Toll-like receptor 2−mediated NF-B activation requires a Rac1-dependent pathway

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http://immunol.nature.com december 2000 volume 1 no 6 nature immunology Laurence Arbibe 1 *, Jean-Paul Mira 1, * ,† , Nicole Teusch 1 , Lois Kline 1 , Mausumee Guha 1 , Nigel Mackman 1 , Paul J. Godowski 2 , Richard J. Ulevitch 1 and Ulla G. Knaus 1 Mammalian Toll-like receptors (TLRs) are expressed on innate immune cells and respond to the membrane components of Gram-positive or Gram-negative bacteria. When activated, they convey signals to transcription factors that orchestrate the inflammatory response. However, the intracellular signaling events following TLR activation are largely unknown.Here we show that TLR2 stimulation by Staphylococcus aureus induces a fast and transient activation of the Rho GTPases Rac1 and Cdc42 in the human monocytic cell line THP-1 and in 293 cells expressing TLR2. Dominant-negative Rac1N17, but not dominant-negative Cdc42N17, block nuclear factor- κB (NF- κB) transactivation. S. aureus stimulation causes the recruitment of active Rac1 and phosphatidylinositol-3 kinase (PI3K) to the TLR2 cytosolic domain.Tyrosine phosphorylation of TLR2 is required for assembly of a multiprotein complex that is necessary for subsequent NF- κB transcriptional activity. A signaling cascade composed of Rac1, PI3K and Akt targets nuclear p65 transactivation independently of IκBα degradation. Thus Rac1 controls a second, I κB–independent, pathway to NF- κB activation and is essential in innate immune cell signaling via TLR2. 1 Department of Immunology,The Scripps Research Institute, La Jolla, CA 92037, USA. 2 Department of Molecular Biology, Genentech, South San Francisco, CA 94080-4990, USA. *These authors contributed equally to this work. Present address: Medical Intensive Care Unit, Cochin University Hospital, 75014 Paris, France. Correspondence should be addressed to R. U. ([email protected]). Toll-like receptor 2–mediated NF-κB activation requires a Rac1-dependent pathway Innate immune cells play an essential role in host defense against invading microorganisms. Detection of infectious organisms by poly- morphonuclear cells or monocytes is accomplished through “pattern recognition receptors” (PRRs) 1,2 . A new class of PRRs, the human Toll-like receptors (TLRs), is related to the Drosophila Toll protein, which is required for ontogenesis and antimicrobial resistance 3,4 . Generally, TLRs are type I transmembrane receptors with leucine-rich repeats in the extracellular domains and cytoplasmic domains that resemble the mammalian IL-1 receptor (IL-1R) 2,3,5 . The specific roles of the various TLRs in the innate immune system are just beginning to be understood. In terms of function, TLR2 and TLR4 are strongly implicated in innate immune recognition. Recent evidence suggests that TLR4 is essential for Gram-negative recognition 6 , whereas TLR2 plays a key role in cell responsiveness to components of Gram-posi- tive bacteria including peptidoglycans 7 , lipoteichoic acid 8 and bac- terial lipoproteins 9 . Initially TLR2 was also linked to responses to lipopolysaccharide (LPS) derived from Gram-negative bacteria 10,11 but recent work shows that LPS preparations that appear to stimu- late via TLR2 contain other bacterial products, which may be removed by phenol extraction 12 . A RTICLES 533 Figure 1. HKSA activates Rac1 and Cdc42 transiently. Time course of Rac1 (left) and Cdc42 (right) activation in (a) monocytic cell line THP1-CD14 or (b) 293-TLR2. Starved cells were stimulated with HKSA, 10 7 colony-forming units (CFU) per ml, in culture medium containing 1% bovine serum albumin (BSA).At the indicated time points activation was quenched with cold PBS and cells were lysed. Lysate (900 µg) was subjected to affinity precipitation with GST-PBD.After washing, proteins bound to GST-PBD beads were separated on SDS-PAGE and immunoblotted for bound Rac1 or Cdc42, then detected by enhanced chemilu- minescence (ECL). Immunoblots (IB) of the whole cell lysates used for the PBD binding analysis are shown in lower panels of a and b. (Results are representative of three independent experiments.) a b © 2000 Nature America Inc. • http://immunol.nature.com © 2000 Nature America Inc. • http://immunol.nature.com

Transcript of Toll-like receptor 2−mediated NF-B activation requires a Rac1-dependent pathway

Page 1: Toll-like receptor 2−mediated NF-B activation requires a Rac1-dependent pathway

http://immunol.nature.com • december 2000 • volume 1 no 6 • nature immunology

Laurence Arbibe1*, Jean-Paul Mira1,*,†, Nicole Teusch1, Lois Kline1, Mausumee Guha1,Nigel Mackman1, Paul J. Godowski2, Richard J. Ulevitch1 and Ulla G. Knaus1

Mammalian Toll-like receptors (TLRs) are expressed on innate immune cells and respond to themembrane components of Gram-positive or Gram-negative bacteria. When activated, they conveysignals to transcription factors that orchestrate the inflammatory response. However, the intracellularsignaling events following TLR activation are largely unknown. Here we show that TLR2 stimulation byStaphylococcus aureus induces a fast and transient activation of the Rho GTPases Rac1 and Cdc42 inthe human monocytic cell line THP-1 and in 293 cells expressing TLR2. Dominant-negative Rac1N17,but not dominant-negative Cdc42N17, block nuclear factor-κB (NF-κB) transactivation. S. aureusstimulation causes the recruitment of active Rac1 and phosphatidylinositol-3 kinase (PI3K) to theTLR2 cytosolic domain.Tyrosine phosphorylation of TLR2 is required for assembly of a multiproteincomplex that is necessary for subsequent NF-κB transcriptional activity. A signaling cascadecomposed of Rac1, PI3K and Akt targets nuclear p65 transactivation independently of IκBαdegradation. Thus Rac1 controls a second, IκB–independent, pathway to NF-κB activation and isessential in innate immune cell signaling via TLR2.

