TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway

Tpl2 knockout mice produce low levels of TNF-alpha when exposed to lipopolysaccharide (LPS) and they are resistant to LPS/D-Galactosamine-induced pathology. LPS stimulation of peritoneal macrophages from these mice did not activate MEK1, ERK1, and ERK2 but did activate JNK, p38 MAPK, and NF-kappaB. The block in ERK1 and ERK2 activation was causally linked to the defect in TNF-alpha induction by experiments showing that normal murine macrophages treated with the MEK inhibitor PD98059 exhibit a similar defect. Deletion of the AU-rich motif in the TNF-alpha mRNA minimized the effect of Tpl2 inactivation on the induction of TNF-alpha. Subcellular fractionation of LPS-stimulated macrophages revealed that LPS signals transduced by Tpl2 specifically promote the transport of TNF-alpha mRNA from the nucleus to the cytoplasm.     

Interleukin-1 stimulated activation of the COT catalytic subunit through the phosphorylation of Thr290 and Ser62

The protein kinase COT/Tpl2 is activated by interleukin-1 (IL-1), TNFalpha and lipopolysaccharide, and its activation by these agonists involves the IkappaB kinase beta (IKKbeta) catalysed phosphorylation of the p105 regulatory subunit. Here, we show that COT activation also requires catalytic subunit phosphorylation, since IL-1beta induced a 5-10-fold activation of a COT mutant unable to bind p105. Activation was paralleled by the phosphorylation of Thr290 and Ser62 and unaffected by the IKKbeta inhibitor PS1145 at concentrations which prevented the degradation of IkappaBalpha. Mutagenesis experiments indicated that COT activation is initiated by Thr290 phosphorylation catalysed by an IL-1-stimulated protein kinase distinct from IKKbeta, while Ser62 phosphorylation is an autophosphorylation event required for maximal activation.     

Tpl2 transduces CD40 and TNF signals that activate ERK and regulates IgE induction by CD40

Macrophages from Tpl2 knockout (Tpl2(-/-)) mice exhibit a defect in ERK activation by lipopolysaccharide (LPS). This impairs the nucleocytoplasmic transport of the tumor necrosis factor alpha (TNF-alpha) mRNA and prevents the induction of TNF-alpha by LPS. As a result, Tpl2(-/-) mice are resistant to LPS/D-galactosamine-induced shock. We demonstrate that Tpl2 is essential for ERK signals transduced by members of the TNF receptor superfamily, such as CD40 and the TNF receptor 1. Thus, ERK activation was impaired in Tpl2(-/-) B cells and macrophages stimulated with agonistic CD40 antibody or TNF-alpha, whereas the induction of other mitogen-activated protein kinases, such as JNK and p38, and the activation of NF-kappaB were unaffected. Tpl2 was recruited to a CD40/TRAF6 complex in response to CD40 stimulation. Moreover, TRAF6, which when overexpressed activates ERK, failed to do so in Tpl2(-/-) cells. The selective signaling defect resulting from the inactivation of Tpl2 allowed us to demonstrate that CD40-mediated ERK activation contributes to immunoglobulin production but is not essential for B-cell proliferation.     

Phosphorylation of threonine 290 in the activation loop of Tpl2/Cot is necessary but not sufficient for kinase activity

Cot/Tpl2/MAP3K8 is a serine/threonine kinase known to activate the ERK, p38, and JNK kinase pathways. Studies of Tpl2 knock-out mice reveal a clear defect in tumor necrosis factor-alpha production, although very little detail is known about its regulation and the signaling events involved. In the present study we demonstrated that phosphorylation of Cot was required for its maximal activity as phosphatase treatment of Cot decreased its kinase activity. The Cot sequence contains a conserved threonine at position 290 in the activation loop of the kinase domain. We found that mutation of this residue to alanine eliminated its ability to activate MEK/ERK and NF-kappaB pathways, whereas a phosphomimetic mutation to aspartic acid could rescue the ability to activate MEK. Thr-290 was also required for robust autophosphorylation of Cot. Antibody generated to phospho-Thr-290-Cot recognized both wild-type and kinase-dead Cot, suggesting that phosphorylation of Thr-290 did not occur through autophosphorylation but via another kinase. We showed that Cot was constitutively phosphorylated at Thr-290 in transfected human embryonic kidney 293T cells as well as human monocytes as this residue was phosphorylated in unstimulated and lipopolysaccharide-stimulated cells to the same degree. Treatment with herbimycin A inhibited Cot activity in the MEK/ERK pathway but did not inhibit phosphorylation at Thr-290. Together these results showed that phosphorylation of Cot at Thr-290 is necessary but not sufficient for full kinase activity in the MEK/ERK pathway.     

