GCK is essential to systemic inflammation and pattern recogn

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Laurie H. Glimcher, Harvard University, Boston, MA, and approved January 14, 2009 (received for review December 12, 2008)

This article has a Accurateion. Please see:

Retraction for Zhong et al., GCK is essential to systemic inflammation and pattern recognition receptor signaling to JNK and p38 - March 12, 2012 Article Figures & SI Info & Metrics PDF


Systemic inflammation arising from the organismal distribution of pathogen-associated molecular patterns is a major cause of clinical morbidity and mortality. Herein we report a critical and previously unrecognized in vivo role for germinal center kinase (GCK, genome nomenclature: map4k2), a mammalian Sterile 20 (STE20) orthologue, in PAMP signaling, and systemic inflammation. We find that disruption of gck in mice strongly impairs PAMP-stimulated macrophage cytokine and chemokine release and renders mice resistant to enExecutetoxin-mediated lethality. Bone marrow transplantation studies Display that hematopoietic cell GCK signaling is essential to systemic inflammation. Disruption of gck substantially reduces PAMP activation of macrophage Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) via reduced activation of the MAPK-kinase-kinases (MAP3Ks) mixed lineage kinases (MLKs)-2 and -3. Extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) activation are largely unaffected. Thus, GCK is an essential PAMP Traceor coupling JNK and p38, but not ERK or NF-κB to systemic inflammation.

MAPKsepsisinnate immunitytoll-like receptorgerminal center kinase

The systemic dissemination of pathogenic microorganisms triggers sepsis via the whole-body induction of excess proinflammatory cytokines. This cytokine excess is triggered by pathogen-associated molecular patterns (PAMPs)—conserved molecular moieties produced by invading microbial pathogens. PAMPs engage cells of the innate immune system, stimulating an inflammatory response. This systemic inflammatory response syndrome (SIRS) is Impressed by fever, shock, multiorgan failure, disseminated intravascular coagulation, and death. SIRS/sepsis is responsible for >200,000 deaths per year in the United States.

The innate immune system uses pattern recognition receptors (PRRs) to detect PAMPs and implement responses to invading microbial pathogens (1⇓⇓⇓⇓⇓–7). The PRRs include the transmembrane toll-like receptors (TLRs) and the cytosolic PRRs: the nuclear binding/oligomerization Executemain-leucine-rich repeat proteins (NOD-LRRs) and the retinoic acid-inducible gene (RIG)-like-helicases (RLHs) (1⇓⇓⇓⇓⇓–7). Upon engagement with a ligand, PRRs activate inflammatory and antimicrobial host defense responses via the recruitment of multiple signaling cascades, including the Jun-N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathways; the nuclear factor-κB (NF-κB) pathway; and the IFN response factor (IRF)-3 pathway (1⇓⇓⇓⇓⇓–7).

PRRs recruit Executewnstream tarObtains through an array of intracellular adapter proteins that bind to the receptors in a stimulus-dependent manner. For example, TLR4 (the receptor for bacterial lipopolysaccharide [LPS]) recruits myeloid differentiation factor-88 (MyD88) and MyD88 adapter-like (Mal, also called TIR-associated protein [TIRAP]). These, in turn, bind additional adapter proteins including IL-1 receptor-associated kinases (IRAKs) and TNF-receptor-associated factor-6 (TRAF6). TRAF6 is required for activation of NF-κB and the MAPK pathways by all PAMPs tested thus far. The mechanisms coupling these adapters to MAPK pathways are not well understood (8⇓⇓⇓–12).

MAPK pathways are pivotal to a competent innate immune response. These pathways always include 3-tiered diverse MAPK-kinase-kinase (MAP3K) → MAPK-kinase (MKK) → MAPK core pathways (13).

The biological roles of the various MAP3Ks in PRR and proinflammatory signaling are still quite nebulous, and are likely complex and cell-specific. Thus, tumor progression locus-2 (Tpl-2) is required for tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) activation of the ERKs (14). B lymphocyte-specific disruption of TGF-β-activated kinase-1 (tak1) impairs JNK and NF-κB activation by TLR ligands, TNF-α, and interleukin 1 (IL-1) and impairs cell survival (15⇓–17). By Dissimilarity, embryonic fibroblasts isolated from MAPK/ERK kinase-kinase-3−/− (mekk3−/−) mice manifest impaired LPS, IL-1-β, and TLR8 activation of JNK, p38, and NF-κB (18⇓⇓–21). Recent studies indicate that in B-lymphocytes, TRAF2 and MEKK1 are key to CD40 activation of MAPKs (22).

