β-PIX and Rac1 GTPase Mediate Trafficking and Negative Regulation of NOD2

Cell culture and materials

Human monocytes were isolated from buffy coat preparations supplied by the German Red Cross. Blood was diluted 1/2 in RPMI 1640 EDTA medium (Life Technologies) and centrifuged over Ficoll (Amersham) for 25 min at 20°C and 800 × g, and cultured in RPMI 1640 supplemented with or without 10% heat-inactivated FCS, 100 IU penicillin, 100 μg/ml streptomycin, and 4.5 mM glutamine. After 2 h (pull-down assay and coimmunoprecipitation) or 3 days of culture (RNA interference (RNAi) experiment), cells were used for experiments. HEK293 and THP-1 cells were obtained from DSMZ and cultured in DMEM and RPMI 1640 (Life Technologies), respectively, supplemented with 10% heat-inactivated FCS. MDP-LD, MDP-DD, and Malp2 were purchased from Alexis Biochemicals and the Rac activation-specific inhibitor NSC23766 (43) was purchased from Calbiochem. All other chemicals used were of analytical grade and obtained from commercial sources.

RNAi in HEK293 cells and human monocytes

Control nonsilencing small interfering RNA (siRNA) (sense, UUCUCCGAACGUGUCACGUtt; antisense, ACGUGACACGUUCGGAGGAGAAtt), siRNA targeting Rac1 (sense, CACCACUGUCCCAACACUCtt; antisense, GAGUGUUGGGACAGUGGUGtt), and siRNA targeting β-PIX (sequence 1, sense, CGACAGGAAUGACAAUCACtt; antisense, GUGAUUGUCAUUCCUGUCGtt; sequence 2, sense, CCAGUGAGAAGUUAAGUUCtt; antisense, GAACUUAACUUCUCACUGGtc) were purchased from MWG-Biotech or Ambion. HEK293 cells were transfected by using RNAiFect (Qiagen) according to the manufacturer’s protocol with 1 μg siRNA per 2 × 10⁵ cells. Primary human monocytes or THP-1 cells were transfected by using Amaxa Nucleofector (Amaxa) according to the manufacturer’s protocol (Human Monocyte Nucleofector Kit, Nucleofector program Y-01, or Nucleofector Solution V, Nucleofector program T-08, respectively) with 3 μg siRNA per 10⁷ cells (primary monocytes) or 2 μg siRNA per 10⁶ cells (THP-1). The efficiency of Rac1 silencing was analyzed 72 h after transfection by immunoblotting. Functional studies examining the role of Rac1 in cell activation by MDP were performed 72 h after siRNA transfection.

Expression plasmids and HEK293 cell overexpression experiments

The expression plasmids pCR3.V72-Met-VSV-RIP2 and pFlag-CMV-TLR2 were provided by J. Tschopp, University of Lausanne, Lausanne, Switzerland, and C. Kirschning, Technical University of Munich, Munich, Germany, respectively. pcDNA3 containing NOD2 was a gift of G. Nuñez, University of Michigan Medical School, Ann Arbor, Michigan, and was subcloned into pCMV-Tag 3C (HindIII, XhoI) to produce pMyc-NOD2. The pRK5-Rac1 expression vector and its mutant forms were generous gifts of A. Ridley, the RSV-β-galactosidase reporter plasmid was provided by W. Hallatschek, Charité-University Medicine, Berlin, Germany, and the IL-8 promoter luciferase reporter plasmid pUHC13-3-IL-8pr (nucleotides 1348–1527 of the IL-8 gene) was a kind gift of M. Kracht, Hannover Medical School, Hannover, Germany. For reporter gene assays, HEK293 cells cultured in 24-well plates were cotransfected using the calcium phosphate method (Clontech) with 0.05 μg NF-κB reporter or 0.05 μg IL-8 reporter, 0.05 μg RSV-β-galactosidase reporter, 0.2 ng of NOD2, 0.025 μg of TLR2, or 0.1 μg of RIP2 expression vectors and 0.1 μg of the different Rac1 expression vectors. For the coimmunoprecipitation experiments, HEK293 cells cultured in 6-well plates were transfected with 2.0 μg of either an empty control vector or Myc-NOD2 expression plasmids, respectively, together with 2 μg of Rac1 expression plasmid by using the Superfect reagent (Qiagen).