1Department of Immunology,The Scripps Research Institute, La Jolla, CA 92037, USA. 2Department of Molecular Biology, Genentech, South San Francisco, CA 94080-4990,USA. *These authors contributed equally to this work. †Present address: Medical Intensive Care Unit, Cochin University Hospital, 75014 Paris, France.

Correspondence should be addressed to R. U. ([email protected]).

Toll-like receptor 2–mediated NF-κB activation requires a

Rac1-dependent pathway

Innate immune cells play an essential role in host defense againstinvading microorganisms. Detection of infectious organisms by poly-morphonuclear cells or monocytes is accomplished through “patternrecognition receptors” (PRRs)1,2. A new class of PRRs, the humanToll-like receptors (TLRs), is related to the Drosophila Toll protein,which is required for ontogenesis and antimicrobial resistance3,4.Generally, TLRs are type I transmembrane receptors with leucine-rich

repeats in the extracellular domains and cytoplasmic domains thatresemble the mammalian IL-1 receptor (IL-1R)2,3,5. The specific rolesof the various TLRs in the innate immune system are just beginning tobe understood. In terms of function, TLR2 and TLR4 are stronglyimplicated in innate immune recognition. Recent evidence suggeststhat TLR4 is essential for Gram-negative recognition6, whereas TLR2plays a key role in cell responsiveness to components of Gram-posi-

tive bacteria including peptidoglycans7, lipoteichoic acid8 and bac-terial lipoproteins9. Initially TLR2 was also linked to responses tolipopolysaccharide (LPS) derived from Gram-negative bacteria10,11

but recent work shows that LPS preparations that appear to stimu-late via TLR2 contain other bacterial products, which may beremoved by phenol extraction12.

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Figure 1. HKSA activates Rac1 and Cdc42 transiently. Time course ofRac1 (left) and Cdc42 (right) activation in (a) monocytic cell line THP1-CD14 or(b) 293-TLR2. Starved cells were stimulated with HKSA, 107 colony-forming units(CFU) per ml, in culture medium containing 1% bovine serum albumin (BSA).Atthe indicated time points activation was quenched with cold PBS and cells werelysed. Lysate (900 µg) was subjected to affinity precipitation with GST-PBD.Afterwashing, proteins bound to GST-PBD beads were separated on SDS-PAGE andimmunoblotted for bound Rac1 or Cdc42, then detected by enhanced chemilu-minescence (ECL). Immunoblots (IB) of the whole cell lysates used for the PBDbinding analysis are shown in lower panels of a and b. (Results are representativeof three independent experiments.)

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Activation of TLR2 by bacterial products triggers several crucialintracellular signaling responses including activation of the transcrip-tion factor nuclear factor-κB (NF-κB) and induction of proinflamma-tory cytokines1,2. Previous work suggested that TLRs and IL-1R initi-ate a similar pathway leading to NF-κB activation that involves sig-naling through MyD88, IRAK and TRAF6 to activate IκB kinases(IKKs) and degrade IκB13,14. NF-κB is composed of Rel family homo-and heterodimers such as p50 and p65. The inactive cytoplasmic NF-κB heterodimer is liberated from inhibitory subunit IκB upon cel-lular stimulation and translocates to the nucleus where it interacts withκB-responsive elements mediating transcriptional gene activation15,16.Phosphorylation of Rel dimer subunits positively controls NF-κB tran-scriptional activity by an IκB-independent pathway17–20.

The Rho family GTPases Rac1 and Cdc42 are key regulators ofvarious cellular functions such as cytoskeletal reorganization, cellu-lar growth and apoptosis21. Rac and Cdc42 are implicated in differ-ent aspects of host defense against bacteria, including leukocytechemotaxis21, pathogen phagocytosis22,23, production of oxygen radi-cals24,25, activation of multiple stress responses cascades21 and NF-κBactivation26,27. Here we provide evidence that Rac1 is a key regulatorof a second, parallel, pathway required for the transcriptional activi-ty of the NF-κB p65 subunit in response to Gram-positive stimuli.

This pathway is initiated by Staphylococcus aureus–stimulated tyrosine phosphorylation of TLR2, leads to the association of TLR2with active Rac1 and PI3K subunit p85 and involves Akt as a down-stream effector. A link between bacterial stimuli, PI3K and NF-κBactivation has been indirectly suggested for some time. Relevant inthis regard is a report showing that B lymphocytes from knockoutmice that are deficient in several PI3K regulatory subunits (p85α,p55α and p50α) fail to respond to LPS28. Our findings now providea mechanistic basis for this and a general paradigm for NF-κB trans-activation by bacterial stimuli that might apply to other TLR familymembers as well.

ResultsTLR2 stimulation by HKSA activates Rac1 and Cdc42The signaling pathways downstream of activated TLRs are still largelyunknown. As RhoGTPases are regulators of multiple cell functions andmight be involved in NF-κB activation, their role in pathways originat-ing from activated TLRs was investigated. The high affinity binding ofRhoGTPases to certain effector kinases is exploited in an assay thatdetects the activation of a specific RhoGTPase family member29. Toassess Rac or Cdc42 activity in lysates of stimulated cells the p21-bind-ing domain (PBD) of p21-activated kinase 1 (Pak1), one of the promi-

nent effectors of RhoGTPases, is used asbinding partner. The identity of the boundGTPase is then determined byimmunoblotting with specific antibodies.Using this approach we investigatedwhether Rac and Cdc42 are activated bybacteria-derived stimuli such as heat-killed S. aureus (HKSA) in TLR2-bearingcells (Fig. 1). Reverse transcription–poly-merase chain reaction (RT-PCR) andimmunoblot analysis revealed that thehuman monocytic cell line THP1 express-es TLR2, which can be stimulated byproducts from Gram-positive bacteria.Stimulation of THP1-CD14 cells byHKSA led to a rapid and transient activa-tion of endogenous Rac1 and Cdc42 witha peak of activation at 1–10 min (Fig. 1a).