Deregulated activation of oncoprotein kinase Tpl2/Cot in HTLV-I-transformed T cells

Protein kinase Tpl2/Cot is encoded by a protooncogene that is cis-activated by retroviral insertion in murine T cell lymphomas. It has remained unclear whether this oncoprotein kinase is mutated or post-translationally activated in human cancer cells. We have shown here that Tpl2/Cot is constitutively activated in human leukemia cell lines transformed by the human T cell leukemia virus type I (HTLV-I). The kinase activity of Tpl2/Cot is normally suppressed through its physical interaction with an inhibitor, the NF-kappaB1 precursor protein p105. Interestingly, a large pool of Tpl2/Cot is liberated from p105 and exhibits constitutive kinase activity in HTLV-I-transformed T cells. In contrast to its labile property in normal cells, the pathologically activated Tpl2/Cot is remarkably stable. Further, whereas the physiological activation of Tpl2/Cot involves its long isoform, the HTLV-activated Tpl2/Cot is predominantly the short isoform. We have also shown that the HTLV-I-encoded Tax protein is able to activate Tpl2/Cot in transfected cells. Finally, Tpl2/Cot participates in the activation of NF-kappaB by Tax. These findings indicate that deregulated activation of Tpl2/Cot may occur in human cancer cells.     

LPS-induced chemokine expression in both MyD88-dependent and -independent manners is regulated by Cot/Tpl2-ERK axis in macrophages

LPS signaling is mediated through MyD88-dependent and -independent pathways, activating NF-κB, MAP kinases and IRF3. Cot/Tpl2 is an essential upstream kinase in LPS-mediated activation of ERKs. Here we explore the roles of MyD88 and Cot/Tpl2 in LPS-induced chemokine expression by studying myd88^?/? and cot/tpl2^?/? macrophages. Among the nine LPS-responsive chemokines examined, mRNA induction of ccl5, cxcl10, and cxcl13 is mediated through the MyD88-independent pathway. Notably, Cot/Tpl2-ERK signaling axis exerts negative effects on the expression of these three chemokines. In contrast, LPS-induced gene expression of ccl2, ccl7, cxcl2, cxcl3, ccl8, and cxcl9 is mediated in the MyD88-dependent manner. The Cot/Tpl2-ERK axis promotes the expression of the first four and inhibits the expression of the latter two. Thus, LPS induces expression of multiple chemokines through various signaling pathways in macrophages.     

Tumor Progression Locus 2 (Tpl2) Kinase Promotes Chemokine Receptor Expression and Macrophage Migration during Acute Inflammation

In autoimmune diseases, the accumulation of activated leukocytes correlates with inflammation and disease progression, and, therefore, the disruption of leukocyte trafficking is an active area of research. The serine/threonine protein kinase Tpl2 (MAP3K8) regulates leukocyte inflammatory responses and is also being investigated for therapeutic inhibition during autoimmunity. Here we addressed the contribution of Tpl2 to the regulation of macrophage chemokine receptor expression and migration in vivo using a mouse model of Tpl2 ablation. LPS stimulation of bone marrow-derived macrophages induced early CCR1 chemokine receptor expression but repressed CCR2 and CCR5 expression. Notably, early induction of CCR1 expression by LPS was dependent upon a signaling pathway involving Tpl2, PI3K, and ERK. On the contrary, Tpl2 was required to maintain the basal expression of CCR2 and CCR5 as well as to stabilize CCR5 mRNA expression. Consistent with impairments in chemokine receptor expression, tpl2^−/− macrophages were defective in trafficking to the peritoneal cavity following thioglycollate-induced inflammation. Overall, this study demonstrates a Tpl2-dependent mechanism for macrophage expression of select chemokine receptors and provides further insight into how Tpl2 inhibition may be used therapeutically to disrupt inflammatory networks in vivo.     

The tyrosine kinase Syk regulates TPL2 activation signals

Tpl2/Cot is a serine/threonine kinase that plays a key physiological role in the regulation of immune responses to pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha). TNF-alpha stimulates the JNK, ERK, and p38 mitogen-activated protein kinases and the NF-kappaB pathway by recruiting RIP1 and TRAF2 to the TNF receptor 1. Here we showed that Tpl2 activation by TNF-alpha signals depends on the integrity of the Tpl2-interacting proteins RIP1 and TRAF2, which are required for the engagement of the ERK mitogen-activated protein kinase pathway. However, neither RIP1 nor TRAF2 overexpression was sufficient to activate Tpl2 and ERK. We also showed that Tpl2 activation by TNF-alpha depends on a tyrosine kinase activity that is detected in TNF-alpha-stimulated cells. Based on both genetic and biochemical evidence, we concluded that in a variety of cell types, Syk is the tyrosine kinase that plays an important role in the activation of Tpl2 upstream of ERK. These data therefore dissect the TNF receptor 1 proximal events that regulate Tpl2 and ERK and highlight a role for RIP1, TRAF2, and Syk in this pathway.     

