Expression of mitogen-activated protein kinase phosphatase 1, a negative regulator of the mitogen-activated protein kinases, in rheumatoid arthritis: up-regulation by interleukin-1beta and glucocorticoids

OBJECTIVE: Mitogen-activated protein kinases (MAPKs) are activated by proinflammatory stimuli. MAPK phosphatases (MKPs), in particular MKP-1, have been identified as endogenous negative regulators of MAPK activation. Since MAPKs are known to be important in rheumatoid arthritis (RA) synoviocyte activation, this study assessed the expression, regulation, and function of MKP-1 in RA. METHODS: MKP-1 expression was measured by Western blotting (WB) and real-time polymerase chain reaction (PCR). RA fibroblast-like synoviocytes (FLS) were treated with interleukin-1beta (IL-1beta), tumor necrosis factor alpha, fetal calf serum, and dexamethasone. Expression of MAPKs in RA FLS was analyzed by WB using phosphospecific antibodies, while IL-6 expression was assessed by real-time PCR. RESULTS: MKP-1 protein and messenger RNA were detected in cultured RA FLS. IL-1beta rapidly up-regulated MKP-1, coinciding with reciprocal down-regulation of ERK, JNK, and p38 MAPK phosphorylation. Dexamethasone rapidly and sustainably up-regulated MKP-1, and this also coincided with down-regulation of ERK, JNK, and p38 MAPK phosphorylation. In addition, dexamethasone augmented IL-1beta-induced up-regulation of MKP-1, and this was associated with inhibition of ERK, JNK, and p38 MAPK phosphorylation and IL-6 expression. Dexamethasone had no effect on the phosphorylation of upstream kinases such as MEKK-3/6. In the presence of glucocorticoid (GC) receptor antagonist RU 486, the dexamethasone-mediated up-regulation of MKP-1 was impaired. Moreover, inhibition of MKP-1 expression impaired dexamethasone-mediated inhibition of MAPK phosphorylation. CONCLUSION: This study demonstrates the expression of MKP-1 in RA FLS. Cytokine and GC regulation of MKP-1 may be important in determining the magnitude of the inflammatory response in RA that is mediated via MAPKs. The effects of GCs in RA may be mediated, in part, via GC receptor-dependent up-regulation of MKP-1.     

Macrophage glucocorticoid receptors regulate Toll-like receptor 4-mediated inflammatory responses by selective inhibition of p38 MAP kinase

To explore the role of glucocorticoids in regulation of kinase pathways during innate immune responses, we generated mice with conditional deletion of glucocorticoid receptor (GR) in macrophages (MGRKO). Activation of toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) caused greater mortality and cytokine production in MGRKO mice than in controls. Ex vivo, treatment with dexamethasone (Dex) markedly inhibited LPS-mediated induction of inflammatory genes in control but not GR-deficient macrophages. We show that Dex inhibits p38 MAPK, but not PI3K/Akt, ERK, or JNK, in control macrophages. Associated with p38 inhibition, Dex induced MAP kinase phosphatase-1 (MKP-1) in control, but not MGRKO, macrophages. Consistent with the ex vivo studies, treatment with a p38 MAPK-specific inhibitor resulted in rescue of MGRKO mice from LPS-induced lethality. Taken together, we identify p38 MAPK and its downstream targets as essential for GR-mediated immunosuppression in macrophages.     

Reciprocal modulation of mitogen-activated protein kinases and mitogen-activated protein kinase phosphatase 1 and 2 in failing human myocardium

