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The Journal of Immunology, 2004, 173: 507-514.
Copyright © 2004 by The American Association of Immunologists

The Low Molecular Weight GTPase RhoA and Atypical Protein Kinase C{zeta} Are Required for TLR2-Mediated Gene Transcription1

Nicole Teusch2, Eleuterio Lombardo2, Jane Eddleston and Ulla G. Knaus3

Departments of Immunology and Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell cycle progression, cell adhesion, and motility. In this study, we report that stimulation of TLR2 in human epithelial and monocytic cells leads to rapid and transient activation of RhoA. RhoA cooperated with the canonical I-{kappa}B kinase-mediated pathway that induces the release of NF-{kappa}B, in regulating the trans activation of the NF-{kappa}B subunit p65/RelA by affecting Ser311 phosphorylation, and subsequent cytokine production. Another consequence of TLR2 stimulation by bacterial derived products was the activation of atypical protein kinase C (PKC) {zeta} and association of this protein kinase with RhoA. Inhibition of PKC{zeta} decreased NF-{kappa}B activation and p65/RelA trans activation without affecting I-{kappa}B{alpha} degradation. The observation of a transient, stimulus-dependent association of RhoA with PKC{zeta} suggests that RhoA mediates at least partially its effect on gene transcription through atypical PKC. In contrast to previous studies, identifying Rac1-PI3K as an upstream element in TLR2-initiated response to NF-{kappa}B, PI3K signaling was not required for RhoA or PKC{zeta} activity. These results indicate that multiple GTPase-regulated pathways emerge from stimulated Toll receptors, controlling different aspects of NF-{kappa}B-mediated gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A remarkable feature of the innate immune system is its capacity to recognize a broad spectrum of pathogens using a repertoire of receptors that are referred to as pattern-recognition receptors (PRRs)4 (1, 2, 3). PRRs recognize conserved molecular patterns, which are invariant products of microbial metabolic pathways and presented to innate immune cells in the context of pathogen-induced breakdown of physiological host barriers. Ten members of a new class of PRRs, the mammalian TLRs, and many of their microbial derived ligands have been identified. One of these receptors, TLR2, has been more extensively studied and is involved in innate immune recognition and subsequent proinflammatory responses. A wide array of molecules has been reported to activate innate immune responses through TLR2, including components from Gram-positive bacteria, such as peptidoglycans and lipoteichoic acid, bacterial lipopeptides, and certain structural variants of LPS. Toll receptors are type 1 transmembrane proteins with extracellular leucine-rich repeat domains and cytoplasmic domains that resemble the mammalian IL-1R. Although induction of certain cellular responses by TLRs seems to conform to paradigms observed in IL-1R signaling, other intracellular signaling cascades mediated by stimulated TLRs, however, appear distinct.

Upon stimulation with bacterial products, TLRs, such as TLR2, mediate several crucial cellular functions, including the production of proinflammatory cytokines, generation of reactive oxygen and NO species, and up-regulation of various cell surface molecules. One major requirement for this response is the activation of the transcription factor NF-{kappa}B. NF-{kappa}B is a collective term referring to Rel family transcription factors, such as p50 and p65, which are maintained in the cytoplasm through association with I-{kappa}Bs. Upon cellular stimulation, phosphorylation of I-{kappa}Bs occurs, resulting in degradation and liberation of Rel heterodimers, which after translocation to the nucleus induce transcription of NF-{kappa}B target genes. Activation of NF-{kappa}B through Toll/IL-1R domains is mediated by the recruitment of cytoplasmic adaptor molecules such as MyD88 and Toll-IL-1R domain-containing adapter protein, IL-1R-associated kinases (IRAKs), and TNFR-associated factor 6 (TRAF6) to activate I-{kappa}B kinases (IKKs), which ultimately leads to I-{kappa}B degradation and NF-{kappa}B translocation (4). Secondary cues, including the phosphorylation of the p65/RelA subunit in the transcriptional complex, are required for NF-{kappa}B-dependent gene transcription. These signals are transmitted by additional signaling pathways, which branch directly from the activated receptor or later from the canonical IKK pathway and are I-{kappa}B independent (5, 6). Hence, the cooperation of several different signaling cascades is required for NF-{kappa}B-dependent gene transcription.

The Rho-family GTPases Rho, Rac, and Cdc42 are well-established regulators of a wide spectrum of cellular functions, such as cytoskeletal reorganization, cell growth, and apoptosis (7). Rho GTPases have been implicated in IL-1R- and TNFR-{alpha}-mediated gene transcription and actin polymerization (8, 9). We described recently the requirement of a Rac1-PI3K-Akt pathway for NF-{kappa}B subunit p65 trans activation by stimulated TLR2 (6). In this study, we report that the GTPase RhoA is also an essential regulator of TLR2-mediated gene transcription. RhoA is required for the transcriptional activity of the NF-{kappa}B p65 subunit, in part by transmitting the signal to protein kinase C (PKC) {zeta}. Although RhoA-PKC{zeta} signaling is activated independently of the previously described TLR2-Rac1-PI3K-mediated pathway promoting cell survival, it seems to connect further downstream to this pathway to control I-{kappa}B{alpha}-independent gene transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents

Human embryonic kidney 293 cells stably transfected with TLR2 (293-TLR2) and parental 293 cells were as described and maintained, as reported previously (6, 10). The monocytic cell line THP-1 stably transfected with CD14 (THP1-CD14) has been described (6). Heat-killed Staphylococcus aureus (HKSA) was provided by J. Mathison (The Scripps Research Institute, La Jolla, CA) and used as 107 CFU/ml. The synthetic lipoprotein Malp-2 and wortmannin were from Alexis (San Diego, CA), Ro-31-8220 was from Calbiochem (La Jolla, CA), and the PKC{zeta} pseudosubstrate (ps) was obtained from BioSource International (Camarillo, CA). Protein G-Sepharose and glutathione agarose beads were from Pharmacia (Uppsala, Sweden). Anti-I-{kappa}B{alpha}, anti-PKC{zeta}, anti-p65, anti-RhoA, and anti-Myc Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho PKC{zeta} (Thr410/403) and anti-phospho RelA Ser536 were from Cell Signaling Techology (Beverly, MA), Anti-Myc (9E10) was from Babco (Richmond, CA), and anti-Rac1 was from Upstate Biotechnology (Lake Placid, NY).

DNA constructs

All plasmids were prepared using endo-free plasmid DNA purification columns (Qiagen, Valencia, CA). DNA eluted from these columns contained contaminating LPS in a concentration range <10 pg/ml. cDNAs for the GTPase constructs RhoA wild type (wt), RhoA T19N, and Rac1wt were subcloned into pRK5 containing an N-terminal Myc-tag (11). An expression vector encoding Rhotekin binding domain (RBD) (Rhotekin aa 7–89) was provided by M. Schwartz (The Scripps Research Institute) (12), and GST p21-activated kinase binding domain (PBD) was used, as described previously (11). pMT-2 expression vectors encoding PKC{zeta}wt and dominant-negative PKC{zeta} (PKC{zeta}dn) were provided by A. Hall (MRC, London, U.K.) and described previously (13). The NF-{kappa}B-responsive luciferase reporter (5x NF-{kappa}B-Luc) and the {beta}-galactosidase plasmid were from Promega (Madison, WI). The plasmid pFA-CMV expressing the GAL4 DNA binding domain and the reporter plasmid pFR-Luc were obtained from Stratagene (La Jolla, CA). The cloning of p65 (aa 346–551) into pFA-CMV to create GAL4-p65 was described previously (6).

Transfection and reporter assays

Transient transfections in 293 cells were done using LipofectAMINE-Plus reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Reporter assays with 5x NF-{kappa}B-Luc or pFR-Luc/GAL4-p65 were done, as previously reported (6). THP-1 cells were transiently transfected with 50 ng of 5x NF-{kappa}B-Luc, 50 ng of {beta}-galactosidase, and 100 ng of various constructs with FuGENE (Roche, Mannheim, Germany), according to the manufacturer’s instructions. After 18 h of expression, cells were starved for 3 h in medium containing 3% FBS. After stimulation with HKSA for 2 h, cell lysates were prepared and analyzed using the Luciferase Assay System (Promega), according to the manufacturer’s instructions. Immunofluorescence was used to assess transfection efficiency (80–90% in 293 cells and ~20% in THP-1 cells, independently of the plasmid).

IL-8 real-time PCR

The 293TLR2 cells were transfected with 700 ng of pcDNA3, enhanced green fluorescence protein-RBD, PKC{zeta}wt, or PKC{zeta}dn 24 h before stimulation with 5 ng/ml Malp-2 for 6 h. Total RNA was isolated, and 1 µg of the total RNA was reverse transcribed using the Omniscript reverse-transcription kit (Qiagen). A one-twentieth aliquot of the reverse-transcription product was used for subsequent real-time, quantitative PCR (iCycler iQ; Bio-Rad, Hercules, CA). The primers were as follows: IL-8 forward primer, 5'-tgccagtgaaacttcaagca-3'; IL-8 reverse primer, 5'-attgcatctggcaaccctac-3'; {beta}-actin forward primer, 5'-tgcgtgacattaaggagaag-3'; {beta}-actin reverse primer, 5'-gtcaggcagctcgtagctct-3'. The following dual-labeled probes were obtained from BioSearch Technologies (Novato, CA): IL-8, 5'-FAM-cagacccacacaatacatgaagtgttga-3' black hole quencher-1; {beta}-actin, 5'-FAM-cacggctgcttccagctcctc-3' black hole quencher-1. Standards, from 10 to 0.0001 attomoles of the PCR product cloned into pGEMTeasy, were run alongside the samples to generate a standard curve. All samples and standards were analyzed in triplicate. The PCR consisted of 1.5 mM Tris-HCl, 5 mM KCl, 4 mM Mg2+, 2 mM dNTPs, 200 ng of sense and antisense primers, and either 2.5 pmol of IL-8 dual-labeled probe or 1.5 pmol of {beta}-actin dual-labeled probe and 0.5 U of AmpliTaq gold (Applied Biosystems, Foster City, CA) in a total volume of 25 µl. The reaction conditions were 95°C for 10 min, followed by 45 cycles of 15 s, 94°C; 15 s, 60°C; and 15 s, 72°C. The starting amount of cDNA in each sample was calculated using the iCycler iQ software package (Bio-Rad).

