The death domain kinase RIP1 is essential for tumor necrosis factor alpha signaling to p38 mitogen-activated protein kinase

The cytokine tumor necrosis factor alpha (TNF-alpha) stimulates the NF-kappaB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting RIP1 and TRAF2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that RIP1 links the TNFR1 to the IkappaB kinase (IKK) complex, whereas TRAF2 couples the TNFR1 to the SAPK/JNK cascade. In transfection studies, RIP1 and TRAF2 stimulate p38 MAP kinase activation, and dominant-negative forms of RIP1 and TRAF2 inhibit TNF-alpha-induced p38 MAP kinase activation. We found TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production impaired in rip1(-/-) murine embryonic fibroblasts (MEF) but unaffected in traf2(-/-) MEF. Yet, both rip1(-/-) and traf2(-/-) MEF exhibit a normal p38 MAP kinase response to inducers of osmotic shock or IL-1alpha. Thus, RIP1 is a specific mediator of the p38 MAP kinase response to TNF-alpha. These studies suggest that TNF-alpha-induced activation of p38 MAP kinase and SAPK/JNK pathways bifurcate at the level of RIP1 and TRAF2. Moreover, endogenous RIP1 associates with the MAP kinase kinase kinase (MAP3K) MEKK3 in TNF-alpha-treated cells, and decreased TNF-alpha-induced p38 MAP kinase activation is observed in Mekk3(-/-) cells. Taken together, these studies suggest a mechanism whereby RIP1 may mediate the p38 MAP kinase response to TNF-alpha, by recruiting the MAP3K MEKK3.     

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.     

Interleukin (IL)-17 enhances tumor necrosis factor-alpha-stimulated IL-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

Inflammatory cytokines are well known to play crucial roles in the pathogenesis of rheumatoid arthritis. Among them, interleukin (IL)-17 is a cytokine that is mainly synthesized by activated T cells and its receptors are present in osteoblasts. The synthesis of IL-6, known to stimulate osteoclastic bone resorption, is reportedly responded to bone resorptive agents such as tumor necrosis factor-alpha (TNF-alpha) in osteoblasts. It has been reported that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We previously showed that sphingosine 1-phosphate (S1-P) mediates TNF-alpha-stimulated IL-6 synthesis in these cells. In the present study, we investigated the mechanism of IL-17 underlying enhancement of IL-6 synthesis in MC3T3-E1 cells. IL-17 induced phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. IL-17 also amplified S1-P-stimulated IL-6 synthesis, and the amplification by IL-17 was suppressed by SB203580. Anisomycin, an activator of p38 MAP kinase, which alone had no effect on IL-6 level, enhanced the IL-6 synthesis stimulated by TNF-alpha. SB203580 and PD169316 inhibited the amplification by anisomycin of the TNF-alpha-induced IL-6 synthesis. Taken together, our results strongly suggest that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

Hck is a key regulator of gene expression in alternatively activated human monocytes

IL-13 is a Th2 cytokine that promotes alternative activation (M2 polarization) in primary human monocytes. Our studies have characterized the functional IL-13 receptor complex and the downstream signaling events in response to IL-13 stimulation in alternatively activated monocytes/macrophages. In this report, we present evidence that IL-13 induces the activation of a Src family tyrosine kinase, which is required for IL-13 induction of M2 gene expression, including 15-lipoxygenase (15-LO). Our data show that Src kinase activity regulates IL-13-induced p38 MAPK tyrosine phosphorylation via the upstream kinases MKK3 or MKK6. Our findings also reveal that the IL-13 receptor-associated tyrosine kinase Jak2 is required for the activation of both Src kinase as well as p38 MAPK. Further, we found that Src tyrosine kinase-mediated activation of p38 MAPK is required for Stat1 and Stat3 serine 727 phosphorylation in alternatively activated monocytes/macrophages. Additional studies identify Hck as the specific Src family member, stimulated by IL-13 and involved in regulating both p38 MAPK activation and p38 MAPK-mediated 15-LO expression. Finally we show that the Hck regulates the expression of other alternative state (M2)-specific genes (Mannose receptor, MAO-A, and CD36) and therefore conclude that Hck acts as a key regulator controlling gene expression in alternatively activated monocytes/macrophages.     

