We have reported that the bacterial LPS induces the activation of NF-κB and inflammatory cytokine gene expression and that this requires the activity of small GTPase, RhoA. In this study, we show that an atypical protein kinase C isozyme, PKCζ, associates functionally with RhoA and that PKCζ acts as a signaling component downstream of RhoA. Stimulation of monocytes and macrophages with LPS resulted in PKCζ activation and that inhibition of PKCζ activity blocks both LPS-stimulated activation of NF-κB and IL-1β gene expression. Our results also indicate that transforming growth factor β-activated kinase 1 acts as a signaling component downstream of PKCζ in cytokine gene transcription stimulated by LPS in human peripheral blood monocytes and macrophages. The specificity of this response suggests an important role for the Rho GTPase/PKCζ/transforming growth factor β-activated kinase 1/NF-κB pathway in host defense and in proinflammatory cytokine synthesis induced by bacterial LPS.
Bacterial LPS is a powerful activator of the innate immune system, stimulating monocytes, and macrophages to synthesize an array of cytokines and chemokines that recruit inflammatory cells to the involved tissue. These cytokines can further activate monocytes, neutrophils, and lymphocytes, initiating cellular injury and tissue damage (1, 2). The inflammatory response produced by infecting bacteria involves leukocyte gene expression that is tightly regulated by the activities of transcription factors such as NF-κB, NF-IL-6, and AP-1 (3, 4, 5). NF-κB is of considerable importance in immune cell function owing to its ability to activate the transcription of many proinflammatory immediate-early genes (6, 7). Numerous stimuli can activate NF-κB, including LPS, as well as other proinflammatory factors, such as TNF-α and IL-1 (8, 9). Indeed, the LPS-induced activation of NF-κB appears essential for proinflammatory cytokine synthesis in human peripheral blood monocytes (10).
The mammalian protein kinase C (PKC)3 family is comprised of at least 10 isoenzymes of serine/threonine protein kinases, which can be divided into three classes: conventional (cPKCα, βΙ, βΙΙ, and γ), novel (nPKCδ, ε, μ, θ, and η), and atypical PKC (aPKC ζ, and λι). This division of PKC isoenzymes is due to their molecular structures and activation mechanisms (11, 12). A number of in vitro and in vivo studies have implicated the role for PKCs in the regulation of host defense and inflammation (13, 14, 15, 16, 17) and it has also been reported that PKCβ may modulate the respiratory burst of both neutrophils and HL-60 cells (18, 19). However, the contribution of each PKC isoform to the regulation of monocytes and macrophage activation has not yet been elucidated. We have reported that the bacterial LPS induces the activation of NF-κB and proinformatory cytokine gene expression, and that this requires the activity of small GTPase, RhoA, and IL-1 receptor-associated kinase (10, 20).
Although the activation of NF-κB has been extensively studied in cell lines of hematopoietic lineage, the signal transduction pathways induced by LPS, in particular the early intracellular events that lead to monocyte/macrophage transcription activation, are still not clearly understood. We therefore investigated the role of PKC in the LPS-stimulated signaling events that lead to the activation of NF-κB. In this study, we report that exposure of monocytes and macrophages to LPS causes increased activity of an atypical PKC isoform, PKCζ, and that inhibition of PKCζ activity blocks both LPS-stimulated activation of NF-κB and IL-1β gene expression. It is also seen that the PKCζ associates functionally with RhoA in LPS-stimulated monocytes and macrophages, and that PKCζ acts as a signaling component upstream of the transforming growth factor β-activated kinase 1 (TAK1) during LPS-induced activation of NF-κB. These findings demonstrate that LPS-induced activation of NF-κB uses a signaling pathway that requires activity of PKCζ, and that PKCζ acts as a signaling component downstream of RhoA and upstream of TAK1 in LPS-induced NF-κB activation and cytokine gene expression in human peripheral blood monocytes and macrophages.
Materials and Methods
Ultra pure LPS (Escherichia coli, 0111:B4) was obtained from InvivoGen. PKCζ inhibitor, myristoylated PKCζ pseudosubstrate inhibitor was purchased from Calbiochem. Recombinant murine TNF-α was purchased from CP Biotech. The cDNAs encoding the constitutively active mutant of PKCζ and the dominant-negative mutants of PKCζ were cloned into the eukaryotic expression vector pEFneo. The constitutively active RhoA mutant, and the recombinant Clostridium botulinum C3 transferase exoenzyme were obtained as previously described (10, 21). The polyclonal Ab against phospho-PKCζ was purchased from Cell Signaling Technology. The mAbs against RhoA and PKCζ were purchased from Santa Cruz Biotechnology.