BACKGROUND: Mitogen-activated protein kinases (MAPKs), consisting of the ERK1/2, JNKs, and p38-kinase families, play a key role in the regulation of myocyte growth and apoptosis in vitro. The activity of MAPKs is regulated by dual-specificity MAPK phosphatases (MKPs). Because myocardial failure is associated with myocyte hypertrophy and apoptosis, MAPKs may play a pathophysiologic role in human myocardial failure. METHODS AND RESULTS: We measured MAPKs activities and the protein levels of MAPKs and MKPs (MKP-1 and MKP-2) in the myocardium explanted at the time of transplantation from patients with end-stage failure caused by idiopathic dilated cardiomyopathy (n = 5-7). Nonfailing donor hearts (n = 5-7) were used for comparison. Although the protein levels for JNK1/2 and p38-kinase in failing hearts were not different from levels in nonfailing hearts, the activities of both were decreased (P <.05). Despite a >3-fold increase in the protein level for ERK1/2 in failing hearts, ERK1/2 activity was not increased. Expression of MKP-2 was significantly increased in failing hearts, while expression of MKP-1 was increased in 5 of 7 failing hearts as measured by Western analysis. CONCLUSIONS: JNK1/2 and p38 activities are decreased in failing human myocardium. Increased expression of MKPs may therefore contribute to decreased MAPKs activity in failing human myocardium.     

Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity- and PI-3K-independent mechanism

Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via the phosphoinositide 3-kinase (PI-3K) pathway by catalyzing the PI-3K product PtdIns-3,4,5P3 (phosphatidylinositol-3,4,5-triphosphate) into PtdIns-3,4P2. However, the role of SHIP1 in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response remains unclear. Here we demonstrate that SHIP1 negatively regulates LPS-induced inflammatory response via both phosphatase activity-dependent and -independent mechanisms in macrophages. SHIP1 becomes tyrosine phosphorylated and up-regulated upon LPS stimulation in RAW264.7 macrophages. SHIP1-specific RNA-interfering and SHIP1 overexpression experiments demonstrate that SHIP1 inhibits LPS-induced tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by negatively regulating the LPS-induced combination between TLR4 and myeloid differentiation factor 88 (MyD88); activation of Ras (p21(ras) protein), PI-3K, extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase (JNK); and degradation of IkappaB-alpha. SHIP1 also significantly inhibits LPS-induced mitogen-activated protein kinase (MAPK) activation in TLR4-reconstitited COS7 cells. Although SHIP1-mediated inhibition of PI-3K is dependent on its phosphatase activity, phosphatase activity-disrupted mutant SHIP1 remains inhibitory to LPS-induced TNF-alpha production. Neither disrupting phosphatase activity nor using the PI-3K pathway inhibitor LY294002 or wortmannin could significantly block SHIP1-mediated inhibition of LPS-induced ERK1/2, p38, and JNK activation and TNF-alpha production, demonstrating that SHIP1 inhibits LPS-induced activation of MAPKs and cytokine production primarily by a phosphatase activity- and PI-3K-independent mechanism.     

The dual-specificity phosphatase MKP-1 limits the cardiac hypertrophic response in vitro and in vivo

Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.     

p38γ and p38δ kinases regulate the Toll-like receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation

On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1β, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNβ production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1β levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses.     

Activation of mitogen-activated protein kinase phosphatase 2 by gonadotropin-releasing hormone

The aim of these studies was to identify the signaling mechanism(s) that contribute to GnRH-induced expression of MAPK phosphatase (MKP)-2, a dual specificity phosphatase that selectively inactivates MAPKs. GnRH receptor activation induced MKP-2 expression in both clonal (alphaT3-1) and primary gonadotropes. Activation of PKC isozymes was sufficient and required for MKP-2 induction. Inhibition of the extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) but not the p38 MAPK cascade was sufficient to block GnRH-induced MKP-2 expression. Induction of MKP-2 by GnRH was dependent on elevation in intracellular Ca(2+). Inhibition of Ca(2+) influx through L-type voltage-gated calcium channels blocked GnRH-induced MKP-2 expression. Depletion of intracellular Ca(2+) stores with thapsigargin blocked MKP-2 activation by GnRH independent of ERK and JNK activity. These results support the conclusion that MKP-2 induction by GnRH occurs via MAPK-dependent and -independent pathways. One mechanism requires GnRH-induced ERK and JNK activation, while a second MAPK-independent pathway requires a thapsigargin-sensitive calcium signal.     