Interleukin-1 beta and tumor necrosis factor-alpha induce MUC5AC overexpression through a mechanism involving ERK/p38 mitogen-activated protein kinases-MSK1-CREB activation in human airway epithelial cells

Mucin hypersecretion is commonly observed in many inflammatory diseases of the respiratory tract. MUC5AC is generally recognized to be a major airway mucin because MUC5AC is highly expressed in the goblet cells of human airway epithelium. Moreover, it is regulated by various inflammatory cytokines. However, the mechanisms by which the interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha induce MUC5AC gene expression in normal nasal epithelial cells, and the signal molecules involved, especially in the downstream signaling of mitogen-activated protein (MAP) kinases, remain unclear. Here we show that pharmacologic or genetic inhibition of either ERK or p38 MAP kinase pathway abolished IL-1beta- and TNF-alpha-induced MUC5AC gene expression in normal human nasal epithelial cells. Our results also indicate that the activation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-response element-binding protein and cAMP-response element signaling cascades via ERK and p38 MAP kinases are crucial aspects of the intracellular mechanisms that mediate MUC5AC gene expression. Taken together, these studies give additional insights into the molecular mechanism of IL-1beta- and TNF-alpha-induced MUC5AC gene expression and enhance our understanding on mucin hypersecretion during inflammation.     

Role of interleukin (IL)-2 receptor beta-chain subdomains and Shc in p38 mitogen-activated protein (MAP) kinase and p54 MAP kinase (stress-activated protein Kinase/c-Jun N-terminal kinase) activation. IL-2-driven proliferation is independent of p38 and p54 MAP kinase activation

We have shown recently that interleukin (IL)-2 activates the mitogen-activated protein (MAP) kinase family members p38 (HOG1/stress-activated protein kinase II) and p54 (c-Jun N-terminal kinase/stress-activated protein kinase I). Furthermore, the p38 MAP kinase inhibitor SB203580 inhibited IL-2-driven T cell proliferation, suggesting that p38 MAP kinase might be involved in mediating proliferative signals. In this study, using transfected BA/F3 cell lines, it is shown that both the acidic domain and the membrane-proximal serine-rich region of the IL-2Rbeta chain are required for p38 and p54 MAP kinase activation and that, as for p42/44 MAP kinase, this activation requires the Tyr338 residue of the acidic domain, the binding site for Shc. It is well established that the acidic domain of the IL-2Rbeta chain is dispensable for IL-2-driven proliferation, and thus our observations suggest that neither p38 nor p54 MAP kinase activation is required for IL-2-driven proliferation of BA/F3 cells. In addition, the tetravalent guanylhydrazone inhibitor of proinflammatory cytokine production, CNI-1493, can block the activation of p54 and p38 MAP kinases by IL-2 but has no effect on IL-2-driven proliferation of BA/F3 cells, activated primary T cells, or a cytotoxic T cell line. Furthermore, our observations provide evidence for the existence of an additional, unknown target of the p38 MAP kinase inhibitor SB203580, the activation of which is essential for mitogenic signaling by IL-2.     

TRAF6 and Src kinase activity regulates Cot activation by IL-1

Cot is one of the MAP kinase kinase kinases that regulates the ERK1/ERK2 pathway under physiological conditions. Cot is activated by LPS, by inducing its dissociation from the inactive p105 NFkappaB-Cot complex in macrophages. Here, we show that IL-1 promotes a 10-fold increase in endogenous Cot activity and that Cot is the only MAP kinase kinase kinase that activates ERK1/ERK2 in response to this cytokine. Moreover, in cells where the expression of Cot is blocked, IL-1 fails to induce an increase in IL-8 and MIP-1betamRNA levels. The activation of Cot-MKK1-ERK1/ERK2 signalling pathway by IL-1 is dependent on the activity of the transducer protein TRAF6. Most important, IL-1-induced ERK1/ERK2 activation is inhibited by PP1, a known inhibitor of Src tyrosine kinases, but this tyrosine kinase activity is not required for IL-1 to activate other MAP kinases such as p38 and JNK. This Src kinases inhibitor does not block the dissociation and subsequently degradation of Cot in response to IL-1, indicating that other events besides Cot dissociation are required to activate Cot. All these data highlight the specific requirements for activation of the Cot-MKK1-ERK1/ERK2 pathway and provide evidence that Cot controls the functions of IL-1 that are mediated by ERK1/ERK2.     