Preparation of monocytes from peripheral blood
Heparinized human peripheral blood from healthy donors was fractionated on Percoll (Amersham Biosciences) density gradients. Mononuclear cells and neutrophils were obtained by centrifugation through a 55/74% discontinuous Percoll gradient. Monocytes were further separated from this population using gelatin/plasma coated flasks (21). The purity of the monocyte preparation was >85–90% when determined by staining with an anti-CD14 mAb (Coulter Immunology), and cell viability was >95% when assessed by trypan blue exclusion. Monocytes were resuspended in RPMI 1640 medium (Irvine Scientific) containing 10% (v/v) heat-inactivated FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and l-glutamine (2 mM; Irvine Scientific).
Electrophoretic mobility shift assay (EMSA)
Immunoprecipitation and immunoblotting
Monocyte lysates were incubated with an appropriate amount of Ab for 3 h and then precipitated following absorption onto protein A-Sepharose (Amersham Biosciences). Precipitates were washed three times, separated by SDS-PAGE and transferred to Hybond-ECL nitrocellulose (Amersham Biosciences). Filter strips were incubated with primary Ab for 30 min at room temperature, followed by addition of peroxidase-conjugated IgG (1/10,000 dilution, 30 min) and then analyzed for peroxidase using ECL reagents (DuPont/NEN Research Products).
Detection of phosphorylated PKCζ
Monocytes or macrophages were incubated in medium ± LPS then lysed in Laemmli buffer. Equal volumes of the lysates from each condition were resolved by SDS-PAGE using 8% gels and transferred to Hybond-ECL nitrocellulose membranes. The membranes were initially probed with a polyclonal anti-phospho PKCζ Ab that at a dilution of 1/2000 detects activated forms of PKCζ. Membranes then were stripped and incubated with a murine anti-PKCζ.
Transfection of cells with PKCζ and TRAF6 small inhibitory RNA (siRNA) and luciferase activity assay
Short interfering RNA was used to reduce PKC or TRAF6 levels in RAW264.7 cells. 21-mer siRNA sequences with a dTdT-3′ overhang (Qiagen-Xeragon) specific for PKCζ or TRAF6 were purchased. RAW264.7 cells were cotransfected with a PKCζ siRNA or TRAF6 siRNA together with the NF-κB-dependent luciferase reporter plasmid (pNF-κB-LUC) following the manufacturer’s protocol. The reporter construct NF-κB-LUC was generated as described (24). It contains three copies of the NF-κB site present in the IL-2 receptor promoter linked to the firefly luciferase gene. The transfected cells were cultivated for 24 h before 16-h LPS stimulation. Luciferase activities were determined using the Dual Luciferase Assay kit (Promega) and Monolight 3010 luminometer (Analytical Luminescence).
LPS induces activation of PKCς in human peripheral blood monocytes and mouse macrophage cell line, RAW 264.7
We first examined whether LPS induced PKCς activation as PKCs plays a key role in intracellular signal processes linked to diverse receptor types. As PKCς is activated through phosphorylation of threonine 410 (25), we were able to use an Ab that strictly recognizes Thr410-phophorylated PKCς to detect the activation of PKCς. Following the stimulation with LPS, monocytes and RAW264.7 cells were lysed and the lysates (20 μg) were analyzed for phosphorylation of PKC by using a Western blot. Exposure to LPS led to a time-dependent increase in phosphorylation of PKCς that was first observed at 10 min and peaked at 30 min of exposure in both human peripheral blood monocytes (Fig. 1,A) and in mouse macrophage cell line, RAW 264.7 (Fig. 1 B).
