The action of nitric oxide to enhance cell survival in chick cardiomyocytes is mediated through a cGMP and ERK1/2 pathway while p38 mitogen-activated protein kinase-dependent pathways do not alter cell death

The objective of this study was to determine whether the dual action of nitric oxide (NO) on cardiomyocyte cell viability is mediated through p38 mitogen-activated protein kinase (MAPK)-induced cell death and extracellular signal-regulated kinase (ERK1/2)-mediated cell survival pathways, and whether either of these is mediated through a cGMP-protein kinase G (PKG) pathway. Cell viability of embryonic chick cardiomyocytes was assessed by the MTT assay, which is based on the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The NO donor sodium nitroprusside (SNP) produced a significant (P < 0.01) concentration-dependent reduction in cell viability or increase in cell death. Sodium nitroprusside induced ERK1/2 phosphorylation, and the mitogen-activated protein kinase (MEK1/2) inhibitor PD 98059 significantly increased cell death. In contrast, SB202190, a relatively selective inhibitor of p38 MAPK, did not affect SNP-induced cell death. The cardioprotective effect of NO was prbably mediated in part via cGMP because 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-sensitive guanylyl cyclase, produced a significant enhancement of SNP-induced cell death. In contrast, the PKG inhibitor KT5823 did not affect cell viability. In summary, these data suggest that NO, via stimulation of soluble guanylyl cyclase, activates MEK1/2 whose product, ERK1/2, protects against cell death. In contrast, SNP-induced p38 MAPK activation does not modulate NO-induced cardiomyocyte cell death. Not all cGMP targets affect NO-induced cell death, since the PKG pathway does not enhance or suppress NO-induced cardiomyocyte cell death. Enhancement of the ERK1/2 responses to NO may permit the beneficial effects of NO to predominate.     

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.     

Opposing Effects of Sodium Salicylate and Haematopoietic Cytokines IL-3, IL-5 and GM-CSF on Mitogen-Activated Protein Kinases and Apoptosis of EOL-1 Cells

Haematopoietic cytokines such as IL-3, IL-5 and GM-CSF not only activate eosinophils but also prolong their life span by inhibiting their apoptotic cell death. We have studied the effects of IL-3, IL-5 and GM-CSF on apoptosis and mitogen-activated protein kinases (MAPKs) in a human eosinophilic leukaemic cell line (EoL-1). Results demonstrated that all three cytokines could trigger the receptor-mediated activation of extracellular signal-regulated kinase (ERK) within one hour but not p38 MAPK activity in EoL-1 cells. In contrast, sodium salicylate (NaSal), a nonsteroidal anti-inflammatory drug (NSAID), could activate p38 MAPK but not ERK within one hour. Both cytokines and specific p38 MAPK inhibitor SB 203580 could partly block the NaSal-induced apoptosis in EoL-1 cells. A specific MAPK/ERK kinase (MEK) inhibitor, PD 098059, could induce apoptosis and eliminate the protective effect of IL-3, IL-5 and GM-CSF against NaSal-induced apoptosis in EoL-1 cells. Taken together, cytokines IL-3, IL-5 and GM-CSF could prolong EoL-1 cells survival through the transient activation of ERK. On the other hand, activation of p38 MAPK in EoL-1 cells by NaSal could lead to apoptosis. Activation of p38 MAPK and the resulting induction of apoptosis in EoL-1 cells may be important to explain the anti-inflammatory action of NSAID in allergic inflammation.     

Opposing effects of sodium salicylate and haematopoietic cytokines IL-3, IL-5 and GM-CSF on mitogen-activated protein kinases and apoptosis of EoL-1 cells

Haematopoietic cytokines such as IL-3, IL-5 and GM-CSF not only activate eosinophils but also prolong their life span by inhibiting their apoptotic cell death. We have studied the effects of IL-3, IL-5 and GM-CSF on apoptosis and mitogen-activated protein kinases (MAPKs) in a human eosinophilic leukaemic cell line (EoL-1). Results demonstrated that all three cytokines could trigger the receptor-mediated activation of extracellular signal-regulated kinase (ERK) within one hour but not p38 MAPK activity in EoL-1 cells. In contrast, sodium salicylate (NaSal), a nonsteroidal anti-inflammatory drug (NSAID), could activate p38 MAPK but not ERK within one hour. Both cytokines and specific p38 MAPK inhibitor SB 203580 could partly block the NaSal-induced apoptosis in EoL-1 cells. A specific MAPK/ERK kinase (MEK) inhibitor, PD 098059, could induce apoptosis and eliminate the protective effect of IL-3, IL-5 and GM-CSF against NaSal-induced apoptosis in EoL-1 cells. Taken together, cytokines IL-3, IL-5 and GM-CSF could prolong EoL-1 cells survival through the transient activation of ERK. On the other hand, activation of p38 MAPK in EoL-1 cells by NaSal could lead to apoptosis. Activation of p38 MAPK and the resulting induction of apoptosis in EoL-1 cells may be important to explain the anti-inflammatory action of NSAID in allergic inflammation.     

