosphorylation
and inactivation of GSK-3β (Fig. 4G). These results indicate that the inhibitory
effect of LiCl on LPS-induced TNFα and TF expression is mediated by
inactivation of GSK-3β and is not mediated by effects on the MAPK pathways.
Inhibition of PI3K enhances LPS-induced DNA binding of NF-κB and AP-1.
It is well documented that NF-κB/Rel and AP-1 transcription factors play a major
role in the LPS induction of TNFα and TF expression in human monocytic cells
(3;12;14). First, we examined the effect of LPS on nuclear translocation of p65
and NF-κB in the presence and absence of LY294002. The level of p65 was
determined in nuclear extracts by Western blot analysis (Fig. 5A). LPS induced
nuclear translocation of p65 that was maximal at 1 hour. LY294002 enhanced
LPS-stimulated p65/Rel nuclear translocation. Next, we examined nuclear
translocation of NF-κB by EMSAs. Similar to the Western blot analysis, LPS
induced nuclear translocation and binding of NF-κB with a peak at 1 hour (Fig.
5B). NF-κB binding was increased when cells were pre-treated with LY294002
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prior to stimulation with LPS (Fig. 5B). Pre-incubation of cells with LY294002
also enhanced LPS-stimulated AP-1 binding at 2 and 4 hours (Fig. 5B).
Inhibition of PI3K had a small stimulatory effect on both basal NF-κB and AP-1
binding (Fig. 5B). Binding of Sp1 to a prototypic site was used as a control to
show that the different nuclear extracts had similar amounts of protein. However,
LiCl did not affect LPS-induced nuclear translocation of NF-κB (Fig. 5C).
Binding of Sp1 was used as a control.
Inhibition of PI3K-Akt enhances LPS-induced Egr-1 expression.
We and others have demonstrated that the TNFα and TF promoters are
regulated in LPS stimulated human monocytic cells by the coordinated binding of
NF-κB, AP-1 and Egr-1 transcription factors (13;15). Since Egr-1 is
synthesized de novo in LPS stimulated monocytic cells, we evaluated the effect
of LY294002 on LPS-induced Egr-1 gene expression. LY294002 enhanced
LPS-induced Egr-1 mRNA expression at 1 hour by 2.1 fold (Fig. 5D). Pre-
incubation with LY294002 also increased LPS-induced Egr-1 protein expression
at 2 hours (Fig. 5D). These results demonstrate that the negative regulation of
the Raf-1/MEK/ERK1/2 pathway by LPS-induced activation of Akt also limits
Egr-1 expression, a target gene of the pathway.
Inhibition of PI3K-Akt enhances LPS induction of NF- κB-, AP-1- and Egr-1-
dependent gene expression.
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The effect of inhibition of the PI3K-Akt pathway on gene expression mediated by
NF-κB, AP-1 or Egr-1 was determined in THP-1 cells transfected with various
reporter plasmids (Fig. 6). LY294002 significantly enhanced LPS-induced NF-
κB- (p=0.024), AP-1- (p=0.003) and Egr-1- (p=0.019) dependent gene
expression. In addition, expression of dominant-negative Akt also enhanced
NF-κB-, AP-1- and Egr-1-dependent gene expression (Fig. 6).
LPS-induced transcriptional activity of p65 is negatively regulated by the PI3K-
Akt pathway via inactivation of GSK-3β.
We have shown that inhibition of the PI3K-Akt pathway blocks the Akt-
dependent inhibitory phosphorylation of GSK-3β whereas LiCl increases the
inactivation of GSK-3β. We determined if the level of activation of GSK-3β
correlated with the transcriptional activity of p65 using a GAL4-p65 chimeric
protein that contains the transactivation domain of p65 (Fig. 7). THP-1 cells
were co-transfected with the reporter plasmid pGAL4-Luc and the expression
plasmid pGAL4-p65. Wortmannin enhanced LPS induction of the transcriptional
activity of p65. In contrast, inactivation of GSK-3β by LiCl reduced p65-
dependent transcription. Treatment of cells with equimolar concentration of NaCl
had no effect on LPS-induced luciferase activity and served as a control for
osmolarity. These results demonstrate that LPS-induced activation of the PI3K-
Akt pathway inactivates GSK-3β and reduces the transactivation activity of p65.