Inhibition of p38 MAPK activation via induction of MKP-1: atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability

The atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory potential due to its inhibitory action on the production of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). The aim of this study was to determine whether ANP is able to attenuate inflammatory effects of TNF-alpha on target cells. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha in the presence or absence of ANP. Changes in permeability, cytoskeletal alterations, phosphorylation of p38 MAPK and HSP27, and expression of MKP-1 were determined by macromolecule permeability assay, fluorescence labeling, RT-PCR, and immunoblotting. Antisense studies were done by transfecting cells with MKP-1 antisense oligonucleotides. Activation of HUVECs with TNF-alpha lead to a significant increase of macromolecule permeability and formation of stress fibers. Treatment of cells with ANP (10(-8) to 10(-6) mol/L) significantly reduced the formation of stress fibers and elevated permeability. Both TNF-alpha-induced effects were shown to be mediated via the activation of p38 using SB203580, a specific inhibitor of p38. ANP significantly reduced the TNF-alpha-induced activation of p38 and attenuated the phosphorylation of HSP27, a central target downstream of p38. ANP showed no effect on p38 upstream kinases MKK3/6. However, a significant induction of the MAPK phosphatase MKP-1 mRNA and protein could be observed in ANP-treated cells. Antisense experiments proved a causal role for MKP-1 induction in the ANP-mediated inhibition of p38. These data show the inhibitory action of ANP on TNF-alpha-induced changes in endothelial cytoskeleton and macromolecule permeability involving an MKP-1-induced inactivation of p38 MAPK. These effects point to an antiinflammatory and antiatherogenic potential of this cardiovascular hormone.     

Downregulation of angiotensin-converting enzyme by tumor necrosis factor-alpha and interleukin-1beta in cultured human endothelial cells.

Angiotensin-converting enzyme (ACE) and cytokines are considered to play an important role in the pathophysiology of cardiovascular diseases such as atherosclerosis. In the present study, the effects of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on ACE in cultured human umbilical vein endothelial cells (HUVECs) was studied. TNF-alpha (0.1-10 ng/ml) and IL-1beta (0.1-10 ng/ml) caused a dose- and time-dependent decrease in the amount of ACE in intact endothelial cell membranes and decreased levels of ACE mRNA. TNF-alpha and IL-1beta activated p44/42 and p38 mitogen-activated protein kinases (MAPKs) in HUVECs; this was inhibited by the specific inhibitors of these kinases, PD98059 and SB202190, respectively. Pretreatment of endothelial cells with the specific p38 MAPK inhibitor SB202190 (5 microM) or hydrocortisone (5 microM) partly reversed the suppression of ACE by TNF-alpha or IL-1beta, whereas the specific p44/42 MAPK inhibitor PD98059 (40 microM) was without effect. Vascular endothelial growth factor (1 ng/ml) caused an increase in membrane-bound ACE and ACE mRNA levels which was inhibited by pretreatment of the cells with TNF-alpha (1 ng/ml) or IL-1beta (1 ng/ml). In summary, the cytokines TNF-alpha and IL-1beta downregulated ACE in cultured human endothelial cells, which effect was probably mediated by the p38 MAPK pathway. Downregulation of ACE by TNF-alpha and IL-1beta locally in the vascular wall may be a counterbalancing mechanism in inflammatory processes such as atherosclerosis, leading to decreased production of angiotensin II and accumulation of bradykinin.     

IL-1β上调大鼠HSCs TIMP-1 mRNA表达与JNK和P38通路的关系

目的探讨IL-1β调控大鼠HSCsTIMP-1mRNA表达与JNK、p38 MAPK通路的关系。方法RT-PCR用于检测大鼠HSCs TIMP-1 mRNA表达；Western blot用于测定IL-1刺激HSCs后JNK、p38的活化程度。结果IL-1β（10ng／mL）作用培养的HSCs 24h后，TIMP-1 mRNA表达（1．191±0．079）明显高于对照组（0．545±0．091），有统计学意义（P〈0．01）；IL-1β以时间依赖方式激活JNK、p38通路。用不同浓度JNK阻断剂-SP600125预处理HSCs后，IL-1β上调HSCs TIMP-1 mRNA表达作用受到抑制，分别为10μmol／L，1．022±0．113;20μmol／L，0．8694±0．070；40μmol／L，0．666±0．123，与对照组相比（1．163±0．107），各浓度组均有显著性差异（P〈0．05，P〈0．01，P〈0．01）。用不同浓度p38阻断剂-SB203580预处理HSCs后，HSCs表达TIMP-1 mRNA逐渐增多，分别为10μmol／L，1．507±0．099；20μmol／L，1．698±0．107；40μmol／L，1．857±0．054。与对照组相比（1．027±0．061），各浓度组均有显著性差异（P均〈0．01）。结论IL-1β可通过上调HSCs TIMP-1 mRNA的表达来加速肝纤维化的发生发展，JNK和p38信号蛋白参与了此过程，HSCs中两条通路均可被IL-1激活，但所起作用完全不同，它们相互作用相互调节，共同调控TIMP-1 mRNA的表达。     