PKCς is required for LPS-induced NF-κB activation and proinflammatory cytokine production
To assess the role of PKCς in LPS-induced proinflammatory cytokine production, we examined the effects of PKCς on IL-1β synthesis. Unstimulated human peripheral blood monocytes produced little IL-1β. Addition of LPS (100 ng/ml) resulted in production of IL-1β as measured by ELISA. Pretreatment with PKCζ pseudosubstrate inhibitor largely inhibited LPS-induced IL-1β protein synthesis (Fig. 2,A, lane 5). In contrast, the PKCζ pseudosubstrate inhibitor treatment only marginally affected TNF-α-induced production of IL-1β (Fig. 2,A, lane 6). We reported that activation of NF-κB is critical for IL-1β gene expression induced by bacterial chemoattractant (5). To assess the mechanisms of LPS-induced IL-1β expression, we examined the role of PKCς in LPS-induced NF-κB activation and cytokine gene expression. Following pretreatment of PKCζ pseudosubstrate inhibitor (ζPS), monocytes (Fig. 2,B, left), or macrophages (Fig. 2,B, right) were stimulated with LPS or TNF-α and the activation of NF-κB was assessed by EMSA and Western blot. Without an inhibitor, activity of NF-κB was strongly induced by both LPS and TNF-α in monocytes and macrophages (Fig. 2,B). This activation was largely prevented in those monocytes and macrophages pretreated with the PKCζ pseudosubstrate inhibitor that were subsequently exposed to LPS (Fig. 2,B, lane 5; Fig. 2,C, lane 9). We further confirmed the requirement for PKCζ in LPS-induced IκBα phosphorylation by knockdown of PKCζ with PKCζ siRNA. As shown in Fig. 2 D, PKCζ siRNA markedly inhibited IκBα phosphorylation by LPS. These results suggest that PKCζ plays important roles in both LPS-induced IκBα phosphorylation, NF-κB activation, and in IL-1β protein synthesis in human peripheral blood monocytes/macrophages.
The necessity of PKCζ for LPS-induced activation of NF-κB was also demonstrated by a dominant-negative form of PKCζ (Fig. 3,A), and PKCζ siRNA (Fig. 3,B). Raw 264.7 cells were cotransfected with expression vectors encoding a dominant-negative PKCζ with the NF-κB-dependent luciferase reporter plasmid (pNF-κB-LUC). They were then exposed to LPS. Fig. 3,A shows that expression of the dominant-negative PKCζ mutant protein was associated with poor induction of luciferase activity promoted by NF-κB (Fig. 3,A, lane 4). The effect of siRNA on PKCζ expression was quantified by immunoblotting cells 96 h post transfection. PKCζ expression was reduced by PKCζ siRNA to 52.43 ± 4.79% compared with cells transfected with control RNA. PKCζ siRNA-transfected cells were then exposed to LPS. PKCζ siRNA reduced NF-κB activity induced by LPS (Fig. 3 B). These results confirmed that PKCζ activity was required for LPS-induced activation of NF-κB leading to expression of cytokines.
PKCζ interacts with RhoA and TRAF6 in LPS-stimulated monocytes
We previously demonstrated that LPS stimulates the activity of RhoA, a small GTPase, and that this activity in monocytes is required for the activation of NF-κB induced by LPS (10). We therefore tested whether the functional interaction between PKCζ and RhoA was important for this LPS-induced activation of NF-κB. We examined whether the biological activity of the PKCζ was based on its ability to interact with low molecular mass GTP-binding proteins and whether PKCζ could was functional when binding to RhoA GTPase in vivo. Human peripheral blood monocytes (Fig. 4,A) or macrophages (Fig. 4,B) were exposed to medium or LPS (37°C, 10 min) and then lysed. Anti-RhoA Ab (Santa Cruz Biotechnology) was used to immunoprecipitate the RhoA GTPase from the lysates, and the components of the immunoprecipitates were detected by immunoblotting with Ab specific for PKCζ. Anti-PKCζ Ab was detected as a coprecipitate with RhoA (Fig. 4). In control experiments, we preincubated the anti-RhoA Ab with blocking peptides (Santa Cruz Biotechnology) before adding it to cell lysates. The blocking peptides eliminated the formation of an immunoprecipitate containing PKCζ (Fig. 4, lanes 3). In addition, we did coimmunoprecipitation experiments with anti-TRAF6 Ab. Immunoblotting analysis of the immunoprecipitation complex showed that TRAF6 associated with PKCζ (Fig. 4 C). These data showed that PKCζ binds to the RhoA GTPase and TRAF6 in LPS stimulated monocytes and macrophages.
