Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.     

Mixed-lineage kinase control of JNK and p38 MAPK pathways

Mixed-lineage kinases (MLKs) are serine/threonine protein kinases that regulate signalling by the c-Jun amino-terminal kinase (JNK) and p38 mitogen-activated-protein kinase (MAPK) pathways. MLKs are represented in the genomes of both Caenorhabditis elegans and Drosophila melanogaster. The Drosophila MLK Slipper regulates JNK to control dorsal closure during embryonic morphogenesis. In mammalian cells, MLKs are implicated in the control of apoptosis and are potential drug targets for many neurodegenerative diseases.     

Human endotoxemia activates p38 MAP kinase and p42/44 MAP kinase, but not c-Jun N-terminal kinase.

BACKGROUND: All three major members of the MAPK family (i.e., p38 MAPK, p42/p44 MAPK, and c-Jun N terminal kinase (JNK)) have been shown to control cellular responses to inflammation in vitro. Therefore these kinases have been designated suitable targets for anti-inflammatory therapy. However, the extent to which these kinases are actually activated during inflammation in humans in vivo has not been investigated. We employed experimental human endotoxemia, a model of systemic inflammation, to address this question. MATERIALS AND METHODS: Male volunteers were intravenously infused with 4 ng/kg bw lipopolysaccharide (LPS). Directly before LPS infusion and up to 24 h thereafter, activation of p38 MAPK, p42/p44 MAPK and JNK was assessed in peripheral blood, using Western blot and in vitro kinase assays. RESULTS: We observed that LPS induced a strong but transient phosphorylation and activation of p38 MAPK and p42/p44 MAPK, maximal activity being reached after 1 hr of LPS infusion. Strikingly, no JNK phosphorylation or activation was detected under these circumstances. CONCLUSIONS: These results suggest that both inhibitors of p38 MAPK and p42/p44 MAPK but not JNK are potentially useful for anti-inflammatory therapy.     

p38 MAPK signalling cascades in inflammatory disease.

Inflammatory mediators released during acute and chronic diseases activate multiple intracellular signalling cascades including the mitogen-activated protein kinase (MAPK) signal transduction pathway, which plays a significant role in the recruitment of leukocytes to sites of inflammation. Stimulation of leukocytes by pro-inflammatory cytokines is known to result in the activation of the MAPK isoform p38. However, the functional consequences of p38 MAPK activation during leukocyte recruitment, including adhesion, migration and effector functions such as oxidative burst and degranulation, are only just beginning to be elucidated. Specific p38 inhibitors aimed at reducing the production of inflammatory mediators are now being developed, and might in the future provide more effective treatment for inflammatory diseases.     

Ferulic acid suppresses expression of tryptophan metabolic key enzyme indoleamine 2, 3-dioxygenase via NFκB and p38 MAPK in lipopolysaccharide-stimulated microglial cells

Ferulic acid (FA) is a phenol compound found in plants that has anti-inflammatory properties. Indoleamine 2, 3-dioxygenase (IDO) is a tryptophan catabolic enzyme induced in immune cells, including glial cells, during inflammation. Enhanced IDO expression leads to reduced tryptophan levels and increased levels of toxic metabolites, including quinolinic acid. Therefore, inhibition of IDO expression may be effective in suppressing progression of neurodegenerative diseases. In this study, we examined the effect of FA in microglial cells on IDO expression levels and related inflammatory signal molecules. FA suppressed LPS-induced IDO mRNA expression and also suppressed nuclear translocation of NF-κB and phosphorylation of p38 MAPK. However, FA did not affect the production of LPS-induced inflammatory mediators and phosphorylation of JNK. Our results indicate that FA suppresses LPS-induced IDO mRNA expression, which may be mediated by inhibition of the NF-κB and p38 MAPK pathways in microglial cells.     

