The interaction between bacteria and macrophages is central to the outcome of Salmonella infections. Salmonella can escape killing by these phagocytes and survive and multiply within them, giving rise to chronic infections. Cytokines produced by infected macrophages are involved in the early gastrointestinal pathology of the infection as well as in the induction and maintenance of the immune response against the invaders. Jun N-terminal kinases (JNK) are activated by inflammatory stimuli and play a role in cytokine production. We have investigated the signaling routes leading to JNK activation in Salmonella-infected macrophages and have discovered that they differ radically from the mechanisms operating in epithelial cells. In particular, activation of the JNK kinase stress and extracellular-activated kinase 1 (SEK1) and of JNK in macrophages occurs independently of actin rearrangements and of the GTPases Cdc42 and Rac, essential mediators in other cells. Activation of JNK is effected by a novel pathway comprising tyrosine kinase(s), phosphoinositide 3-kinase and, likely, atypical protein kinase C ζ. SEK1 is stimulated by a distinct mechanism involving phosphatidylcholine-phospholipase C and acidic sphingomyelinase. Dominant-negative SEK1 can block JNK activation by LPS, but not by Salmonella. These data demonstrate that SEK1 and JNK are activated independently in Salmonella-infected macrophages and offer experimental support for the concept that incoming signals can direct the selective coupling of downstream pathways to elicit highly specific responses. Inhibitors of stress kinase pathways are receiving increasing attention as potential anti-inflammatory drugs. The precise reconstruction of stimulus-specific pathways will be instrumental in predicting/evaluating the effects of the inhibitors on a given pathological condition.

0almonellae are facultative intracellular pathogens that cause a variety of illnesses from localized gastroenteritis to more overt, host-specific ones, such as typhoid fever. In the host’s intestine, Salmonella adheres to specialized epithelial cells (M cells) and cause cytoskeletal and membrane rearrangements that result in its uptake (1). By destroying the infected M cells (2), the bacteria gain access to the mesenteric lymph follicles, where they encounter and infect macrophages. Recently, the induction of macrophage apoptosis by invasive Salmonella has been documented in vitro (3, 4, 5, 60) and in vivo (6). Both epithelial cells invasion (7) and the induction of macrophage apoptosis (8) depend on bacterial virulence determinants translocated into the eukaryotic cell by a specialized, host-dependent secretion system (9, 10).

The analysis of the biochemical cross-talk between Salmonella and epithelial cells has shown that the proteins secreted by the type III secretion system have the capacity to trigger host cell signaling pathways (11). The bacterial product SopE, for instance, activates the small GTPases Cdc42 and Rac-1 (7) and by so doing induces the cytoskeletal changes required for membrane ruffling and for the macropinocytosis of Salmonella (12). A functional type III secretion system is also a prerequisite for the activation of the mitogen-activated protein kinase (MAPK)4 subgroups extracellular-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 and for the production of proinflammatory cytokines by epithelial cells infected with Salmonella (13).

For Salmonella as for many other facultative intracellular pathogens the key to a successful infection lies in the outcome of their encounter with the host’s macrophages. Besides playing a crucial role in the immune response against the bacteria, the pro-inflammatory cytokines produced by these cells mediate the early gastrointestinal pathology of the infection (14). Still, much less is known about the molecular mechanisms operating during the interaction of Salmonella typhimurium with macrophages than about the signaling events taking place during epithelial cell invasion. We have previously addressed the question of Salmonella-mediated ERK activation, showing that LPS is the major determinant responsible for ERK stimulation by this pathogen (15). In the present study, we focus on reconstructing the mechanism of JNK activation.

JNK can be stimulated by cellular stress signals like irradiation, heat shock, osmotic stress, and protein synthesis inhibitors (16), and also by growth factors (17, 18). Relevant to our study, this pathway is activated by inflammatory stimuli (19, 20, 21) and upon infection of cultured cells by pathogens, including Gram-negative (13, 22) and Gram-positive bacteria (23). Targeted distruption of the JNK kinase stress and extracellular signal-activated kinase 1 (SEK1) causes defects in the activity of AP-1 (24), a transcription factor implicated in the regulation of cytokine genes (25). Recent data also implicate JNK in the stabilization (26, 27) and translation (28, 29) of cytokine mRNAs. Consistently, T cell differentiation is defective in Jnk-1- (30) and Jnk-2-deficient mice (31).

