Calcineurin antagonists differentially affect mediator secretion, p38 mitogen-activated protein kinase and extracellular signal-regulated kinases from immunologically activated human basophils

BACKGROUND: Basophils participate in allergic diseases by invading affected tissues and secreting histamine, leukotriene (LT)C4, IL-4 and IL-13 following FcepsilonRI cross-linking. A reduction of basophil mediator production is therefore of considerable therapeutical interest. Macrolactam derivatives, which inhibit calcineurin activation, may be candidates for antiallergic therapy as they reduce both symptoms of inflammatory skin disease in animal models and mast cell degranulation. OBJECTIVE: To investigate the effects of the calcineurin antagonists ascomycin and cyclosporin A on IgE-dependent mediator release from human basophils. METHODS: Basophils were purified by Ficoll density centrifugation, elutriation and negative selection. Histamine release was measured spectrofluorometrically; LTC4, IL-4 and IL-13 secretions were assayed by enzyme-linked immunosorbent assay (ELISA). Lysed cells were subjected to Western blotting using specific antibodies to phospho-p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)-1 and -2. RESULTS: Ascomycin (0.01 nm to 1 micro m) and cyclosporin A (0.1 nm to 10 micro m) strikingly inhibited (maximally 100%) anti-IgE-induced histamine and cytokine release from basophils, and these actions were unaffected by IL-3 priming. Ascomycin, however, was less potent at blocking LTC4 secretion, whereas cyclosporin A was unable to block production of this mediator. In immunoblotting studies, ascomycin and cyclosporin A reduced IgE-dependent p38 MAPK activation but were less potent at reducing ERK phosphorylation in basophils. CONCLUSION: Calcineurin antagonists like ascomycin and cyclosporin A block IgE-dependent basophil degranulation and cytokine synthesis. Calcineurin may target p38 MAPK activation, but seems to have less activity on ERK phosphorylation. This is paralleled by a reduced or even absent effect of calcineurin antagonists on eicosanoid production.     

Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells

BACKGROUND: Flavonoids have a variety of activities including anti-allergic activities, and are known to inhibit histamine release from human basophils and murine mast cells. OBJECTIVE: The effects of luteolin, a flavone, on the immunoglobulin (Ig) E-mediated allergic mediator release from human cultured mast cells (HCMCs) were investigated and compared with those of baicalein and quercetin. METHODS: HCMCs were sensitized with IgE, and then treated with flavonoids before challenge with antihuman IgE. The amount of released mediators was determined as was mobilization of intracellular Ca2+ concentration, protein kinase C (PKC) translocation and phosphorylation of intracellular proteins were detected after anti-IgE stimulation. RESULTS: Luteolin, baicalein and quercetin inhibited the release of histamine, leukotrienes (LTs), prostaglandin D2 (PGD2), and granulocyte macrophage-colony stimulating factor (GM-CSF) from HCMC in a concentration-dependent manner. Additionally, the three flavonoids inhibited A23187-induced histamine release. As concerns Ca2+ signalling, luteolin and quercetin inhibited Ca2+ influx strongly, although baicalein did slightly. With regard to PKC signalling, luteolin and quercetin inhibited PKC translocation and PKC activity strongly, although baicalein did slightly. The suppression of Ca2+ and PKC signallings might contribute to the inhibition of mediator release. The activation of extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinase (JNK), that were activated just before the release of LTs and PGD2 and GM-CSF mRNA expression in IgE-mediated signal transduction events, were clearly suppressed by luteolin and quercetin. In contrast, the flavonoids did not affect the activation of p38 mitogen-activated protein kinase (p38 MAPK) pathway. CONCLUSION: These results indicate that luteolin is a potent inhibitor of human mast cell activation through the inhibition of Ca2+ influx and PKC activation.     