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Figure 2. Rac1 controls NF-κB activationafter TLR2 stimulation. (a) Parental 293(open bars) and 293-TLR2 cells (shaded bars)were analyzed for NF-κB activation bycotransfection of the reporter plasmid 5×NF-κB–Luc and β-galactosidase plasmid either untreated (–) or stimulated for 6 h with HKSA (107 CFU/ml) or IL-1β (10 ng/ml). Activation is expressed as relative luciferaseunits (RLU). (b,c) 293-TLR2 cells were transfected with vector (–) or the indicated constructs and either not stimulated (shaded bars) or stimulated with HKSA (filled bars).Cells were analyzed for NF-κB activation as reported in a. In b luciferase activities are expressed as percentage activation for each construct relative to cells transfectedwith vector alone.

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Figure 3. TLR2 cytosolic domain is associ-ated with active Rac1. (a) 293-TLR2 cellswere treated for the indicated time periods withHKSA. Cell extracts (300 µg) were incubatedwith anti-Rac1 and immune complexes were thenprobed for TLR2 or Rac1 by immunoblotting (IB).(b) Cell extracts (70–100 µg) from 293 cellstransfected with different Myc-Rac1 or Myc-Cdc42 plasmids (adjusted to comparable expression) were incubated withGST-TLR2cd. Proteins bound to GST-TLR2cd beads were separated on SDS-PAGE and immunoblotted with anti-Myc,followed by ECL detection. (Results represent five independent experiments.)

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To confirm these results we used human embryonic kidney 293 cellsstably transfected with TLR2 (293-TLR2)7,11. HKSA activated endoge-nous Rac1 and Cdc42 with a time course similar to that for THP1-CD14 cells. Stimulation of the 293 parental cell line with HKSA didnot activate these GTPases, which demonstrated that TLR2 is requiredto transduce the signal (data not shown). Rac3, another member of theRac family, which is present in both THP-1 and 293-TLR2 cells, wasnot activated after HKSA challenge, emphasizing the specificity ofRac1 and Cdc42 activation (data not shown).

Rac1 controls NF-κB–dependent gene expressionTo evaluate whether HKSA will trigger NF-κB activation in 293-TLR2 cells we monitored the activity of a transfected luciferasereporter gene driven by five NF-κB–responsive enhancers (5×NF-κB–Luc). A 15 to 20-fold increase in reporter gene activity wasobserved after IL-1 treatment both in parental 293 and in 293-TLR2cells (Fig. 2a). In contrast, HKSA stimulation resulted in a 20-foldincrease in NF-κB–mediated gene activation in 293-TLR2 but not inparental 293 cells, which lacked TLR2. These results confirmed theinvolvement of TLR2 in recognition and signaling by Gram-positivebacteria11. Coexpression of the NF-κB reporter gene with plasmidswhose gene products inhibit Rac1 and/or Cdc42 signaling was usedto establish the involvement of these GTPases in TLR2 signaling to NF-κB. Initially the PBD of Pak1 was used, which encompasses theCdc42-Rac interactive binding (CRIB) domain and the Pak inhibito-ry domain (PID) and will block active Cdc42 and Rac as well as theactivity of the effector Pak itself 30,31. PBD coexpression reducedNF-κB activation in HKSA-stimulated 293-TLR2 cells by 80%(Fig. 2b).

To analyze whether the PBD effect was due to Pak1 inhibition, wecoexpressed 5×NF-κB–Luc and PID. Overexpression of PID did notalter HKSA-induced NF-κB activation, which indicated that Pak

kinase activity was not required for NF-κB transactiva-tion. Expression of PBD F107, which binds to activeRac1 and Cdc42 but cannot inhibit Pak kinase activity,was used to confirm the requirement for Rac1 andCdc42 in NF-κB activation. PBD F107 coexpressiondecreased NF-κB activation to a similar extent as PBD.To determine whether Rac1 or Cdc42 regulate NF-κBactivation, dominant-negative mutants of theseGTPases (Rac1N17 and Cdc42N17) were introduced.In 293-TLR2 cells, Rac1N17 substantially decreased(by 70%) HKSA-stimulated NF-κB activation. In con-trast, Cdc42N17 did not affect HKSA-activated NF-κB–dependent gene expression. Likewise, dominant-negative Ha-Ras did not block NF-κB activation sug-gesting that in HKSA-stimulated 293-TLR2 cells, Rac1regulates NF-κB–dependent gene expression by a Ras-independent pathway. The expression of dominant-neg-ative Rac1, Cdc42 and Ha-Ras proteins in transient293-TLR2 transfections were consistently high andsimilar as judged by epitope tag immunoblotting (datanot shown). The role of Rac1 in HKSA-mediated NF-κB activation was emphasized by the effect of a consti-tutively active Rac1 mutant (Rac1V12). Only Rac1V12potentiated HKSA-stimulated NF-κB activity in 293-TLR2 cells (18-fold increase in NF-κB activity for vec-tor-transfected cells versus a 48-fold increase forRac1V12).