Rac1 activation assay

Rac1 activation in stimulated cells was determined with the Rac1 activation kit (Chemicon) according to the manufacturer’s instructions. In brief, THP-1 cells or primary monocytes were grown to ∼10⁷ cells per sample, serum-starved for 3 h, and stimulated with MDP (0.1–10 μg/ml) for 5 to 60 min. Cells were washed twice with ice-cold PBS and resuspended in the assay buffer. Cell lysates were mixed with the PAK-1 PBD agarose slurry (50%) for 1 h at 4°C. Beads were collected by centrifugation, washed with the assay buffer, and resuspended in SDS sample buffer. Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland). Proteins were detected using the Odyssey infrared imaging system (Li-Cor).

Luciferase assay

Luciferase activity was measured by using the luciferase reporter-gene assay (Promega), and results were normalized with values obtained by RSV-β-galactosidase as described previously (44).

IL-8 ELISA

IL-8 concentrations in the THP-1 or primary monocyte supernatants were quantified using a commercially available sandwich ELISA kit (R&D Systems).

Immunoblotting

Cell extracts were separated by SDS-PAGE and blotted on Hybond-ECL membranes (Amersham Biosciences). Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), β-Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively. Subsequently, membranes were incubated with secondary Abs (IRDye 800-labeled anti-rabbit or Cy5.5-labeled anti-mouse, Rockland). Proteins were detected by using an Odyssey infrared imaging system (Li-Cor).

Immunoprecipitation

For immunoprecipitation, cells were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodiumdeoxycholat) containing protease and phosphatase inhibitors. Lysates were cleared for 15 min at 14,000 g at 4°C. Protein lysates (500 μg/500 μl) were precleared with 0.25 μg isotype specific Ab and Protein-G-Agarose (Upstate Biotechnology) for 1 h at 4°C and the supernatants were recovered. Subsequently, 2 μg of primary Ab was added and incubated on an “end-to-end” shaker for 1.5 h at 4°C. Protein G-Agarose slurry (50 μl) was added and incubated overnight at 4°C. The beads were precipitated by centrifugation steps and washed five times in RIPA buffer before SDS loading buffer was added.

Confocal laser scanning microscopy

For indirect immunofluorescence microscopy, HEK293 were seeded on coverslips, transfected with NOD2 and Rac1 expression vectors using the Superfect reagent (Qiagen) as described above, fixed in 3% paraformaldehyde in PBS, and permeabilized with 1% Triton X-100 for 15 min at room temperature. Cells were incubated in 5% goat serum and staining was done by subsequent incubation of primary and secondary Abs in 1% goat serum. Primary Abs were rabbit anti-NOD2 (1/500; ProSci) and mouse anti-Rac1 (1/500; Transduction Laboratories). Primary Abs were detected with Alexa 488-conjugated goat anti-rabbit IgG and Alexa 546-conjugated goat anti-mouse IgG (1/8000; Invitrogen Molecular Probes) secondary Abs, respectively. Fluorescent images were acquired using an Axioskop 2 mot (objective: PlanNeoFluar 60×, NA 1.4) confocal laser scanning microscope (Zeiss).