In this study we show that SEK1 and JNK are activated in a phagocytosis-independent manner upon infection of macrophages by Salmonella. Stimulation of JNK, but not of SEK1, involves the activation of tyrosine kinases, phosphoinositide 3-kinase (PI 3-K), and, likely, protein kinase C ζ (PKCζ); on the other hand, SEK1 activation depends on the stimulation of phosphatidylcholine phospholipase C (PC-PLC) and acidic sphingomyelinase (ASMase). Cdc42 and Rac, which mediate JNK activation by a variety of stimuli in many cell types, are not required for SEK1 or JNK stimulation in macrophages. Thus, Salmonella activates the JNK pathway in macrophages by a mechanism that bypasses the canonical activators Cdc42/Rac and SEK1.

S. typhimurium strain LT2 (virulent, wild type (wt)) and SB111 (32) were grown in Luria-Bertani (LB) broth (1% bactotryptone, 0.5% yeast extract, and 1% sodium chloride) at 37°C overnight under agitation (poorly invasive). To obtain highly invasive bacteria, bacteria from overnight cultures were diluted to an OD600 of 0.02 in 50 ml fresh LB and incubated for 5 h under agitation 60 .

BAC-1.2F5 cells (33) were cultured in DMEM supplemented with 10% FCS and 20% L cell conditioned medium as a source of CSF-1. Confluent cells (about 5 × 106 cells/100-mm-diameter tissue culture dish) were cultured for 16 h in medium without CSF-1, and then stimulated with 1.5 μg/ml bacterial LPS (from S. typhimurium; Sigma, St. Louis, MO) or infected with bacterial cultures as previously described (15). A multiplicity of infection (moi; bacteria per macrophage) of 25 was used.

Actin polymerization was blocked by pretreatment with 10 μM cytochalasin B (30 min; Sigma). Tyrosine kinases were inhibited by pretreatment with herbimycin A (4 μg/ml for 4 h; Sigma). Activation of PI 3-K was blocked by pretreatment with wortmannin (100 nM, 20 min; Sigma). Rho-family small GTPases (RhoA, Rac1, and Cdc42) were inhibited by a 60-min preincubation with toxin B (34) at a final concentration of 10 or 100 ng/ml. Inhibition of PC-PLC activity was performed by preincubating the cells for 60 min with 10 μM xanthogenate tricyclodecan-9-yl (D609, Alexis Biochemicals, Laufelfingen, Switzerland). PKC was inhibited by treating the cells with 10 μM bisindoleylmaleimide (BIM; Calbiochem, La Jolla, CA) for 60 min before stimulation (35). Diacylglycerol (DAG)-dependent PKC isoforms were down-regulated by a 24-h treatment with 5 μM PMA (Sigma).

The SuperFect reagent (Qiagen, Basel, Switzerland) was used according to the manufacturer’s instructions. Macrophages were transfected with an epitope-tagged JNK1 (HA-JNK1, 2 μg/100-mm-diameter culture dish) together either with an epitope-tagged (GST-) SEK wt, or with GST-SEK KR (kinase defective mutant, in which the nucleophilic lysine was substituted for by an arginine), or with pEBG vector as a control (each 8 μg/100-mm-diameter culture dish). Twenty-four hours after transfection, the cells were infected with Salmonella as described above.

Colony counting assays were performed to assess phagocytosis of S. typhimurium. Briefly, cells (0.05 × 106) were seeded in 96-well plates and infected (moi of 25). Cells were allowed to phagocytose for 30 min and then washed three times with PBS. Fresh medium containing 50 μg/ml gentamicin was added and the cells were incubated for further 60 min to kill residual extracellular bacteria. Thereafter, cells were lysed in PBS supplemented with 0.5% sodium deoxycholate. Serial dilutions of the lysates were prepared in PBS and plated onto the Luria-Bertani agar plates. Colonies were allowed to develop for 18 h before counting. Assays were conducted in triplicates.