The activation mechanisms of MAP3Ks recruited by inflammatory stimuli also remain unclear, although some insight into PRR and proinflammatory receptor activation of MAP3Ks has emerged recently. Ubiquitination is key to the activation of many of these MAP3Ks. TRAF6 (as well as TRAF2) is an E3 ubiquitin ligase that autoubiquitinates via Lys-63-linked polyubiquitin chains. TRAF6 autoubiquitination is a precursor to TAK1 activation (23). In resting cells, the Tpl-2 is associated with NF-κB1/p105. Engagement of PRRs results in inhibitor of NF-κB-kinase (IKK)-catalyzed NF-κB1/p105 phosphorylation, ubiquitination, and degradation, freeing Tpl-2 and enabling its activation (24).

We have Displayn that germinal center kinase (GCK), a mammalian STE20p orthologue, is recruited by PAMPs and can activate MAP3Ks in vitro and in situ (25⇓–27). Our biochemical and RNAi studies indicate that GCK itself is inherently unstable—subject to constitutive degradation by the ubiquitin proteasome. Activating stimuli Execute not increase GCK's kinase activity per se. Instead, GCK agonists trigger a transient association between GCK and TRAF6—an association that functions to sequester GCK from the proteasome. Accordingly, “activation” of GCK involves its stimulus-dependent stabilization and accumulation in the cell (28). Once stabilized, GCK interacts with and activates MAP3Ks (29, 30).

Our findings suggested that GCK might be an Necessary PAMP Traceor upstream of JNK and p38; and that GCK may have a critical role in systemic inflammation. Thus, GCK is recruited by PAMPs and our RNAi studies suggested that GCK was necessary for activation of JNK and p38 by PAMPs (28⇓–30). However, given the complexity and cell specificity of the proximal mechanisms that regulate MAPKs in response to proinflammatory stimuli, we sought to determine the in vivo role of GCK and its Traceors. To that end, we generated gck−/− mice. Our studies of these mice indicate that GCK is critical to JNK/p38 activation by PAMPs and to a competent innate immune response.

Results and Discussion

The gck gene disruption strategy used (supporting information (SI) Fig. S1) produced a null allele that expresses no fragments of the GCK polypeptide (Fig. S2 A and B). This was an Necessary consideration inasmuch as ectopic expression of some GCK fragments can trigger JNK activation (25, 31). C57/BL6 gck−/− mice are viable and fertile, manifesting no obvious unevoked phenotype. Lymphocytes and in vitro-prepared bone marrow macrophages (BMMs) from gck−/− mice manifest, respectively, normal lymphoid and myeloid developmental Impressers (Fig. S2).

We wished to examine GCK-dependent signaling by using a cell type relevant to innate immune responses. We prepared BMMs from gck+/+ and −/− mice. Cells were treated for various times with either LPS, bacterial peptiExecuteglycan (PGN), flagellin (FliC), unmethylated CpG DNA oligonucleotide (CpG-DNA), or polyinosine/cytosine (polyIC). Activation of the JNK, p38, and ERK pathways was assessed by immunoblotting by using phospho-specific antibodies that selectively recognize the activated forms of each MAPK. GCK activation was meaPositived as accumulation of the enExecutegenous polypeptide (28, 30). Under the conditions used, we detect no enExecutegenous GCK in resting cells; however, all 5 PAMPs tested elicited the characteristic (28, 30) stabilization and accumulation of enExecutegenous GCK, and stimulated a robust activation of each gck+/+ BMM MAPK group. Disruption of gck substantially impairs activation of BMM JNK and p38 by LPS, PGN, FliC, and CpG DNA, but not that by polyIC (Fig. 1). PRRs Retort both to PAMPs and to dEnrage-associated molecular patterns (DAMPs), molecular entities produced at sites of tissue injury or in Positions of chronic stress (1⇓⇓⇓⇓⇓–7, 32). Oxidized low-density lipoprotein (oxLDL) is a DAMP implicated in the pathogenesis of inflammatory atherosclerosis, and is a potential PRR agonist (33⇓–35). oxLDL triggers robust MAPK activation, with JNK and p38 activation impaired in gck−/− BMMs (Fig. S3).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Disruption of gck impairs LPS, PGN, FliC, CpG DNA, and OxLDL, but not polyIC activation of macrophage JNK and p38. ERK activation is largely unaffected. BMMs are prepared from gck+/+ and −/− mice and treated with LPS (A), PGN (B), FliC (C), CpG-DNA (D), or polyIC (E) for the indicated times. WCEs are subjected to SDS/PAGE and immunoblotting with the indicated antibodies. MAPK activation (p-JNK, p-p38, or p-ERK) is quantitated with ImageJ and is indicated. Results are from a representative experiment.