Regulation of suppressor of cytokine signaling 3 (SOCS3) mRNA stability by TNF-alpha involves activation of the MKK6/p38MAPK/MK2 cascade

The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-alpha. In contrast to IL-6, TNF-alpha increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38(MAPK)-cascade is required for TNF-alpha-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38(MAPK) cascade, basal SOCS3-expression is strongly reduced and TNF-alpha-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38(MAPK)-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3' untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-alpha. In summary, these data indicate that TNF-alpha regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38(MAPK) cascade and that the activation of MK2, a downstream target of p38(MAPK), is important for the regulation of SOCS3 expression.     

p38 mitogen-activated protein kinase regulates human T cell IL-5 synthesis.

Involvement of p38 mitogen-activated protein (MAP) kinase in human T cell cytokine synthesis was investigated. p38 MAP kinase was clearly induced in human Th cells activated through the TCR. SB203580, a highly selective inhibitor of p38 MAP kinase, inhibited the induction of p38 MAP kinase in human Th cells. Major T cell cytokines, IL-2, IL-4, IL-5, and IFN-gamma, were produced by Der f 2-specific Th clones upon stimulation through the TCR. IL-5 synthesis alone was significantly inhibited by SB203580 in a dose-dependent manner, whereas the production of IL-2, IL-4, and IFN-gamma was not affected. The proliferation of activated T cells was not affected. IL-5 synthesis of human Th clones induced upon stimulation with rIL-2, phorbol ester plus anti-CD28 mAb, and immobilized anti-CD3 mAb plus soluble anti-CD28 mAb was also suppressed by SB203580 in the same concentration response relationship. The results clearly indicated that IL-5 synthesis by human Th cells is dependent on p38 MAP kinase activity, and is regulated distinctly from IL-2, IL-4, and IFN-gamma synthesis. Selective control of IL-5 synthesis will provide a novel treatment devoid of generalized immune suppression for bronchial asthma and atopic dermatitis that are characterized by eosinophilic inflammation.     

Rho-kinase regulates endothelin-1-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts

We have previously reported that endothelin-1 (ET-1) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the ET-1-stimulated IL-6 synthesis in MC3T3-E1 cells. ET-1 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific inhibitor of Rho-kinase, significantly suppressed the IL-6 synthesis induced by ET-1 as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, reduced the ET-1-stimulated IL-6 synthesis. Y27632 as well as fasudil attenuated the ET-1-induced phosphorylation of p38 MAP kinase but not p44/p42 MAP kinase. These results strongly suggest that Rho-kinase regulates ET-1-stimulated IL-6 synthesis through p38 MAP kinase activation in osteoblasts.     

p38 mitogen-activated protein kinase and c-jun-NH2-terminal kinase regulate RANTES production by influenza virus-infected human bronchial epithelial cells

Airway epithelial cells which are the initial site of influenza virus (IV) infection are suggested to participate in airway inflammatory response by expressing various cytokines including RANTES; however, the intracellular signal that regulates RANTES expression has not been determined. In the present study, we examined the role of p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (Erk), and c-Jun-NH2-terminal kinase (JNK) in RANTES production by IV-infected human bronchial epithelial cells. The results showed that IV infection induced increases in p38 MAP kinase, and Erk and JNK phosphorylation and activity. SB 203580, PD 98059, and CEP-1347 attenuated IV-infection induced p38 MAP kinase activity, Erk activity, and JNK activity, respectively. SB 203580 and CEP-1347 attenuated RANTES production by 45.3% and 45.2%, respectively, but a combination of these inhibitors additively attenuated by 69.1%. In contrast, PD 98059 did not attenuate. Anti-IL-1alpha mAb, anti-IL-1beta mAb, anti-TNF-alpha mAb, anti-IL-8 mAb, anti-IFN-beta mAb, anti-RANTES mAb, and a combination of these mAbs did not affect IV infection-induced increases in p38 MAP kinase, Erk, and JNK phosphorylation, indicating that each cytokine neutralized by corresponding Ab was not involved in IV infection-induced phosphorylation of MAP kinases. N-acetylcysteine (NAC) did not affect IV infection-induced increases in MAP kinase phosphorylation, whereas NAC attenuated RANTES production by 18.2%, indicating that reactive oxygen species may act as a second messenger leading to RANTES production via p38 MAP kinase- and JNK-independent pathway. These results indicate that p38 MAP kinase and JNK, at least in part, regulate RANTES production by bronchial epithelial cells.     