Cells were lysed in solubilization buffer (10 mM Tris-base, 50 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, and 1% Triton X-100, pH 7.0) supplemented with 1 mM PMSF, 100 μM sodium vanadate, 1 mM DTT, and protease inhibitors (aprotinin (3 μg/ml), pepstatin and leupeptin (each at 0.5 μg/ml)). For immunoblotting, 30–40 μg of whole cell extracts were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. For immunoprecipitation, 500–600 μg of whole cell extracts were incubated in the presence of protein A beads (Amersham, Arlington, Heights, IL) with anti-PKCζ (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-HA Abs for 16–18 h at +4°C. The beads were collected and washed three times with lysis buffer before elution of the immunocomplexes by boiling in SDS sample buffer. Membranes were blocked for 8–16 h at 4°C in TTBS (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween 20) supplemented with 4% BSA (fraction V; Sigma), and probed with the appropriate primary Abs in 1% BSA in TTBS before incubation with peroxidase-conjugated secondary Abs and detection by the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL). The primary Abs used in this study recognize selectively phosphorylated JNK1/2 (anti-phJNK, Thr183/Tyr185), SEK1 (anti-phSEK, Thr223; all from New England BioLabs, Schwalbach, Germany) and PKCζ (36) or their unmodified forms (Santa Cruz Biotechnology).

PC-PLC and ASMase activity of whole cell extracts was determined as previously described (37). Briefly, cells (2.5 × 106) were scraped in 2 ml ice-cold PBS and centrifuged for 10 min at 400 rpm at 4°C. A total of 300 μl of Triton X 100 (0.01% for PC-PLC, 0.2% for ASMase activity measurements) were added to the pellet, and the samples were incubated on ice for 10 min before sonication. A total of 15 μg of lysate were incubated for 2 h at 37°C either in PC-PLC buffer (50 mM Tris-HCl (pH 7.3), 6.3 mM CaCl2, 150 mM ammonium sulfate, plus 50 nCi L-3-phosphatidyl[N-methyl-14C]choline ([14C]PC); 80 μl total volume) or in ASMase buffer (250 mM sodium acetate (pH 5.0), 0.2% Triton X 100, plus 50 nCi [methyl-14C]sphingomyelin; 50 μl total volume). Labeled lipids were from Amersham. The PC-PLC assay was terminated by extracting the lipids with CHCl3:CH3OH (1:2 v/v, 180 μl), 0.9% NaCl (60 μl), and CHCl3 (60 μl). The ASMase assay was terminated by extracting the lipids with CHCl3:CH3OH (1:1 v/v, 400 μl) and water (180 μl). The aqueous and organic phases were separated and quantitated by liquid scintillation. The amount of substrate hydrolyzed was quantitated by liquid scintillation counting. PC-PLC and ASMase activity were expressed as percentage of control.

Quiescent BAC-1.2F5 cells were infected with Salmonella for different time periods. The activation state of the kinases was assessed in whole cell lysates by immunoblotting with Abs that specifically recognize the phosphorylated, activated form of each enzyme (Fig. 1,A). All kinases were activated by Salmonella infection of BAC-1.2F5 cells with fast activation/inactivation kinetics. Peak activation occurred after 25 min and then decayed. Inactivation was complete by 1 h, and no further changes were observed over a period of 4 h (data not shown). These kinetics of activation resembled those of the other MAPK subfamily, ERK (15). Highly invasive Salmonella, which causes apoptosis in macrophages, activates JNK less efficiently than the poorly invasive form (Fig. 1,A) and than an invasion-defective mutant (Fig. 1 B).

FIGURE 1.

Activation of SEK1 and JNK is an early event in Salmonella-induced signal transduction. A, Quiescent BAC-1.2F5 cells were infected with wt Salmonella (S.t., moi of 25) at 37°C for different times before solubilization. The presence of the phosphorylated, active forms of SEK1 (phSEK1) and JNK (phJNK) was detected by immunoblotting with the corresponding Abs. An anti-JNK1 immunoblot is shown as a loading control. In B, cells were infected with highly invasive Salmonella (wt) and with an invA mutant.

FIGURE 1.

Activation of SEK1 and JNK is an early event in Salmonella-induced signal transduction. A, Quiescent BAC-1.2F5 cells were infected with wt Salmonella (S.t., moi of 25) at 37°C for different times before solubilization. The presence of the phosphorylated, active forms of SEK1 (phSEK1) and JNK (phJNK) was detected by immunoblotting with the corresponding Abs. An anti-JNK1 immunoblot is shown as a loading control. In B, cells were infected with highly invasive Salmonella (wt) and with an invA mutant.