RBD assay

The 293 or 293-TLR2 cells were transfected with Myc-RhoAwt 24 h before stimulation with HKSA. Cells were stimulated in culture medium containing 1–3% FBS after a starvation period of at least 3 h. At the indicated time points, activation was quenched with cold PBS. Cells were lysed in 50 mM Tris-HCL (pH 7.5), 500 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 10% glycerol, 5 mM NaF, 1 mM DTT, 1% Nonidet P-40, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Protein concentrations were quantified by bicinchoninic acid assay. Cell lysates (400–1000 µg of protein) were incubated with 20 µg of rGST-RBD (human Rhotekin aa 7–89) for 1 h at 4°C. Afterward, beads were washed four times with lysis buffer, and bound proteins were analyzed by SDS-PAGE, immunoblotting with anti-RhoA or anti-Myc Abs, and detection by ECL.

PBD assay

Cell lysates from 293-TLR2 cells were incubated with 10 µg of rGST-PBD (human p21-activated kinase 1 aa 67–150) for 1 h at 4°C, as described (11), followed by immunoblotting with anti-Rac1 Ab.

Immunoprecipitation

For some experiments, 293-TLR2 cells were transfected with 50–100 ng of Myc-RhoAwt 24 h before stimulation with HKSA. Cell lysates were incubated with primary Ab for 1 h at 4°C, followed by incubation for another hour with 30 µl of protein A/G-Sepharose beads. The beads were washed four times with lysis buffer, and samples were analyzed by SDS-PAGE and immunoblotting with anti-Myc or anti- PKC{zeta} Abs. PKC{zeta} immunoprecipitates were used for kinase assays and immunoblots, as described previously (14).

p65 Phosphorylation

The 293TLR2 cells were transfected with 700 ng of pcDNA3, enhanced green fluorescence protein-RBD, PKC{zeta}wt, or PKC{zeta}dn 24 h before stimulation with 5 ng/ml Malp-2 or 20 ng/ml TNF-{alpha} for different times, after which cells were lysed with RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris-HCl pH 8.0) containing phosphatase and protease inhibitors. Cell lysates were incubated with anti-p65 Ab and protein G-Sepharose beads at 4°C. The beads were washed four times with lysis buffer, and samples were analyzed by SDS-PAGE and immunoblotting with anti-phospho p65 (S536) or anti-phospho p65 (S311) (kindly provided by J. Moscat, Centro de Biologia Molecular "Severo Ochoa," Madrid, Spain). Total p65 was determined by reblotting the membrane with anti-p65 Ab.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of TLR2 activates the GTPase RhoA

To detect the activation of low m.w. GTPases by extracellular stimuli, biochemical assays take advantage of the fact that only the GTP-bound, but not the GDP-bound forms of GTPases specifically interact with downstream effectors. For example, the Rho binding domain (RBD) of the effector protein Rhotekin is used to precipitate active, GTP-bound Rho from cell lysates. Using this approach, we investigated whether RhoA was activated after stimulation of TLR2 with Gram-positive stimuli such as HKSA or the lipoprotein Malp-2 in two cell types expressing TLR2. Stimulation with these ligands led to rapid and transient activation of RhoA in human embryonic kidney cells that were stably transfected with TLR2 (293-TLR2) (Fig. 1). RhoA activation was an immediate event and continued for ~15 min. However, stimulation of 293 parental cells with S. aureus did not activate RhoA, demonstrating that this GTPase is part of a signaling cascade specifically induced by ligand-activated TLR2. Gram-positive stimuli induced RhoA activity with a similar time course in the human monocytic cell line THP1-CD14 expressing endogenous TLR2 and RhoA (Fig. 1).



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  		FIGURE 1. Stimulation of TLR2 activates RhoA. Time course of RhoA activation in 293 cells stably expressing TLR2 (293-TLR2), parental 293 cells, and monocytic THP-1 cells. The 293 and 293-TLR2 cells were transiently transfected with Myc-RhoAwt (100 ng) 24 h before stimulation with HKSA or Malp-2 (data not shown) in 1% FBS-containing medium. THP-1 cells were kept for 12 h in culture medium containing 4% FBS before stimulation with HKSA or Malp-2 (data not shown). Upper panels, Lysates of each time point were subjected to affinity precipitation with GST-RBD. Proteins bound to GST-RBD beads were separated on SDS-PAGE, immunoblotted for RhoA or Myc-RhoA, and analyzed using ECL. Lower panels, Immunoblots (IB) of RhoA in the cell lysates used for the binding assay. Results shown are representative of at least three independent experiments.