4-O-carboxymethylascochlorin protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS–induced neuroinflammation and death by inhibiting MAPK,NF-κB,and Akt pathways

In our previous studies, structurally similar compounds of ascochlorin and ascofuranone exhibited anti-inflammatory activity. Neural inflammation plays a significant role in the commence and advancement of neurodegenerative diseases. It is not known whether 4-O-carboxymethylascochlorin (AS-6) regulates the initial stage of inflammatory responses at the cellular level in BV2 microglia cells. We here investigated the anti-inflammatory effects of AS-6 treatment in microglia cells with the microglial protection in neurons. We found that the lipopolysaccharide (LPS)-stimulated production of nitric oxide, a main regulator of inflammation, is suppressed by AS-6 in BV2 microglial cells. In addition, AS-6 dose-dependently suppressed the increase in COX-2 protein and messenger RNA levels in LPS-stimulated BV2 cells. Moreover, AS-6 inhibited the expression and secretion of proinflammatory cytokines in BV2 microglial cells. At the intracellular level, AS-6 inhibited LPS-activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in BV2 microglial cells. AS-6 negatively affected mitogen-activated protein kinases (MAPK) and Akt phosphorylation: Phosphorylated forms of ERK, JNK, p38, and Akt decreased. To check whether AS-6 protects against inflammatory inducer-mediated neurotoxicity, neuronal SH-SY5Y cells were coincubated with BV2 cells in conditioned medium. AS-6 exerted a neuroprotective effect by suppressing microglial activation by LPS or amyloid-β peptide. AS-6 is a promising suppressor of inflammatory responses in LPS-induced BV2 cells by attenuating NF-κB and MAPKs signaling. AS-6 protected against microglial-mediated neurotoxicity in SH-SY5Y and BV2 cocultured cells from LPS–induced neuroinflammation and death via inhibiting MAPK, NF-κB, and Akt pathways.     

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.     

p38丝裂原活化蛋白激酶抑制剂研究进展

p38丝裂原活化蛋白激酶（MAPK）通路是细胞内应激反应信号通路,与炎症反应密切相关。炎症反应失控是很多疾病产生的重要原因之一,传统抗炎药物的严重副作用使得寻找强效、安全的抗炎药物极为迫切。通过抑制剂调节信号通路的炎症药物研发成为目前发展的趋势,而p38MAPK的中心地位使其成为首选靶点。p38MAPK抑制剂和p38MAPK的研究进展相辅相成,发展迅速。已报道的100多种不同化学结构的p38MAPK特异性抑制剂中已有20多种进入临床试验阶段,但至今尚没有一种化合物被批准应用于临床治疗。我们讨论了p38MAPK抑制剂的研究现状和研究策略。     

Conference highlight: do T cells care about the mitogen-activated protein kinase signalling pathways?

Mitogen-activated protein (MAP) kinases, which include the extracellular response kinases, p38 and c-Jun amino terminal kinases (JNK), play a significant role in mediating signals triggered by cytokines, growth factors and environmental stress. The JNK and p38 MAP kinases have been involved in growth, differentiation and cell death in different cell types. In the present paper, we describe how the JNK and p38 MAP kinase signalling pathways are regulated and their role during thymocyte development and the activation and differentiation of T cells in the peripheral immune system. The results from these studies demonstrate that the JNK and p38 MAP kinase signalling pathways regulate different aspects of T-cell mediated immune responses.     

Pathological roles of MAPK signaling pathways in human diseases

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH₂-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.     

A low molecular weight copper chelator crosses the blood-brain barrier and attenuates experimental autoimmune encephalomyelitis

Increasing evidence suggests that enhanced production of reactive oxygen species (ROS) activates the MAP kinases, c-Jun N-terminal protein kinase (JNK) and mitogen-activated protein kinase MAPK (p38). These phosphorylated intermediates at the stress-activated pathway induce expression of matrix metalloproteinases (MMPs), leading to inflammatory responses and pathological damages involved in the etiology of multiple sclerosis (MS). Here we report that N-acetylcysteine amide (AD4) crosses the blood-brain barrier (BBB), chelates Cu(2+), which catalyzes free radical formation, and prevents ROS-induced activation of JNK, p38 and MMP-9. In the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, oral administration of AD4 drastically reduced the clinical signs, inflammation, MMP-9 activity, and protected axons from demylination damages. In agreement with the in vitro studies, we propose that ROS scavenging by AD4 in MOG-treated animals prevented MMP's induction and subsequent damages through inhibition of MAPK pathway. The low toxicity of AD4 coupled with BBB penetration makes this compound an excellent potential candidate for the therapy of MS and other neurodegenerative disorders.     