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Discussion
Our study demonstrates that LPS-induced expression of TNFα and TF in human
monocytic cells is regulated by both positive and negative pathways. We provide
multiple lines of evidence and new insights to support the contention that LPS-
induced activation of the PI3K-Akt pathway produces a “limiting” effect on TNFα
and TF gene expression in PBMCs and THP-1 monocytic cells. We show that
LPS-induced activation of PI3K-Akt negatively regulates the transcription factors
Egr-1, AP-1 and NF-κB. The net inhibitory effect on the activation of all three
transcription factors reduces LPS induction of TNFα and TF expression in
monocytic cells, which is a key cell type in sepsis.
This study demonstrates that LPS-induced activation of PI3K-Akt in monocytic
cells negatively regulates Raf-1. Two targets of the Raf-1 pathway (ERK1/2 and
Egr-1) were also negatively regulated by LPS-induced activation of PI3K-Akt.
Egr-1 is required for maximal induction of both TNFα and TF in human
monocytic cells treated with LPS (13; 15). Enhancement of LPS-induced TF
mRNA expression in the presence of LY294002 was delayed (2h) relative to the
enhancement of TNFα mRNA expression (1h). This difference may be due to a
greater contribution of Egr-1 to the induction of TF gene expression because the
TF promoter contains three Egr-1 sites. We also found that inhibition of PI3K
enhanced LPS-induced activation of p38 and JNK. Our data is consistent with a
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recent study demonstrating that inhibition of the PI3K-Akt pathway in endothelial
cells enhanced TF expression by increasing the activation of p38 (27). We found
that LY294002 and dnAkt increased LPS-induced AP-1-dependent gene
expression. Gratton and colleagues (49) have recently shown that Akt-
dependent phosphorylation of MEKK3 reduces its kinase activity and inhibits the
MKK3/6-p38 pathway. This data suggests that Akt can negatively regulate
multiple signaling pathways.
Studies on the role of the PI3K-Akt pathway in NF-κB-dependent gene
expression are controversial. The PI3K-Akt pathway has been shown to act
both positively and negatively on NF-κB-dependent gene expression. These
differences may reflect the use of different cell types and different agonists. In
addition, overexpression of a constitutively active form of Akt may override
normal regulatory pathways. Our study demonstrates that the PI3K-Akt pathway
negatively regulates NF-κB in LPS-stimulated monocytic cells. Inhibition of
PI3K-Akt enhanced LPS-induced nuclear translocation of p65, increased NF-κB
binding and increased NF-κB-dependent gene expression.
Recent studies showed that both TLR2 and TLR4 signaling activates the PI3K-
Akt pathway in human monocytic cells and macrophages (35; 50). We show that
LPS-TLR4 signaling in human monocytic cells activates Akt. Importantly,
macrophages exhibited different patterns of cellular responses after stimulation
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with TLR2 and TLR4 agonists, which suggested that different intracellular
signaling pathways were activated by TLR2 and TLR4 (50-54). In human
monocytic cells, TLR2-dependent activation of the PI3K-Akt pathway positively
regulated the transactivational activity of p65 (35). In contrast, we show that
TLR4-dependent activation of the PI3K-Akt pathway negatively regulates the
transactivational activity of p65. These differences probably reflect the activation
of different signaling pathways that modulate the effect of the PI3K-Akt pathway
on the transactivational activity of p65.
The increased transactivational activity of p65 in the presence of wortmannin
correlated with the inhibition of LPS-induced, Akt-dependent inactivation of
GSK-3β. In parallel, we showed that inhibition of GSK-3β with LiCl reduced LPS
induction of TNFα and TF in monocytic cells, suggesting that GSK-3β positively
regulates these genes by increasing NF-κB activity. LiCl did not affect LPS-
induced nuclear translocation of NF-κB but decreased the transactivational
activity of p65. Therefore, inhibition of GSK-3β at later times via Akt-dependent
phosphorylation may represent at least one mechanism by which monocytic cells
limit the expression of NF-κB-dependent genes.
Several kinases have been implicated in the control of p65 transcriptional activity
but the most compelling data is derived from studies of various knockout mice.
GSK-3β and T2K are both necessary for TNFα and IL-1 signaling whereas NIK
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is selective to the LT-βR pathway (42; 55; 56; 57). Interestingly, PKCζ
deficiency impairs p65 transcriptional activity in response to TNFα, IL-1 and
lymphotoxin-β, which suggests that PKCζ may be downstream of GSK-3β, NIK
and T2K (58). Indeed, PKCζ is the only kinase that has been shown to interact
directly with p65.