 
RhoA controls TLR2-mediated NF-{kappa}B-dependent gene transcription

We have previously reported the HKSA-induced NF-{kappa}B activation in 293-TLR2 cells by monitoring the activity of a transfected luciferase reporter gene (5x NF-{kappa}B-Luc) (6). Although with less efficiency (20% transfection), we introduced this reporter gene together with control or dominant-negative plasmids into THP-1 cells. Stimulation of 293-TLR2 and THP1-CD14 cells with heat-killed S. aureus resulted in a 15- to 20-fold increase in reporter gene activity (Fig. 2A). To evaluate the role of RhoA in TLR2 signaling leading to NF-{kappa}B activation, we expressed the NF-{kappa}B reporter together with either dominant-negative RhoA (RhoAN19) or the Rho binding domain (RBD), which binds activated RhoA and abrogates its interaction with downstream effectors. In both cell types, 293-TLR2 and THP-1 cells, RhoA activity was required for NF-{kappa}B-dependent gene transcription. Inhibition of RhoA activation or RhoA-GTP signaling substantially decreased luciferase activity (70–90%, depending on cell type and concentration of the plasmid). Expression of the vector (control) or wt RhoA did not affect NF-{kappa}B activity. The expression levels of all constructs used were for 293-TLR2 cells consistently similar, as judged by immunoblotting (see Figs. 2, 3, and 4). Expression of Rho inhibitory proteins in THP-1 cells was assessed by {beta}-galactosidase cotransfection to normalize for transfection efficiency.



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  		FIGURE 2. RhoA regulates TLR2-mediated NF-{kappa}B trans activation. A, 293-TLR2 (left) and THP-1 cells (right) were analyzed for NF-{kappa}B activation by cotransfection of the reporter plasmid 5x NF-{kappa}B-Luc with {beta}-galactosidase, pRK5 (control vector), or the indicated constructs. The cells were either unstimulated ({square}) or stimulated for 2–4 h with HKSA ({blacksquare}). Activation is expressed as relative luciferase units. Three to five independent experiments were done. B, Time course of I-{kappa}B{alpha} degradation in 293-TLR2 cells. Cells were either transfected with vector (first panel) or the indicated constructs (middle panels) 24 h before stimulation with HKSA. The lysates of each time point (50 µg) were subjected to SDS-PAGE and immunoblotted for I-{kappa}B{alpha}. Immunoblots (IB) show expression of the transfected constructs in whole cell lysates (lower panels). C, To analyze p65 trans activation, 293-TLR2 cells were cotransfected with pFR-Luc, pGAL4-p65, and control vector or the indicated constructs. Cells were either unstimulated ({square}) or stimulated for 5 h with HKSA ({blacksquare}) and analyzed for GAL4-p65 trans activation by measuring luciferase activity. Luciferase activities are expressed as percentage of activation for each construct relative to cells transfected with vector alone (100%). Expression of proteins was analyzed by immunoblot. At least four independent experiments were done.

 


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  		FIGURE 3. TLR2-mediated RhoA signaling involves PKC{zeta}. A, 293-TLR2 cells transfected with Myc-RhoAwt were stimulated with HKSA. Anti-RhoA immunoprecipitates were separated by SDS-PAGE and immunoblotted for PKC{zeta} (upper panel). Immunoblots (IB) show total RhoA precipitated from the lysates and PKC{zeta} expression in whole cell lysates. B, THP-1 and 293-TLR2 cells were stimulated with HKSA (data not shown) or Malp-2 and analyzed for PKC{zeta} activation. Upper panels (phospho PKC{zeta} (pPKC{zeta})), Depict anti-phospho PKC{zeta} immunoblots, which were reprobed for total PKC{zeta} protein. In THP-1 cells, the lower band, recognized by the PKC Ab, corresponds to the pPKC{zeta} band in the panel above. Activation of PKC{zeta} was also confirmed with PKC{zeta} immunoprecipitates in kinase assays using myelin basic protein as substrate.

 


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  		FIGURE 4. RhoA and PKC{zeta} regulate p65 phosphorylation at position 311. The 293-TLR2 cells transfected with RBD, PKC{zeta}wt, PKC{zeta}dn, or vector were stimulated with Malp-2 or TNF-{alpha} for the indicated times, and endogenous p65 was immunoprecipitated with anti-p65 Ab. A, p65 phosphorylation at Ser536 was determined by immunoblotting with anti-phospho p65 (S536) Ab (upper panel). B, p65 phosphorylation at Ser311 was detected by immunoblotting with anti-phospho p65 (S311) Ab (upper panel). Total p65 was determined by reprobing of the same membrane with anti-p65 (lower panels).