Active Rac1 and TLR2 form a signaling complexAs Rac1 can be an integral part of signaling complexes at cellular mem-branes, we analyzed whether Rac1 and TLR2 could interact either con-stitutively or upon TLR2 stimulation. In 293-TLR2 cells, TLR2 andRac1 were not associated in the absence of stimulation (Fig. 3a).However, HKSA treatment of 293-TLR2 led to a rapid and transientassociation of the two proteins, as detected by immunoblotting usingantibody to TLR2 after immunoprecipitation of Rac1. The associationof TLR2 and Rac1 was at its maximum at 3–5 min, which represents atime interval similar to maximum Rac1 activation (Fig. 1b). In contrast,no association between Cdc42 and TLR2 was detected in Cdc42immunoprecipitates. Control experiments using an unrelated mono-clonal antibody (mAb), anti-Myc, for immunoprecipitation showed noTLR2 binding.

Interaction between Rac1 and TLR2 was also analyzed by using aglutathione S-transferase (GST) fusion protein that contained thecytosolic domain of TLR2 (TLR2cd). Binding experiments with cellu-lar extracts derived from 293 cells expressing Rac or Cdc42 wild-typeor mutant proteins confirmed that only the active form of Rac1,Rac1V12, bound to GST-TLR2cd (Fig. 3b). We analyzed the associa-tion between TLR2cd and active Rac1 in more detail, testing for directbinding by using recombinant Rac1, purified from Escherichia coli orSf9 cells and labeled with GTP-γS in vitro, together with purified GST-TLR2cd in pulldown and overlay assays. Direct interaction betweenactive Rac1 and TLR2cd could not be detected under the conditionsused, which suggested that these two proteins might interact through anintermediate protein (data not shown).

Rac1 regulates NF-κB transactivation through PI3KRac1-regulated signaling can be mediated by a variety of downstreameffectors21. We have shown that Pak, one of the major targets of Rac, isnot required for HKSA-induced NF-κB activation in the 293-TLR2 cell

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Figure 4. PI3K is recruited by TLR2 andacts downstream of Rac1 in NF-κB acti-vation. (a) PI3K activity was inhibited in 293-TLR2 cells either by pretreatment for 30 minwith the inhibitors wortmannin (50 nM) orLY294002 (10 µM) or by transfection withp85αDN.After stimulation with HKSA (filled bars) NF-κB–dependent luciferase expression was analyzedas reported in Fig. 2 and recorded as a percentage of the controls. DMSO pretreatment or vector trans-fection were the controls. (b) 293-TLR2 cells were transiently transfected with Myc-Rac1 WT (100 ng) andpreincubated with wortmannin (100 nM) before stimulation with HKSA (107 CFU/ml). At the indicatedtime points activation was quenched with cold PBS and cells were lysed. Lysate (100 µg) was used for theGST-PBD binding assay and proteins bound to the beads were separated on SDS-PAGE and immunoblot-ted for Myc, followed by ECL detection. (Results are representative of three independent experiments.)(c) 293-TLR2 cells were transfected with vector (–) or the indicated constructs and stimulated with HKSA.Cells were analyzed for NF-κB–dependent gene expression by analyzing luciferase activity.

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line. PI3K represents another potential effector of Rac1 in TLR2 sig-naling. PI3K is a heterodimer consisting of a catalytic subunit (p110)associated with a regulatory polypeptide (p85)32. The p85 subunit caninteract directly with active Rac133,34. To study the involvement of PI3Kin TLR2 signaling to NF-κB, PI3K-specific pharmacological inhibitorsand a dominant-negative form of the p85α subunit of PI3K (p85DN)were used. Both approaches have been frequently used to demonstratethe role of PI3K in various signal transduction pathways. Pretreatmentof 293-TLR2 with either wortmannin or LY294002 or expression ofp85DN caused a 70–80% decline in HKSA-stimulated luciferaseexpression from the NF-κB–dependent reporter. This shows that PI3Kactivity is required for TLR2-mediated signaling to NF-κB (Fig. 4a).

PI3K can serve as an activator or effector of Rac1 in a stimulus- andcell-dependent manner. Lipid products derived from activated PI3K canactivate guanine nucleotide-exchange factors for Rac1, thereby activat-ing Rac1, or active Rac1 can regulate the activation and localization ofPI3K by binding to the p85 subunit. The role of PI3K in HKSA-medi-ated Rac1 activation was analyzed in 293-TLR2 cells pretreated withwortmannin (Fig. 4b) or LY294002 (data not shown). Pharmacologicalinhibitors of PI3K did not block Rac1 activation, as determined by PBDbinding assays. As shown earlier, transfection of Rac1V12 potentiatedNF-κB–dependent luciferase activity in stimulated 293-TLR2 cells(Fig. 2c). Coexpression of the dominant-negative p85 subunit (p85DN)with Rac1V12 completely inhibited this potentiation, decreasing it to

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Figure 6. Rac1 regulates Akt activation by HKSA and subsequent NF-κB activation. (a) THP-1 cells were treated with vehicle (DMSO) or wortmannin (50 nM)for 60 min and stimulated with HKSA for the indicated times. (b) 293-TLR2 cells were transfected with either vector or Myc-Rac1N17 and stimulated with HKSA for theindicated time period.To assess Akt activation 150 µg of cell extracts were separated on SDS-PAGE and immunoblotted with anti-phospho Thr308 (p-Akt,T308), anti-phos-pho Ser473 (p-Akt, S473), Akt and anti-Akt, followed by ECL detection.To determine Rac1 expression, 30 µg of 293-TLR2 cell lysates were separated on SDS-PAGE andimmunoblotted for Myc. (c) 293-TLR2 cells were cotransfected with the reporter plasmid 5×NF-κB–Luc, the β-galactosidase plasmid, vector (–) and the indicated plasmids.Cells were left either untreated (open bars) or stimulated for 6 h with HKSA (107 Cfu/ml) (filled bars) and analyzed for NF-κB activation via luciferase expression. (Threeindependent experiments were done.)