Hepatitis C virus core protein suppresses NF-kappaB activation and cyclooxygenase-2 expression by direct interaction with IkappaB kinase beta

In addition to hepatocytes, hepatitis C virus (HCV) infects immune cells, including macrophages. However, little is known concerning the impact of HCV infection on cellular functions of these immune effector cells. Lipopolysaccharide (LPS) activates IkappaB kinase (IKK) signalsome and NF-kappaB, which leads to the expression of cyclooxygenase-2 (COX-2), which catalyzes production of prostaglandins, potent effectors on inflammation and possibly hepatitis. Here, we examined whether expression of HCV core interferes with IKK signalsome activity and COX-2 expression in activated macrophages. In reporter assays, HCV core inhibited NF-kappaB activation in RAW 264.7 and MH-S murine macrophage cell lines treated with bacterial LPS. HCV core inhibited IKK signalsome and IKKbeta kinase activities induced by tumor necrosis factor alpha in HeLa cells and coexpressed IKKgamma in 293 cells, respectively. HCV core was coprecipitated with IKappaKappabeta and prevented nuclear translocation of IKKbeta. NF-kappaB activation by either LPS or overexpression of IKKbeta was sufficient to induce robust expression of COX-2, which was markedly suppressed by ectopic expression of HCV core. Together, these data indicate that HCV core suppresses IKK signalsome activity, which blunts COX-2 expression in macrophages. Additional studies are necessary to determine whether interrupted COX-2 expression by HCV core contributes to HCV pathogenesis.     

Artemisolide is a typical inhibitor of IkappaB kinase beta targeting cysteine-179 residue and down-regulates NF-kappaB-dependent TNF-alpha expression in LPS-activated macrophages

Nuclear factor (NF)-kappaB regulates a central common signaling for immunity and cell survival. Artemisolide (ATM) was previously isolated as a NF-kappaB inhibitor from a plant of Artemisia asiatica. However, molecular basis of ATM on NF-kappaB activation remains to be defined. Here, we demonstrate that ATM is a typical inhibitor of IkappaB kinase beta (IKKbeta), resulting in inhibition of lipopolysaccharide (LPS)-induced NF-kappaB activation in RAW 264.7 macrophages. ATM inhibited the kinase activity of highly purified IKKbeta and also LPS-induced IKK activity in the cells. Moreover, the effect of ATM on IKKbeta activity was completely abolished by substitution of Cys-179 residue of IKKbeta to Ala residue, indicating direct targeting site of ATM. ATM could inhibit IkappaBalpha phosphorylation in LPS-activated RAW 264.7 cells and subsequently prevent NF-kappaB activation. Further, we demonstrate that ATM down-regulates NF-kappaB-dependent TNF-alpha expression. Taken together, this study provides a pharmacological potential of ATM in NF-kappaB-dependent inflammatory disorders.     

Inhibition of interleukin-1beta -induced NF-kappa B activation by calcium/calmodulin-dependent protein kinase kinase occurs through Akt activation associated with interleukin-1 receptor-associated kinase phosphorylation and uncoupling of MyD88

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) and Akt are two multifunctional kinases involved in many cellular responses. Although Akt and Ca(2+) signals have been implicated in NF-kappaB activation in response to certain stimuli, these results are still controversial, and the mechanism(s) involved remains unknown. In this study, we show the roles that CaMKK and Akt play in regulating interleukin-1beta (IL-1beta)-induced NF-kappaB signaling. In human embryonic kidney 293 cells, IL-1beta induces IkappaB kinase beta (IKKbeta) activation, IkappaBalpha degradation, NF-kappaB transactivation, and weak Akt activation. A CaMKK inhibitor (KN-93) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002) do not inhibit IL-1beta-induced NF-kappaB activation. However, IL-1beta-induced NF-kappaB activity is attenuated by increased intracellular calcium in response to ionomycin, UTP, or thapsigargin or by overexpression of CaMKKc and/or Akt. Ionomycin and CaMKKc overexpression increases Akt phosphorylation on Thr(308) and enzyme activity. Under these conditions or upon overexpression of wild type Akt, IL-1beta-induced IKKbeta activity is diminished. Furthermore, a dominant negative mutant of Akt abolishes IKKbeta inhibition by CaMKKc and ionomycin, suggesting that Akt acts as a mediator of CaMKK signaling to inhibit IL-1beta-induced IKK activity at an upstream target site. We have also identified a novel interaction between CaMKK-stimulated Akt and interleukin-1 receptor-associated kinase 1 (IRAK1), which plays a key role in IL-1beta-induced NF-kappaB activation. CaMKKc and Akt overexpression decreases IRAK1-mediated NF-kappaB activity and its association with MyD88 in response to IL-1beta stimulation. Furthermore, CaMKKc and Akt overexpression increases IRAK1 phosphorylation at Thr(100), and point mutation of this site abrogates the inhibitory effect of Akt on IRAK1-mediated NF-kappaB activation. Taken together, these results indicate a novel regulatory mechanism for IL-1beta signaling and suggest that CaMKK-dependent Akt activation inhibits IL-1beta-induced NF-kappaB activation through interference with the coupling of IRAK1 to MyD88.