Cell fractionation (membrane/cytosol) experiments

THP-1 cells or primary monocytes were seeded on 6-well plates at a density of ∼10⁷ cells/3 ml and serum-starved for 3 h. Subsequently, MDP was added for 30 to 120 min. Protein extraction was conducted as previously described (19). In brief, cells were pelleted for 5 min at 800 × g, medium was removed and 200 μl of lysis buffer (1% Triton X-100, 0.1 M NaCl, 10 mM HEPES (pH 5.6), 2 mM EDTA, 4 mM Na₃VO₄, and 40 mM NaF), supplemented with protease inhibitors, was added. Lysates were harvested and passed through a 21-gauge needle ten times. The cytosolic fraction was obtained by centrifugation at 10,000 × g for 30 min at 4°C. The pellet was resuspended in 150 μl of lysis buffer containing 1% SDS and was sonicated twice for 20 s. After centrifugation for 5 min at 10,000 × g, the resulting supernatant was collected. The protein concentration was determined by using the Bio-Rad Protein Assay (Bio-Rad).

Statistical analysis

Data are shown as means ± SD of three independent experiments. A one-way ANOVA was used for data of Figs. 2, B–D; 3, B–G; and 6, E and F. Main effects were then compared by a Newman-Keul‘s posttest. Throughout the figures, p < 0.01 is indicated by double asterisks.

NOD2 stimulation by MDP activates Rac1

Two considerations led us to examine the role of Rac1 in NOD2 signaling: the fact that Rac1 has been implicated in different NF-κB-regulating pathways, and secondly its involvement in actin cytoskeletal reorganization which might contribute to the membrane recruitment of NOD2 (19, 24). A possible activation of Rac1 in NOD2-mediated signal transduction was determined by pull-down assays using PAK-1 agarose to detect the activated form of Rac1, Rac1-GTP. Stimulation of THP-1 monocytes (Fig. 1⇓, A and C) and primary human monocytes (Fig. 1⇓B) with the NOD2 agonist MDP-LD (hereafter referred to as MDP) resulted in activation of endogenous Rac1 in a time- and dose-dependent manner. In contrast, stimulation of THP-1 cells with the inactive control compound MDP-DD did not activate Rac1 (Fig. 1⇓A), suggesting that Rac1 activation was not simply related to unspecific uptake of the components used.

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FIGURE 1.

Activation of Rac1 by the NOD2 agonist MDP in THP-1 cells and primary human monocytes. THP-1 cells (A) were stimulated with either the NOD2 agonist MDP-LD (10 μg/ml) or with the inactive compound MDP-DD (10 μg/ml), or primary human monocytes (B) were treated with MDP-LD for the indicated time intervals, and Rac1 activation was determined by the amount bound to the GST-PAK Rac1 interaction binding site (PD, pulldown). C, THP-1 cells were stimulated with MDP-LD in concentrations as indicated for 30 min and Rac1 activation was determined by Rac1 pulldown assay. Activated GTPase levels were normalized to the amount of total Rac1 in cell lysates (IB, immunoblot) as analyzed by Western blotting. The intensity of the bands was quantified and the values are the mean ± SD (error bars) of three independent experiments normalized to the response of untreated cells.

Rac1 inhibition by siRNA or NSC23766 increased the NOD2-mediated IL-8 production but suppressed the TLR2-dependent IL-8 secretion

To assess the impact of Rac1 in NOD2-dependent gene expression, first we made use of RNAi experiments in primary human monocytes. Rac1-specific siRNA but not an unspecific control siRNA reduced Rac1 expression in these cells (Fig. 2⇓A). Rac1 siRNA enhanced the NOD2-mediated IL-8 production but reduced IL-8 release induced by TLR2 stimulation (Fig. 2⇓B). Next, we made use of the Rac1 inhibitor NSC23766 and examined its effect on NOD2-dependent IL-8 production in THP-1 monocytes. The TLR2-dependent chemokine induction was tested in parallel as a control. In line with results shown above, Fig. 2⇓C shows that preincubation with the Rac1 inhibitor strongly increased the MDP-stimulated IL-8 secretion. In contrast to the NOD2-dependent IL-8 expression and consistent with published studies, IL-8 release stimulated by the TLR2 ligand Malp2 was inhibited by NSC23766 (Fig. 2⇓D). According to the hypothesis that Rac1 might influence the NOD2 pathway via actin cytoskeletal reorganization (30), disruption of actin cytoskeleton by cytochalasin D also led to an enhanced IL-8 secretion in human monocytes (data not shown). These results suggest that Rac1, via actin regulation, can function as a negative regulator of NOD2-dependent IL-8 production but positively contributes to TLR2-dependent gene expression.

