TRAF6 is a critical signal transducer in IL-33 signaling pathway

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.     

Rho-kinase mediates lysophosphatidic acid-induced IL-8 and MCP-1 production via p38 and JNK pathways in human endothelial cells

Lysophosphatidic acid (LPA), an inflammatory mediator that is elevated in multiple inflammatory diseases, is a potent activator of Rho kinase (ROCK) signaling and of chemokine production in endothelial cells. In this study, LPA activated ROCK, p38, JNK and NF-κB pathways and induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) mRNA and protein expression in human endothelial cells. We mapped signaling events downstream of ROCK, driving chemokine production. In summary, MCP-1 production was partly regulated by ROCK acting upstream of p38 and JNK and mediated downstream by NF-κB. IL-8 production was largely driven by ROCK through p38 and JNK activation, but with no involvement of NF-κB.     

Ubiquitination and translocation of TRAF2 is required for activation of JNK but not of p38 or NF-kappaB

TRAF2 is a RING finger protein that regulates the cellular response to stress and cytokines by controlling JNK, p38 and NF-kappaB signaling cascades. Here, we demonstrate that TRAF2 ubiquitination is required for TNFalpha-induced activation of JNK but not of p38 or NF-kappaB. Intact RING and zinc finger domains are required for TNFalpha-induced TRAF2 ubiquitination, which is also dependent on Ubc13. TRAF2 ubiquitination coincides with its translocation to the insoluble cellular fraction, resulting in selective activation of JNK. Inhibition of Ubc13 expression by RNAi resulted in inhibition of TNFalpha-induced TRAF2 translocation and impaired activation of JNK but not of IKK or p38. TRAF2 aggregates in the cytoplasm, as seen in Hodgkin-Reed-Sternberg lymphoma cells, resulting in constitutive NF-kappaB activity but failure to activate JNK. These findings demonstrate that the TRAF2 RING is required for Ubc13-dependent ubiquitination, resulting in translocation of TRAF2 to an insoluble fraction and activation of JNK, but not of p38 or NF-kappaB. Altogether, our findings highlight a novel mechanism of TRAF2-dependent activation of diverse signaling cascades that is impaired in Hodgkin-Reed-Sternberg cells.     

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.     

Implications of proteasome inhibition: an enhanced macrophage phenotype

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis

Two key features of atherosclerotic plaques that precipitate acute atherothrombotic vascular occlusion ("vulnerable plaques") are abundant inflammatory mediators and macrophages with excess unesterified, or "free," cholesterol (FC). Herein we show that FC accumulation in macrophages leads to the induction and secretion of two inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The increases in TNF-alpha and IL-6 mRNA and protein were mediated by FC-induced activation of the IkappaB kinase/NF-kappaB pathway as well as activation of MKK3/p38, Erk1/2, and JNK1/2 mitogen-activated protein kinases (MAPK). Activation of IkappaB kinase and JNK1/2 was needed for the induction of both cytokines. However, MKK3/p38 signaling was specifically involved in TNF-alpha induction, and Erk1/2 signaling was required for IL-6. Most interestingly, activation of all of the signaling pathways and induction of both cytokines required cholesterol trafficking to the endoplasmic reticulum (ER). The CHOP branch of the unfolded protein response, an ER stress pathway, was required for Erk1/2 activation and IL-6 induction. In contrast, one or more other ER-related pathways were responsible for activation of p38, JNK1/2, and IkappaB kinase/NF-kappaB and for the induction of TNF-alpha. These data suggest a novel scenario in which cytokines are induced in macrophages by endogenous cellular events triggered by excess ER cholesterol rather than by exogenous immune cell mediators. Moreover, this model may help explain the relationship between FC accumulation and inflammation in vulnerable plaques.     

TGF-β-activated kinase 1 mediates mechanical stress-induced IL-6 expression in osteoblasts

Mechanical stress plays a key role in bone remodeling. Previous studies showed that loading of mechanical stretch induces a rapid Ca²⁺ influx and subsequent activation of stress-activated protein kinase pathways in osteoblasts. However, the activation mechanism and its significance in bone remodeling have not been fully elucidated. Here we show that TAK1 MAPKKK was activated by cyclic stretch loading of MC3T3-E1 cells. Knockdown of TAK1 attenuated the stretch-induced activation of JNK, p38, and NF-κB. Extracellular (EGTA) or intracellular (BAPTA/AM) Ca²⁺ chelator prevented the stretch-induced activation of TAK1. Activation of TAK1 and its associated downstream signaling pathways were also suppressed by CaMKII inhibitors (KN-93 and KN-62). Furthermore, TAK1-mediated downstream pathways cooperatively induced the expression of IL-6 mRNA in the stretched MC3T3-E1 cells. We also confirmed that TAK1 mediates cyclic stretch-induced IL-6 protein synthesis in the cells using immunoblotting and ELISA. Finally, stretch loading of murine primary osteoblasts induced the expression of IL-6 mRNA via TAK1. Collectively, these data suggest that stretch-dependent Ca²⁺ influx activates TAK1 via CaMKII, leading to the enhanced expression of IL-6 through JNK, p38, and NF-κB pathways in osteoblasts.     

Modulation of the activation of extracellular signal-regulated kinase (ERK) and the production of inflammatory mediators by ADP-ribosylation inhibitors

ADP-ribosylation is involved in nuclear factor kappaB (NF-kappaB)-dependent gene expression induced by lipopolysaccharide in murine macrophages. Here we have investigated the mechanism by which ADP-ribosylation inhibitors block signaling pathways induced in macrophages. In RAW264.7 macrophages the inducers of NF-kappaB activate the production of reactive oxygen species and three mitogen-activated protein kinases (MAPK), the extracellular signal regulated kinase (ERK), the c-jun N-terminal kinase/stress-activated protein kinase (JNK), and p38. We demonstrate that ADP-ribosylation inhibitors specifically inhibit ERK MAPK activation and reduce the release of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha), IL-6 and nitrite.