The ups and downs of MEK kinase interactions

MEK kinases (MEKKs) comprise a family of related serine–threonine protein kinases that regulate mitogen-activated protein kinase (MAPK) signalling pathways leading to c-Jun NH₂-terminal kinase (JNK) and p38 activation, induced by cellular stress (e.g., UV and γ irradiation, osmotic stress, heat shock, protein synthesis inhibitors), inflammatory cytokines (e.g., tumour necrosis factor , TNF, and interleukin-1, IL1) and G protein-coupled receptor agonists (e.g., thrombin). These stress-activated kinases have been implicated in apoptosis, oncogenic transformation, and inflammatory responses in various cell types. At present, the signalling events involving MEKKs are not well understood. This review summarises our current knowledge concerning the regulation and function of MEKK family members, with particular emphasis on those factors capable of directly interacting with distinct MEKK isoforms.     

Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.     

In the cellular garden of forking paths: how p38 MAPKs signal for downstream assistance

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes which connect cell-surface receptors to regulatory targets within cells and convert receptor signals into various outputs. In mammalian cells, four distinct MAPKs have been identified: the extracellular signal-related kinases (ERK)-1/2, the c-jun N-terminal kinases or stress-activated protein kinases 1 (JNK1/2/3, or SAPK1s), the p38 MAPKs (p38 alpha/beta/gamma/delta, or SAPK2s), and the ERK5 or big MAP kinase 1 (BMK1). The p38 MAPK cascade is activated by stress or cytokines and leads to phosphorylation of its central elements, the p38 MAPKs. Downstream of p38 MAPKs there is a diversification and extensive branching of signalling pathways. For that reason, we will focus in this review on the different signalling events that are triggered by p38 activity, and analyse how these events contribute to specific gene expression and cellular responses.     

Selenium suppressed the LPS-induced inflammation of bovine endometrial epithelial cells through NF-κB and MAPK pathways under high cortisol background

The bovine uterus is susceptible to infection, and the elevated cortisol level due to stress are common in cows after delivery. The essential trace element selenium plays a pivotal role in the antioxidant and anti-inflammatory defence system of body. This study investigated whether selenium supplementation protected endometrial cells from inflammation in the presence of high-level cortisol. The primary bovine endometrial epithelial cells were subjected to Escherichia coli lipopolysaccharide to establish cellular inflammation model. The gene expression of inflammatory mediators and proinflammatory cytokines was measured by quantitative PCR. The key proteins of NF-κB and MAPK signalling pathways were detected by Western blot and immunofluorescence. The result showed that pre-treatment of Na₂SeO₃ (1, 2 and 4 μΜ) decreased the mRNA expression of proinflammatory genes, inhibited the activation of NF-κB and suppressed the phosphorylation of extracellular signal-regulated kinase, P38MAPK and c-Jun N-terminal kinase. This inhibition of inflammation was more apparent in the presence of high-level cortisol (30 ng/mL). These results indicated that selenium has an anti-inflammatory effect, which is mediated via NF-κB and MAPK signalling pathways and is augmented by cortisol in bovine endometrial epithelial cells.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

Mitogen-activated protein kinase p38 and MK2, MK3 and MK5: Ménage à trois or ménage à quatre?

The mitogen-activated protein kinase (MAPK) signalling pathways play pivotal roles in cellular processes such as proliferation, apoptosis, gene regulation, differentiation, and cell motility. The typical mammalian MAPK pathways ERK1/2, JNK, p38^MAPK, and ERK5 operate through a concatenation of three successive phosphorylation events mediated by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs phosphorylate substrates with distinct functions, including other protein kinases referred to as MAPK-activated protein kinases. One family of related MAPK-activated protein kinases includes MK2, MK3, and MK5. While it is generally accepted that MK2 and MK3 are bona fide substrates for p38^MAPK, the genuineness of MK5 as a p38^MAPK substrate is disputed. This review summarizes the findings pro and contra an authentic p38^MAPK-MK5 relationship, discusses possible explanations for these discrepancies, and proposes experiments that may help to unequivocally clarify whether MK5 is indeed a substrate for p38^MAPK.