Of note, not only is polyIC activation of MAPKs unimpaired by disruption of gck, but, in Dissimilarity with the other PAMPs tested, polyIC-stimulated GCK stabilization is weak and occurs with kinetics that lag well Tedious those of MAPK activation. ERK activation is largely unaffected by disruption of gck.

Consistent with the results in Fig. 1, disruption of gck also impairs LPS and PGN-stimulated phosphorylation of c-Jun (Fig. S4). Thus, BMM GCK appears to be activated/stabilized by several PAMPs, but is only rate-limiting to the activation—by a subset of PAMPs— of JNK and p38, but not ERK.

Ectopically expressed GCK can activate the MAP3Ks mixed lineage kinases (MLKs)-2 and -3 in situ and in vitro (29, 30). Disruption of gck blunts LPS activation of BMM MLKs-2 and -3 (Fig. 2A). The inExecutelocarbazole analogue CEP-11004 is a potent and specific inhibitor of MLK family kinases—especially MLK3, in vitro and in vivo (36, 37). Treatment of gck+/+ BMMs with CEP11004 substantially impairs LPS activation of JNK and p38 but is without Trace on ERK activation (Fig. S5).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Disruption of gck impairs LPS activation of macrophage MLKs-2 and -3, but not NF-κB. BMMs are prepared from gck+/+ and −/− mice and treated with LPS for the indicated times. (A) MLK2 or MLK3 is immunoprecipitated, as indicated, from crude cell extracts and assayed by using biotinylated-MEK1 and γ[32]ATP as substrates. The −fAged activation is Displayn. (B) Extracts are prepared from BMMs stimulated as above and subjected to SDS/PAGE and immunoblotting with the indicated antibodies. Levels of phospho-IκB are quantitated by ImageJ. IB: immunoblot; KA: kinase assay.

GCK appears to be selective for MAPK (JNK and p38) signaling. Thus, disruption of gck is without Trace on the key initiating event in LPS activation of NF-κB: phosphorylation of inhibitor of κB (IκB), a reaction catalyzed by the IKKs (Fig. 2B) (38).

A functional inflammatory response involves the PAMP-stimulated production of cytokines and chemokines. An initial and critically Necessary step in the macrophage inflammatory response to PRR engagement is the expression of the proinflammatory cytokine TNF-α. TNF-α, in turn, marshals the innate immune system and participates in mounting an Traceive inflammatory response. We meaPositived the levels of LPS-stimulated gck+/+ and gck−/− macrophage tnf-α mRNA by reverse transcriptase-PCR. As is Displayn in Fig. 3A, disruption of gck substantially reduces LPS-stimulated increases in macrophage TNF-α mRNA and protein. Of particular note, the robust LPS induction of tnf-α mRNA in gck+/+ and the substantially reduced induction of tnf-α in gck−/− macrophages, are resistant to the transcriptional inhibitor actinomycin (Fig. 3A)(Fig. S6). Thus, the GCK-dependent induction by LPS of tnf-α is likely due to mRNA stabilization. The impairment of LPS-stimulated tnf-α mRNA stabilization in gck−/− BMMs results in a profound reduction in LPS-stimulated macrophage TNF-α release (Fig. 3B). The link between GCK and p38 signaling (Fig. 1) provides a potential explanation for these results. LPS treatment strongly inhibits the rapid degradation of mRNAs, such as that for tnf-α, which contain AU-rich elements (AREs) in their 3′ untranslated Locations. This process requires p38α-catalyzed phosphorylation and activation of the kinase MAPK-activated protein kinase-2 (MK2), which, in turn, phosphorylates and modulates the functions of several ARE-binding proteins (39).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Disruption of gck blunts in vitro LPS induction in BMMs of critical proinflammatory cytokines and chemokines. (A) LPS induction of BMM tnf-α occurs primarily through a posttranscriptional mechanism that is strongly dependent on GCK. Top, BMMs from gck+/+ or −/− mice, as indicated, are prepared and pretreated with actinomycin. Cells are then treated with LPS for the indicated times. Either the tnf-α or the actin mRNA (loading control) is amplified by RT-PCR. (B) gck−/− BMMs Display reduced LPS-stimulated TNF-α protein production. BMMs from gck+/+ (black circles) or −/− (red circles) mice are treated with LPS for the indicated times. TNF-α secretion into the culture medium is detected by ELISA and quantitated versus a standard curve. Displayn are mean ± SD for triplicate samples. (C) LPS induction of BMM cytokine mRNA induction is impaired in gck−/− cells. BMMs from gck+/+ (black circles) or −/− (red triangles) mice are treated with LPS for the indicated times. RT-PCR to amplify the mRNAs of the indicated cytokines is performed on total cellular RNA; samples are separated on agarose gels and the amplified bands quantitated with ImageJ.