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The time frame investigated (5–45 min) allowed phagocytosis of Salmonella by the macrophages. We therefore examined whether phagocytosis was involved in JNK activation. Phagocytosis of latex beads of the approximate size of the bacteria did not stimulate any of the kinases at study (data not shown). However, the mechanisms involved in the phagocytosis of bacteria and inert particles likely differ. We therefore analyzed the effect of inhibitors of specific signal transduction pathways on Salmonella phagocytosis and JNK activation (Fig. 2,A). As a positive control we used cytochalasin B, which destroys the actin cytoskeleton and totally abrogated phagocytosis. The PI 3-K inhibitor wortmannin, which blocks epithelial cell invasion by Listeria monocytogenes but not by Salmonella (24), fully blocked phagocytosis, as did toxin B (1), which glucosylates and inactivates the small GTPases Cdc42, Rac and Rho (Ref. 34 ; and data not shown). Cytochalasin B (Fig. 2,B) did not suppress Salmonella-mediated activation of SEK1 or JNK. Wortmannin, on the other hand, decreased Salmonella-stimulated activation of JNK, but not SEK1 (Fig. 2 C). Identical results were obtained using a second PI 3-K inhibitor, LY294002 (data not shown). Thus, phagocytosis and kinase activation by Salmonella are independent events. Furthermore, the involvement of PI 3-K in JNK, but not SEK1 activation, suggests that these kinases are regulated independently during infection of macrophages by Salmonella.

FIGURE 2.

Salmonella-induced kinase activation is independent of phagocytosis. Cells were treated with cytochalasin B (cyto B, 10 μM for 30 min; A and B), with wortmannin (WM, 100 nM for 20 min; A and C), or with toxin B (tox B, 100 ng/ml for 60 min; A and D) before infection with Salmonella (S.t., moi of 25) for 15 min. A, The phagocytosis assay was conducted as described in Materials and Methods. The plot represents the mean of three independent experiments, and vertical bars indicate the SEs of the mean. B–D, The presence of phosphorylated forms of SEK1 and JNK, as well as of JNK1 as a loading control, was detected by immunoblotting with the corresponding Abs.

FIGURE 2.

Salmonella-induced kinase activation is independent of phagocytosis. Cells were treated with cytochalasin B (cyto B, 10 μM for 30 min; A and B), with wortmannin (WM, 100 nM for 20 min; A and C), or with toxin B (tox B, 100 ng/ml for 60 min; A and D) before infection with Salmonella (S.t., moi of 25) for 15 min. A, The phagocytosis assay was conducted as described in Materials and Methods. The plot represents the mean of three independent experiments, and vertical bars indicate the SEs of the mean. B–D, The presence of phosphorylated forms of SEK1 and JNK, as well as of JNK1 as a loading control, was detected by immunoblotting with the corresponding Abs.

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Neither SEK1 nor JNK activation were blocked by toxin B pretreatment (Fig. 2 D). Thus, a novel pathway independent of functional Cdc42/Rac operates in macrophages infected with Salmonella. This was all the more unexpected because PI 3-K-mediated JNK activation has been shown to be dependent on Rac (18, 38).

Herbimycin-dependent kinases have previously been implicated in the activation of PI 3-K (39) and JNK (19, 21) by LPS in monocytes. Salmonella-mediated JNK activation was also efficiently reduced by herbimycin A (Fig. 3). However, SEK1 activation was herbimycin-insensitive, confirming that the pathways targeting SEK1 and JNK differ in Salmonella-infected macrophages.

FIGURE 3.

Herbimycin A treatment decreases Salmonella-induced activation of JNK, but not SEK1. Quiescent BAC-1.2F5 cells were treated with herbimycin A (1 μg/ml for 4 h) before infection with wt Salmonella (S.t., moi of 25 for 15 min). The presence of phosphorylated forms of SEK1 and JNK, as well as of JNK1 as a loading control, was detected by immunoblotting with the corresponding Abs.

FIGURE 3.