 
RhoA regulates the trans activation of the NF-{kappa}B subunit p65

To examine the role of RhoA in TLR2-mediated NF-{kappa}B activation in more detail, we investigated the effect of this GTPase on I-{kappa}B{alpha} degradation. Incubation of 293-TLR2 cells with heat-killed S. aureus resulted in a significant reduction of I-{kappa}B{alpha} 40 min after stimulation. This time course was not altered in the presence of the RBD or dominant-negative RhoA (Fig. 2B; 80–90% transfection efficiency). In contrast, I-{kappa}B{alpha} degradation can be strongly inhibited by overexpression of dominant-negative I-{kappa}B kinase 2 or by preincubation with the proteasome inhibitor MG132 in these cells (6). NF-{kappa}B-dependent gene transcription is not only dependent on I-{kappa}B{alpha} degradation and translocation of p65/p50 to the nucleus, but requires the inducible activity of the NF-{kappa}B subunit p65. To evaluate the effect of HKSA-induced Rho signaling on p65 trans activation, we transfected 293-TLR2 cells with a GAL4-dependent promotor driven by the chimeric protein GAL4-p65. Stimulation with heat-killed S. aureus induced a 10- to 15-fold increase in p65 transcriptional activity. Dominant-negative RhoA as well as the Rho binding domain RBD inhibited p65 trans activation by 70–90% (Fig. 2C). The vector control as well as wt RhoA did neither attenuate nor trigger p65-dependent gene transcription. Thus, the TLR2-mediated Rho-dependent signaling pathway regulates NF-{kappa}B-dependent gene transcription by directly affecting the p65 transcriptional complex.

RhoA signaling is connected to PKC{zeta}

Studies in Saccharomyces cerevisiae revealed that Pkc1, a homologue of mammalian PKC, associates with the small GTP-binding protein Rho1 and acts as a downstream target (15). Additionally, the atypical PKC (aPKC) isoform {zeta} has been implicated in association and phosphorylation of p65 in PKC{zeta}-deficient animals (16, 17). Analysis of 293-TLR2 and THP-1 cell lysates indicated that aPKC isoforms were expressed. We investigated whether RhoA interacts with PKC family members in 293-TLR2 cells upon TLR2 stimulation. Western blot analysis of proteins bound to RhoA upon HKSA stimulation revealed that only the aPKC isoform {zeta} (Fig. 3A), but not other PKC isoforms such as PKC{delta} or PKC{theta} associated with activated RhoA (data not shown). The interaction of RhoA with PKC{zeta} was strictly stimulus dependent. The association was observed during a time course that was slightly delayed when compared with RhoA activation. Control experiments using an unrelated control Ab for immunoprecipitation showed no PKC{zeta} binding (data not shown). From these experiments, it would be reasonable to predict that PKC{zeta} activation will occur when analyzed in the same conditions. In fact, TLR2 stimulation led in THP-1 and 293-TLR2 cells to PKC{zeta} activation, as determined by immunoblotting with an Ab, which detects the activated form of PKC{zeta} or by using kinase assays after PKC{zeta} immunoprecipitation (Fig. 3B). The time course of activation is slightly delayed when compared with RhoA, and mirrors the presence of the RhoA-PKC{zeta} complex. In contrast to rapid RhoA inactivation, PKC{zeta} activity can be observed for up to 2 h (data not shown).

PKC{zeta} regulates I-{kappa}B{alpha}-independent NF-{kappa}B trans activation

Because aPKC isoforms such as PKC{zeta} have been shown to play a critical role in the regulation of various NF-{kappa}B activation pathways (18, 19), we investigated whether aPKCs mediate NF-{kappa}B-dependent gene transcription in 293-TLR2 cells (Fig. 5A). Blocking the activity of conventional PKC isoforms with the selective inhibitor Ro-31-8220 did not show any effect on NF-{kappa}B-dependent gene expression compared with the control cells. Furthermore, pretreatment with phorbol ester (PMA) for an extended period, resulting in removal of conventional and novel PKC pools, did not decrease NF-{kappa}B activation. These findings suggest that conventional and novel PKCs are not involved in TLR2 signaling to NF-{kappa}B in 293-TLR2 cells. To evaluate whether the aPKC isoform {zeta} plays a role in HKSA-induced NF-{kappa}B activation, we pretreated 293-TLR2 cells with a specific pseudosubstrate (PKC{zeta} ps) or transfected with PKC{zeta}dn. Both approaches led to a substantial and reproducible decrease in NF-{kappa}B-dependent gene expression. In contrast, expression of PKC{zeta}wt did not alter TLR2-mediated NF-{kappa}B activation.



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  		FIGURE 5. PKC{zeta} regulates TLR2-mediated NF-{kappa}B trans activation. A, 293-TLR2 cells were analyzed for NF-{kappa}B activation by cotransfection of the reporter plasmid 5x NF-{kappa}B-Luc with the {beta}-galactosidase plasmid. The cells were either pretreated with the conventional PKC inhibitor Ro-31-8220 (5 µM) for 1 h, or with phorbol ester (PMA, 1 µM) overnight, or with PKC{zeta} ps (30 µM) for 1 h, or by transfection of the indicated constructs for 24 h. DMSO treatment or vector transfection served as controls. The cells were either unstimulated ({square}) or stimulated for 5 h with HKSA ({blacksquare}). NF-{kappa}B activation is expressed as relative luciferase units (RLU). Three independent experiments were done. B, 293-TLR2 cells were either left untreated (control) or preincubated for 1 h with PKC{zeta} ps (30 µM) (lower panel). After stimulation with HKSA, cells were lysed at the indicated time points, and lysates were subjected to SDS-PAGE and immunoblotted for I-{kappa}B{alpha}. C, To analyze p65 trans activation, 293-TLR2 cells were transfected with pFR-Luc and pGAL4-p65. Cells were either preincubated for 1 h with PKC{zeta} ps, as in B, or cotransfected with control vector or the indicated constructs. Cells were then either not stimulated ({square}) or stimulated for 5 h with HKSA ({blacksquare}) and analyzed for GAL4-p65 trans activation by measuring luciferase activity. Activation is expressed as relative luciferase units (RLU). Three independent experiments were done. D, IL-8 gene expression in Malp-2-stimulated 293-TLR2 cells. Real-time RT-PCR analysis was performed in 293-TLR2 cells expressing RBD, PKC{zeta}wt, PKC{zeta}dn, or vector following stimulation with Malp-2 for 6 h. Data are expressed as percentage of IL-8 RNA induction upon stimulation relative to vector-transfected cells. Figure shows a representative experiment made in triplicate (three experiments were performed).