Opposing Effects of Sodium Salicylate and Haematopoietic Cytokines IL-3, IL-5 and GM-CSF on Mitogen-Activated Protein Kinases and Apoptosis of EOL-1 Cells

Haematopoietic cytokines such as IL-3, IL-5 and GM-CSF not only activate eosinophils but also prolong their life span by inhibiting their apoptotic cell death. We have studied the effects of IL-3, IL-5 and GM-CSF on apoptosis and mitogen-activated protein kinases (MAPKs) in a human eosinophilic leukaemic cell line (EoL-1). Results demonstrated that all three cytokines could trigger the receptor-mediated activation of extracellular signal-regulated kinase (ERK) within one hour but not p38 MAPK activity in EoL-1 cells. In contrast, sodium salicylate (NaSal), a nonsteroidal anti-inflammatory drug (NSAID), could activate p38 MAPK but not ERK within one hour. Both cytokines and specific p38 MAPK inhibitor SB 203580 could partly block the NaSal-induced apoptosis in EoL-1 cells. A specific MAPK/ERK kinase (MEK) inhibitor, PD 098059, could induce apoptosis and eliminate the protective effect of IL-3, IL-5 and GM-CSF against NaSal-induced apoptosis in EoL-1 cells. Taken together, cytokines IL-3, IL-5 and GM-CSF could prolong EoL-1 cells survival through the transient activation of ERK. On the other hand, activation of p38 MAPK in EoL-1 cells by NaSal could lead to apoptosis. Activation of p38 MAPK and the resulting induction of apoptosis in EoL-1 cells may be important to explain the anti-inflammatory action of NSAID in allergic inflammation.     

Endogenous nitric oxide downregulates the Bcl-2 expression of eosinophils through mitogen-activated protein kinase in bronchial asthma

BACKGROUND: Eosinophil apoptosis might play a crucial role in the maintenance of persistent airway inflammation in asthma. Nitric oxide (NO) synthase activity is upregulated in eosinophils of asthmatic patients. Mitogen-activated protein kinase (MAPK) is implicated in regulating eosinophil apoptosis by modulating Bcl-2 expression. NO-induced cell apoptosis is associated with an inhibition of Bcl-2 expression. OBJECTIVE: We sought to study whether NO might induce eosinophil apoptosis through extracellular signal-regulated protein kinase (ERK) or p38 MAPK pathways and Bcl-2 expression. METHODS: Eosinophils were freshly isolated from peripheral blood of 16 asthmatic patients and 12 healthy subjects and then cultured with or without the NO synthase inhibitor N-nitro-l-arginine methyl ester (L-NAME) at 1 and 10 mmol/L for 16 hours. The expression of Bcl-2 and induced NO synthase on eosinophils was analyzed by means of flow cytometry. Apoptosis was assessed by means of propidium iodide and DNA ladder. The activity of ERK and p38 MAPK was determined by means of Western blotting. RESULTS: The induced NO synthase immunoreactivities and the spontaneous release of nitrite from the eosinophils of asthmatic patients were higher compared with those of healthy subjects. Eosinophils of asthmatic patients were found to express more highly constitutive Bcl-2 than those of healthy subjects. After incubation for 16 hours, the expression of Bcl-2 on eosinophils from patients with asthma was significantly enhanced by L-NAME. The percentage of apoptosis was decreased by the addition of 1 mmol/L L-NAME in patients with asthma. The activity of p38 MAPK and ERK in eosinophils from patients with asthma was enhanced in the presence of L-NAME. An inhibition of MAPK reduced the Bcl-2 expression and increased eosinophil apoptosis in patients with asthma. CONCLUSION: We concluded that inhibition of endogenous NO might increase the expression of Bcl-2 in eosinophils from patients with asthma through the signaling pathway of ERK or p38 MAPK, which in turn decrease the apoptosis.     