Herbimycin A treatment decreases Salmonella-induced activation of JNK, but not SEK1. Quiescent BAC-1.2F5 cells were treated with herbimycin A (1 μg/ml for 4 h) before infection with wt Salmonella (S.t., moi of 25 for 15 min). The presence of phosphorylated forms of SEK1 and JNK, as well as of JNK1 as a loading control, was detected by immunoblotting with the corresponding Abs.

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The results described above suggest that a pathway comprising herbimycin-sensitive tyrosine kinases and PI 3-K, but independent of Cdc42/Rac, targets JNK during Salmonella infection. PKC isoforms can act as PI 3-K downstream effectors both in vitro and in vivo (36, 40). BAC-1.2F5 cells express the novel DAG-dependent PKC isoforms δ and ε, and the atypical PKCζ (35), all of which can be inhibited by BIM (41). Activation of JNK was completely suppressed after pretreatment with BIM (Fig. 4,A). In agreement with the hypothesis that distinct pathways cause SEK1 and JNK stimulation by Salmonella, SEK activation was insensitive to BIM treatment. Sustained treatment (up to 24 h) with 5 μM PMA, which causes down-regulation of DAG-dependent PKC isoforms, did not affect JNK activation (Fig. 4,B). This finding indicates that DAG-dependent isoforms δ and ε, which were efficiently degraded under these conditions (Fig. 4 C), are not involved in Salmonella stimulation of JNK. Therefore, the effect of BIM on JNK activation must be due to the inhibition of an atypical PKC.

FIGURE 4.

Involvement of PKCζ in Salmonella-stimulated SEK1 and JNK activation. Quiescent BAC-1.2F5 cells were treated with the PKC inhibitor BIM (10 μM for 60 min) before infection with wt Salmonella (S.t., moi of 25 for 15 min; A). Alternatively, DAG-dependent PKC isoforms were down-regulated by prolonged PMA treatment (5 μM; B and C). The presence of phosphorylated SEK1/JNK, of unmodified JNK1 (as a loading control), and of PKCδ and PKCε was detected by immunoblotting with the corresponding Abs. The presence of phosphorylated PKCζ was assessed by immunoblot analysis of either whole cell extracts (D) or of anti-PKCζ immunoprecipitates (E). Anti-PKCζ blots of whole cell extracts (D) or immunoprecipitates (E) are shown as a loading control.

FIGURE 4.

Involvement of PKCζ in Salmonella-stimulated SEK1 and JNK activation. Quiescent BAC-1.2F5 cells were treated with the PKC inhibitor BIM (10 μM for 60 min) before infection with wt Salmonella (S.t., moi of 25 for 15 min; A). Alternatively, DAG-dependent PKC isoforms were down-regulated by prolonged PMA treatment (5 μM; B and C). The presence of phosphorylated SEK1/JNK, of unmodified JNK1 (as a loading control), and of PKCδ and PKCε was detected by immunoblotting with the corresponding Abs. The presence of phosphorylated PKCζ was assessed by immunoblot analysis of either whole cell extracts (D) or of anti-PKCζ immunoprecipitates (E). Anti-PKCζ blots of whole cell extracts (D) or immunoprecipitates (E) are shown as a loading control.

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Atypical PKCs would be natural targets of the phosphoinositides (PI) generated by PI 3-K. PKCλ expression cannot be detected in BAC-1.2F5 macrophages. We monitored the stimulation of the only other known atypical PKC, PKCζ, by S. typhimurium. PKCζ can be activated by PI(3, 4, 5)P3, PI(4, 5)P2, and PI(3, 4)P2 directly (42) as well as indirectly via phosphorylation by PI-dependent kinase 1 (36, 40). Activation was monitored by immunoblotting with Abs that specifically recognize phosphorylation of the PDK1 site Thr410 in the C-loop of the PKCζ isoform (36). Western blot analysis of whole cell extracts showed that the phosphorylation reached a maximum 10 min after infection and then slowly decayed (Fig. 4,D). We confirmed the identity of the PKC isoform activated by analyzing the phosphorylation status of immunoprecipitated PKCζ (Fig. 4 E). Taken together, the data identify PKCζ as a target of Salmonella downstream of PI 3-K and PDK1. Because the only other known kinase inhibited by BIM (albeit at higher concentrations) is protein kinase A (PKA), and this enzyme has never been connected with JNK activation, the data implicate PKCζ in relaying the Salmonella signal to the JNK module.