 
Because we have shown earlier that RhoA is controlling TLR2-mediated NF-{kappa}B activation by targeting nuclear p65 trans activation (Fig. 2), the effect of PKC{zeta} on I-{kappa}B{alpha} degradation and p65 trans activation was analyzed (Fig. 5, B and C). The time course of I-{kappa}B{alpha} degradation and resynthesis was not altered in the presence of the PKC{zeta} ps, suggesting that PKC{zeta} regulates NF-{kappa}B trans activation independently of I-{kappa}B{alpha} degradation. In contrast, transfection of dominant-negative IKK2 caused inhibition of I-{kappa}B{alpha} degradation and NF-{kappa}B activation, as expected (6) (data not shown). To confirm that PKC{zeta} is controlling NF-{kappa}B by affecting the p65 transcriptional complex, 293-TLR2 were transfected with the chimeric protein GAL4-p65. Pretreatment with PKC{zeta} ps led to a decrease in p65 transcriptional activity of ~40% (Fig. 5C). In accord, PKC{zeta}dn, but not PKC{zeta}wt, inhibited p65 trans activation by 50%. Thus, PKC{zeta} regulates NF-{kappa}B-dependent gene transcription by affecting p65 activity. Expression of wt or constitutively active RhoA or PKC{zeta}wt did not trigger an enhancement of gene transcription. Real-time RT-PCR confirmed that inhibition of RhoA or PKC{zeta} activity decreases substantially TLR2-mediated IL-8 expression (75 and 50%, respectively; Fig. 5D). Malp-2-induced up-regulation of IL-8 expression levels in vector or PKC{zeta}wt-expressing 293-TLR2 cells was ~1000-fold. Collectively, our data demonstrate that the aPKC isoform {zeta} is a component of a Rho-initiated signaling cascade targeting the p65 transcriptional complex upon stimulation of TLR2.

The RelA phosphorylation site S311 is a mutual target for RhoA and PKC{zeta}

Previous studies illustrated that the phosphorylation of RelA on serine residues located either in the Rel homology domain (S276, S311) or in the trans activation domain (S529, S536) is critical for NF-{kappa}B transcriptional activity. In particular, Duran et al. (20) reported recently that PKC{zeta} is responsible for RelA S311 phosphorylation, which in turn is essential for RelA-CREB binding protein interaction. Because RhoA-dependent signals downstream of TLR2 are most likely connected to RelA phosphorylation events, we sought to determine whether inhibition of RhoA activity affects the phosphorylation of certain RelA serine residues. Stimulation of 293-TLR2 cells with the lipopeptide Malp-2 or TNF-{alpha} caused phosphorylation of RelA Ser536 and Ser311 (Fig. 4). In contrast to phosphoserine on position 536 in RelA, Ser311 phosphorylation was dependent on RhoA activity, and as expected on PKC{zeta} activity.

RhoA signaling is distinct from the Rac1-dependent NF-{kappa}B activation pathway

To analyze the signaling cascade initiated by activated TLR2 in more detail, we blocked PKC{zeta} activation with PKC{zeta} ps and analyzed HKSA-mediated RhoA activation. Pretreatment with the pseudosubstrate did not block RhoA activation in 293-TLR2, as determined by RBD-binding assay (Fig. 6A). Thus, PKC{zeta} is not regulating RhoA activity, but represents a downstream target for RhoA.



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  		FIGURE 6. RhoA activation is upstream of PKC{zeta} and independent of the Rac1-PI3K pathway. A, RhoA activation is independent of PKC{zeta} activity. The 293-TLR2 cells were transfected with Myc-RhoAwt for 24 h and preincubated for 1 h with PKC{zeta} ps (30 µM) before HKSA stimulation. Upper panel, Lysates were subjected to affinity precipitation with GST-RBD. Bound proteins were separated by SDS-PAGE and immunoblotted for RhoA. Lower panel, Depicts RhoA expression levels in whole cell lysates at various time points (IB). B, RhoA activation by HKSA after inhibition of PI3K. The 293-TLR2 cells were transfected with Myc-RhoAwt 24 h before stimulation. To inhibit PI3K activity, cells were incubated with wortmannin (50 nM) or DMSO for 30 min prior to HKSA stimulation. Upper panel, Each lysate was subjected to affinity precipitation with GST-RBD. Bound proteins were separated by SDS-PAGE and immunoblotted for RhoA. Lower panel, Immunoblot (IB) of RhoA expression in whole cell lysates used for the binding assay. C, Rac1 activation by HKSA is independent of RhoA activity. The 293-TLR2 cells were transfected with Myc-RhoA N19 (or as control with GFP) 24 h before stimulation with HKSA. Lysates derived from various time points were subjected to affinity precipitation with GST-PBD. Proteins bound to GST-PBD were separated by SDS-PAGE and immunoblotted for Rac1 (upper panel). Immunoblots of Rac1 and RhoA N19 in cell lysates used for the binding assay (IB).