Role of caspases in dexamethasone-induced apoptosis and activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase in human eosinophils

Eosinophils are the principal effector cells for the pathogenesis of allergic inflammation. Glucocorticoids such as dexamethasone have long been used therapeutically for eosinophilia in allergic inflammation by inducing eosinophil apoptosis, but little is known about the intracellular mechanisms mediating dexamethasone-induced apoptosis. In the present study, we investigated the effect of dexamethasone on three mitogen-activated protein kinases (MAPK) involved in the intracellular signalling pathway: c-Jun NH2-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK). We found that dexamethasone could activate JNK and p38 MAPK in a time-dependent manner but not ERK. Further, SB 203580, a specific p38 MAPK inhibitor, was additive with dexamethasone in inducing eosinophil apoptosis, while JNK1/2 antisense phosphorothioate oligodeoxynucleotides did not show any significant effect. These suggest that dexamethasone-induced JNK1/2 and p38 MAPK activation are not crucial to the induction of apoptosis. Pretreatment of eosinophils with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.FMK), a broad-spectrum caspase inhibitor, could inhibit dexamethasone-induced apoptosis in eosinophils dose-dependently. Moreover, Z-VAD.FMK partially inhibited dexamethasone-activated JNK and p38 MAPK activities. However, dexamethasone treatment did not activate specific caspase-3, -8 activity in eosinophils compared with spontaneous apoptosis. We therefore conclude that dexamethasone-induced apoptosis and activation of JNK and p38 MAPK activity in eosinophils are regulated by caspases but not through the common apoptosis-related caspase-3, -8 as in other cell types. Elucidation of the important role of caspases in eosinophil apoptosis may facilitate the development of more specific and effective treatment for allergic inflammation.     

Cyclic Adenosine Monophosphate Inhibits Nitric Oxide‐Induced Apoptosis of Cardiac Muscle Cells in a c‐Jun N‐Terminal Kinase‐Dependent Manner

Cyclic adenosine monophosphate (cAMP) modulates various agent‐induced apoptosis. In this study, we observed that cAMP had a significantly protective effect on nitric oxide (NO)‐induced cytotoxicity in H9c2 cardiac muscle cells. Pretreatment with DBcAMP (cAMP analogue) or forskolin (adenylyl cyclase activator) also significantly prevented the SNP‐induced apoptosis in H9c2 cells. In contrast, H‐89 or KT5720 (PKA inhibitor) reversed the protective effects of DBcAMP. In this study, DBcAMP or forskolin reduced SNP‐induced JNK/SAPK activation to the basal level, but KT5720 reversed the inhibitory effects of these two agents. In contrast to JNK/SAPK activation, DBcAMP and forskolin significantly enhanced SNP‐activated p38 MAPK phosphorylation and did not affect SNP‐mediated ERK activation. KT5720 reversed the effects of DBcAMP and forskolin on p38 MAPK phosphorylation. The inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK/SAPK markedly reduced the extent of SNP‐induced cell death. Taken together, we suggest that JNK/SAPK is related to cAMP‐protective effect in SNP‐induced apoptosis. In addition, c‐AMP relating agents protected SNP‐induced cell death in neonatal rat ventricular cardiomyocytes. The cAMP‐relating agent‐induced protective effect is not resricted in H9c2 cardiac muscle cells.     

p38 MAPK as a negative regulator of VEGF/VEGFR2 signaling pathway in serum deprived human SK-N-SH neuroblastoma cells