MAPK phosphatase-1 is required for regulatory natural autoantibody-mediated inhibition of TLR responses

Naturally arising IgM antibodies, which recognize neo-determinants on apoptotic cell (AC) membranes, are present from birth and can be further induced by AC challenge. Such naturally arising IgM antibodies can suppress proinflammatory responses to purified agonists for Toll-like receptors (TLRs), as well as block the induction of IgG immune complex-induced in vitro and in vivo pathogenic responses. To investigate the responsible mechanisms, we studied the regulatory effects of IgM anti-AC antibody on responses in bone marrow-derived dendritic cells mediated by a range of different TLRs and found that addition of IgM anti-AC inhibited the activation of the primary MAPKs: ERK1/2, JNK, and particularly p38. This was dependent on the recruitment of either C1q or mannose-binding lectin, which are both early complement factors that tag ACs for innate immune recognition. Strikingly, MAPK inhibition of responses to TLR agonists, and to lupus IgG autoantibody-chromatin immune complexes, was found to correlate with, and had an absolute requirement for, the induction and nuclear localization of MAPK phosphatase-1, a factor known to mediate glucocorticoid suppression of immune responses. Further experiments showed that natural IgM antibodies in serum exhibited the same inhibitory properties. These studies elucidate a novel homeostatic pathway by which natural antibodies, which are products of the adaptive immune system, can directly blunt inflammatory responses by recruitment and coordination of a primitive regulatory pathway of the innate immune system.     

Reactive oxygen species-mediated pancreatic beta-cell death is regulated by interactions between stress-activated protein kinases, p38 and c-Jun N-terminal kinase, and mitogen-activated protein kinase phosphatases

Pancreatic beta-cells are susceptible to reactive oxygen species (ROS), which are known to be generated by high or low glucose (LG), hypoxic, or cytokine-producing conditions. When we cultured mouse beta-cell-derived MIN6 cells in a LG condition, we detected a significant generation of ROS, including hydrogen peroxide, which was comparable to the ROS production in hypoxic or cytokine-treated conditions. ROS accumulation induced by the LG culture led to cell death, which was prevented by the ROS scavengers N-acetylcysteine and manganese(III)tetrakis(4-benzoic acid) porphyrin. We next investigated the mechanism of stress-activated protein kinases (SAPKs), c-jun N-terminal kinase (JNK) and p38, in ROS-induced MIN6 cell death. Activation of p38 occurred immediately after the LG culture, whereas JNK activation increased slowly 8 h later. Adenoviral p38 expression decreased MIN6 cell death, whereas the JNK expression increased it. Consistently, blocking p38 activation by inhibitors increased beta-cell death, whereas JNK inhibitors decreased it. We then examined the role of MAPK phosphatases (MKPs) specific for stress-activated protein kinases in beta-cell death. We found that MKP-1 presented an increase in its oxidized product after the LG culture. ROS scavengers prevented the appearance of this oxidized product and JNK activation. Thus, ROS-induced MKP inactivation causes sustained activation of JNK, which contributes to beta-cell death. Adenoviral overexpression of MKP-1 and MKP-7 prevented the phosphorylation of JNK at 36 h after the LG culture, and decreased MIN6 beta-cell death. We suggest that beta-cell death is regulated by interactions between JNK and its specific MKPs.     

