Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in macrophages

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-kappaB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1(-/-) macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1(-/-) macrophages. TPL-2(S400A) expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8(-/-) macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-kappaB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.     

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.     

Suppression of cytokine production in lipopolysaccharide-stimulated mouse macrophages by novel cationic glucosamine derivative involves down-regulation of NF-kappaB and MAPK expressions

Exposure of macrophages to bacterial lipopolysaccharide (LPS) induces release of proinflammatory cytokines that play crucial roles in chronic inflammation. Glucosamine has reported to possess anti-inflammatory properties and currently is the oral supplement of choice for the management of inflammation related complications including osteoarthritis. In this study, quaternized amino glucosamine (QAGlc), a newly synthesized cationic glucosamine (Glc) derivative was found to inhibit LPS-stimulated production of IL-1beta, IL-6, TNF-alpha, and PGE(2) in RAW264.7, mouse macrophages more potently than its starting material Glc. Since production of cytokines is regulated mainly via activation of NF-kappaB and regulation of mitogen-activated protein kinases (MAPKs), we examined if QAGlc could be responsible for the suppression of NF-kappaB pathway and MAPKs. We used reporter gene assay and Western blotting to examine the effects of QAGlc on activation and translocation of NF-kappaB. Further, QAGlc-mediated inhibition of NF-kappaB was accompanied with a suppression of its translocation. Apparently, QAGlc was shown to attenuate LPS-induced activation of p38 MAPK and JNK in RAW264.7 cells suggesting that inhibition of MAPK-mediated LPS signaling also contribute to suppression of cytokine production following stimulation of macrophages with LPS.     

Integrin-linked kinase regulates inducible nitric oxide synthase and cyclooxygenase-2 expression in an NF-kappa B-dependent manner.

Nitric oxide (NO) and prostaglandins are produced as a result of the stimulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, respectively, in response to cytokines or lipopolysaccharide (LPS). We demonstrate that the activity of integrin-linked kinase (ILK) is stimulated by LPS activation in J774 macrophages. Inhibition of ILK activity by dominant-negative ILK or a highly selective small molecule ILK inhibitor, in epithelial cells or LPS-stimulated J774 cells and murine macrophages, resulted in inhibition of iNOS expression and NO synthesis. LPS stimulates the phosphorylation of IkappaB on Ser-32 and promotes its degradation. Inhibition of ILK suppressed this LPS-stimulated IkappaB phosphorylation and degradation. Similarly, ILK inhibition suppressed the LPS-stimulated iNOS promoter activity. Mutation of the NF-kappaB sites in the iNOS promoter abolished LPS- and ILK-mediated regulation of iNOS promoter activity. Overexpression of ILK-stimulated NF-kappaB activity and inhibition of ILK or protein kinase B (PKB/Akt) suppressed this activation. We conclude that ILK can regulate NO production in macrophages by regulating iNOS expression through a pathway involving PKB/Akt and NF-kappaB. Furthermore, we also demonstrate that ILK activity is required for LPS stimulated cyclooxygenase-2 expression in murine and human macrophages. These findings implicate ILK as a potential target for anti-inflammatory applications.     

Mechanisms of endotoxin tolerance in human intestinal microvascular endothelial cells

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6-60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-kappaB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24-48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-kappaB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.     

Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells

Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS restimulation, known as endotoxin tolerance. Induction of LPS tolerance has been reported to correlate with decreased cell surface expression of the LPS receptor complex, Toll-like receptor 4 (TLR4)/MD-2. However, other results have underscored the existence of mechanisms of LPS tolerance that operate downstream of TLR4/MD-2. In the present study we sought to delineate further the molecular basis of LPS tolerance by examining the TLR4 signaling pathway in endotoxin-tolerant cells. Pretreatment of human monocytes with LPS decreased LPS-mediated NF-kappaB activation, p38 mitogen-activated protein kinase phosphorylation, and TNF-alpha gene expression, documenting the induction of endotoxin tolerance. FACS and Western blot analyses of LPS-tolerant monocytes showed increased TLR2 expression, whereas TLR4 expression levels were not affected. Comparable levels of mRNA and protein for myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase 1 (IRAK-1), and TNFR-associated factor-6 were found in normal and LPS-tolerant monocytes, while MD-2 mRNA expression was slightly increased in LPS-tolerant cells. LPS induced the association of MyD88 with TLR4 and increased IRAK-1 activity in medium-pretreated cells. In LPS-tolerant monocytes, however, MyD88 failed to be recruited to TLR4, and IRAK-1 was not activated in response to LPS stimulation. Moreover, endotoxin-tolerant CHO cells that overexpress human TLR4 and MD-2 also showed decreased IRAK-1 kinase activity in response to LPS despite the failure of LPS to inhibit cell surface expression of transfected TLR4 and MD-2 proteins. Thus, decreased TLR4-MyD88 complex formation with subsequent impairment of IRAK-1 activity may underlie the LPS-tolerant phenotype.     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]     