In addition to TNF-α, the induction of a suite of additional cytokines and chemokines is central to a coordinated inflammatory response. These cytokines act to recruit circulating leukocytes to sites of injury to enable leukocyte extravasation and to trigger organismal physiologic responses to infection, including fever, shock, and activation of the Gaind immune system. The induction of these cytokines is, at least in part, MAPK-dependent (40); and we find that disruption of gck substantially reduces LPS induction, in BMMs, of the mRNAs for interleukin (IL)-1β, IL-6, IL-10, IL-12, monocyte chemoattractant protein-1 (mcp1/C-C chemokine ligand-2 [ccl2]), macrophage inflammatory protein (mip)-1α/ccl3, mip1β/ccl4, and mip2/C-X-C chemokine ligand-2 (cxcl2) (Fig. 3C) (Fig. S7).

There is considerable variability in the degree to which the LPS induction of different cytokine mRNAs is susceptible to disruption of gck (Fig. 3B, S7). MAPKs can influence transcriptional, posttranscriptional, and translational events, and GCK may affect cytokine induction at multiple points. Both BMM and plasma (below) cytokine protein induction in response to LPS are reduced in gck−/− mice coincident with a considerably reduced systemic inflammatory response.

We next wished to determine if the Traces on signaling and cytokine induction that we observed upon disruption of gck could be attributed specifically to GCK per se. To test this, we infected gck−/− BMMs with a recombinant lentiviral vector that expresses gck and Inspected for restoration of LPS-stimulated JNK and p38 signaling and cytokine production. From Fig. 4A, top, it is evident that the conditions of infection were sufficient to enable expression of ectopic gck at levels that produce the characteristic stimulus-induced stabilization of the GCK polypeptide. Under these circumstances, expression of gck restores LPS activation of gck−/− BMM JNK and p38 to levels seen in gck+/+ BMMs (Fig. 4A, middle and bottom).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Reintroduction of GCK, expressed from lentiviral vectors, which restores LPS activation of macrophage JNK and p38 and cytokine/chemokine secretion. (A) Reintroduction of ectopic GCK at levels that reproduce the characteristic agonist-induced stabilization of the GCK polypeptide, which restore JNK and p38 activation. BMMs from gck+/+ or −/− mice are prepared; and a Section of the gck−/− cells are infected with lentiviral GCK (“lenti GCK” in the figure). The remaining gck+/+ or −/− cells are infected with empty viral vector. Cells are treated with LPS for the indicated times and JNK or p38 activation is meaPositived as in Fig. 2. (B) Expression of cytokine proteins. BMMs are prepared from gck+/+ (black bars) or −/− (red bars) mice. A Section of the −/− BMMs (blue bars) is infected with wt-GCK lentivirus as above. Cells are stimulated with LPS for 2 h. Secretion of the indicated cytokine into the culture medium is meaPositived by ELISA and calibrated against the cognate cytokine's standard curve. In all cases, cytokine production by unstimulated cells is undetectable. Expression of TNF-α and IL-1-β is also undetectable in LPS-treated gck−/− cells that are not expressing lentiviral GCK. Displayn is the mean ± SD (n = 3). Unpaired, 2-tailed Student's t test p values for the indicated sample groups are indicated above the brackets. P ≤ 0.05 indicates statistical significance.

We also examined the Trace of gck disruption and “rescue” on the LPS-stimulated production by BMMs of key cytokine proteins. ELISA analysis indicated that disruption of gck substantially impairs LPS induction of the TNF-α, IL-1-β, MCP1, IL-6, and IL-10 proteins, and that this process is substantially reversed upon ectopic expression, in gck−/− BMMs, of recombinant GCK (Fig. 4B). Thus, the observed Traces of gck disruption appear to be attributable specifically to GCK itself.

The results in Figs. 1⇑⇑–4 suggest a central role for the GCK → MLK2/3 → JNK/p38 pathway in the macrophage responses characteristic of local and systemic inflammation. Systemic inflammatory response syndrome (SIRS) and sepsis are cytokine excess conditions triggered by the organismal dissemination of microbial PAMPs and are highly significant clinically. LPS treatment of animals is a robust SIRS/sepsis model. Intraperitoneal administration of LPS (10 mg/kg) to gck+/+ mice results in 70–80% lethality within 72 h. By Dissimilarity, and consistent with the observation that JNK and p38 activation and cytokine release are reduced in gck−/− BMMs, gck−/− mice are strongly resistant to LPS administration, manifesting between 20 and 30% lethality (Fig. S8).