Evidence suggests that vascular endothelial growth factor (VEGF) mediates neuroprotection to prevent an apoptotic cell death. The p38 mitogen-activated protein kinase (MAPK) pathway is implicated as an important mediator of neuronal apoptosis but its role in VEGF-mediated neuroprotection is unclear. Herein, we show that treatments with the p38 MAPK inhibitor, SB202190, enhanced VEGF-mediated survival in serum deprived SK-N-SH neuroblastoma cells by decreasing caspase-3/7 activation while increasing the phosphorylation of the extracellular signal-regulated kinase (ERK1/2) and Akt signaled through the VEGF receptor, VEGFR2. A blockade of VEGFR2 signaling with a selective inhibitor, SU1498 or gene silencing with VEGFR2 siRNA in SB202190 treated cells abrogated this prosurvival response and induced high activation levels of caspase-3/7. These findings suggested that the protection elicited by p38 MAPK inhibition in serum starved cells was dependent on a functional VEGF/VEGFR2 pathway. However, p38 MAPK inhibition attenuated caspase-3 cleavage in SU1498/SB202190 treated cells, indicating that p38 MAPK and caspase-3 only contributed in part to the total levels of caspase-3/7 induced by VEGFR2 inhibition. Pretreatments with the pan caspase inhibitor, z-VAD-fmk, prevented the apoptosis induced by VEGFR2 inhibition and promoted survival in serum starved cells irrespective of p38 MAPK inhibition. Collectively, our findings suggest that p38 MAPK exerts a negative effect on VEGF-mediated signaling through VEGFR2 in serum starved neuroblastoma cells. Furthermore, VEGF signals protection against a caspase-mediated cell death that is regulated by p38 MAPK-dependent and -independent mechanisms.     

Selective COX-2 inhibitor regulates the MAP kinase signaling pathway in human osteoarthritic chondrocytes after induction of nitric oxide

The purpose of this study was to examine the effect of cyclooxygenase-2 (COX-2) inhibitors on the mitogen-activated protein (MAP) kinase signaling pathway and synthesis of glucosaminoglycan after nitric oxide (NO) induction in articular human chondrocytes. After NO induction, the cells were divided into three groups that were treated with either ethanol (control); a selective COX-2 inhibitor (Celecoxib), or no additive, and evaluated. There were no differences in the effect of the selective COX-2 inhibitor on mitochondrial membrane potential or Annexin V levels. However, Celecoxib significantly decreased prostaglandin E2 (PGE2) production. Celecoxib also decreased the phosphorylation state of p38 and p44/42 of MAP kinase. The ratio of chondroitin-6 sulfate (C6S)/C4S was increased in response to the exposure to Celecoxib. Celecoxib did not affect apoptosis, but decreased the activation of MAP kinase in osteoarthritic chondrocytes after NO induction. NO-induced OA chondrocytes were associated with the p38 and the p44/42 MAPK signaling pathways, in a pathway that is distinct from PGE2-mediated apoptosis.     

Role of PKC-delta during hypoxia in heart-derived H9c2 cells

In the present study, we investigated the role of protein kinase C (PKC) isoforms during hypoxia in heart-derived H9c2 cells. Hypoxia caused a rapid translocation of PKC-delta from soluble to particulate fraction and a downregulation of PKC-epsilon and PKC-zeta, whereas PKC-alpha and PKC-beta I remained unaltered. When H9c2 cells were pretreated with PKC-delta inhibitor rottlerin (3 microM), hypoxia-induced apoptotic and necrotic cell death were significantly increased. Hypoxic insult also caused an activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK with no change in c-Jun NH(2)-terminal protein kinase (JNK) phosphorylation. Hypoxia-induced cell death was increased by treatment with ERK1/2 inhibitor U0126 (10 microM), but attenuated by p38 MAPK inhibitor SB202190 (10 microM). Treatment with rottlerin completely blocked the hypoxia-induced ERK phosphorylation, whereas it significantly increased p38 MAPK phosphorylation. The hypoxia-induced translocation of PKC-delta was not altered by U0126 and/or SB202190. From these results, it is suggested that hypoxia causes a rapid translocation of PKC-delta and subsequently ERK activation and p38 inactivation, rendering H9c2 cells resistant to hypoxia-induced cell death.     

Sodium salicylate-induced apoptosis of human peripheral blood eosinophils is independent of the activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase

BACKGROUND: It has been shown that the inhibition of eosinophilic apoptosis is an important mechanism for the development of blood and tissue eosinophilia in allergic diseases. Considerable attention has recently been focused on the role played by different intracellular kinase cascades in the control of apoptosis. In the present study, we investigated the effect of sodium salicylate (NaSal), a nonsteroidal anti-inflammatory drug, on mitogen-activated protein kinases (MAPK) and apoptosis of human eosinophils. METHODS: Human blood eosinophils were purified from buffy coat. NaSal-induced apoptosis of eosinophils was assessed by morphological changes and Annexin-V binding assay. Changes of MAPK activity upon treatment with NaSal were measured by kinase activity assay and Western blot. RESULTS: NaSal could induce apoptosis of human blood eosinophils in a dose- and time-dependent manner. It could also activate c-Jun N-terminal kinase (JNK) and p38 MAPK but not extracellular signal-regulated protein kinase (ERK) activity within 1 h. Pretreatment of eosinophils with p38 MAPK and JNK anti-sense (AS) phosphorothioate oligodeoxynucleotides (ODN) or specific p38 MAPK inhibitor SB 203580 did not have any significant effect on NaSal-induced apoptosis. However, ERK AS ODNs could trigger the apoptosis of normal eosinophils. CONCLUSION: There is no direct relationship between the activation of JNK and p38 MAPK pathways and NaSal-induced apoptosis in human peripheral blood eosinophils.     