 
Our previous studies showed that TLR2-mediated NF-{kappa}B activation requires a pathway controlled by the Rho GTPase Rac1, which forms transiently a complex with activated TLR2 (6). In contrast to these observations, association between RhoA and TLR2 was not detected in immunoprecipitations or in vitro binding studies (data not shown). Similar binding experiments revealed that TLR2-activated RhoA did not interact with IRAK1, IRAK4, or TRAF6 in Malp-2-stimulated cells. Potential interaction of RhoA with MyD88 could not be analyzed, because coexpression of the adapter MyD88 and RhoA, even at very low protein expression levels, affects cell viability significantly, prohibiting further analysis of these cells.

As RhoA signaling affects the p65 transcriptional complex analogous to Rac1, we analyzed whether RhoA activation is independent of the Rac-dependent signaling cascade to NF-{kappa}B. PI3K has been identified as a downstream effector of Rac1 in TLR2-mediated NF-{kappa}B activation (6). PI3K can fulfill a dual role in a stimulus- and cell-dependent manner, either serving as an effector of GTPase function or as activator of Rho GTPases via phosphatidylinositol 3,4,5-triphosphate-stimulated activation of guanine nucleotide exchange factors. To analyze the role of PI3K in TLR2-induced RhoA activation, we used the PI3K-specific pharmacological inhibitor wortmannin. Pretreatment with wortmannin did not block RhoA activation in 293-TLR2 (Fig. 6B) or in THP-1 cells(data not shown), as determined by RBD-binding assays. We have shown earlier that wortmannin treatment abolishes the phosphorylation and activation of the protein kinase Akt in HKSA-stimulated human embryonic kidney 293-TLR2 cells (6). These results indicate that RhoA activation is not regulated by a PI3K-dependent pathway. Similarly, TLR2-mediated PKC{zeta} activation in THP-1 and 293-TLR2 cells was not inhibited by preincubation with PI3K inhibitors (data not shown).

To evaluate whether the GTPase Rho may play a role in TLR-2-mediated Rac1 activation, we transfected 293-TLR2 cells with dominant-negative RhoA (RhoAN19) and determined HKSA-mediated Rac1 activation by using the p21 binding domain (PBD) of p21-activated kinase 1, followed by immunoblotting with anti-Rac1 Ab. The time course of Rac1 activation by TLR2, as previously reported by us (6), was not altered by expression of dominant-negative RhoA (Fig. 6C), RhoAwt, or GFP expression (data not shown). Thus, our data indicate that, at least initially, the RhoA- and Rac1-initiated pathways commence independently before targeting nuclear p65 trans activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TLRs initiate via their cytoplasmic Toll/IL-1R domains a signaling cascade composed of adapters such as MyD88 or Toll-IL-1R domain-containing adapter protein, IRAKs, TRAF6, and IKKs to release NF-{kappa}B from the inhibitor I-{kappa}B to stimulate subsequently NF-{kappa}B-dependent gene transcription (1, 21, 22). However, evidence is accumulating that additional parallel pathways, emanating from stimulated TLRs, are essential for NF-{kappa}B-dependent gene transcription. In respect to TLR2, our study assigns a prominent role for the GTPase Rho in the control of this cellular response. Rho family proteins can coordinate multiple signaling pathways through their ability to regulate both gene transcription and the actin cytoskeleton. RhoA and Rac1 were implicated in NF-{kappa}B activation for agonists such as TNF-{alpha} and IL-1{beta} (9, 23). Rho was also described as an enhancer of AP-1 transcription in T cells (24). Furthermore, studies have shown that inhibitors of Rho GTPases block the stimulation of macrophages with a mycoplasmal lipoprotein (25). A role for RhoA in TLR4-induced cytokine expression was postulated by two groups using different experimental approaches and cell types (26, 27). We show in this study that RhoA controls a signaling cascade, which regulates NF-{kappa}B trans activation without affecting I-{kappa}B degradation.