A model of LPS induction of TNFα and TF in monocytic cells is shown in Figure
8. The current study demonstrates that LPS stimulation of monocytic cells leads
to an activation of the PI3K-Akt pathway, which inactivates MAPK kinase
pathways (ERK1/2, p38 and JNK) and the NF-κB pathway by phosphorylation of
Raf-1, IKK, GSK-3β and other upstream kinases, such as MEKK3. Inhibition of
these pathways limits the activation of the transcription factors NF-κB, AP-1 and
Egr-1, all of which co-operatively regulate TNFα and TF gene expression.
Thus, the PI3K-Akt pathway imposes a “braking“ mechanism to limit the
expression of TNFα and TF in LPS stimulated monocytes and ensure transient
expression of these inflammatory mediators.
Acknowledgments
We would like to thank C. Johnson for preparing the manuscript, D. Navamani for
technical help and R. Pawlinski, M. Riewald and U. Knaus for critical reading of
the manuscript. This work was supported by a grant from the National Institutes
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Figure Legends
Figure 1: Regulation of LPS-induced TNF α and TF protein expression in human
monocytic cells. PBMCs (A) or THP-1 cells (B) were preincubated with vehicle
(0.2% Me2SO3), LY294002 (10 µM) or wortmannin (100 nM) for 60 minutes prior
to stimulation with LPS for 5 hours. PBMCs and THP-1 cells were stimulated
with either 10 ng/ml or 10 µg/ml of LPS, respectively. (C) THP-1 cells were left
untreated or preincubated with LiCl (20 or 50 mM) or NaCl (50 mM) for 60
minutes prior to stimulation with LPS (10 µg/mL) for 5 hours. TNFα antigen in
cell culture supernatants was determined by ELISA and TF activity was
determined using a one-step clotting assay. This data represents mean ± SD
from three independent experiments. Black bars represent TNFα levels and
white bars represent TF levels.
Figure 2: Activation of the PI3K-Akt pathway negatively regulates LPS induction
of TNFα and TF mRNA expression. Total RNA was extracted from THP-1 cells
preincubated with vehicle or with LY294002 (10 µM) for 60 minutes prior to
stimulation with LPS (10 µg/ml) for various times. TNFα, TF and G3PDH mRNA
levels were determined by Northern blotting. The asterisk shows the
differentially-spliced TF transcript.
Figure 3: Activation of the PI3K-Akt pathway negatively regulates LPS induction
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of TNFα and TF promoter activity. (A) pTNFα-LUC (3 µg) or pTF-LUC (3 µg)
were transiently transfected into THP-1 cells. After 46 hours, transfected cells
were preincubated with vehicle or LY294002 (10 µM) for 60 minutes prior to
stimulation with LPS for 5 hours at 37ºC. Luciferase activity in cell lysates was
determined and normalized to Renilla luciferase. (B) THP-1 cells were
cotransfected with pTNFα-LUC (3 µg) and either pcDNA3 (2 µg) or a plasmid
expressing dnAkt. Similar experiments were performed with pTF-LUC.
Transfected cells were treated with or without LPS for 5 hours at 37ºC.
Luciferase activity in cell lysates was determined, and the results were expressed
as a percentage of control induction (n=3). Data (mean ± SD) is from 3
independent experiments. The black bar represents pTNFα-LUC activity and
the white bar represents pTF-LUC activity.
Figure 4: LPS-induced phosphorylation of various kinases in the presence of
either LY294002 or LiCl. (A) THP-1 cells were preincubated with either
LY294002 (10 µM) or vehicle for 60 minutes prior to stimulation with LPS (10
µg/ml) for the various times as indicated. Whole cell lysates were prepared and
phospho-Akt levels measured by Western blotting using an anti-phospho-
Ser473 antibody. Whole cell lysates (B-D) or cytosolic extracts (E-F) were analyzed
by Western blotting using an anti-phospho-Ser338 Raf-1 antibody (B), an anti-
phospho-p38 antibody (C), an anti-phospho-Ser9 GSK-3β antibody (D), an
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anti-phospho-ERK1/2 antibody (E), and an anti-phospho-JNK antibody (F).