Figure 5. TLR2 and PI3K form a signalingcomplex that is dependent on TLR2 tyrosineresidues. (a) HKSA-stimulated 293-TLR2 cellswere lysed at different time points to immunopre-cipitate TLR2 with anti-TLR2.The precipitates wereprobed first with anti-phosphotyrosine (IB pY) andthen with anti-TLR2 (IB TLR2). (b) Cell lysates (600µg) from 293-TLR2 cells stimulated with HKSAwere used for immunoprecipitation. After incuba-tion with anti-TLR2, immune complexes wereprobed for the presence of p85 and TLR2 byimmunoblot. (c) NH2-terminal Flag-tagged TLR2WT as well as several TLR2 mutants were tran-siently transfected into parental 293 cells andlysates probed for expression by anti-Flagimmunoblots. (d) Parental 293 cells were transfect-ed with either vector (pCVM6) or the indicatedconstructs. Cells were left either untreated (openbars) or stimulated with HKSA for 6 h (filled bars)and analyzed for NF-κB activation as described in

Fig. 2. (Three independent experiments were done.) (e) After transfection with TLR2 WT (upper),TLR2-A616 (middle) or TLR2-A616,761 (lower) parental 293 cells werestimulated with HKSA for the indicated times and lysed.Anti-Flag immunoprecipitates were probed for the presence of p85 and Flag-TLR2 by immunoblotting (IB).

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the same level as p85DN expression alone (Fig. 4c). These data indi-cate PI3K as a Rac1 effector in the signaling pathway to NF-κB inHKSA-stimulated 293-TLR2 cells.

TLR2 signaling dependent on phosphotyrosine residuesActivation of PI3K induces a ligand-dependent interaction betweenthe SH2 domains of the p85 subunit and phosphotyrosine-containingmotifs present on several cytokine/growth factors receptors32.Although TLR2 by itself has no tyrosine kinase activity, it possessesseveral phosphotyrosine residues in the cytosolic domain. HKSA stim-ulation of 293-TLR2 and THP-1 cells led to transient tyrosine phos-phorylation of the TLR2 cytosolic domain (Fig. 5a, THP-1 data notshown). Because the p85 subunit of PI3K associates with tyrosinephosphorylated motifs on receptors or signaling molecules, TLR2immunoprecipitates from stimulated 293-TLR2 and THP-1 cells wereprobed for the presence of p85. Transient complex formation betweenp85 and TLR2 upon HKSA stimulation was observed (Fig. 5b, THP-1 data not shown). Probing of Rac1 immunoprecipitates that were pre-pared under the same conditions revealed that not only TLR2, but alsothe p85 subunit of PI3K, was bound (data not shown). Thus we haveshown that Rac1, p85 and the cytosolic domain of TLR2 form a stim-ulus-dependent signaling complex.

The TLR2 cytosolic domain contains several tyrosine residues thatcould serve as tyrosine-phosphorylated p85 docking sites. We preparedseveral Y→A TLR2 single and double mutations as well as a COOH-terminal TLR2 deletion mutant (TLR2∆) that were similarly expressedon the surface of parental 293 cells as on wild-type TLR2 cells (Fig. 5c,FACS data not shown). Mutation of Tyr616 (YXXM) or Tyr761 (YXXW)

alone did not alter HKSA-dependent NF-κB activation or associationwith p85 (Fig. 5d,e). Mutation of both residues (TLR2-A616,761) orCOOH-terminal truncation of TLR2 corresponding to a region requiredfor IL-1 receptor signaling completely abolished NF-κB transcription-al activity (Fig. 5d) and p85 association (Fig. 5e). As anticipated, incor-poration of phosphate into the TLR2-A616,761 mutant after HKSAstimulation was diminished (data not shown). Our results indicate thattyrosine phosphorylation of TLR2 is important for signaling to NF-κB.

TLR2 signaling involves AktThe phosphorylated lipid products generated by PI3K act as secondmessengers to activate protein kinases such as Akt (PKB)35. After stim-ulation, Akt becomes phosphorylated at two major sites, Thr308 in thekinase domain and Ser473 in the COOH-terminal tail35. We used Thr308

and Ser473 phosphospecific antibodies to analyze Akt activity afterHKSA treatment in THP-1 and 293-TLR2 cells. Endogenous Akt isactivated after TLR2 stimulation by HKSA in both cell lines (Fig.6a,b). Phosphorylation of Thr308 precedes the enhanced autophospho-rylation at Ser473 (THP-1). Pretreatment of THP-1 with the PI3Kinhibitors wortmannin or LY294002 abolished basal and stimulatedAkt autophosphorylation. Expression of dominant-negative Rac1 in293-TLR2 cells blocked Akt activation, which showed that HKSA-activated Akt is regulated by a Rac1- PI3K pathway. The involvementof Akt in NF-κB transactivation was evaluated by using plasmid con-centrations, 250–500 µg of dominant-negative Akt (AktDN), that didnot interfere with cell viability. AktDN prevented NF-κB–dependentgene transcription in HKSA-stimulated 293-TLR2 cells and inhibitedthe potentiation of HKSA-induced NF-κB activation by Rac1V12