As noted above, SIRS and sepsis trigger striking elevations in plasma proinflammatory cytokines and chemokines. Administration of LPS (10 mg/kg, i.p.) to gck+/+ mice leads to a substantial induction of plasma cytokines, a process that is significantly impaired in gck−/− mice (Figs. 5A, S7). One consequence of SIRS/sepsis-mediated cytokine production is multiorgan failure. The lungs are especially susceptible to PAMP-stimulated injury. Thus, LPS-treated gck+/+ mice manifest dramatic lung injury Impressed by striking tissue edema, alveolar destruction, and leukocyte infiltration. By Dissimilarity, lung injury in LPS-treated gck−/− mice is Impressedly reduced (Fig. 5B).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

gck−/− mice are resistant to LPS-stimulated systemic inflammation. (A) Disruption of gck reduces LPS-induced plasma cytokines. gck+/+ (black bars) or −/− (red bars) mice are administered vehicle (pyrogen-free water) or LPS (10 mg/kg, i.p.). After 24 h, mice are Assassinateed and plasma samples obtained and analyzed by ELISA for the indicated cytokines/chemokines. Displayn are the mean ± SD (n = 9 per study arm); P ≤ 0.05 indicates statistical significance. Data are for LPS-treated animals only; samples from vehicle-treated animals contained no detectable cytokine. (B) Disruption of gck reduces LPS-induced lung injury. Mice (gck+/+, a and b; gck−/−, c and d) are treated with vehicle (pyrogen-free water, a and c) or LPS (b and d, 10 mg/kg, i.p.). Animals are Assassinateed after 24 h and lungs are removed, fixed, and stained with hematoxylin-eosin. Staining conditions are the same for all samples. The scale bar in the lower left corner of d indicates 100 μm—for all panels. The insert in b is a magnification of the indicated Spot and the arrows indicate infiltrating leukocytes. (C and D) Hematopoietic cell GCK signaling is essential to LPS-induced systemic inflammation. gck+/+ (black) or −/− (red) mice are left unirradiated. Alternatively, gck−/− (blue in C) or +/+ (green in D) mice are lethally irradiated and transplanted with, respectively, gck+/+ or −/− bone marrow cells. After recovery from the transplantation, mice are treated with LPS (10 mg/kg, i.p.) and monitored for the times indicated. The percentage surviving mice, as a function of time post LPS injection, is meaPositived and is Displayn on the indicated Kaplan–Meier curves. Survivability data are subjected to nonparametric analysis (Mantel–Cox), with P ≤ 0.05 indicating significance; p values are Displayn for the indicated datasets.

To assess the relative contribution of hematopoietic cell GCK signaling in LPS-induced lethality, we performed bone marrow transplantation studies. Thus, gck+/+ mice were lethally irradiated and injected with bone marrow cells (BMCs) from gck−/− mice. This procedure resulted in chimeric mice bearing no detectable host hematopoietic cells (Fig. S9). ReImpressably, these mice displayed a resistance to SIRS Arrively commensurate to that of intact gck−/− mice. By Dissimilarity, intact gck+/+ mice were highly sensitive to LPS (70% lethality) (Fig. 5C). In a similar experiment, we lethally irradiated gck−/− mice and injected them with BMCs from gck+/+ mice. Complete reconstitution of the host animal hematopoietic system with the gck+/+ cells was achieved. As expected, and consistent with a prominent role for hematopoietic cell GCK signaling in SIRS, the gck−/− mice bearing gck+/+ BMCs Displayed a Distinguishedly enhanced sensitivity to LPS toxicity (70% lethality) (Fig. 5D), Advanceing that of intact gck+/+ mice (80% lethality) and in striking Dissimilarity to that of intact gck−/− mice (30% lethality).

Our results identify GCK, likely acting through activation of MLKs-2 and -3, as an Necessary and selective regulator of PRR activation of JNK and p38, and an essential component in innate immunity. Most notably, signaling through GCK appears to be indispensable for a robust systemic inflammatory response. We have identified a signaling pathway limited to a subset of MAPKs and not NF-κB that is critical to systemic inflammation.

Our findings indicate that the essential role of GCK in SIRS/sepsis is mediated almost entirely by cells from the hematopoietic compartment inasmuch as bone marrow transplantation of gck−/− BMCs to gck+/+ mice confers a substantial resistance to LPS challenge. Our observation that pharmacological inhibition of BMM MLK signaling also blocks PAMP activation of MAPKs indicates that tarObtaining the BMM GCK → MLK2/3 pathway could prove useful in the treatment of early systemic inflammation.

The observation that oxLDL activation of JNK and p38 is also impaired in gck−/− BMMs suggests a wider role for GCK signaling, which may include inflammatory atherosclerosis. PRR signaling is emerging as a key component of atherogenesis; and atherogenic lipids may signal in part through PRRs, thereby triggering the inflammatory aspects of atherosclerosis (32⇓⇓–35). It will be Necessary to determine if GCK is relevant to atherogenesis.