Ethanol-induced oxidative stress is mediated by p38 MAPK pathway in mouse hippocampal cells

It has been known that ethanol causes neuronal cell death through oxidative stress. Ethanol itself and reactive oxygen species (ROS) produced by ethanol modulate intracellular signaling pathways including mitogen-activated protein kinase (MAPK) cascades. This study was conducted to examine the impact of ethanol on MAPK signaling in HT22 cells. Ethanol (100 and 400 mM) caused activation of ERK, p38 MAPK, and JNK. ERK activation occurred in early time and p38 MAPK activation was evident when ERK activation was diminished. Specific inhibitor of p38 MAPK (SB203580) protected HT22 cells against ethanol, which was accompanied by an inhibition of ROS accumulation. However, inhibitors of ERK (U0126) and JNK (SP600125) had no effects on ethanol-induced neuronal cell death when they are treated with ethanol for 24 h. These results suggest that p38 MAPK may have important roles in ROS accumulation during ethanol-induced oxidative stress in HT22 cells.     

Apoptosis induced by pneumolysin in human endothelial cells involves mitogen-activated protein kinase phosphorylation

Pneumolysin (Ply) is an essential virulence factor of S.?pneumoniae, which can induce apoptosis in a variety of host cells to facilitate infection of pathogenic bacteria by as yet unclear mechanisms. To confirm the apoptosis-inducing properties of pneumolysin in endothelial cells, human umbilical vein endothelial cells (HUVECs) were exposed to pneumolysin. The proliferation of HUVECs was inhibited by pneumolysin in a dose- and time-dependent manner. Flow cytometry analysis and ultrastructural changes of the cells indicated the apoptotic response. Exposure to pneumolysin significantly increased the number of apoptotic cells and the activities of caspases-3 and -8. This change was associated with activation of p38 mitogen-activated protein kinase (MAPK) and suppression of extracellular signaling regulation kinase (ERK)1/2. Pre-exposure to the p38 MAPK inhibitor SB-203850 prevented human endothelial cells from apoptosis induced by pneumolysin. In conclusion, these findings demonstrate that pneumolysin induces apoptosis in endothelial cells and the involvement of p38 MAPK activation and ERK1/2 deactivation.     

TGF-β1激活的p38MAPK在TGF-β1上调人卵巢癌细胞PAI-1表达中的作用

目的:纤溶酶原激活物抑制剂1(PAI-1)在凝血、创伤修复、炎症和肿瘤转移中起重要作用。已有报道转化生长因子β1(TGF-β1)能通过Smad通路诱导PAI-1表达,但TGF-β1能否通过激活非Smad通路诱导PAI-1表达尚不清楚,因此本研究探讨了在卵巢癌细胞中TGF-β1激活的非Smad通路p38丝裂原活化蛋白激酶(p38MAPK)和细胞外信号调节激酶(ERK)与TGF-β1上调PAI-1表达的关系。方法:用10μg/L TGF-β1处理卵巢癌SKOV3细胞和HO-8910细胞后,采用real-time PCR和Western blotting的方法检测PAI-1的表达,用磷酸化p38MAPK的抗体和磷酸化ERK的抗体检测p38 MAPK和ERK的激活情况,用p38 MAPK和ERK的特异性抑制剂SB203580和PD98059分别抑制其活性后,检测PAI-1的表达。结果:TGF-β1在卵巢癌细胞中可明显上调PAI-1mRNA和蛋白的表达,并可快速激活p38 MAPK和ERK。用p38 MAPK的抑制剂可以明显抑制TGF-β1上调PAI-1表达,但是抑制ERK活性对TGF-β1上调PAI-1表达没有明显影响。结论:TGF-β1激活的p38 MAPK通路参与了TGF-β1上调PAI-1的表达。     

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.     