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FIGURE 2.

Effect of Rac1 siRNA and the Rac1 inhibitor NSC23766 on NOD2- and TLR2-mediated IL-8 secretion. Primary human monocytes were transfected with control nonsilencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed (A). Membranes were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. B, Primary human monocytes were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP or Malp2 for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. THP-1 cells were untreated (none) or preincubated overnight with the Rac1 inhibitor NSC23766 (200 μM). Subsequently, the cells were either stimulated with MDP (C) or Malp2 (D) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data presented are mean ± SD of three different experiments performed in duplicates (∗∗, p < 0.01).

Rac1 inhibition enhanced IL-8 and NF-κB reporter activities mediated by NOD2, but reduced TLR2-dependent NF-κB reporter activation

Next, we more closely examined the involvement of Rac1 in NOD2-dependent IL-8 induction and also in NF-κB activation which is a hallmark event downstream of NOD2 and is crucial for IL-8 expression. Transfection of HEK293 cells with Rac1 siRNA specifically attenuated the Rac1 expression compared with untransfected cells or cells which were transfected with control siRNA as shown by immunoblot analysis (Fig. 3⇓A). Then, HEK293 cells were transfected with control or Rac1-specific siRNAs or left untreated, subsequently transfected with NOD2 and IL-8 or NF-κB luciferase reporters, and stimulated with MDP. Treatment with MDP stimulated reporter activity in untransfected and in control siRNA-transfected cells. Importantly, Rac1 siRNA clearly increased the NOD2-mediated IL-8 and NF-κB reporter activities (Fig. 3⇓, B and C). In contrast to the NOD2-dependent signaling, Rac1 inhibition by RNAi abrogated the NF-κB activation by Malp2 in TLR2-transfected HEK293 cells (Fig. 3⇓D), confirming results shown above and published studies (34). Moreover, cotransfection of wild-type Rac1 slightly and of constitutive active Rac1L61 markedly reduced the NOD2-mediated IL-8 promoter activation, whereas the dominant negative Rac1N17 mutant dramatically enhanced the IL-8 and NF-κB reporter activities in HEK293 cells stimulated with MDP (Fig. 3⇓, E and F). According to our RNAi data, dominant-negative Rac1 reduced the TLR2-mediated NF-κB activation as shown by us previously (37).

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FIGURE 3.

Influence of Rac1 on NOD2- or TLR2-mediated IL-8 and NF-κB activation. A, HEK293 cells were left untreated, or were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed in duplicates. Membranes were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. B–D, HEK293 cells were left untreated (ctrl), or were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 48 h, the cells were additionally cotransfected with NOD2 (B and C) or TLR2 (D) expression plasmids, together with an IL-8-reporter (B) or NF-κB reporter construct (C and D) and a β-galactosidase reporter plasmid. Cells were either left untreated (−) or stimulated with MDP (B and C) or Malp2 (D) and relative luciferase activities were obtained. E and F, HEK293 cells seeded in 24-well plates were transiently transfected with a control vector (ctrl) or NOD2 expression plasmid along with an IL-8 luciferase reporter plasmid (E) or a NF-κB-driven luciferase reporter (F), respectively, and a β-galactosidase reporter plasmid. Additionally, the cells were cotransfected with wild-type Rac1 (Rac1wt), dominant negative Rac1N17 or constitutively active Rac1L61. Cells were either stimulated with 10 μg/ml MDP (MDP) or left untreated (−), and relative luciferase activities were obtained the next day. G, HEK293 cells seeded in 24-well plates were transiently transfected with a control vector (ctrl), or a RIP2 expression plasmid along with a NF-κB-driven luciferase reporter and a β-galactosidase plasmid. The influence of Rac1 was tested by additionally introducing wild-type Rac1 (Rac1wt), dominant negative Rac1 (Rac1N17), or constitutive active Rac1 (Rac1L61) mean ± SD; ∗∗, p < 0.01.