High concentrations of the PC-PLC inhibitor D609 decrease LPS-mediated stimulation of Raf, MEK, and ERK (35). To investigate whether phospholipase activation was important for the stimulation of SEK1 or JNK by Salmonella, we treated quiescent BAC-1.2F5 cells with low concentrations of D609 (10 μM) before infection. The inhibitor severely blunted SEK1 activation, but had only a minor impact on JNK stimulation (Fig. 5 A). The concentration of D609 used is reportedly specific for PC-PLC, and does not affect phospholipase D (PLD) (43). Furthermore, Salmonella-induced SEK1 activation was not affected by the presence of 1% 1-butanol (competitive inhibitor of PLD; Ref. 44 and data not shown). This finding indicated that PC-PLC, and not PLD, mediated SEK1 activation during infection.

FIGURE 5.

Involvement of PC PLC and ASMase in Salmonella-induced kinase activity. Quiescent BAC-1.2F5 cells were pretreated with the PC-PLC inhibitor D609 (10 μM for 60 min) before infection wt Salmonella (S.t., moi of 25; 15 min). To assess the contribution of PLD to kinase activation, infection was conducted in the presence of PLD inhibitor 1-butanol (1%). The presence of phosphorylated SEK1 and JNK was detected by immunoblotting with the corresponding Abs (A). Neither of the inhibitors had any effect on the basal level of kinase activation. An anti-JNK1 blot is shown as loading control. PC-PLC (B) and ASMase (C) activity were determined in whole cell extracts as described in Materials and Methods. In C, cells were pretreated with D609 (10 μM for 60 min) to block PC-PLC. The results are expressed as percentage increase with respect to the control values. The plot in B represents the mean of three independent experiments, and vertical bars represent the SEs of the mean. The 100% value represents 455.33 cpm (±18.98) and corresponds to 0.47% (±0.023) substrate hydrolized. The plot in C represents the mean of two independent experiments, and vertical bars represent the range of the samples. The 100% value represents 45,023.5 cpm (range 39,390–50,657) and corresponds to 44.25% (range 32.6–55.9) substrate hydrolized.

FIGURE 5.

Involvement of PC PLC and ASMase in Salmonella-induced kinase activity. Quiescent BAC-1.2F5 cells were pretreated with the PC-PLC inhibitor D609 (10 μM for 60 min) before infection wt Salmonella (S.t., moi of 25; 15 min). To assess the contribution of PLD to kinase activation, infection was conducted in the presence of PLD inhibitor 1-butanol (1%). The presence of phosphorylated SEK1 and JNK was detected by immunoblotting with the corresponding Abs (A). Neither of the inhibitors had any effect on the basal level of kinase activation. An anti-JNK1 blot is shown as loading control. PC-PLC (B) and ASMase (C) activity were determined in whole cell extracts as described in Materials and Methods. In C, cells were pretreated with D609 (10 μM for 60 min) to block PC-PLC. The results are expressed as percentage increase with respect to the control values. The plot in B represents the mean of three independent experiments, and vertical bars represent the SEs of the mean. The 100% value represents 455.33 cpm (±18.98) and corresponds to 0.47% (±0.023) substrate hydrolized. The plot in C represents the mean of two independent experiments, and vertical bars represent the range of the samples. The 100% value represents 45,023.5 cpm (range 39,390–50,657) and corresponds to 44.25% (range 32.6–55.9) substrate hydrolized.

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To strengthen this conclusion, we measured PC-PLC activity in extracts of untreated and infected cells. PC-PLC was strongly activated by Salmonella with kinetics consistent with the ones observed for SEK1/JNK activation (Fig. 5,B). The activity of ASMase, the PC-PLC downstream target responsible for ceramide generation (45), increased steadily during the first 30 min of infection. Like SEK1 stimulation, ASMase activation was abrogated by pretreatment with the PC-PLC inhibitor D609 (Fig. 5 C). Taken together, the data strongly suggest that infection of macrophages generates ceramide via a PC-PLC/ASMase pathway and that in this specific setting the lipid second messenger is important for SEK1, but not for JNK activation.