Effects of KGF on alveolar epithelial cell transdifferentiation are mediated by JNK signaling

Rat alveolar epithelial cells (AEC) in primary culture transdifferentiate from a type II (AT2) toward a type I (AT1) cell-like phenotype, a process that can be both prevented and reversed by keratinocyte growth factor (KGF). Microarray analysis revealed that these effects of KGF are associated with up-regulation of key molecules in the mitogen-activated protein kinase (MAPK) pathway. To further explore the role of three key MAPK (i.e., extracellular signal-related kinase [ERK] 1/2, c-Jun N-terminal kinase [JNK] and p38) in mediating effects of KGF on AEC phenotype, primary rat AEC cultivated in minimal defined serum-free medium (MDSF) were treated with KGF (10 ng/ml) from Day 4 for intervals up to 48 hours. Exposure to KGF activated all three MAPK, JNK, ERK1/2, and p38. Inhibition of JNK, but not of ERK1/2 or p38, abrogated the ability of KGF to maintain the AT2 cell phenotype, as evidenced by loss of expression of lamellar membrane protein (p180) and increased reactivity with the AT1 cell-specific monoclonal antibody VIIIB2 by Day 6 in culture. Overexpression of JNKK2, upstream kinase of JNK, increased activation of endogenous c-Jun in association with increased expression of p180 and abrogation of AQP5, suggesting that activation of c-Jun promotes retention of the AT2 cell phenotype. These results indicate that retention of the AT2 cell phenotype by KGF involves c-Jun and suggest that activation of c-Jun kinase may be an important determinant of maintenance of AT2 cell phenotype.     

Regulation of mediator secretion in human basophils by p38 mitogen-activated protein kinase: phosphorylation is sensitive to the effects of phosphatidylinositol 3-kinase inhibitors and calcium mobilization

Although human basophils modulate allergic diseases by secreting histamine, leukotriene C(4), interleukin (IL)-4, and IL-13, the intermediary signals controlling the release of these mediators are poorly understood. Here, we show that p38 mitogen-activated protein kinase (MAPK) crucially affects basophil activation following stimulation with various secretagogues. Phosphorylation of p38 MAPK occurred within 5 min following anti-immunoglobulin (Ig)E stimulation, but was more rapidly activated in basophils stimulated with formyl-Met-Leu-Phe or A23187. Additionally, activation of p38 MAPK to the above stimuli was dependent on extracellular influx and intracellular mobilization of calcium. SB 203580, a specific p38 MAPK inhibitor, blocked anti-IgE-induced secretion of all basophil mediators and reduced not only p38 MAPK, but also extracellular signal-regulated kinases 1 and 2 activity, whereas the MAPK antagonist, PD 098059, did not affect p38 MAPK. IgE-dependent activation of p38 MAPK and MKK3/6 was affected by LY 294002 and wortmannin, suggesting that these kinases are targets for phosphatidylinositol 3 kinase (PI 3-K). We conclude that p38 MAPK is a pivotal regulator of basophil function downstream of PI 3-K activation and calcium mobilization.     

Comparison of mechanisms of IL-3 induced histamine release and IL-3 priming effect on human basophils

We have investigated the mechanisms by which interleukin-3 (IL-3) induces histamine release and primes basophils for increased histamine release in response to anti-IgE- and N-formyl-methionyl-leucyl-phenylalanine (fMLP). The responsiveness of basophils from atopic donors was variable, only 5/11 subjects showing release of > 10%, to IL-3 in the range 0.1-100 ng/ml. IL-3-induced histamine release required both extracellular Ca2+ and cell membrane IgE, removal of membrane IgE by lactate stripping or desensitization of basophils by incubation with anti-IgE in a Ca2+-free medium blocking IL-3-induced histamine release. IL-3 also primed basophils for histamine release by anti-IgE and fMLP in the same concentration range as it evoked histamine release. When IL-3 and either anti-IgE or fMLP were combined, the result was additive or supra-additive depending on the basophil donor. Unlike IL-3-evoked histamine release, IL-3 priming of basophils for fMLP-induced histamine release was shown to be independent of the presence of both cell surface IgE and of extracellular calcium. The protein kinase C (PKC) inhibitor, staurosporine (10 nM), inhibited anti-IgE induced histamine release, but neither IL-3 induced histamine release nor IL-3 priming of IgE- and fMLP-induced histamine release. Pertussis toxin (1.0 microg/ml) inhibited fMLP-induced histamine release but not anti-IgE-induced histamine release, IL-3-evoked histamine release or IL-3 priming. These results indicate that IL-3 modulates mediator release from human basophils by two mechanisms; a direct release of histamine which involves cell surface IgE and the influx of extracellular calcium but which is unlikely to proceed by the same mechanism as cross-linkage of IgE, and a priming effect which is independent of IgE and extracellular Ca2+ and which enhances the secretory effects of a wide range of unrelated secretagogues.     

