Role of mitogen-activated protein kinases in Thy-1-induced T-lymphocyte activation

Thy-1 (CD90) crosslinking by monoclonal antibodies (mAb) in the context of costimulation causes the activation of mouse T-lymphocytes; however, the associated signal transduction processes have not been studied in detail. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in Thy-1-mediated T-lymphocyte activation using mAb-coated polystyrene microspheres to crosslink Thy-1 and costimulatory CD28 on murine T-lymphocytes. Concurrent Thy-1 and CD28 crosslinking induced DNA synthesis by T-lymphocytes, as well as interleukin (IL)-2 and IL-2 receptor (IL-2R) α chain (CD25) expression. Increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal protein kinase (JNK) was also observed. Pharmacologic inhibition of ERK1/2 or JNK activation inhibited Thy-1-induced DNA synthesis and IL-2 production by T-lymphocytes. p38 MAPK inhibition also decreased DNA synthesis in Thy-1-stimulated T-lymphocytes; however, IL-2 production was increased in these cells. Inhibition of JNK, but not ERK1/2 or p38 MAPK, caused a marked reduction in Thy-1-induced CD25 expression. In addition, inhibition of p38 MAPK or JNK, but not ERK1/2, impaired the growth of IL-2-dependent CTLL-2 T-lymphocytes but did not substantially affect CD25 expression. Finally, exogenous IL-2 reversed the inhibitory effect of ERK1/2 or JNK inhibition on Thy-1-stimulated DNA synthesis by T-lymphocytes but did not substantially reverse JNK inhibition of CD25 expression. Collectively, these results suggest that during Thy-1-induced T-lymphocyte activation, ERK1/2 and JNK promoted IL-2 production whereas p38 MAPK negatively regulated IL-2 expression. JNK signalling was also required for CD25 expression. IL-2R signalling involved both p38 MAPK and JNK in CTLL-2 cells, whereas p38 MAPK was most important for IL-2R signalling in primary T-lymphocytes. MAPKs are therefore essential signalling intermediates for the Thy-1-driven proliferation of mouse T-lymphocytes.     

Formyl peptide receptor like 1 differentially requires mitogen-activated protein kinases for the induction of glial fibrillary acidic protein and interleukin-1alpha in human U87 astrocytoma cells

Mitogen-activated protein kinases (MAPKs) are not only pivotal mediators of signal transduction but they also regulate diverse biological processes ranging from survival, proliferation and differentiation to apoptosis. By using human U87 astrocytoma and transfected FPRL1/CHO cells, we have demonstrated that activation of FPRL1 with WKYMVM effectively phosphorylated JNK and ERK. Interestingly, p38 MAPK activation was only seen with FPRL1/CHO cells. The MAPK phosphorylations in response to WKYMVM were blocked by WRW(4) (a selective FPRL1 antagonist), but not cyclosporine H (a well-known FPR antagonist). The key signaling intermediates in the MAPK pathways were also delineated. G(i)/G(o) proteins, Src family tyrosine kinases, but not phosphatidylinositol-3 kinase, protein kinase C and calmodulin-dependent kinase II, were required to transmit signals from FPRL1 toward JNK, ERK and p38 MAPK. Furthermore, phospholipase Cbeta was distinctively involved in the regulation of JNK but not the other MAPKs. Importantly, WKYMVM-stimulated U87 cells triggered noticeable increases in glial fibrillary acidic protein (GFAP) and interleukin-1alpha (IL-1alpha), which are correlated with reactive astrocytosis. In contrast, GFAP expression was not altered following stimulation with N-formyl-methionyl-leucyl-phenylalanine. Moreover, inhibitions of G(i)/G(o) proteins and JNK completely abolished both GFAP and IL-1alpha upregulations by FPRL1, while blockade of the MEK/ERK cascade exclusively suppressed the GFAP production. Consistently, overexpression of MEK1 and constitutively active JNKK in U87 cells led to ERK and JNK activation, respectively, which was accompanied with markedly increased GFAP production. We have thus identified a possible linkage among FPRL1, MAPKs, astrocytic activation and the inflammatory response.     

Cutting edge: IL-4 induces suppressor of cytokine signaling-3 expression in B cells by a mechanism dependent on activation of p38 MAPK

The signaling cascade initiated by IL-4 is classically divisible into two major pathways: one mediated by STAT6, and the other by insulin receptor substrates-1 and -2 via activation of PI3K. In murine splenic B cells, the suppressor of cytokine signaling (SOCS)3 is inducible by IL-4 via a mechanism independent of STAT6 and PI3K. SOCS3 expression increases 9-fold within 5 h of IL-4 treatment. This induction occurs normally in B cells deficient in STAT6 and is unaffected by pretreatment with the PI3K inhibitor wortmannin, or with the ERK pathway inhibitor, PD98059. However, the IL-4 induction of SOCS3 is blocked by inhibitors of either the JNK or p38 MAPK pathways (SP600125 and SB203580, respectively). Direct examination of these pathways reveals rapid, IL-4-directed activation of p38 MAPK, uncovering a previously unappreciated pathway mediating IL-4 signal transduction.     

Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery

Paclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1-1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery.     

Polysaccharide isolated from seeds of Plantago asiatica L. induces maturation of dendritic cells through MAPK and NF-κB pathway

Plantago species are used as traditional medicine in Asian and Europe. Polysaccharide isolated from the seeds of Plantago asiatica L. could stimulate maturation transformation of bone-marrow derived dendritic cells (DCs). We found that blocking p38, ERK1/2 and JNK MAPK signal transduction could significantly decreased the PLP-2 induced expression of MHC II, CD86 surface molecules on DCs. Blocking p38 and JNK signal also significantly inhibited the cytokine secretion of TNF-α and IL-12p70 as well, while blocking ERK1/2 signal only decreased the secretion of TNF-α. Meanwhile, DCs in the three MAPK signal-blocking groups showed dramatically attenuated effects on stimulating proliferation of T lymphocytes. Similarly, blocking signal transduction of NF-κB pathway also significantly impaired the phenotypic and functional maturation development of DCs induced by PLP-2. These data suggest that MAPK and NF-κB pathway mediates the PLP-induced maturation on DCs. Especially, among the three MAPK pathways, activation of JNK signal transduction is the most important for DCs development after PLP-2 incubation. And PLP-2 may activate the MAPK and NF-κB pathway by triggering toll-like receptor 4 on DCs.     

p38 MAPK autophosphorylation drives macrophage IL-12 production during intracellular infection

The intracellular protozoan Toxoplasma gondii triggers rapid MAPK activation in mouse macrophages (Mphi). We used synthetic inhibitors and dominant-negative Mphi mutants to demonstrate that T. gondii triggers IL-12 production in dependence upon p38 MAPK. Chemical inhibition of stress-activated protein kinase/JNK showed that this MAPK was also required for parasite-triggered IL-12 production. Examination of upstream MAPK kinases (MKK) 3, 4, and 6 that function as p38 MAPK activating kinases revealed that parasite infection activates only MKK3. Nevertheless, in MKK3(-/-) Mphi, p38 MAPK activation was near normal and IL-12 production was unaffected. Recently, MKK-independent p38alpha MAPK activation via autophosphorylation was described. Autophosphorylation depends upon p38alpha MAPK association with adaptor protein, TGF-beta-activated protein kinase 1-binding protein-1. We observed TGF-beta-activated protein kinase 1-binding protein-1-p38alpha MAPK association that closely paralleled p38 MAPK phosphorylation during Toxoplasma infection of Mphi. Furthermore, a synthetic p38 catalytic-site inhibitor blocked tachyzoite-induced p38alpha MAPK phosphorylation. These data are the first to demonstrate p38 MAPK autophosphorylation triggered by intracellular infection.     

Iron chelation acutely stimulates fetal human intestinal cell production of IL-6 and VEGF while decreasing HGF: the roles of p38, ERK, and JNK MAPK signaling

Bacteria have developed mechanisms to sequester host iron via chelators such as deferoxamine (DFO). Interestingly, DFO has been shown to stimulate acute intestinal epithelial cell inflammatory cytokine production in the absence of bacteria; however, this mechanism has not been elucidated. Intestinal epithelial cell production of IL-6 and TNF-alpha is elevated in various gastrointestinal pathologies, including acute intestinal ischemia. Similarly, VEGF and HGF are essential to intestinal epithelial cell integrity. Therapeutic strategies that decrease IL-6 and TNF-alpha while increasing VEGF and HGF therefore have theoretical appeal. We hypothesized that 1) fetal human intestinal epithelial cells acutely produce increased IL-6, TNF-alpha, VEGF, and HGF during iron chelation and 2) the MAPK pathway mediates these effects. Fetal human intestinal epithelial cells were stimulated by iron chelation (1 mM DFO) with and without p38 MAPK, ERK, or JNK inhibition. Supernatants were harvested after 24 h of incubation, and IL-6, TNF-alpha, VEGF, and HGF levels were quantified by ELISA. Activation of MAPK pathways was confirmed by Western blot analysis. DFO stimulation resulted in a significant increase in epithelial cell IL-6 and VEGF production while yielding a decrease in HGF production (P<0.05). Unexpectedly, TNF-alpha was not detectable. p38 MAPK, ERK, and JNK inhibition significantly decreased IL-6, VEGF, and HGF production (P<0.05). In conclusion, DFO acutely increases fetal human intestinal epithelial cell IL-6 and VEGF expression while causing an unexpected decrease in HGF expression and no detectable TNF-alpha production. Furthermore, chelator-induced intestinal epithelial cell cytokine expression depends on p38, ERK, and JNK MAPK pathways.     