The experiments reported above argue that Salmonella-mediated activation of SEK1 and of JNK are regulated independently. To address this question directly, we cotransfected cells with either wt or KR GST-SEK1 and HA-tagged JNK1. Twenty-four hours after transfection cells were either left untreated, treated with LPS, or infected with Salmonella. HA-JNK1 was immunoprecipitated and its activation state was assessed by Western blotting with anti-phJNK Abs.

HA-JNK1 was activated by LPS or Salmonella in cells cotransfected with wt GST-SEK1. Expression of GST-SEK1 KR significantly reduced JNK stimulation by LPS but had no effect on Salmonella-induced JNK activation (Fig. 6, top panel). Equal amounts of HA-JNK1 were present in the immunoprecipitates (Fig. 6, middle panel). Thus, Salmonella stimulated JNK by a SEK1-independent mechanism.

FIGURE 6.

Dominant-negative SEK1 blocks LPS-induced, but not Salmonella-induced, JNK activation. BAC-1.2F5 cells were transfected with a plasmid encoding HA-tagged JNK1 together with GST-tagged wt or KR SEK1. HA-tagged JNK1 was immunoprecipitated from untreated cell and from cells stimulated with either LPS (1 μg/ml for 20 min) or Salmonella (20 min). The presence of phosphorylated (top panel) and unmodified HA-JNK1 (loading control, middle panel) as well the expression of GST-SEK wt and KR (bottom panel) was detected by immunoblotting with the corresponding Abs.

FIGURE 6.

Dominant-negative SEK1 blocks LPS-induced, but not Salmonella-induced, JNK activation. BAC-1.2F5 cells were transfected with a plasmid encoding HA-tagged JNK1 together with GST-tagged wt or KR SEK1. HA-tagged JNK1 was immunoprecipitated from untreated cell and from cells stimulated with either LPS (1 μg/ml for 20 min) or Salmonella (20 min). The presence of phosphorylated (top panel) and unmodified HA-JNK1 (loading control, middle panel) as well the expression of GST-SEK wt and KR (bottom panel) was detected by immunoblotting with the corresponding Abs.

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The interaction of Salmonella with the host’s macrophages is a key event in the early phases of infection. Still, the signaling steps taking place during this interaction are largely unknown.

In this paper we describe the mechanisms that lead to the activation of stress-induced kinases after infection of macrophages with Salmonella. We find that a pathway involving the sequential activation of PI 3-K/PDK1 and PKCζ is responsible for JNK activation. PC-PLC and ASMase, also activated upon infection, support SEK1 but not JNK activation. Unexpectedly, stimulation of both SEK1 and JNK is independent of the function of Cdc42 and Rac.

Herbimycin A and PI 3-K inhibitors severely blunted Salmonella-mediated activation of JNK. PI 3-K has been reported previously to mediate JNK activation by tyrosine kinase and G-protein coupled receptors (17, 18, 38). However, in all cases in which this has been investigated, dominant-negative forms of Cdc42/Rac blocked PI 3-K-dependent JNK activation. In contrast, toxin B, a bacterial inhibitor of these GTPases, does not prevent the stimulation of JNK by Salmonella. At the same time, the ability of the macrophages to phagocytose and, on longer incubations, their adherence to the substrate (data not shown) were blocked efficiently, demonstrating that the inhibitor functions to prevent Rho family-directed cytoskeletal rearrangements in macrophages. Our results were all the more surprising in view of the fact that both cytoskeletal reorganization and JNK activation during invasion of epithelial cells with Salmonella depend on the function of Cdc42 and Rac-1 (7, 12). The most likely explanation for this discrepancy is that JNK activation is implemented by the bacteria in a cell-type-specific manner. In favor of this hypothesis, noninvasive Salmonella mutants are incapable of initiating JNK activation in epithelial cells (13), whereas they do so in macrophages. Furthermore, highly invasive Salmonella, which induces apoptosis in macrophages, activates JNK less efficiently than poorly invasive bacteria (Fig. 1 B). This phenomenon is reminiscent of the results obtained with Yersinia spp., in which invasive, virulent bacteria caused both macrophage apoptosis and the suppression of MAPK activity (46, 47). It is possible that Salmonella, like Yersinia (48), produces an inhibitor of eukaryotic MAPK and translocates it into the host cell via the type III secretion system.