Moreover, we evaluated the effect of Rac1 on NF-κB activation induced by RIP2, a key protein of the NOD2 signaling pathway. Overexpression of RIP2 in HEK293 cells activated the NF-κB reporter as shown before (16, 45). Cotransfection of dominant negative Rac1N17 strongly increased, whereas wild-type Rac1 or the constitutive active mutant Rac1L61 only slightly influenced the NF-κB activation induced by RIP2 (Fig. 3⇑G). In an analog experiment, IKKβ induced NF-κB activation was not enhanced but even moderately reduced by dominant negative Rac1 (data not shown), indicating that Rac1 interacts with the NOD2 pathway at a level upstream of IKKβ, possibly at the level of the NOD2/RIP2 complex. Taken together, Rac1 seems to play a differential role in NOD2- and TLR-dependent signaling: it negatively regulates NOD2-mediated cell activation but positively affects TLR2-induced NF-κB activation.

NOD2 and activated Rac1 form a signaling complex

To verify the hypothesis that Rac1 effects NOD2 signaling at the level of NOD2 itself, confocal microscopy to look for colocalization was performed. HEK293 cells were transiently transfected with NOD2 and (wild-type) Rac1 expression plasmids and the proteins were visualized by confocal microscopy using the indicated Abs. As shown in Fig. 4⇓A, both proteins are enriched close to the plasma membrane suggesting colocalization and possibly interaction. Although this membrane localization of overexpressed but otherwise unstimulated NOD2 seems somehow unexpected, it is in agreement with recent studies (17, 24) and is likely caused by the constitutive activation of ectopically expressed NOD2. Next, possible NOD2 and Rac1 interaction was analyzed. HEK293 cells were transiently transfected with either expression vectors encoding Myc-tagged NOD2 and Rac1 (wt) or Myc-NOD2 and a control plasmid. Overexpression of both NOD2 and Rac1 in HEK293 cells led to an association of the two proteins, as detected by immunoblotting using Ab to Rac1 after immunoprecipitation of Myc-tagged NOD2 (Fig. 4⇓B). In a vice versa approach, immunoprecipitation of Rac1 and immunoblotting using anti-Myc Ab also showed an interaction of the two proteins (Fig. 4⇓B). Control experiments using an unrelated IgG-Ab for immunoprecipitation yielded in negative results (data not shown).

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FIGURE 4.

Rac1 and NOD2 colocalized and interacted at the plasma membrane. A, Immunofluorescence micrographs of HEK293 cells cotransfected with NOD2 and Rac1 expression plasmids. Fixed cells were stained with anti-NOD2 and anti-Rac1 Abs. Signals obtained with the two Abs (left and middle panels) are presented and a merged image is shown in the right panel. B, Western blot analysis of coimmunoprecipitations from HEK293 cells are shown. Myc-NOD2 and Rac1 wild-type were ectopically expressed in HEK293 cells and lysates were precipitated using anti-c-myc Ab (IP) and coprecipitated Rac1 was detected using anti-Rac1 Ab (IB). Vice versa, lysates were precipitated with anti-Rac1 (IP) and coprecipitated myc-NOD2 was detected using an anti-c-myc Ab (IB). MDP-stimulated THP-1 cells (C) or primary monocytes (D) were lysed at different time points, immunoprecipitations of endogenous NOD2 or endogenous Rac1 with the respective Abs were performed, and immune complexes were probed for the presence of Rac1 or NOD2, respectively. Equal loading was confirmed by blotting whole cell lysates with anti-Rac1 Ab (Input). E, MDP-stimulated THP-1 cells were immunoprecipitated with Abs directed against Nalp3 and immune complexes were probed for the presence of Rac1 or Nalp3. All experiments were repeated three times.