Endogenous superallergen protein Fv interacts with the VH3 region of IgE to induce cytokine secretion from human basophils

BACKGROUND: Protein Fv is an endogenous protein, synthesized in liver and largely released in the digestive tract during acute and chronic viral hepatitis, that binds to immunoglobulin (Ig) from various mammalian and nonmammalian species. METHODS: Basophils obtained from normal subjects were purified by a double Percoll gradient and elutriation. The secretion of histamine induced by protein Fv was assayed by a fluorometric technique, the extracellular protein levels of IL-4 and IL-13 were measured by ELISA, and IL-4 mRNA levels were evaluated by RT-PCR. RESULTS: Protein Fv concentration-dependently induced histamine and IL-4 and IL-13 release from purified basophils. IL-4 mRNA, constitutively present in basophils, was increased after stimulation by protein Fv. Histamine and IL-4 secretion from basophils, but not histamine and IL-13 release, activated by protein Fv was significantly correlated (rs = 0.70, p<0.001). Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released IL-4 in response to protein Fv and anti-IgE. Two preparations of human VH3(+) monoclonal IgM inhibited protein Fv-induced secretion of IL-4 and histamine from basophils. In contrast, VH6(+) monoclonal IgM did not inhibit the release of mediators caused by protein Fv. CONCLUSIONS: These results indicate that protein Fv, which acts as an endogenous superallergen, interacts with the VH3 domain of IgE to induce the synthesis and release of IL-4, IL-13 and the secretion of histamine from basophils.     

Identification of caspase-independent PKCepsilon-JNK/p38 MAPK signaling module in response to metabolic inhibition in H9c2 cells

To understand the molecular mechanism of ischemia-induced cardiac myocyte cell death, H9c2 cells were studied by chemical hypoxia (CH), using metabolic inhibition buffer. CH suppressed the activities of caspase-3, -8, and -9. c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were activated, whereas extracellular regulated kinase (ERK) was inactivated. Only protein kinase Cepsilon (PKCepsilon) among PKC isotypes was translocated to the membrane fraction implying its activation. Moreover, the administration of PKCepsilon inhibitor suppressed the phosphorylations of JNK/p38 MAPK and reduced CH-induced cell death. An administration of JNK/p38 MAPK inhibitors also decreased CH-induced cell deaths, implying JNK/p38 MAPK's causative roles in the deaths. Collectively, this study identified a novel caspase-independent PKCepsilon-JNK/p38 MAPK signaling module induced by CH in cardiac myocytes. Our data show that the PKCepsilon-JNK/p38 MAPK signaling module contributes to CH-induced H9c2 cell death. This contrasts with previous notions, i.e., PKCepsilon's protective effect against ischemic death. Thus our data suggest that PKCepsilon can mediate alternative signals, i.e., beneficiary or deleterious signals, depending on the cell type, intensity, and/or type of injury.     