Conventional, novel, and atypical isoforms of PKC have been previously implicated in MAPK stimulation (49, 50, 51, 52), and are downstream targets of PI 3-K (36, 40). During infection of macrophages with Salmonella, DAG-dependent PKC isoforms are in fact activated as a result of PI 3-K-mediated stimulation of PLD and act as intermediates in ERK activation (15). Salmonella also activates the atypical PKCζ (Fig. 4, D and E) via phosphorylation of Thr410 in its activation loop by PDK1, a phosphoinositide-dependent kinase acting downstream of PI-3K (36, 40). Activation of PKCζ has mostly been connected with the stimulation of the ERK pathway (49, 50, 52, 53), although this enzyme has been shown to modulate the JNK pathway target AP-1 (54, 55). Our data represent the first demonstration that PKCζ is activated downstream of PI 3-K/PDK1 during infection of macrophages by Salmonella. The participation of this enzyme in the activation of JNK is supported by the inhibitory effect of BIM on JNK activation.

SEK1 activation by Salmonella was resistant to all inhibitors that efficiently prevented JNK stimulation by the bacterium. Besides supporting the specificity of these substances, this indicated that distinct mechanisms target SEK1 and JNK during infection. PC-PLC and ASMase were activated by Salmonella, and treatment with the PC-PLC inhibitor D609 abolished both ASMase and SEK1 activation, leaving JNK stimulation undisturbed. We propose that, as shown in Neisseria gonorrhoeae-infected epithelial cells (56) and in LPS-stimulated macrophages (57), the activation of the PC-PLC/ASMase pathway by Salmonella leads to the generation of ceramide and thereby to SEK1 stimulation. The actual link between ceramide generation and SEK1 activation remains unidentified. The TAK-1 kinase, a SEK1 activator stimulated by endogenous and exogenous ceramides (58), would be a suitable candidate.

The data discussed above demonstrate that distinct mechanisms implement SEK1 and JNK activation in Salmonella-infected macrophages. Cross-talk between these two signal transduction pathways was not observed, and Salmonella-induced JNK activation was not blocked by a dominant-negative SEK1 mutant. In contrast, the SEK1 dominant-negative mutant inhibited the bulk of JNK activation by LPS in macrophages (Fig. 6). These data demonstrate the ability of extracellular signals to choose the signaling route which leads to MAPK activation in distinct situations. It is tempting to speculate that they might do so by modulating the composition of MAPK modules comprising the dual specificity kinase, its activator, and its substrate (59). This hypothesis could help explain why many extracellular signals elicit highly specific responses despite their apparent use of similar intracellular pathways.

The nature of the JNK activator and of the SEK1 substrate(s) in Salmonella-infected macrophages is at present under investigation.

The data reported here extend our understanding of the biochemical events induced by the infection of macrophages with Salmonella, and show for the first time that a pathway comprising PI 3-K and PKCζ leads to JNK activation independently of Cdc42/Rac and SEK1. Stress kinase pathways involved in cytokine production are emerging targets for the therapy of a variety of inflammatory conditions. Understanding the alignment of stimulus-specific pathways will be important for selecting the appropriate kinase inhibitor(s) and for the evaluation of their effects in the context of a given pathological situation.

We thank Dr. Silvo Gutkind (National Institute on Dental Research, National Institutes of Health, Bethesda, MD) for the gift of the plasmids encoding HA-JNK1, GST-SEK wt, and GST-SEK KR. We also thank Pavel Kovarik, Thomas Decker, and Alexander von Gabain (Vienna Biocenter) for constructive discussions and continuous support.

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This work was supported by Grants P10766-MED and P13252-MOB of the Austrian Research Fund (to M.B.) and by a grant from the Associazione Italiana per la Ricerca sul Cancro (to R.T.).

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Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ASMase, acidic sphingomyelinase; BIM, bisindoleylmaleimide; DAG, diacylglycerol; ERK, extracellular-regulated kinases; JNK, Jun N-terminal kinases; PC-PLC, phosphatidylcholine phospholipase C; PDK1, phosphoinositide-dependent kinase 1; PI, phosphoinositides; PI 3-K, phosphoinositide 3-kinase; PKC, protein kinase C; SEK1, stress and extracellular signal-activated kinase 1; moi, multiplicity of infection; PLD, phospholipase D; wt, wild type; KR, kinase-defective; HA, hemagglutinin.

1
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