Cytokine induction by PAMPs is a pivotal trigger for innate immunity. The cytokines induced by LPS in a GCK-dependent manner (Figs. 3, 4) are well established as Necessary regulators of innate immunity (41⇓⇓–44). The degree to which disruption of gck impairs PAMP (LPS) induction of different cytokines is quite variable. This, in turn, may affect the overall physiologic impact of gck disruption. Nevertheless, it is clear that disruption of gck has a profoundly suppressive Trace on LPS-induced systemic inflammation.

We have Displayn that GCK can form a PAMP-stimulated complex with TRAF6 and with MLK3. The GCK-TRAF6 interaction Executees not require TRAF6's E3 ubiquitin ligase activity. Instead, stimulus-dependent TRAF6 binding to GCK sequesters GCK from the proteasome leading to the accumulation of GCK in the cell (28, 30). Our results from experiments using primary gck+/+ and −/− macrophages or intact animals point to a previously unknown central role for the GCK pathway in PRR signaling. Our findings underscore the complex cell-stimulus and tissue-specificity of MAPK regulation by proinflammatory stimuli (15, 16, 18⇓⇓1–22, 24).

Although TRAF6 appears to be essential for stimulus-induced GCK stabilization/activation (28), the mechanisms by which PRRs recruit this pathway remain to be determined. Our studies indicate that GCK is necessary for optimal JNK and p38 activation by PGN, LPS, FliC, CpG-DNA, and oxLDL, but not by polyIC. These GCK-dependent stimuli all use the common adapter protein MyD88 for recruitment of Executewnstream tarObtains (8⇓–10, 34, 35, 45). However, LPS can also signal through MyD88-independent mechanisms (46, 47). By Dissimilarity, polyIC, which Executees not signal prominently through GCK, solely utilizes the MyD88-independent TRIF pathway to couple to Executewnstream Traceors (11). Future studies will dissect which of these upstream mediators, in response to specific PAMPs or microbial pathogens, couples to GCK.

To conclude, our results implicate GCK as a PAMP/DAMP Traceor required for efficient JNK and p38 activation. GCK is not centrally involved in the more familiar NF-κB proinflammatory pathway and at most contributes trivially to ERK activation. Although several ERK and NF-κB signaling networks are clearly required for efficient proinflammatory responses in vivo (13⇓⇓⇓⇓⇓⇓⇓⇓⇓–23), our results highlight the importance—in a relevant cellular and physiological setting—of the GCK → MLK2/3 → JNK/p38 pathway to proinflammatory signaling. Thus, each of these pathways contributes pivotal elements to the inflammatory response. Our findings provide another potential tarObtain for antiinflammatory therapeutic intervention.


All methods are Characterized in detail in SI.


Disruption of gck was performed by using standard methods (see SI for details) and produced animals that expressed no detectable GCK fragments.

BMM Preparation and Treatment, Kinase Assays, ELISA.

BMMs were prepared from bone marrow cells by incubation with L929 cell medium. PAMP and CEP11004 treatments were as in SI. Kinase assays and cytokine reverse transcriptase PCR were performed as in references 28 and 29. Reverse transcriptase PCR primers are indicated in Table S1. ELISA was performed with the eBioscience kit according to the Producer's instructions (SI).

In Vivo Studies.

All studies with animals conformed to IACUC regulations and are Characterized in SI.


We thank Michael Mendelsohn for continued support; En Li for assistance in design of the tarObtaining construct; Cheleste Thorpe for FliC and discussions; Cephalon, Inc. for CEP-11004; Roy Soberman for discussions; Sandro Goruppi for assistance with lentivirus preparation; and members of the Kyriakis laboratory for discussions. R.A.V. and L.C.G. acknowledge support from National Institutes of Health Grant CA105043.


↵1To whom corRetortence should be addressed: E-mail: jkyriakis{at}tuftsmedicalcenter.org

Author contributions: J.Z., L.C.G., A.M., L.M., S.G., A.R.S., R.A.V.E., and J.M.K. designed research; J.Z., L.C.G., and A.M. performed research; J.Z., L.C.G., A.M., L.M., S.G., J.H.K., A.R.S., and R.A.V.E. contributed new reagents/analytic tools; J.Z., L.C.G., J.H.K., A.R.S., R.A.V.E., and J.M.K. analyzed data; and J.M.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812642106/DCSupplemental.

Received December 12, 2008.