Alveolar macrophage activation after trauma-hemorrhage and sepsis is dependent on NF-kappaB and MAPK/ERK mechanisms

The acute respiratory distress syndrome (ARDS) is a major cause of morbidity after injury. We hypothesized that alveolar macrophage (AMPhi) chemokine and cytokine release after hemorrhage and sepsis is regulated by NF-kappaB and MAPK. Adult male rats underwent soft tissue trauma and hemorrhagic shock (~90 min) followed by crystalloid resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) 20 h after resuscitation. AMPhi were harvested, and TNF-alpha, IL-6, and macrophage inflammatory protein (MIP)-2 release and serum IL-6 and TNF-alpha levels were measured at 5 h after HCLP. Lung tissues were analyzed for activation of NF-kappaB, myeloperoxidase activity, and wet/dry weight ratio. In control animals, AMPhi were stimulated with LPS with or without inhibitors of NF-kappaB and MAPK. Serum TNF-alpha and IL-6 levels and spontaneous AMPhi TNF-alpha and MIP-2 release were elevated (P < 0.05) after HCLP, concomitantly with the development of lung edema and leukocyte activation. Activation of NF-kappaB increased in lungs from the hemorrhage and CLP group compared with shams. Inhibition of NF-kappaB or the upstream MAPK significantly decreased LPS-stimulated AMPhi activation. Because enhanced release of inflammatory mediators by AMPhi may contribute to ARDS after severe trauma, inhibition of intracellular signaling pathways represents a target to attenuate organ injury under those conditions.     

Regulation of carotenoid biosynthesis genes in response to light in Chlamydomonas reinhardtii

As we had found previously that thapsigargin, an endomembrane Ca2+-ATPase inhibitor, induces production of intracellular platelet-activating factor (PAF) [Br. J. Pharmacol. 116 (1995) 2141], we decided to investigate the possible roles of intracellular PAF in nuclear factor (NF)-kappaB activation of thapsigargin-stimulated rat peritoneal macrophages. When rat peritoneal macrophages were stimulated with thapsigargin, the level of inhibitory protein of NF-kappaB-alpha (IkappaB-alpha) was decreased and the nuclear translocation of NF-kappaB was increased. The thapsigargin-induced activation of NF-kappaB was inhibited by the PAF synthesis inhibitor SK&F 98625 and the PAF antagonist E6123. Structurally unrelated PAF antagonists such as E5880 and L-652,731 also inhibited the thapsigargin-induced activation of NF-kappaB. Lipopolysaccharide (LPS)-induced activation of NF-kappaB was also suppressed by these drugs. In a culture of rat peritoneal macrophages, exogenously added PAF did not induce degradation of IkappaB-alpha. These findings suggest that the intracellular PAF produced by the stimulation with thapsigargin or LPS is involved in activation of the NF-kappaB pathway.     

Extracellular Signal-regulated Kinase Mediates Expression of Arginase II but Not Inducible Nitric-oxide Synthase in Lipopolysaccharide-stimulated Macrophages

The mitogen-activated protein kinases (MAPK) have been shown to participate in iNOS induction following lipopolysaccharide (LPS) stimulation, while the role of MAPKs in the regulation of arginase remains unclear. We hypothesized that different MAPK family members are involved in iNOS and arginase expression following LPS stimulation. LPS-stimulated RAW 264.7 cells exhibited increased protein and mRNA levels for iNOS, arginase I, and arginase II; although the induction of arginase II was more robust than that for arginase I. A p38 inhibitor completely prevented iNOS expression while it only attenuated arginase II induction. In contrast, a MEK1/2 inhibitor (ERK pathway) completely abolished arginase II expression while actually enhancing iNOS induction in LPS-stimulated cells. Arginase II promoter activity was increased by ∼4-fold following LPS-stimulation, which was prevented by the ERK pathway inhibitor. Arginase II promoter activity was unaffected by a p38 inhibitor or JNK pathway interference. Transfection with a construct expressing a constitutively active RAS mutant increased LPS-induced arginase II promoter activity, while transfection with a vector expressing a dominant negative ERK2 mutant or a vector expressing MKP-3 inhibited the arginase II promoter activity. LPS-stimulated nitric oxide (NO) production was increased following siRNA-mediated knockdown of arginase II and decreased when arginase II was overexpressed. Our results demonstrate that while both the ERK and p38 pathways regulate arginase II induction in LPS-stimulated macrophages, iNOS induction by LPS is dependent on p38 activation. These results suggest that differential inhibition of the MAPK pathway may be a potential therapeutic strategy to regulate macrophage phenotype.     

A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production.