Initial Fc epsilon RI-mediated signal strength plays a key role in regulating basophil signaling and deactivation

BACKGROUND: Mediator releases after high-affinity IgE receptor (Fc(epsilon)RI) cross-linking in basophils and mast cells crucially govern the symptoms of allergic disease and amplify underlying T(H)2-type responses. Interestingly, the dose-response curve for Fc(epsilon)RI activation is bell-shaped, with supraoptimal stimulation leading to reduced mediator release. OBJECTIVE: We sought to characterize the mechanisms responsible for this control of Fc(epsilon)RI-triggered basophil activation. METHODS: Human basophils were purified by means of Ficoll density centrifugation, elutriation, and negative selection with immunomagnetic beads. Various intracellular signal protein activities were assessed by means of Western blotting, and mediator releases were analyzed either spectrofluorometrically (histamine) or by means of ELISA (IL-4 and IL-13). RESULTS: Supraoptimal anti-IgE concentrations led to lower mediator release than optimal concentrations but simultaneously to considerably faster histamine release kinetics. In parallel, basophil signaling proteins (Syk, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinases 1 and 2) were more rapidly phosphorylated at higher anti-IgE concentrations but more transiently activated in the supraoptimal range. This endogenous regulation most likely involved src homology 2 domain-containing inositol 5' phosphatase (SHIP), which was highly phosphorylated after supraoptimal anti-IgE triggering compared with lower stimulus concentrations. Conversely, N-formyl-methionyl-leucyl-phenylalanine-stimulated basophils failed to phosphorylate SHIP in the supraoptimal concentration range and did not display a bell-shaped dose-response curve. CONCLUSION: The kinetics of IgE-mediated signaling and mediator release in primary human Fc(epsilon)RI(+) cells varies substantially according to the magnitude of stimulation, and SHIP most likely plays an important role in terminating these events. CLINICAL IMPLICATIONS: The speed of allergic symptom generation depends on the degree of IgE receptor triggering, which is downregulated by SHIP, a potential target for allergy therapy.     

Stress-activated protein kinases mediate cell migration in human airway epithelial cells

Airway epithelial cell (AEC) repair immediately after injury requires coordinated cell spreading and migration at the site of injury. Stress-activated protein kinases such as p38 MAPK and c-Jun N-terminal Protein Kinase (JNK) modulate several responses to cell stress and injury, but their role in AEC migration is not clear. We examined migration in confluent 16HBE14o(-) human AEC lines and in primary AEC grown on collagen-IV. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Inhibitors of either p38 extracellular signal-regulated kinase (ERK)1/2 (PD98059), mitogen-activated protein kinase (MAPK) (SB203580), or JNK (SP600125) could block cell migration substantially. Inhibiting JNK but not p38 MAPK or ERK1/2 blocked extension of cells into the wound region from the original line of injury. Initial migration was associated with phosphorylation of ERK, p38 MAPK, and JNK within 5-15 min. The downstream effector of p38, heat shock protein 27, also was phosphorylated rapidly after injury; phosphorylation could be blocked by prior treatment with SB203580 but not SP600125. The downstream effector of JNK, c-Jun, likewise was phosphorylated rapidly after injury and could be blocked by inhibiting JNK. Our data demonstrate that p38 MAPK, JNK, and ERK1/2 participate in the early stages of AEC migration.     

Role of mitogen-activated protein kinases in influenza virus induction of prostaglandin E2 from arachidonic acid in bronchial epithelial cells