The physical interaction of endogenous NOD2 and Rac1 was subsequently demonstrated in THP-1 cells and primary monocytes. In this more physiological situation, an association of endogenous NOD2 with Rac1 was detected which was further enhanced upon MDP stimulation with a maximum after ∼15–30 min, which represents a time interval similar to the time point in which maximum Rac1 activation occurred as seen above (Fig. 4⇑, C and D, see also Fig. 1⇑). In contrast to the NOD2-Rac1 interaction, the NLR protein Nalp3 did not co-precipitate with Rac1 (Fig. 4⇑E).

Rac1 inhibition abrogates membrane recruitment of NOD2

As both NOD2 and Rac1 were shown to be associated at the cell membrane after stimulation with MDP, we hypothesized that Rac1 might mediate recruitment of the NOD2/RIP2 complex to the membrane. To test this, THP-1 cells were left untreated or stimulated with MDP for 40 min. The membrane fractions were separated and subsequently immunoblotted with Rac1 and NOD2 Abs. After stimulation with MDP, recruitment of both NOD2 and Rac1 to the plasma membrane was observed (Fig. 5⇓A). Preincubation with the Rac1 inhibitor NSC23766 clearly decreased membrane association of NOD2 and Rac1 compared with untreated cells. We also observed recruitment of Rip2 to the membrane, which was abrogated by the Rac1 inhibitor (data not shown). Next, levels of Rac1 in primary human monocytes were reduced using siRNA, and membrane and cytosol fractions were separated and immunoblotted with NOD2 Abs. Rac1 knock-down led to reduced membrane association of NOD2 (Fig. 5⇓B), whereas in the cytosolic fraction, no major differences in NOD2 levels were observed. Moreover, cytochalasin D also abrogated membrane recruitment of Rac1 in human monocytes (data not shown). Overall, these data indicate that Rac1, possibly via actin organization, is involved in the recruitment of NOD2 to the plasma membrane.

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FIGURE 5.

Influence of Rac1 inhibition on recruitment of NOD2 and Rac1 to the plasma membrane. A, THP-1 cells were left untreated or preincubated with the Rac1 inhibitor NSC23766 (NSC) overnight. The next day, the cells were left untreated (−) or stimulated with 10 μg/ml MDP (MDP) for 40 min. Membrane fractions were separated and immunoblotted with anti-NOD2 or anti-Rac1 Abs. The intensity of the bands was quantified and the values are the mean ± SD (error bars) of three independent experiments normalized to untreated cells and indicated as fold activation. B, Primary human monocytes were mock-transfected (none), transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1) and, after 72 h, stimulated with MDP for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti-NOD2 Abs. All experiments were repeated three times.

β-PIX is involved in regulation of NOD2 signaling by Rac1

β-PIX is a Rac/Cdc42GEF that has been shown to directly bind Rac1, thereby mediating Rac1 translocation to the plasma membrane as well as Rac1 activation (40). To test if β-PIX plays a role in Rac1’s regulatory role on NOD2 signaling, we first tested whether endogenous β-PIX binds to endogenous NOD2 upon stimulation with MDP. Our coimmunoprecipitation experiments in THP-1 cells and primary monocytes demonstrated interaction of NOD2 and β-PIX after MDP stimulation in a time dependent manner (Fig. 6⇓B and data not shown). According to the recent finding of Hordijk and colleagues (40), we also observed interaction of β-PIX and Rac1, which was enhanced after MDP stimulation (Fig. 6⇓A). Next, we conducted β-PIX gene silencing experiments. Human THP-1 monocytes were transfected with control siRNA or β-PIX-specific siRNA, which led to a reduced level of endogenous β-PIX expression (Fig. 6⇓C). Moreover, β-PIX knock-down cells showed reduced membrane translocation of NOD2 after MDP treatment (Fig. 6⇓D), suggesting that the NOD2-Rac1 complex can be targeted to the membrane by β-PIX. Most importantly, β-PIX siRNA enhanced the NOD2-mediated IL-8 production but had no effect on IL-8 release induced by TLR2 stimulation (Fig. 6⇓, E and F). Thus, β-PIX appears to play a role in Rac1-mediated negative regulation of NOD2 signaling.