PKCζ acts as a signaling component downstream of RhoA and TRAF6
The results presented above demonstrated that there was an interaction between PKCζ and RhoA in monocytes after their exposure to LPS. We next examined whether RhoA mediated the activation of NF-κB via a downstream effect on PKCζ. This question was addressed by using Clostridium botulinum C3 transferase, which is an exotoxin that inhibits the Rho GTPases (Rho A, B, and C) but does not inhibit Rac or Cdc42 GTPases (26). Following incubation with and without 10 μg/ml of recombinant C3 transferase for 16 h, human monocytes (Fig. 5,A, left) or macrophages (Fig. 5,B, right) were stimulated with LPS for 10 min and PKCζ activity was assessed. As shown in Fig. 5 A, the PKCζ activity was significantly inhibited by C3 transferase, indicating that PKCζ is a signaling component that becomes activated downstream of RhoA.
The relationship between RhoA activity and subsequent PKCζ activation was further investigated using macrophages that had been cotransfected with expression vectors encoding a constitutively active form of RhoA (Fig. 5,B, lanes 2 and 3), a dominant-negative PKCζ (lane 3), and a NF-κB-dependent luciferase reporter plasmid. The constitutively active RhoA induced a strong luciferase activity but this activity was inhibited by coexpression of the dominant-negative PKCζ (Fig. 5 C, lane 3). These results confirm that RhoA activity is required for the activation of PKCζ induced by LPS and that PKCζ acts as a signaling component downstream of RhoA.
We further confirmed the requirement for TRAF6 in LPS-induced PKCζ activation by inhibition of TRAF6, either a dominant-negative form of TRAF6 (Fig. 5,C, left) or knockdown of TRAF6 with TRAF6 siRNA (Fig. 5,C, right). As shown in Fig. 5,C, the dominant-negative TRAF6 significantly reduced LPS-induced luciferase activity promoted by NF-κB (Fig. 5,C, left, lane 4). The effect of siRNA on TRAF6 expression was quantified by immunoblotting cells 96 h post transfection. TRAF6 siRNA markedly inhibited PKCζ phosphorylation by LPS (Fig. 5 C, right, lane 4). These results indicate that TRAF6 is required for the activation of PKCζ induced by LPS.
PKCζ signals LPS-induced activation NF-κB activation via TAK1
TAK1 has been shown to initiate NF-κB activation (27). We therefore sought to determine the involvement of TAK1 in the PKCζ-mediated activation of NF-κB induced by LPS. LPS-induced phosphorylation of TAK1 was time-dependent, so it was detectable as early as 15 to 20 min in both human peripheral blood monocytes (Fig. 6,A) and in the mouse macrophage cell line, RAW 264.7 (Fig. 6,B). Macrophages were cotransfected with NF-κB-LUC in combination with TAK1 siRNA. This largely prevented the activation of NF-κB (Fig. 6 C, lane 3), indicating a requirement for TAK1 in the response.
We also addressed the function of TAK1 in PKCζ-mediated activation of NF-κB. Pretreatment with PKCζ pseudosubstrate inhibitor largely inhibited LPS-induced TAK1 activation (Fig. 7,A, lane 3). Although expression of the constitutively active form of PKCζ induces NF-κB activity in the absence of TAK1 siRNA, this NF-κB activity was inhibited by cotransfection of the TAK1 siRNA (Fig. 7B, lane 3). Thus, it appears that PKCζ mediates NF-κB activation via the molecule of TAK1.
We previously demonstrated that LPS stimulates small GTPase RhoA activity in human monocytes and this activity is required for the activation of NF-κB that is induced by LPS (10). In the present study, we used multiple approaches to establish the role of PKCζ in LPS-induced activation of NF-κB in human blood monocytes because PKCζ has not previously been implicated in the signaling pathway. Exposure of monocytes and macrophages to LPS stimulated a time-dependent increase in phosphorylation of PKCζ. Preincubation of monocytes and macrophages with PKCζ inhibitor significantly inhibited the LPS-induced activation of NF-κB. The requirement for PKCζ was then confirmed by cotransfecting macrophages with either plasmids encoding dominant-negative forms of PKCζ or siRNA to reduce the expression of PKCζ. Because there was only partial inhibition of NF-κB activity, both after incubation with PKCζ inhibitor and by expression of dominant negative PKCζ, it is possible that other PKC isoforms, such as PKCδ and PKCθ, are also involved because PKCδ and PKCθ can activate NF-κB as well (15, 16).