BACKGROUND: Influenza virus (IV) infection causes airway inflammation; however, it has not been determined whether IV infection could catabolize arachidonic acid cascade in airway epithelial cells. In addition, the responsible intracellular signalling molecules that catabolize arachidonic acid cascade have not been determined. OBJECTIVE: In the present study, to clarify these issues, we examined the cyclooxygenase (COX) expression, cytosolic phospholipase A2 (cPLA2) phosphorylation and prostaglandin E2 (PGE2) release in human bronchial epithelial cells (BEC) upon IV infection, and the role of mitogen-activated protein kinase (MAPK) including extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun-NH2-terminal kinase (JNK) in catabolizing arachidonic acid cascade in BEC. METHODS: COX-2 expression, phosphorylation of cPLA2 and phosphorylation of ERK, JNK and p38 MAPK were determined by Western blot. The concentrations of PGE2 were determined by ELISA. PD 98059 as a specific inhibitor of MAPK kinase-1 (MEK-1), an up-stream kinase of ERK, SB 203580 as a specific inhibitor of p38 MAPK and CEP-11004 as a specific inhibitor of JNK cascade were used to investigate the role of ERK, p38 MAPK and JNK in catabolizing arachidonic acid cascade in BEC. RESULTS: The results showed that (1) IV infection increases COX-2 expression, cPLA2 phosphorylation and PGE2 release, (2) ERK, p38 MAPK and JNK were phosphorylated, (3) CEP-11004 and PD 98059 predominantly attenuated COX-2 expression and cPLA2 phosphorylation, respectively, (4) SB 203580 did not remarkably affect COX-2 expression and cPLA2 phosphorylation, and (5) each inhibitor dose-dependently attenuated PGE2 release by various extents. CONCLUSION: These results indicate that IV infection activates three distinct MAPKs, ERK, p38 MAPK and JNK, to participate to various extents in the induction of PGE2 synthesis from arachidonic acid in BEC.     

Immunolocalization of the mitogen-activated protein kinase signaling pathway in Hassall's corpuscles of the human thymus

In the present study, we investigated the immunohistochemical localization of mitogen-activated protein kinase (MAPK) signaling pathway in the human thymus. Three members of MAPK, the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK) and the p38 kinase, showed differential expression patterns in the thymus medulla. The phosphorylated form of ERK (p-ERK) was abundantly present in the outer layer of Hassall's corpuscles, and the phosphorylated form of p38 kinase (p-p38 kinase) was present in the entire Hassall's corpuscles. The phosphorylated form of JNK (p-JNK) was expressed in medullary thymocytes. We also examined localization of MAPK kinases (MAPKK or MEK) which specifically activate MAPK. MEK1, an activator of ERK, was found in the outer layer of Hassall's corpuscles where p-ERK was expressed. MEK3, an activator of p38 kinase, was also expressed in the outer layer. MEK4 and MEK7, which are activators of JNK, were present in the entire Hassall's corpuscles. Thus, differential expression of MAPK in the thymus supports the concept that the MAPK signaling pathway controls the specificity of functional thymic responses to extracellular stimuli. Furthermore, the abundant expression of various elements of the pathway in Hassall's corpuscles suggests that the pathway is involved in thymic medullary epithelial maturation.     

Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3

Interleukin 1 receptor (IL-1R) and Toll-like receptors (TLRs) induce inflammatory genes through the complex of MyD88, IL-1R-associated protein kinase (IRAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6), which is believed to function 'upstream' of the cascades of IkappaB kinase (IKK) and nuclear factor-kappaB (NF-kappaB); extracellular signal-regulated protein kinase (ERK); c-Jun N-terminal kinase (JNK); and p38 mitogen-activated protein kinase (MAPK). Here we show that MAPK-ERK kinase kinase (MEKK3) is an essential signal transducer of the MyD88-IRAK-TRAF6 complex in IL-1R-TLR4 signaling. MEKK3 forms a complex with TRAF6 in response to IL-1 and lipopolysaccharide (LPS) but not CpG, and is required for IL-1R- and TLR4-induced IL-6 production. Furthermore, MEKK3 is crucial for IL-1- and LPS-induced activation of NF-kappaB and JNK-p38 but not ERK, indicating that MAPKs are differentially activated during IL-1R-TLR4 signaling. These data demonstrate that MEKK3 is crucial for IL-1R and TLR4 signaling through the IKK-NF-kappaB and JNK-p38 MAPK pathways.*Note: In the version of this article originally published online, the third author's name was incorrect. The correct author name should be Yong Lin. This error has been corrected for the HTML and print versions of this article.     