Leishmania donovani promastigotes evade the activation of mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase-1/2 during infection of naive macrophages

The protozoan parasite Leishmania fails to activate naive macrophages for proinflammatory cytokines production, and selectively impairs signal transduction pathways in infected macrophages. Because mitogen-activated protein kinases (MAPK)- and NF-kappaB-dependent signaling pathways regulate proinflammatory cytokines release, we investigated their activation in mouse bone marrow-derived macrophages (BMM) exposed to Leishmania donovani promastigotes. In naive BMM, the parasite failed to induce the phosphorylation of p38 MAPK, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)1/2, as well as the degradation of IkappaB-alpha. The use of L. donovani mutants defective in the biosynthesis of lipophosphoglycan revealed that evasion of ERK1/2 activation requires surface expression of the repeating unit moiety of this virulence determinant. In IFN-gamma-primed BMM, L. donovani promastigotes strongly induced the phosphorylation of p38 MAPK and ERK1/2, and the use of selective inhibitors for ERK (PD98059) and p38 MAPK (SB203580) revealed that both kinases are required for L. donovani-induced TNF-alpha but not NO(2)(-) release. Collectively, these data suggest that both p38 MAPK and ERK1/2 pathways participate in some Leishmania-induced responses in IFN-gamma-primed BMM. The ability of L. donovani promastigotes to avoid MAPK and NF-kappaB activation in naive macrophages may be part of the strategy evolved by this parasite to evade innate immune responses.     

Arginase induction promotes Trypanosoma cruzi intracellular replication in Cruzipain-treated J774 cells through the activation of multiple signaling pathways

Given that arginase activation may effectively influence nitric oxide (NO) production in macrophages, we have investigated the intracellular signals that regulate L-arginine metabolism and its influence on Trypanosoma cruzi growth. We demonstrate that cruzipain (Cz), a parasite antigen, induces arginase I expression in J774 cells, and the pretreatment of Cz-treated cells with N-omega-hydroxy-L-arginine (arginase inhibitor) leads to a dramatic decrease in amastigote growth. The study of intracellular signals shows that genistein [tyrosine kinase (TK) inhibitor], KT5720 [protein kinase (PK) A inhibitor] and SB203580 [p38 mitogen-activated protein kinase (MAPK) inhibitor] significantly decrease Cz-induced arginase activation. However, calphostin C (PKC inhibitor) and PD98059 [p44/p42 MAPK kinase (MEK) inhibitor] did not cause a significant change. To determine if signaling pathways triggered by Cz were involved in the T. cruzi growth, we studied the effect of those inhibitors. In Cz-treated cells--pre-incubated with TK, PKA or p38 MAPK inhibitors--the balance of NO/urea was biased towards NO, and the amastigote growth was diminished. Besides, genistein and mainly KT5720 induced down-regulation of arginase I expression in Cz-treated cells. Thus, activation of TK, PKA and p38 MAPK by Cz induces an increase of arginase activity in macrophages and the subsequent T. cruzi growth.     

Fibroblast growth factor-10 prevents asbestos-induced alveolar epithelial cell apoptosis by a mitogen-activated protein kinase-dependent mechanism

Asbestos induces alveolar epithelial cell (AEC) DNA damage and apoptosis by the mitochondria-regulated death pathway and oxidative stress. Fibroblast growth factor-10 (FGF-10), an alveolar epithelial type II cell mitogen that is required for the lung development, prevents H(2)O(2)-induced AEC DNA damage by a mitogen activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)-dependent mechanism. In this study, we show that FGF-10 attenuates asbestos-induced AEC DNA strand break formation and apoptosis. MAPK/ERK kinase (MEK) inhibitors, U0126 or PD98059, each blocked the protective effect of FGF-10 against asbestos-induced DNA damage and apoptosis, whereas a p38-MAPK inhibitor had a negligible effect, suggesting a crucial role for MEK/ERK activation in mediating the protective effects of FGF-10. Further, we show that FGF-10 attenuates asbestos-induced change in AEC mitochondrial membrane potential and caspase 9 activation, both of which are blocked by U0126. We conclude that FGF-10 decreases asbestos-induced AEC DNA damage and apoptosis in part by mechanisms involving MEK/ERK-dependent signaling that affects the mitochondria-regulated death pathway.