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Figure 7. The Rac1 pathway regulates p65 transactiva-tion independently of IκBα degradation. (a) THP-1 cellswere either untreated or pretreated with ethanol, LY294002(10 µM) or MG132 (10 µM) for 60 min before stimulation withHKSA for the indicated times. (b) 293-TLR2 cells were trans-fected with vector or dominant-negative Rac1N17, AktDN orIKK2DN and stimulated 30 h later with HKSA in 1% FBS con-taining medium. Each cell lysate (50 µg) was subjected to SDS-PAGE and immunoblotted for IκBα. (c) THP-1 cells were prein-cubated with DMSO (–) or wortmannin (50 or 100 nM) for 60min followed by 60 min HKSA stimulation. 293-TLR2 cellstransfected with (d) Rac1N17 and (e) vector, AktDN orTRAF6DN were also stimulated with HKSA for 60 min. Nuclearextracts were prepared and incubated with radiolabeled κB-dependent probe (EMSA). (Results are representative of DNA-protein complexes from three independent experiments.) (f) To

analyze p65 transactivation, 293-TLR2 cells were transfected with pFR-Luc, pGAL4-p65 and vector (-) or the indicated constructs.The cells were either not stimulated (shad-ed bars) or stimulated for 5h with HKSA (filled bars) and analyzed for GAL4-p65 transactivation by measuring luciferase activity. Luciferase activities are expressed as per-centage activation for each construct relative to cells transfected with vector alone (100%).Wortmannin-treated cells were plotted as percentage of DMSO control (100%).(For PBD F107 two, and for all others three, independent experiments were done but one representative experiment is shown.)

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(Fig. 6c). Thus Akt is part of the signaling cascade that originates fromstimulated TLR2 leading to Rac1 and PI3K in order to stimulate NF-κB transcriptional activity.

Rac1 signaling regulates p65 transactivationTo examine the effect of TLR2-mediated Rac1 signaling on NF-κBactivation in more detail, the effect of Rac1, PI3K and Akt on IκBαdegradation and on NF-κB nuclear translocation and DNA binding wasevaluated in THP-1 and 293-TLR2 cells. The time course of IκBαdegradation was not altered in the presence of PI3K inhibitors(LY294002 shown) or dominant-negative Rac1 (Rac1N17) (Fig. 7a,b).293-TLR2 cells transfected with AktDN showed HKSA-stimulatedIκBα degradation that was comparable to cells transfected with thevector alone, but displayed delayed IκBα resynthesis. In contrast, IκBαdegradation was strongly inhibited by transfection of dominant-nega-tive IKK2 in 293-TLR2 cells and by preincubation with the proteasomeinhibitor MG132 in THP-1 cells.

To assess the effect of the Rac1-initiated pathway on HKSA-inducedNF-κB, DNA-binding electrophoretic mobility shift assays (EMSA)were done on inhibitor-treated THP-1 or transfected 293-TLR2 cells. In

contrast to TRAF6DN, the dominant-negative forms of Rac1 and Aktdid not alter NF-κB DNA binding in 293-TLR2 cells (Fig. 7d,e).HKSA-stimulated THP-1 cells showed no change in nuclear transloca-tion and DNA binding of NF-κB when pretreated with wortmannin orLY294002 (Fig. 7c). This suggested that Rac1 controls a signalingpathway triggered by HKSA that is important for NF-κB transactiva-tion but distinct from IκBα degradation, NF-κB nuclear translocationand DNA binding.

Regulation of NF-κB activation is not only dependent on phospho-rylation of IκBs but is also dependent on the inducible phosphorylationand transactivation activity of p65. We tested whether HKSA-inducedsignaling through the Rac1 cascade leads directly to stimulated tran-scription from a GAL4-dependent promoter driven by the chimeric pro-tein GAL4-p65 in 293-TLR2 cells. HKSA increased the transcription-al activity of p65 by 10–15-fold. Dominant-negative Rac1, as well asthe PBD F107 fragment that binds activated Rac1 and impedes its inter-action with downstream effectors, inhibited p65 transactivation by65–80% (Fig. 7f). Similar inhibitory effects were observed for thePI3K inhibitor wortmannin and dominant-negative forms of p85α andAkt. Thus, TLR2-mediated Rac1-PI3K signaling regulates NF-κB acti-vation by affecting the p65 transcriptional complex.

DiscussionHuman TLRs are involved in the recognition and response to Gram-negative and Gram-positive bacterial cell wall components and play akey role in innate immunity. Although certain consequences of stimu-lated TLR signaling, including activation of transcription factors andstress-activated mitogen-activated protein kinases (MAPKs), areknown, the molecular details of intracellular pathways are largely unde-fined. Similarities between the signaling pathways triggered by IL-1and microbial products have been suggested. Others have shown thatactivated IL-1R and TLRs share a common signaling pathway leadingto NF-κB nuclear translocation involving MyD88, IL-1R–associatedkinase (IRAK), tumor necrosis factor receptor–associated factor 6(TRAF6) and IKKs11,36. Our results indicate that TLR2 signaling to NF-κB requires a second, parallel, pathway that is regulated by the smallGTPase Rac1 (Fig. 8).

Stimulation of TLR2 with S. aureus results in Rac1 activation, asso-ciation of active Rac1 with TLR2 and initiates NF-κB–dependent genetranscription. A role for RhoGTPases in NF-κB activation has beenreported for agonists including IL-1 and TNF-α20,37–39. In cell typeswhere active Rac1 induced the production of reactive oxygen interme-diates, NF-κB nuclear translocation was blocked by dominant-negativeRac126. It was suggested that Rac1 might activate a redox-dependentpathway involved in IκB phosphorylation. NF-κB activation of S.aureus–stimulated human neutrophils was insensitive to several antiox-idants40. Our study defines a Rac1-dependent pathway in the TLR2-mediated immune cell response that is independent of IκB degradationand NF-κB release but essential for activation of NF-κB–regulated genetranscription.