The finding that PKCζ was critical to the activation of NF-κB led us to address its role in LPS-induced signaling in monocytes and macrophages. Despite an apparent role of the Rho GTPases in cytokine gene expression, relatively little is known about the intracellular signaling pathways linking RhoA activation to cytokine gene transcription in leukocytes. The Rho family of small G proteins has been shown to activate several serine/threonine kinases, which can mediate downstream effects. These appear mainly to act on the actin cytoskeleton. Naumann and colleagues (28) reported that p21-activating kinase 1 (PAK1), the immediate downstream effector of Rac1 and Cdc42, bound and activated NF-κB-inducing kinase, leading to degradation of inhibitory κB and increasing the binding of NF-κB to DNA in gastric epithelial cells infected by Helicobacter pylori. Furthermore, Frost et al. (29) showed that a constitutively active PAK1 stimulated NF-κB activity in fibroblasts and macrophages, and that activation of PAK1 was required for the stimulation of NF-κB activity both by Rac1 in fibroblasts and following exposure of mouse macrophages to LPS. These results suggest that PAK1 may play a role in the activation of NF-κB and the induction of inflammatory cytokine expression mediated by both Rac1 and Cdc42 but not by RhoA (30).
Activated RhoA has been shown to be involved in TLR2-mediated activation of PKC in HEK 293 cells (31). Recently, several studies have indicated the role of PKCζ in NF-κB activation (32, 33). Our data suggest that in human peripheral blood monocyte LPS activates PKCζ, which provides signal acting downstream of GTPase RhoA and which mediates LPS-induced NF-κB activation. We further determined the mechanisms by which PKCζ and its downstream effector, TAK1, contributed to NF-κB activity. Studies have shown that TAK1 activity is required for NF-κB activation (34, 35). We observed that transfection of TAK1 siRNA prevented the development of NF-κB activity induced by LPS and by the expression of a constitutively active form of PKCζ. Although we find that TAK1 is required for PKCζ-mediated NF-κB activation induced by LPS, the underlying mechanism is yet to be defined. One possible mechanism is that TAK1 may phosphorylate IKKβ directly. This suggestion is supported by the observation that TAK1 directly phosphorylates IKKα and IKKβ in vitro in HeLa cells (27). However, we have been unable to detect a physical association between TAK1 and the IKK complexes in monocytes (data not shown). An alternative explanation is that TAK1 may indirectly activate IKK through one or more intermediate components. One such signaling intermediate might be TAK1-binding proteins, which has been indicated in the signaling pathway whereby IL-1 stimulation leads to the activation of NF-κB through activation of IKKβ (36).
Several studies have demonstrated that production of cytokines by IL-1 and thymosin α1 involves TRAF6/PKCζ/IKK/NF-κB pathway (37, 38). Our work has shown that: 1) exposure of monocytes and macrophages to LPS leads to activation of PKCζ and that inhibition of PKCζ abrogates LPS-induced activation of NF-κB and proinflammatory cytokine production; 2) PKCζ becomes functionally associated with RhoA GTPase and TRAF6 in LPS-stimulated monocytes and macrophages; 3) PKCζ acts as a signaling component downstream of TRAF6 and RhoA; and 4) PKCζ mediates NF-κB activity via activation of TAK1 in monocytes and macrophages induced by LPS (Fig. 8). The specificity of this response suggests an important role for the Rho GTPase/PKCζ/TAK1/NF-κB pathway in host defense and in inflammation induced by bacterial LPS in human peripheral blood monocytes and macrophages. The function of small GTPase RhoA in cytokine gene expression induced by IL-1 and thymosin α1, however, remains to be determined.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by United States Public Health Service Grant AI43524 (to Z.K.P.) and National Institutes of Health Training Grant T32 AI 07469 (to L.-Y.C.). This work was also supported in part by the Sam and Ross Stein Charitable Trust and National Institutes of Health Grant M01RR00833 provided to the General Clinical Research Center of the Scripps Research Institute.
Abbreviations used in this paper: PKC, protein kinase C; aPKC, atypical PKC; EMSA, electrophoretic mobility shift assay; ζPS, PKCζ pseudosubstrate inhibitor; PAK1, p21-activating kinase 1; TAK1, transforming growth factor β-activated kinase 1; siRNA, small inhibitory RNA.