Close association of p38 and JNK with plasminogen-dependent upregulation of PAI-1 in rat astrocytes in vitro

As reported previously, stimulation of astrocytes with plasminogen (PLGn) remarkably enhances their production/release of plasminogen activator inhibitor-1 (PAI-1). In addition, both p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) are activated in these astrocytes. However, it remains to be determined whether the MAPK activation is associated with the PAI-1 induction in PLGn-stimulated astrocytes. In the present study, we investigated the relationship between MAPK activity and PAI-1 induction in PLGn-stimulated astrocytes. PLGn stimulation led to definitive phosphorylation of three MAPKs: external signal regulated kinase (ERK), JNK and p38. These results suggest that all of these MAPKs, either alone or in combination, are involved in PAI-1 induction. To verify this association, an inhibition experiment was carried out by using inhibitors specific for each MAPK. The results of the immunoblotting analysis indicated that 20 μM SB203580 (the p38 inhibitor) or SP600125 (the JNK inhibitor) suppressed approximately 85% or 40% of PLGn-inducible PAI-1, respectively. Only 20% inhibition was achieved by pretreatment of astrocytes with 20 μM PD98059 (the inhibitor of MEK1/2, an upstream kinase of ERK). In conclusion, p38 and JNK were shown to be the major MAPKs involved in the signaling cascade leading to PAI-1 induction in astrocytes stimulated with PLGn.     

Signal transduction for interleukin-3-dependent leukotriene synthesis in normal human basophils: opposing role of tyrosine kinase and protein kinase.

The intracellular signaling pathways regulating the synthesis of leukotrienes by myeloid cells are largely unknown. In addition, the signal transduction mechanisms utilized by the cytokine receptor family are still poorly understood. The fact that in mature human basophils the synthesis of leukotriene C4 (LTC4) induced by C5a is strictly dependent on a short preincubation with the cytokine interleukin-3 (IL-3), allowed us to investigate the metabolic requirements for LTC4 synthesis, and also to provide some information on early signal transduction mechanisms of IL-3 in these differentiated, non-dividing blood leukocytes. IL-3 itself does not alter intracellular free calcium concentration ([Ca2+]i) in basophils, whereas C5a induces a transient rise independent of IL-3 pretreatment, indicating that the priming effect of IL-3 cannot be explained by alterations in [Ca2+]i changes. The protein kinase C inhibitor staurosporine did not inhibit C5a-induced histamine release nor IL-3-dependent LTC4 formation in contrast to the IgE receptor-dependent basophil response. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) induced histamine release without leukotriene formation. PMA-treated basophils did not produce LTC4 in response to C5a. Rather, PMA blocked the IL-3 effect on C5a-induced LTC4 synthesis. Only the C5a signal but not the IL-3 effect was pertussis toxin sensitive. Two unrelated tyrosine kinase inhibitors, tyrphostin RG-50864 and herbimycin A, were both very efficient blockers of IL-3-dependent lipid mediator formation whereas C5a-induced histamine release was preserved. Thus LTC4 formation does not require activation of a staurosporine-sensitive serine/threonine kinase. To the contrary, IL-3-dependent LTC4 formation appears to be regulated by serine/threonine and tyrosine phosphorylation in an antagonistic manner.     

Regulation and kinetics of platelet-activating factor and leukotriene C4 synthesis by activated human basophils