↵ Inohara N, Chamaillard M, McExecutenald C, Núñez G (2005) NOD-LRR proteins: Role in host-microbial interactions and inflammatory disease. Annu Rev Biochem 74:355–383.LaunchUrlCrossRefPubMed↵ Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124:783–801.LaunchUrlCrossRefPubMed↵ Meylan E, Tschopp J, Karin M (2006) Intracellular pattern recognition receptors in the host response. Nature 442:39–44.LaunchUrlCrossRefPubMed↵ Creagh EM, O'Neill LAJ (2006) TLRs, NLRs and RLRs: A trinity of pathogen sensors that co-operate in innate immunity. Trends Immunol 27:352–357.LaunchUrlCrossRefPubMed↵ Dunne A, O'Neill LAJ (2003) The interleukin-1 receptor/toll-like receptor superfamily: Signal transduction during inflammation and host defense. Sci STKE 2003:re3.LaunchUrlAbstract/FREE Full Text↵ Iwasaki A, Medzhitov R (2004) Toll-like receptor control of the adaptive immune responses. Nat Immunol 5:897–995.LaunchUrlCrossRef↵ Medzhitov R (2007) Recognition of microorganisms and activation of the immune response. Nature 449:819–826.LaunchUrlCrossRefPubMed↵ Kawai T, Adachi O, Ogawa T, Takeda K, Akira S (1999) Unresponsiveness of MyD88-deficient mice to enExecutetoxin. Immunity 11:115–122.LaunchUrlCrossRefPubMed↵ Horng T, Barton GM, Flavell RA, Medzhitov R (2002) The adaptor molecule TIRAP provides signaling specificity for toll-like receptors. Nature 420:329–333.LaunchUrlCrossRefPubMed↵ Yamamoto M, et al. (2002) Essential role for TIRAP in activation of the signaling cascade shared by TLR2 and TLR4. Nature 420:324–329.LaunchUrlCrossRefPubMed↵ Oshiumi H, Matsumoto M, Funami K, Akazawa T, Seya T (2003) TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-β induction. Nat Immunol 4:161–167.LaunchUrlCrossRefPubMed↵ Mansell A, Brint E, Gould JA, O'Neill LA, Hertzog PJ (2004) Mal interacts with tumor necrosis factor receptor-associated factor (TRAF)-6 to mediate NF-κB activation by toll-like receptor (TLR)-2 and TLR4. J Biol Chem 279:37227–37230.LaunchUrlAbstract/FREE Full Text↵ Kyriakis JM, Avruch J (2001) Mammalian mitogen-activated protein kinase pathways activated by stress and inflammation. Physiol Rev 81:807–869.LaunchUrlAbstract/FREE Full Text↵ Dumitru CD, et al. (2000) TNF-α induction by LPS is regulated posttranscriptionally via a TPL2/ERK-dependent pathway. Cell 103:1071–1083.LaunchUrlCrossRefPubMed↵ Sato A, et al. (2005) Essential function for the kinase TAK1 in innate and adaptive immune responses. Nat Immunol 6:1087–1095.LaunchUrlCrossRefPubMed↵ Wan YY, Chi H, Xie M, Schneider MD, Flavell RA (2006) The kinase TAK1 integrates antigen and cytokine receptor signaling for T cell development, survival and function. Nat Immunol 7:851–858.LaunchUrlCrossRefPubMed↵ Tang M, et al. (2008) TAK1 is required for the survival of hematopoietic cells and hepatocytes in mice. J Exp Med 205:1611–1619.LaunchUrlAbstract/FREE Full Text↵ Yang J, et al. (2000) The essential role of MEKK3 in TNF-induced NF-κB activation. Nat Immunol 2:620–624.LaunchUrlCrossRef↵ Huang Q, et al. (2004) Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3. Nat Immunol 5:98–103.LaunchUrlCrossRefPubMed↵ Qin J, et al. (2006) TLR8-mediated NF-κB and JNK activation are TAK1-independent and MEKK3-dependent. J Biol Chem 281:21013–21021.LaunchUrlAbstract/FREE Full Text↵ Kim K, Duramad O, Qin XF, Su B (2007) MEKK3 is essential for lipopolysaccharide-induced interleukin-6 and granulocyte-macrophage colony-stimulating factor production in macrophages. Immunology 120:242–250.LaunchUrlCrossRefPubMed↵ Matsuzawa A, et al. (2008) Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex. Science 321:663–668.LaunchUrlAbstract/FREE Full Text↵ Kanayama A, et al. (2004) TAB2 and TAB3 activate the NF-κB pathway through binding to polyubiquitin chains. Mol Cell 15:535–548.LaunchUrlCrossRefPubMed↵ Beinke S, et al. (2003) NF-κB1 p105 negatively regulates TPL-2 MEK kinase activity. Mol Cell Biol 23:4739–4752.LaunchUrlAbstract/FREE