Recently, a pathway that leads to enhanced transactivation potential,once NF-κB is bound to its consensus sequence, was defined19,20.Phosphorylation of the p65 subunit, which promotes the interaction withthe coactivator proteins p300 and CBP, is a key element. Potential kinases implicated in p65 phosphorylation include casein kinase II41, theMAPKs p38, p42 and p4418 and Akt (PKB)19,42. However, it is unlikelythat they act on p65 directly. Most likely, the Rac1→PI3K→Akt signal-ing cascade exerts control of the p65 transcriptional complex by induc-ing p65 phosphorylation thereby cooperating with the MyD88→IKKpathway in NF-κB–dependent gene transcription.

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Figure 8. Proposed model for Toll signaling to NF-κB.The diagram shows twosignaling cascades that flow from membrane-bound TLR2 to the activation of thetranscription factor NF-κB. Gram-positive bacterial stimuli lead to TLR2 activationand recruitment of a signaling complex, which initiates two pathways for NF-κB acti-vation. Activation of the RhoGTPase Rac1 by stimulated exchange factors (GEF)leads to association of Rac1, the PI3K subunit p85 and the TLR2 cytosolic domainand regulates NF-κB transactivation through Akt. IκBα degradation and NF-κBrelease are independent of this pathway and rely on IKK signaling.

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The distinct role of the Rac1 GTPase in TLR2 signaling is empha-sized by the formation of a signaling complex containing Rac1, theTLR2 cytosolic domain and the p85 subunit of PI3K. With wild-typeand mutant forms of Rac1, Cdc42 and RhoA in TLR2-binding experi-ments, only active Rac1 was bound. In cells, the association betweenTLR2 and Rac1 was dependent on activation of the receptor, as we didnot detect complex formation in the absence of stimulation. TLR2 stim-ulation activates Cdc42 in addition to Rac1. Although we cannotexclude Cdc42 as potential regulator of NF-κB activation in THP-1cells, only Rac1 controlled the PI3K→Akt pathway to NF-κB transac-tivation in 293-TLR2 cells. Cdc42 has been implicated in other aspectsof host defense against microorganisms, including phagocytosis23,organization of the actin cytoskeleton21,39 or activation of MAPKs21.

In addition to Rac1, the p85 subunit of PI3K is recruited to the TLR2signaling complex and PI3K activity is essential for NF-κB signaling.Lipid products generated by activated PI3K can mediate effects eitherupstream or downstream of Rac1. Rac1 activation in HKSA-stimulatedTLR2 signaling is independent of PI3K activity. The active GTP-boundform of Rac1 can bind directly to p85 and increase PI3K activity33,34.The participation of PI3K in LPS signaling and NF-κB activation hasbeen suggested28,43,44. PI3K was implicated either in regulating nucleartranslocation and DNA binding of NF-κB or NF-κB transactiva-tion19,42,44, presumably reflecting cell type and stimulus-dependent dif-ferences. TLR2-mediated responses in 293-TLR2 cells favor PI3K-dependent up-regulation of the NF-κB transactivation potential bydirectly targeting p65. Additionally, Akt, a downstream effector ofPI3K that has been implicated in NF-κB translocation45,46 or NF-κBtransactivation19, is activated by Gram-positive microbial products andis involved in mediating Rac1-regulated p65 transactivation.

It is currently unknown whether other members of the TLR familysignal to NF-κB via a Rac1-regulated pathway. Macrophages chal-lenged with Pseudomonas aeruginosa, a Gram-negative bacteria thatis thought to activate TLR4, requires Rac1 and Cdc42 for phagocyto-sis but not for NF-κB–dependent gene expression23. Differences in thecytosolic TLR domains might contribute to specific signalingresponses. The cytosolic domain of TLR2 contains a PI3K bindingmotif (YXXM), which is present in TLR1, TLR2 and TLR6 but miss-ing in TLR3, TLR4 and TLR5. IL-1R, the IL-1R accessory proteinand MyD88 also possess a putative p85-binding site. Tyrosine phos-phorylation of TLR2 induced by HKSA stimulation is necessary forp85 association. As TLR2 does not have any intrinsic tyrosine kinaseactivity, the cytoplasmic domain has to be phosphorylated by anothertyrosine kinase. This could be accomplished by activated nonreceptortyrosine kinases or by dimerization with a tyrosine kinase receptor.Elucidating new targets within the TLR signaling cascades has impor-tant implications for anti-inflammatory strategies aimed at inhibitingNF-κB transcription in human disease states where dysregulation ofthe innate immune response contributes to morbidity or mortality.

MethodsCells and reagents. Human embryonic kidney 293 cells stably transfected with TLR2 (293-TLR2) or parental 293 were as described11. Cells were maintained in low glucoseDulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivatedfetal bovine serum (FBS), HEPES (10 mM), L-glutamine (2 mM), penicillin (100 U/ml),streptomycin (100 µM) and puromycin (1 µg/ml). The monocytic cell line THP-1 stablytransfected with human CD14 (THP1-CD14) has been described previously and was main-tained thus47. HKSA was provided by C. Fearns. Contaminating LPS in HKSA preparationswas determined using a quantitative Limulus Lysate Assay (BioWhittaker, Walkersville,MD) and were <1 pg/ml at final concentration. LY294002 and wortmannin were from theAlexis Corporation (San Diego, CA), MG132 from Calbiochem (La Jolla, CA). Protein Gsepharose and glutathione agarose beads were from Pharmacia (Uppsala), Protein A agarosefrom Sigma (St. Louis, MO). Polyclonal anti-p85, anti-Cdc42, anti-Myc and anti-IκBαwere from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-phospho-S473-Akt,

anti-phospho T308 and anti-Akt were from New England Biolabs (Beverly, MA); mAbs toMyc (9E10) and HA were from Babco (Richmond, CA); mAbs to Rac1 and pY were fromUBI (Lake Placid, NY) and mAb to Flag from Sigma. mAbs to 2392 for human TLR2 andmAbs 5B6 that recognize the glycoprotein D (gD) epitope on stably expressed human TLR2were as described11.