Recent studies demonstrated that phosphorylation of the transcriptional subunit p65 positively controls NF-{kappa}B trans activation via an I-{kappa}B-independent pathway. It has been suggested that phosphorylation of p65 may be important not only for its transcriptional activity, but also for the interaction with specific enhancer elements in the promoter. Potential kinases implicated in p65 phosphorylation include casein kinase II; protein kinase A; glycogen synthase kinase 3{beta}; the MAPKs p38, p42, and p44; IKK{beta}; Akt; and the aPKC isoform {zeta} (5, 17, 28, 29). However, among these kinases, only PKC{zeta} has been shown to interact directly with p65 and is responsible for RelA Ser311 phosphorylation (20). Embryonic fibroblasts derived from mice deficient in PKC{zeta} displayed a significant reduced cytokine-stimulated {kappa}B-dependent transcriptional activity with no defect on I-{kappa}B degradation or the nuclear translocation of NF-{kappa}B (17). We found a transient association of PKC{zeta} and RhoA upon stimulating TLR2 with stimuli derived from Gram-positive bacteria. The time course of RhoA activation preceded slightly the formation of this complex, which dissociated when RhoA deactivation occurs. Moreover, TLR2-mediated responses in 293-TLR2 cells caused PKC{zeta} activation and PKC{zeta}-dependent up-regulation of the NF-{kappa}B trans activation potential by directly targeting p65, indicating that aPKC{zeta} represents a downstream target of RhoA in signaling to p65. Additionally, our results illustrate that one of the targets of this pathway is the TLR2-mediated phosphorylation of RelA Ser311. Recent evidence indicates that aPKCs, but not conventional or novel isoforms, bind selectively to the scaffold protein p62 (16, 19). The direct interaction between p62 and the adapter protein TRAF6, which play a crucial role in signaling through IL-1{beta} and TLR2, may account for the involvement of aPKCs in different NF-{kappa}B activation pathways. Hu et al. (30) showed direct interaction of IRAK1, a serine/threonine kinase upstream of TRAF6, with PKC{zeta} when monocytic cells were stimulated with LPS. Using a TLR2-dependent stimulus, we did not detect an association of PKC{zeta} with IRAK1, IRAK4, or TRAF6.

The GTPase RhoA is well recognized for its role in cytoskeletal reorganization, inducing cell rounding and stress fiber assembly. Many of these effects are promoted through the Rho effectors p160Rock and PKC-related kinase 2 (31, 32). Our data indicate that both of these RhoA targets are not involved in NF-{kappa}B activation. Both the p160Rock inhibitor Y27632 or dominant-negative PKC-related kinase 2 had no effect on NF-{kappa}B or p65 trans activation. Because inhibition of RhoA activity with either a dominant-negative RhoA mutant or a RhoA-GTP binding domain affected NF-{kappa}B activation more efficiently than introduction of a dominant-negative PKC{zeta} or incubation with PKC{zeta} ps, we conclude RhoA may be required for transmitting the signal to PKC{zeta} and another, yet unidentified target. It is important to note that the RhoGTPase Cdc42 has been recently linked to aPKCs in regulating cell polarity (33, 34).

Previous work from our laboratory demonstrated that TLR2 signaling to NF-{kappa}B uses also a Rac1-PI3K-dependent pathway, which targets the transcriptional subunit p65/RelA independent of I-{kappa}B degradation (6). However, RhoA does not serve as an activator or effector for Rac1 in TLR2-mediated signaling to NF-{kappa}B, indicating that RhoA targets nuclear p65 trans activation at least partially independently of Rac1. A distinction between Rac1- or RhoA-regulated gene transcription was apparent because active Rac1, but not active RhoA, triggered a synergistic increase of stimulated NF-{kappa}B activation. Interestingly, we could not confirm a signaling pathway PI3K-3-phosphoinositide-dependent protein kinase 1-PKC{zeta} initiated by TLR2 in 293-TLR2 and THP-1 cells. The PI3K target PDK1 has been shown previously to phosphorylate and activate PKC{zeta} (35). Most likely, the TLR2-mediated RhoA-PKC{zeta} signaling pathway cooperates with the Rac1-PI3K cascade by affecting different phosphorylation sites on RelA or other elements of the transcriptional complex, which are equally crucial for gene transcription. The coordination of both of these pathways together with the MyD88-IKK-I-{kappa}B cascade is vital to regulate NF-{kappa}B-dependent gene transcription and provide host cells with an effective immunity.


   Acknowledgments
 
We thank A. Hall, P. Godowski, R. Ulevitch, J. Moscat, and L. Quilliam for providing materials. We also acknowledge the contributions of J.-P. Mira and A. Reilly, and thank P. Rutledge and K. Hawkins for excellent administrative and graphical assistance.


   Footnotes
 
1 This work was supported by U.S. Public Health Service grants (to U.G.K.), a Boehringer Ingelheim Fonds fellowship (to N.T.), and an award from the American Heart Association (to E.L.). Back

2 N.T. and E.L. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Ulla G. Knaus, Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: uknaus{at}scripps.edu Back

4 Abbreviations used in this paper: PRR, pattern-recognition receptor; aPKC, atypical PKC; HKSA, heat-killed Staphylococcus aureus; IKK, I-{kappa}B kinase; IRAK, IL-1R-associated kinase; PBD, p21-activated kinase binding domain; PKC, protein kinase C; PKC{zeta}dn, dominant-negative PKC{zeta}; ps, pseudosubstrate; RBD, Rhotekin binding domain; TRAF, TNFR-associated factor; wt, wild type. Back

Received for publication October 17, 2003. Accepted for publication April 19, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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