Activated protein C,an anticoagulant polypeptide,ameliorates severe acute pancreatitis via regulation of mitogen-activated protein kinases

Background Our aim was to investigate the changes of mitogen-activated protein kinases (MAPKs) by activated protein C (APC) treatment in rats with severe acute pancreatitis (SAP), and relate them to changes in SAP severity, thus providing evidence for developing clinical therapies. Methods Sprague-Dawley rats were given an intravenous injection of saline (SAP group), APC (50 μg/kg or 10 μg/kg), or CNI1493 just before SAP induction. One group of rats underwent a sham operation (control group). Experimental samples were harvested 16 h after SAP induction. The gene expression of pancreatic MAPKs was evaluated by cDNA microarrays. The mRNA and protein/phosphorylated protein levels of p38 MAPK, extracellular signal-regulated protein kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK) and the protein levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were determined in pancreatic tissue. The severity of disease was evaluated by pancreatic histology, the pancreatic wet/dry weight ratio, and the serum amylase level. Results In rats treated with APC (50 μg/kg) or CNI1493, the severity of pancreatitis and expression of pancreatic TNF-α and IL-1β proteins were attenuated by the decreased expression and activity of p38 MAPK and JNK (vs. the SAP group, P < 0.01). The expression and activity of ERK1/2 were increased in APC-treated rats, especially in the group treated with APC 50 μg/kg (vs. the SAP or CNI1493-treated group, P < 0.01, respectively). Conclusions Inhibition of expression of pancreatic p38 MAPK and JNK and upregulation of ERK1/2 expression by APC treatment may protect against pancreatic injury, thus ameliorating severity of the disease.     

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration

Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor alpha, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IkappaB kinase signaling cascade.     

Inactivation of JNK1 enhances innate IL-10 production and dampens autoimmune inflammation in the brain

Environmental insults such as microbial pathogens can contribute to the activation of autoreactive T cells, leading to inflammation of target organs and, ultimately, autoimmune disease. Various infections have been linked to multiple sclerosis and its animal counterpart, autoimmune encephalomyelitis. The molecular process by which innate immunity triggers autoreactivity is not currently understood. By using a mouse model of multiple sclerosis, we found that the genetic loss of the MAPK, c-Jun N-terminal kinase 1 (JNK1), enhances IL-10 production, rendering innate myeloid cells unresponsive to certain microbes and less capable of generating IL-17-producing, encephalitogenic T cells. Moreover, JNK1-deficient central nervous system myeloid cells are unable to respond to effector T cell inflammatory cytokines, preventing further progression to neuroinflammation. Thus, we have identified the JNK1 signal transduction pathway in myeloid cells to be a critical component of a regulatory circuit mediating inflammatory responses in autoimmune disease. Our findings provide further insights into the pivotal MAPK-regulated network of innate and adaptive cytokines in the progression to autoimmunity.     

NF-kappaB activation by the Toll-IL-1 receptor domain protein MyD88 adapter-like is regulated by caspase-1

Toll-like receptors (TLRs)-2 and -4 are important proteins in innate immunity, recognizing microbial products and eliciting host defense responses. Both use the adapter proteins MyD88 and MyD88 adapter-like (Mal) to activate signaling pathways. Here we report that Mal but not MyD88 interacts with caspase-1, the enzyme that processes the precursors of the proinflammatory cytokines IL-1beta and IL-18. The interaction was found in a yeast two-hybrid screen and was confirmed by reciprocal GST pull-downs and coimmunoprecipitation of endogenous proteins. We were unable to implicate Mal in regulating caspase-1 activation. However, we found that Mal was cleaved by caspase-1 and that inhibition of caspase-1 activity blocked TLR2- and TLR4-mediated NF-kappaB and p38 MAP kinase activation but not IL-1 or TLR7 signaling, which are Mal independent. These responses, and the induction of TNF, were also attenuated in caspase-1-deficient cells. Finally, unlike wild-type Mal, a mutant Mal, which was not cleaved by caspase-1, was unable to signal and acted as a dominant negative inhibitor of TLR2 and TLR4 signaling. Our study therefore reveals a role for caspase-1 in the regulation of TLR2 and TLR4 signaling pathways via an effect on Mal. This functional interaction reveals an important aspect of the coordination between TLRs and caspase-1 during the innate response to pathogens.