Activation of extracellular signal-regulated kinase (ERK)1/2, but not p38 and c-Jun N-terminal kinase, is involved in signaling of a novel cytokine, ML-1

A novel cytokine, ML-1, was recently discovered, which shares a similar sequence homology with, but is functionally distinct from, IL-17 (Kawaguchi, M., Onuchic, L., Li, X. D., Essayan, D. M., Schroeder, J., Xiao, H. Q., Liu, M. C., Krishnaswamy, G., Germino, G., and Huang, S. K. (2001) J. Immunol. 167, 4430-4435). To determine the signaling mechanisms of ML-1, we investigated activation of mitogen-activated protein (MAP) kinases induced by ML-1. Results show that ML-1 induces in a time-dependent fashion the expression of IL-6 and IL-8 in both primary bronchial epithelial cells (PBECs) and human umbilical vein endothelial cells (HUVECs). ML-1 activated a MAP kinase and an extracellular signal-regulated kinase (ERK)1/2 but not p38 or the c-Jun N-terminal kinase (JNK) in both cell types. Selective MAP kinase kinase (MEK)1/2 inhibitors, PD98059 and U0126, inhibited, in a dose-dependent manner, ML-1-induced expression of IL-6 and IL-8. These findings suggest that ML-1-induced IL-6 and IL-8 production is mediated through the activation of ERK1/2 in both cell types.     

IL-1beta stimulates IL-6 production in cultured skeletal muscle cells through activation of MAP kinase signaling pathway and NF-kappa B

Recent studies suggest that the skeletal muscle may be a significant site of IL-6 production in various conditions, including exercise, inflammation, hypoperfusion, denervation, and local muscle injury. The mediators and molecular mechanisms regulating muscle IL-6 production are poorly understood. We tested the hypothesis that IL-6 production in muscle cells is regulated by IL-1beta and that mitogen-activated protein (MAP) kinase signaling and NF-kappaB activation are involved in IL-1beta-induced IL-6 production. Cultured C2C12 cells, a mouse skeletal muscle cell line, were treated with different concentrations (0.1-2 ng/ml) of IL-1beta in the absence or presence of the p38 MAP kinase inhibitor SB-208350 or the p42/44 inhibitor PD-98059. Protein and gene expression of IL-6 were determined by ELISA and real-time PCR, respectively. NF-kappaB DNA binding activity was determined by electrophoretic mobility shift assay and by transfecting myocytes with a luciferase reporter plasmid containing a promoter construct with multiple repeats of NF-kappaB binding site. Treatment of myotubes with IL-1beta resulted in a dose- and time-dependent increase of IL-6 production accompanied by an approximately 25-fold increase in IL-6 mRNA levels. IL-1beta stimulated NF-kappaB DNA binding activity and gene activation. SB-208350 and PD-98059 inhibited the increase in IL-6 production induced by IL-1beta. The present results support the concept that skeletal muscle is an important site of IL-6 production. In addition, the results suggest the IL-1beta regulates muscle IL-6 production at least in part by activating the MAP kinase pathway and NF-kappaB.     

(-)-Epigallocatechin gallate amplifies interleukin-1-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells

We previously reported that interleukin-1 (IL-1), a potent bone resorptive cytokine, stimulates the synthesis of interleukin-6 (IL-6) via activation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that AMP-activated protein kinase (AMPK) negatively regulates the IL-1-induced IL-6 synthesis through the inhibitor of κB (IκB)/nuclear factor-κB (NF-κB) pathway. On the other hand, it is recognized that catechin possesses a beneficial property for bone metabolism. Among them, (-)-epigallocatechin gallate (EGCG) is an abundant and major bioactive component. In the present study, we investigated the effect of EGCG on the IL-1 stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. EGCG significantly enhanced the IL-1-stimulated IL-6 synthesis in a dose-dependent manner in the range between 50 and 100 μM. EGCG increased the mRNA levels of IL-6 stimulated by IL-1. IL-1-induced phosphorylation of IκB and NF-κB were suppressed by EGCG. On the other hand, EGCG failed to affect the IL-1-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and AMPK. These results strongly suggest that EGCG enhances IL-1-stimulated IL-6 synthesis through inhibiting the AMPK-IκB/NF-κB pathway at the point between AMPK and IκB/NF-κB in osteoblasts.     

The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.