Modulation of the activation of extracellular signal-regulated kinase (ERK) and the production of inflammatory mediators by ADP-ribosylation inhibitors

ADP-ribosylation is involved in nuclear factor kappaB (NF-kappaB)-dependent gene expression induced by lipopolysaccharide in murine macrophages. Here we have investigated the mechanism by which ADP-ribosylation inhibitors block signaling pathways induced in macrophages. In RAW264.7 macrophages the inducers of NF-kappaB activate the production of reactive oxygen species and three mitogen-activated protein kinases (MAPK), the extracellular signal regulated kinase (ERK), the c-jun N-terminal kinase/stress-activated protein kinase (JNK), and p38. We demonstrate that ADP-ribosylation inhibitors specifically inhibit ERK MAPK activation and reduce the release of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha), IL-6 and nitrite.     

The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage

Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll-like receptors (TLR). Although mitogen-activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP approximately 35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF-alpha, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF-alpha, MCP-1, and KC production by Kupffer cells were significantly increased following trauma-hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG-stimulated TLR9 signaling increased TNF-alpha and IL-6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, -4, and -9 signaling. In contrast, TNF-alpha and IL-6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma-hemorrhage.     

Up-regulation of IL-23 p19 expression in human periodontal ligament fibroblasts by IL-1β via concurrent activation of the NF-κB and MAPKs/AP-1 pathways

Interleukin (IL)-23 is an essential cytokine involved in the expansion of a novel CD4(+) T helper subset known as Th17, which has been implicated in the pathogenesis of periodontitis recently. Our previous study first identified specialized human periodontal ligament fibroblasts (hPDLFs) as an important production source of IL-23. The present study was undertaken to investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-1β on hPDLFs-mediated IL-23 p19 production, and the molecular mechanism involved. IL-23 p19 expression was in situ detected in IL-1β-stimulated hPDLFs. IL-1β was capable of stimulating the expression of IL-23 p19 mRNA and protein in cultured hPDLFs, which was attenuated by IL-1 receptor antagonist (IL-1Ra) or myeloid differentiation primary response gene 88 (MyD88) inhibitor. Meanwhile, inhibitors of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), activator protein-1 (AP-1), or nuclear factor-kappaB (NF-κB) significantly suppressed IL-23 p19 production from IL-1β-stimulated hPDLFs. Moreover, IL-1β-initiated AP-1 activation was blocked by p38 MAPK, ERK 1/2, or JNK inhibition, whereas NF-κB activity remained unaltered by all the above pathway specific inhibitors. Thus, these results provide evidence that Th17-polarizing mediator IL-1β up-regulated the expression of IL-23 p19 in hPDLFs via NF-κB signaling and MAPKs-dependent AP-1 pathways. Taken together, our findings indicate that IL-1Ra may be used therapeutically to inhibit Th17-driven inflammatory diseases including periodontitis.     

Induction of signaling pathways by herpes simplex virus type 1 through glycoprotein H peptides

Eukaryotic cells respond to extracellular stimuli, such as viruses, by recruiting signal transduction pathways, many of which are mediated through activation of distinct mitogen-activated protein kinase (MAPK) cascades and activation of transductional regulation factors. The best characterized of this pathway are the extracellular signal regulated kinase (ERK), the c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK), and the p38 MAPK cascade. Herpes simplex virus type 1 (HSV-1) encodes at least 11 envelope glycoproteins, which alone or in concert play different roles in viral adsorption, entry, cell-to-cell spread, and immune evasion. Of these proteins, three are designated glycoprotein B (gB), glycoprotein D (gD), and the gH/gL heterodimer, are clearly involved in attachment and entry, and therefore possible candidates in inducing early cellular activation.Nevertheless, the precise role of each glycoprotein and the cellular factor involved remain elusive. The signal transduction pathways involved, and the outcome of cellular activation on viral entry or postentry events, are still to be elucidated. To better understand the role of signal transduction pathways and phosphorylation events in HSV-1 entry, synthetic peptides modeled on HSV-1 gH were synthesized and tested for MEK1-MEK2/MAPK cascade activation. Our results show a major involvement of the JNK pathway in the intracellular signal transmission after stimulation with gH HSV-1 peptides.     

