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: nmackman@scripps.edu

Running title: Regulation of LPS Signaling by PI3K-Akt

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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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