Endotoxin (lipopolysaccharide, LPS) is a component of the outer membrane of Gram-negative bacteria and promotes the activation of macrophages and microglia. Although these cells are highly LPS-responsive, they serve unique tissue-specific functions and exhibit different LPS sensitivities. Accordingly, it was of interest to evaluate whether these biological differences reside in variations within LPS signaling pathways between these two cell types. Because the mitogen-activated protein kinases ERK-1 and ERK-2 have been implicated in the control of many immune responses, we tested the concept that they are a key indicator for differences in cellular LPS sensitivity. We observed that murine RAW 264.7 macrophages and murine BV-2 microglial cells both respond to LPS by exhibiting increased IkappaBalpha degradation, enhanced NF-kappaB DNA binding activity, and elevated nitric oxide and interleukin-1beta production. Although LPS potently stimulates ERK activation in RAW 264.7 macrophages, it does not activate ERK-1/-2 in BV-2 microglia. Moreover, antagonism of the MEK/ERK pathway potentiates LPS-stimulated nitric oxide production, suggesting that LPS-stimulated ERK activation can exert inhibitory effects in macrophage-like cells. These data support the idea that ERK activation is not a required function of LPS-mediated signaling events and illustrate that alternative/additional pathways for LPS action exist in these cell types.     

Ca2+- and protein kinase C-dependent signaling pathway for nuclear factor-kappaB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-alpha production in lipopolysaccharide-stimulated rat peritoneal macrophages

Lipopolysaccharide (LPS)-activated macrophages are pivotal in innate immunity. With LPS treatment, extracellular signals are transduced into macrophages via Toll-like receptor 4 and induce inflammatory mediator production by activating signaling pathways, including the nuclear factor-kappaB (NF-kappaB) pathway and the mitogen-activated protein kinase (MAPK) pathway. However, the mechanisms by which the intracellular free Ca2+ concentration ([Ca2+]i) increases and protein kinase C (PKC) is activated remain unclear. Therefore, we investigated the signaling pathway for Ca2+- and PKC-dependent NF-kappaB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-alpha (TNF-alpha) production in LPS-stimulated rat peritoneal macrophages. The results demonstrated that the LPS-induced transient [Ca2+]i increase is due to Ca2+ release and influx. Extracellular and intracellular Ca2+ chelators inhibited phosphorylation of PKCalpha and PKCbeta. A PKCbeta-specific and a general PKC inhibitor blunted phosphorylation of serine in mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) 1. Moreover, a MEKK inhibitor reduced activation of inhibitorykappaB kinase and NF-kappaB. Upstream of the [Ca2+]i increase, a protein-tyrosine kinase inhibitor reduced phosphorylation of phospholipase C (PLC) gamma. Furthermore, a PLC inhibitor eliminated the transient [Ca2+]i increase and decreased the amount of activated PKC. Therefore, these results revealed the following roles of Ca2+ and PKC in the signaling pathway for NF-kappaB activation in LPS-stimulated macrophages. After LPS treatment, protein-tyrosine kinase mediates PLCgamma1/2 phosphorylation, which is followed by a [Ca2+]i increase. Several PKCs are activated, and PKCbeta regulates phosphorylation of serine in MEKK1. Moreover, MEKKs regulate inhibitory kappaB kinase activation. Sequentially, NF-kappaB is activated, and inducible nitric-oxide synthase and tumor necrosis factor-alpha production is promoted.     

Relevance of the MEK/ERK signaling pathway in the metabolism of activated macrophages: a metabolomic approach

The activation of immune cells in response to a pathogen involves a succession of signaling events leading to gene and protein expression, which requires metabolic changes to match the energy demands. The metabolic profile associated with the MAPK cascade (ERK1/2, p38, and JNK) in macrophages was studied, and the effect of its inhibition on the specific metabolic pattern of LPS stimulation was characterized. A [1,2-[(13)C](2)]glucose tracer-based metabolomic approach was used to examine the metabolic flux distribution in these cells after MEK/ERK inhibition. Bioinformatic tools were used to analyze changes in mass isotopomer distribution and changes in glucose and glutamine consumption and lactate production in basal and LPS-stimulated conditions in the presence and absence of the selective inhibitor of the MEK/ERK cascade, PD325901. Results showed that PD325901-mediated ERK1/2 inhibition significantly decreased glucose consumption and lactate production but did not affect glutamine consumption. These changes were accompanied by a decrease in the glycolytic flux, consistent with the observed decrease in fructose-2,6-bisphosphate concentration. The oxidative and nonoxidative pentose phosphate pathways and the ratio between them also decreased. However, tricarboxylic acid cycle flux did not change significantly. LPS activation led to the opposite responses, although all of these were suppressed by PD325901. However, LPS also induced a small decrease in pentose phosphate pathway fluxes and an increase in glutamine consumption that were not affected by PD325901. We concluded that inhibition of the MEK/ERK cascade interferes with central metabolism, and this cross-talk between signal transduction and metabolism also occurs in the presence of LPS.