Regulation of IL-1beta -induced GM-CSF production in human airway smooth muscle cells by carbon monoxide

Asthma, a chronic inflammatory disease of the airways, involves the increased expression of inflammatory mediators, including granulocyte-monocyte colony-stimulating factor (GM-CSF). Heme oxygenase-1 (HO-1), a stress-response protein, confers protection against oxidative stress. We hypothesized that carbon monoxide (CO), a byproduct of HO-1-dependent heme catabolism, regulates GM-CSF synthesis in human airway smooth muscle cells (HASMC). IL-1beta treatment induced a time-dependent induction of GM-CSF in HASMC. Furthermore, IL-1beta stimulated the major MAPK pathways, including ERK1/ERK2, JNK, and p38 MAPK. Exposure of HASMC to CO at low concentration (250 ppm) markedly inhibited IL-1beta-induced GM-CSF synthesis (>90%) compared with air-treated controls. CO treatment inhibited IL-1beta-induced ERK1/2 activation but did not inhibit JNK and p38 MAPK. Furthermore, CO increased cGMP levels in HASMC. Inhibition of guanylate cyclase by IH-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-1 (ODQ) abolished the inhibitory effects of CO on GM-CSF synthesis and ERK1/2 activation. Collectively, these data demonstrate that the inhibitory effect of CO on GM-CSF synthesis depends on ERK1/2 MAPK and guanylate cyclase/cGMP-dependent pathways.     

Carbon monoxide inhibits IL-17-induced IL-6 production through the MAPK pathway in human pulmonary epithelial cells

Interleukin (IL)-17 is a proinflammatory cytokine that is produced by activated memory CD4 T cells, which regulates pulmonary neutrophil emigration by the induction of CXC chemokines and cytokines. IL-17 constitutes a potential target for pharmacotherapy against exaggerated neutrophil recruitment in airway diseases. As a cytoprotective and anti-inflammatory gaseous molecule, carbon monoxide (CO) may also regulate IL-17-induced inflammatory responses in pulmonary cells. Herein, we examine the production of cytokine IL-6 induced by IL-17 and the effect of CO on IL-17-induced IL-6 production in human pulmonary epithelial cell A549. We first show that IL-17 can induce A549 cells to release IL-6 and that CO can markedly inhibit IL-17-induced IL-6 production. IL-17 activated the ERK1/2 MAPK pathway but did not affect p38 and JNK MAPK pathways. CO exposure selectively attenuated IL-17-induced ERK1/ERK2 MAPK activation without significantly affecting either JNK or p38 MAPK activation. Furthermore, in the presence of U0126 and PD-98059, selective inhibitors of MEK1/2, IL-17-induced IL-6 production was significantly attenuated. We conclude that CO inhibits IL-17-stimulated inflammatory response via the ERK1/2-dependent pathway.     

Intracellular Signaling MAPK Pathway After Cerebral Ischemia–Reperfusion Injury

The MAPK/ERK/p38 are signal transduction pathways that couple intracellular responses to the external stimuli. Contrary to ERK protein which is part of the survival route, presence of p38 could have an impact on cell injury. Tolerance induced by ischemic preconditioning (IPC) is a phenomenon of tissue adaptation, which results in increased tolerance to lethal ischemia-reperfusion injury (IRI). Paper describes changes in MAPK protein pathways after brain IPC. Ischemia was induced by 4-vessels occlusion and rats were preconditioned by sub-lethal ischemia. Western blot and immunohistochemistry identified ERK/p38 proteins in injured areas. The highest level of the pERK was detected at 24 h in IPC groups. A contrary pattern of MAPK/p38 activation was observed in this group, where the lowest level of p38 was displayed at 24 h after ischemia. This suggests that the MAPK signal transduction might have a potential role in tissues response subjected to IRI and in the phenomenon of tolerance.     

Canonical and Alternative MAPK Signaling

The archetype of MAPK cascade activation is somewhat challenged by the most recent discovery of unexpected phosphorylation patterns, alternative activation mechanisms and sub-cellular localization, in various members of this protein kinase family. In particular, activation by autophosphorylation pathways has now been described for the three best understood MAPK subgroups: ERK1/2; JNK1/2 and p38α/β. Also, a form of dosage compensation between homologues has been shown to occur in the case of ERK1/2 and JNK1/2. In this paper we summarize the MAPK activation pathway, with an emphasis on non-canonical examples. We use this information to propose a model for MAPK signal transduction that considers a cross-talk between MAPKs with different activation loop sequence motifs and unique C-terminal extensions. We highlight the occurrence of non-canonical substrate specificity during MAPK auto-activation, in strong connection with MAPK homo- and hetero-dimerization events.