THP-1 cells were preincubated with or without LiCl (50 nM) for 60 minutes prior
to stimulation with LPS (10 µg/ml) for various times. Whole cell lysates were
analyzed by Western blotting using an anti-phospho-Ser9 GSK-3β antibody
(G). Each blot was stripped and reprobed with an antibody against the non-
phosphorylated form of each kinase to monitor loading. The blots are
representative of three independent experiments.
Figure 5: Inhibition of PI3K enhances LPS-induced DNA binding of NF- κB and
AP-1 and Egr-1 expression (A) THP-1 cells were preincubated with either
LY294002 (10 µM) or vehicle for 60 minutes prior to stimulation with LPS (10
µg/ml) for the various times indicated. p65 was detected in nuclear extracts by
Western blotting using the N-terminal anti-p65 antibody. The blots were
stripped and reprobed with anti-histone antibody to monitor loading. (B) EMSAs
were performed by incubating nuclear extracts with double-stranded
radiolabeled oligonucleotide containing an NF-κB site, an AP-1 site or a Sp1
site. (C) THP-1 cells were preincubated with either LiCl (50 nM) or vehicle for
60 minutes prior to stimulation with LPS. Levels of nuclear NF-κB and Sp1 were
determined by ELISA. (D) Total RNA was extracted from THP-1 cells
preincubated with either LY294002 (10 µM) or vehicle for 60 minutes prior to
stimulation with LPS at 37ºC for various times. Egr-1 mRNA levels were
determined by Northern blotting. The membrane was reprobed with a G3PDH
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probe to assess loading. Egr-1 protein levels were analyzed at 2 hours by
Western blotting using an anti-Egr-1 antibody.
Figure 6: Inhibition of PI3K-Akt enhances LPS induction of NF- κB-, AP-1- and
Egr-1-dependent gene expression . (A, C, E) THP-1 cells were transiently
transfected with 3µg of p(κB)5-LUC, p(AP-1)4-LUC, or pEgr-1-LUC. Forty-six
hours post transfection, cells were preincubated with either LY294002 or vehicle
for 60 minutes prior to stimulation with LPS for 5 hours at 37ºC. (B, D, F)
p(κB)5-LUC, p(AP-1)4-LUC or pEgr-1-LUC were cotransfected with either pcDNA3 or
a plasmid expressing dnAkt. Forty-six hours post transfection cells were
stimulated with LPS. Luciferase activity is presented as a percentage of control.
Data is shown for 3 independent experiments and is expressed as mean ± SD.
Figure 7: LPS-induced transcriptional activity of p65 is regulated by GSK-3 β.
(A) THP-1 cells were cotransfected with pGal4-p65TA (5 µg) and pGal4-LUC
(1.5 µg). Forty-six hours post transfection cells were preincubated with either
control or with wortmannin (100 nM) for 60 minutes prior to stimulation with LPS
(10 µg/ml) for 5 hours at 37ºC. Luciferase activity in cell lysates was determined,
and results were expressed as fold induction. (B) THP-1 cells were transfected
as above. Forty-six hours post transfection cells were preincubated with either
LiCl (50 mM), NaCl (50 mM) or left untreated (control) prior to stimulation with
LPS for 5 hours at 37ºC. Luciferase activity in cell lysates was determined, and
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results were expressed as fold induction. Data (mean ± SD) is shown for 3
independent experiments.
Figure 8: Activation of the PI3K-Akt pathway in monocytic cells limits LPS-
induced TNFα and TF gene expression. Binding of LPS to the CD14 and
TLR4/MD2 complex activates the PI3K-Akt signaling pathway. Akt directly or
indirectly inactivates the MAPK (ERK1/2, p38 and JNK) and the NF-κB pathway
by negatively regulating upstream kinases including Raf-1, MEKK3 and IKK.
LPS activation of PI3K-Akt also inactivates GSK-3β which reduces the
transactivational activity of p65. Akt-dependent inactivation of these pathways
limits the activation of the transcription factors NF-κB, AP-1 and Egr-1 all of
which co-operatively regulate TNFα and TF gene expression. Wort,
wortmannin; LY, LY294002.
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Mausumee Guha and Nigel Mackmaninflammatory mediators in human monocytic cells
The PI3K-Akt pathway limits LPS activation of signaling pathways and expression of
published online June 6, 2002J. Biol. Chem.
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