BACKGROUND: Allergic disease is the result of an interplay of many different cell types, including basophils and mast cells, in combination with various inflammatory lipid mediators, such as platelet-activating factor (PAF) and leukotrienes (LT). LTC4 synthesis by human basophils has been studied quite extensively. However, not much is known about the synthesis of PAF by human basophils. OBJECTIVE: In this study, we have made a comprehensive comparison between the kinetics of PAF and LTC4 synthesis, in highly purified basophils, activated with different stimuli or with combinations of stimuli. METHODS : Synthesis of PAF and LTC4 by human basophils was determined with commercially available assay kits. The basophils were activated with C5a, fMLP, PMA, allergen or anti-IgE, in the absence and presence of IL-3 and/or in combination with elevation of cytosolic free Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. RESULTS: Most stimuli were found to induce both PAF and LTC4 synthesis. PAF synthesis and LTC4 release were enhanced by preincubation of the basophils with IL-3 or by elevation of cytosolic free Ca2+ by thapsigargin. Incubation of human basophils with IL-3 alone or thapsigargin alone did not result in detectable synthesis of PAF and LTC4, whereas the combination of the two resulted in high amounts of PAF and LTC4 synthesis. Depending on the stimulus used, LTC4 release was 5-100-fold higher than PAF synthesis. In addition, PAF, but not LTC4, was transiently detected, probably due to PAF degradation. LTC4 and PAF synthesis was strongly blocked by inhibitors of cytosolic phospholipase A2, indicating that this enzyme is involved in PAF and LTC4 synthesis by activated human basophils. CONCLUSION: This study provides a first comprehensive comparison of PAF and LTC4 synthesis in highly purified human basophils, stimulated with a variety of stimuli.     

Mitogen-activated protein kinase and activator protein-1 dependent signals are essential for Bacteroides fragilis enterotoxin-induced enteritis

The approximately 20-kDa heat-labile toxin produced by enterotoxigenic Bacteroides fragilis is known to be associated with the development of enteritis. However, the molecular mechanism involved is not yet fully understood. In this study, we assessed whether B. fragilis enterotoxin (BFT)-induced enteritis is related to mitogen-activated protein kinase (MAPK) signaling pathways. In human colon epithelial cells, BFT activated three major MAPK cascades. The activation of p38 was sustained for a relatively long period, while the stimulation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) was transient. BFT stimulation also activated AP-1 signals composed of c-Jun/c-Fos heterodimers. The p38 inhibitor SB203580 and the ERK inhibitor U0126 reduced not only AP-1 activity, but also decreased IL-8 and MCP-1 expression. In addition, the overexpression of superrepressors for c-Jun and Ras induced by BFT stimulation decreased the levels of IL-8 and MCP-1 production. Furthermore, SB203580 prevented BFT-induced colitis in the mouse ileum, as evidenced by significant decreases in villous destruction, neutrophil infiltration, and mucosal congestion. These results suggest that a pathway, including Ras, MAPK, and subsequent AP-1 activation, is required for IL-8 and MCP-1 expression in intestinal epithelial cells exposed to BFT, and can be involved in the development of enteritis.     

Low concentrations of nitric oxide (NO) induced cell death in PC12 cells through activation of p38 mitogen-activated protein kinase (p38 MAPK) but not via extracellular signal-regulated kinases (ERK1/2) or c-Jun N-terminal protein kinase (JNK)

Nitric oxide (NO), a highly reactive gaseous molecule, has been previously reported to induce apoptosis-like cell death even at a low concentration in PC12 cells. In this study, we examined NO-induced activation of members of the mitogen-activated protein kinase (MAPK) family, i.e., p38 MAPK, extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal protein kinase (JNK). Following the exposure of PC12 cells to an NO donor, (+)-(E)-4-ethyl-2-[hydroxyimino]-5-nitro-3-hexenamide (NOR3; 100 muM), the phosphorylation level of p38 MAPK increased time dependently from 2 to 6 h, but that of both ERK1/2 and JNK did not. Treatment with a p38 MAPK inhibitor SB203580 partially blocked the NOR3-induced cell death. Neither PD98059, U0126 (inhibitors of ERK1/2) nor SP600125 (a specific inhibitor of JNK) treatments had any significant effect on the NOR3-induced cell death. These findings suggest that the activation of a p38 MAPK pathway, but not that of ERK1/2 or JNK, plays an essential role in the apoptosis-like cell death induced by low concentrations of NO.