Plasmids. Plasmids were prepared using endo-free plasmid DNA purification columns(Qiagen, Valencia, CA). DNA was eluted from the columns using LPS-free buffers and con-taminating LPS was found to be <10 pg/ml. cDNAs for Rac1 wild-type (WT), Rac1N17,Rac1V12, Cdc42 WT, Cdc42N17 and Cdc42L61 were subcloned into pRK5 containing anNH2-terminal Myc tag31. Dominant-negative IKK2 was as described48. PCMV6 expressionvectors encoding PBD (Pak1 aa 67–150), PBD F107 and PID (Pak1 aa 83–149) were asdescribed31. Expression vectors encoding Akt WT, Akt A308A473, Ha-RasN17 and p85α∆(aa 478–513) were provided by C. King and G. Bokoch. Myc-tagged TRAF6∆ (aa288–522) was subcloned into pCVM6. Human TLR2 mutants (Y616A, Y761A,Y616,761A) were obtained using the Quick Exchange Mutagenesis kit (Stratagene, LaJolla, CA). TLR2∆ (aa 1–745) was generated by PCR. All TLR2 constructs were confirmedby sequencing and cloned into the expression vector pCVM6 containing an NH2-terminalFlag tag. The control plasmid pFA-CMV expresses the GAL4 DNA-binding domain aloneand was obtained from Stratagene. The plasmid pFR-Luc contained 5×GAL4 upstream ofa minimal promoter that drives expression from the firefly luciferase reporter gene. The bac-terial expression plasmid for GST-p65 (aa 346–551) was provided by T. Sakurai (TanabeSeiyaku Co., Osaka)49. PCR fragments of 600-bp were generated from this plasmid andcloned in-frame into pFA-CMV to create pGAL4-p65.

Transfection and reporter assays. Transient transfections were done using Lipofectamine-Plus reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer’s instructions.All transfections utilized 40 ng of 5×NF-κB–Luc (the NF-κB responsive luciferase reporter)and 40 ng of β-galactosidase plasmids (Promega, Madison, WI) or 80 ng of pFR–Luc and160 ng of pGAL4-p65 together with plasmid DNA consisting of expression vectors or vec-tor plasmid alone. After 20 h of expression, cells were starved for 3–12 h in 1% FBS. Forstimulation, individual wells were left untreated or were stimulated with HKSA as indicat-ed. Pretreatment with PI3K inhibitors LY294002 (10 µM) and wortmannin (50–100 nM)was initiated for 30–60 min at 37 °C before stimulation. Cells were incubated for addition-al 6 h before collection. Luciferase assays were done using the Luciferase Assay System(Promega) according to the manufacturer’s instructions. Luciferase activity was normalizedto β-galactosidase to standardize transfection efficiency. The viability of each transfectedcell population was measured by Trypan blue exclusion at the time of collecting.Immunofluorescence was used to assess transfection efficiency (about 65–80%, indepen-dently of the plasmid). Transfection experiments were repeated at least three times.

EMSA. Transiently transfected 293-TLR2 or THP-1 cells were stimulated with HKSA for30 or 60 min. Nuclear extracts were prepared and EMSA reactions done as described20.Three independent experiments were done.

Immunoblotting and immunoprecipitation. Stimulation of 75–85% confluent cells wasquenched with cold PBS. Cells were lysed in Tris-HCl (25 mM, pH 7.5), EDTA (1 mM),MgCl2 (5 mM), DTT (0.1 mM), EGTA (0.1 mM), NaCl (100 mM), 10% glycerol, 1% non-idet P-40, phenylmethylsulfonylfluoride (1 Mm), leupeptin (1 µg/ml), aprotinin (1 µg/ml).Protein concentrations were quantified by Lowry assay. For immunoprecipitations, cellextracts were incubated with primary antibody for 2 h followed by incubation for 1 h with10–25 µl of protein G sepharose or protein A agarose beads. The beads were washed fourtimes with lysis buffer and samples analyzed by SDS-PAGE and immunoblotting. At leastthree independent experiments were done.

PBD assay. Cell lysates (150–900 µg), obtained from either THP1-CD14 or 293-TLR2before or after stimulation with HKSA were incubated with 10 µg of recombinant GST-PBD (human Pak1 aa 67–150) for 1 h at 4 °C as described29,31. Immunoblotting was donewith anti-Rac1, anti-Cdc42 or anti-Myc.

Binding analysis with GST-TLR2cd. The full-length TLR2cd (human TLR2 aa 609–784)was expressed as GST fusion protein in E. coli and affinity purified. Lysates derived fromRac1 and Cdc42 wild-type and mutant protein–overexpressing 293 cells (70–100 µg),adjusted to equivalent GTPase protein expression, were incubated with 5 µg of recombinantGST-TLR2cd for 2 h at 4 °C, then washed four times in lysis buffer. Immunoblotting wasdone with anti-Myc. Five different experiments using two independent preparations of GST-TLR2cd and GTPase lysates were done.

AcknowledgementsWe thank P. Rutledge for excellent secretarial support. Supported by NIH grants GM37696 (to U. G. K. and to R. J. U.), GM 28485 and AI 15136 (to R. J. U.), HL48872 (toN.M.). N.T. is the recipient of a Boehringer Ingelheim Fonds fellowship.

Received 12 September 2000; accepted 19 October 2000

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