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FIGURE 6.

Involvement of β-PIX in NOD2-mediated signaling. MDP-stimulated primary monocytes or THP-1 cells (A and B) were lysed at different time points, immunoprecipitations of endogenous Rac1 (A) or endogenous NOD2 (B) with the respective Abs were performed, and immune complexes were probed for the presence of β-Pix. Equal protein amounts in the lysates were confirmed by blotting total cell lysates with an ERK2 Ab (Input). All experiments were repeated three times. C, THP-1 cells were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting β-Pix (si-β-Pix_S1 (sequence 1), si-Pix_S2 (sequence 2)). After 72 h, cells were lysed and Western blots using anti-β-Pix Abs were performed. Western blots were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. D, THP-1 cells were transfected as indicated, incubated for 72 h, and stimulated with MDP (10 μg/ml) for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti-NOD2 Abs. E and F, THP-1 cells were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP (E) or Malp2 (F) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data presented are mean ± SD of three different experiments performed in duplicates (∗∗, p < 0.01).

Rac1 inhibition abrogated the interaction of NOD2 with its negative regulator Erbin

The fact that NOD2’s negative regulator Erbin has been shown to interact with NOD2 at the plasma membrane (24, 25), together with our observations that β-PIX-Rac1 influenced the membrane recruitment of NOD2 and negatively regulated the NOD2 signal cascade prompted us to the hypothesis that Rac1 somehow effects the interaction of NOD2 with Erbin. To test this, coimmunoprecipitation experiments with NOD2 and Erbin Abs were performed in THP-1 cells and primary monocytes, which were pretreated with or without the Rac1 inhibitor and subsequently stimulated with MDP. After stimulation with MDP, interaction of NOD2 and Erbin was observed in the cells which were not treated with the inhibitor (Fig. 7⇓A), consistent with published results (24, 25). Interestingly, preincubation with the Rac1 inhibitor NSC23766 abrogated the interaction of NOD2 with its negative regulator in THP-1 (data not shown) and primary monocytes (Fig. 7⇓A). Experiments using Rac1 RNAi confirmed these data, showing that Rac1 knock-down reduced NOD2- and Erbin-interaction in THP-1 (data not shown) and primary monocytes (Fig. 7⇓B). Furthermore, β-PIX knock-down also diminished the NOD2 and Erbin, as well as Rac1 and Erbin coimmunoprecipitations (Fig. 7⇓C). Overall, these findings might explain the aforementioned results demonstrating that Rac1 together with β-PIX negatively regulate NOD2 signaling.

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FIGURE 7.

Rac1 and β-PIX siRNAs as well as the Rac1 inhibitor NSC23766 inhibit interaction of NOD2 with Erbin. Primary monocytes (A and B) or THP-1 cells (C) were either preincubated with the Rac1 inhibitor NSC23766 (NSC) or were transfected with Rac1 siRNA or β-PIX siRNA, as indicated, and were stimulated with 10 μg/ml MDP (MDP) for 40 min. Subsequently, immunoprecipitations with an Erbin Ab and subsequent immunoblots with NOD2 and ERK2 Abs (A) or NOD2, Rac1, and Erbin Abs (B and C) were performed. One representative Western blot out of three is shown.