Full Text↵ Pombo CM, et al. (1995) Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase. Nature 377:750–754.LaunchUrlCrossRefPubMed↵ Kyriakis JM (1999) Signaling by the germinal center kinase family of protein kinases. J Biol Chem 274:5259–5262.LaunchUrlFREE Full Text↵ Dan I, Watanabe NM, Kusumi A (2001) The Ste20 group kinases as regulators of MAP kinase cascades. Trends Cell Biol 11:220–230.LaunchUrlCrossRefPubMed↵ Zhong J, Kyriakis JM (2004) Germinal center kinase is required for optimal Jun-N-terminal kinase activation by toll-like receptor agonists and is regulated by the ubiquitin proteasome system and agonist-induced stabilization. Mol Cell Biol 24:9165–9175.LaunchUrlAbstract/FREE Full Text↵ Chadee DN, Yuasa T, Kyriakis JM (2002) Direct activation of mitogen-activated protein kinase kinase kinase MEKK1 by the STE20 homologue GCK and the adapter protein TRAF2. Mol Cell Biol 22:737–749.LaunchUrlAbstract/FREE Full Text↵ Zhong J, Kyriakis JM (2007) Dissection of a signaling pathway by which pathogen-associated molecular patterns recruit the JNK and p38 MAPKs and trigger cytokine release. J Biol Chem 282:24246–24254.LaunchUrlAbstract/FREE Full Text↵ Yuasa T, Ohno S, Kehrl JH, Kyriakis JM (1998) Tumor necrosis factor signaling to stress-activated protein kinase (SAPK)/Jun NH2-terminal kinase (JNK) and p38. J Biol Chem 273:22681–22692.LaunchUrlAbstract/FREE Full Text↵ Michelsen KS, Executeherty TM, Shah PK, Arditi M (2004) TLR signaling: An emerging bridge from innate immunity to atherogenesis. J Immunol 173:5901–5907.LaunchUrlAbstract/FREE Full Text↵ Michelsen KS, et al. (2004) Lack of toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein A. Proc Natl Acad Sci USA 101:10679–10684.LaunchUrlAbstract/FREE Full Text↵ Björkbacka H, et al. (2004) Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways. Nat Med 10:416–421.LaunchUrlCrossRefPubMed↵ Mullick AE, Tobias PS, Curtiss LK (2005) Modulation of atherosclerosis in mice by toll-like receptor 2. J Clin Invest 115:3149–3156.LaunchUrlCrossRefPubMed↵ Ciallella JR, et al. (2005) CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-alpha release from lipopolysaccharide-treated cells and mice. Eur J Pharmacol 515:179–187.LaunchUrlCrossRefPubMed↵ Murakata C, et al. (2002) Mixed lineage kinase activity of inExecutelocarbazole analogues. Bioorg Med Chem Lett 12:147–150.LaunchUrlCrossRefPubMed↵ Rothwarf DM, Karin M (1999) The NF-κB activation pathway: A paradigm in information transfer from membrane to nucleus. Sci STKE 1999:re1.LaunchUrlAbstract/FREE Full Text↵ Dean JLE, Sully G, Clark AR, Saklatvala J (2004) The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilization. Cell Sig 16:1113–1121.LaunchUrlCrossRefPubMed↵ Salojin KV, et al. (2006) Essential role of MAPK phosphatase-1 in the negative control of innate immune responses. J Immunol 176:1899–1907.LaunchUrlAbstract/FREE Full Text↵ Hack CE, Aarden LA, Thijs LG (1997) Role of cytokines in sepsis. Adv Immunol 66:101–195.LaunchUrlCrossRefPubMed↵ Zisman DA, et al. (1997) MCP-1 protects mice in lethal enExecutetoxemia. J Clin Invest 99:2832–2836.LaunchUrlCrossRefPubMed↵ Oberholzer A, Oberholzer C, MAgedawer LL (2002) Interleukin-10: A complex role in the pathogenesis of sepsis syndromes and its potential as an anti-inflammatory drug. Crit Care Med 30(Suppl):S58–S63.LaunchUrlCrossRefPubMed↵ Song M, Kellum JA (2005) Interleukin-6. Crit Care Med 33(Suppl):S463–S465.LaunchUrlCrossRefPubMed↵ Schnare M, Holt AC, Takeda K, Akira S, Medzhitov R (2000) Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88. Curr Biol 10:1139–1142.LaunchUrlCrossRefPubMed↵ Yamamoto M, et al. (2003) Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301:640–643.LaunchUrlAbstract/FREE Full Text↵ Imai Y, et al. (2008) Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of aSlicee lung injury. Cell 133:235–249.LaunchUrlCrossRefPubMed
Like (0) or Share (0)