Superoxide dismutase expression and H2O2 production by hemocytes of the trematode intermediate host Lymnaea stagnalis (Gastropoda)

Snail hemocytes mobilise ROS-generating enzymes during oxidative burst similar to those of mammalian leukocytes. We report herein the identification of an inducible Cu/Zn superoxide dismutase, which converts O2- to H2O2, in hemocytes of the pond snail Lymnaea stagnalis. The deduced amino acid sequence with all characteristic residues (His44,46,61,69,78 and 118, Asp81, Cys55/144, Arg141 and the Greek Key loop region Glu119-Leu/Val142) includes an open reading frame of 155 AA. Changes in Cu/ZnSOD gene expression induced by stimulation with Zymosan or trematode larvae were examined in a time course. Activated hemocytes significantly up-regulate Cu/ZnSOD expression during 2-48 h upon stimulation with the maximal induction at 45 min during phagocytosis and at 12 h during encapsulations. This increase in Cu/ZnSOD expression paralleled the increasing production of hydrogen peroxide by hemocytes. Thus, intracellular or extracellular targets elicit an induced expression of Cu/ZnSOD and the generation of elevated amounts of hydrogen peroxide by L. stagnalis hemocytes, reflecting a significant activation of their host defense function.     

Down-regulation of p38 mitogen-activated protein kinase activation and proinflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)‐α, a known agonist of the mitogen‐activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho‐p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex‐ and age‐matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF‐α, interleukin (IL)‐1β and IL‐6 were measured in the organ and cell culture supernatants by enzyme‐linked immunosorbent assay. We found higher levels of phospho‐p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF‐α, IL‐1β and IL‐6 from IBD LPMCs and biopsies. Activated p38α MAPK is up‐regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down‐regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.     

Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation

The molecular details of mammalian stress-activated signal transduction pathways have only begun to be dissected. This, despite the fact that the impact of these pathways on the pathology of chronic inflammation, heart disease, stroke, the debilitating effects of diabetes mellitus, and the side effects of cancer therapy, not to mention embryonic development, innate and acquired immunity, is profound. Cardiovascular disease and diabetes alone represent the most significant health care problems in the developed world. Thus it is not surprising that understanding these pathways has attracted wide interest, and in the past 10 years, dramatic progress has been made. Accordingly, it is now becoming possible to envisage the transition of these findings to the development of novel treatment strategies. This review focuses on the biochemical components and regulation of mammalian stress-regulated mitogen-activated protein kinase (MAPK) pathways. The nuclear factor-kappa B pathway, a second stress signaling paradigm, has been the subject of several excellent recent reviews (258, 260).     

Macrophage migration inhibitory factor promotes innate immune responses by suppressing glucocorticoid-induced expression of mitogen-activated protein kinase phosphatase-1

The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) acts as a physiological counter-regulator of the immuno-suppressive effects of glucocorticoids. However, the mechanisms whereby MIF exerts its counter-balancing effect remain largely unknown. Here we report that MAPK phosphatase 1 (MKP-1), an archetypal member of dual specificity phosphatase that inactivates MAPK activity in response to pro-inflammatory stimuli, is a critical target of MIF-glucocorticoid crosstalk. Recombinant MIF counter-regulated in a dose-dependent fashion dexamethasone inhibition of TNF and IL-8 production by RAW 264.7 macrophages and U-937 promonocytes stimulated with lipopolysaccharides (LPS) or with LPS plus phorbol 12-myristate 13-acetate. Stimulation of RAW 264.7 macrophages with dexamethasone or dexamethasone plus LPS led to a robust up-regulation of MKP-1 mRNA and protein expressions that were counter-regulated by addition of recombinant MIF. Antisense MIF macrophages expressing reduced levels of endogenous MIF produced higher amount of MKP-1 and lower amount of TNF after exposure to dexamethasone and dexamethasone plus LPS, indicating that endogenous MIF acts in an autocrine fashion to override glucocorticoid-induced MKP-1 expression and inhibition of cytokine production. Taken together, these data identify MKP-1 as a molecular target of MIF-glucocorticoid crosstalk and provide a molecular basis for the control of macrophage responses by a pair of physiological regulators of innate immunity.     

Immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC, and ERK kinase families

Various hemocyte cell types have been described in invertebrates, but for most species a functional characterization of different hemocyte cell types is still lacking. In order to characterize some immunological properties of mussel (Mytilus galloprovincialis) hemocytes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. Phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) inhibitors were also used to biochemically characterize these cell responses. Four morphologically distinct subpopulations, designated R1-R4, were detected. R1, R2, and R3 cells presented different levels of phagocytosis towards zymosan, latex beads, and two bacteria species. Similarly, R1 to R3, but not R4, cells produced ROS, while all subpopulations produced NO, in response to zymosan. Internalization of all phagocytic targets was blocked by PI 3-K inhibition. In addition, internalization of latex particles, but not of bacteria, was partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan was impaired by PKC, or ERK inhibitors, only in R2 cells. Zymosan-induced ROS production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1 and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the existence of profound functional and biochemical differences in mussel hemocytes and indicate that M. galloprovincialis hemocytes are specialized cells fulfilling specific tasks in the context of host defense.     

Cochlear protection from acoustic injury by inhibitors of p38 mitogen-activated protein kinase and sequestosome 1 stress protein

This study evaluated the protective role of p38 mitogen-activated protein kinase (p38 MAPK) inhibitors and sequestosome 1 (Sqstm1/A170/p62), a stress-induced signal modulator, in acoustic injury of the cochlea in mice. Two weeks after the exposure of mice to acoustic stress, threshold shifts of the auditory brainstem response (ABR) from the pre-exposure level and hair cell loss were evaluated. The activation of p38 MAPK was observed in cochlea by immunostaining 4 h after acoustic stress. To examine the role of p38 MAPK in tissue injury, its inhibitors were i.p. injected into male wild-type C57BL mice before the acoustic overexposure. The inhibitors SB202190 and SB203580 but not the inactive analogue SB202474 dose-dependently decreased the auditory threshold shift and outer hair cell loss induced by acoustic overexposure, suggesting the involvement of p38 MAPK in ototoxicity. We found that acoustic overexposure induced the up-regulation of Sqstm1 mRNA expression in the cochlea of wild-type mice and that SQSTM1-deficient mice exhibited an enhanced ABR threshold shift and hair cell loss, suggesting a role of SQSTM1 in the protection of tissue from acoustic stress.     

Differential activation of mitogen-activated protein kinase signalling pathways by isometric contractions in isolated slow- and fast-twitch rat skeletal muscle

Activation of mitogen-activated protein (MAP) kinases has been implicated in the signal transduction pathways linking exercise to adaptive changes of muscle protein expression. In the present study, we investigated whether contractions of isolated muscles induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 MAPK in a fibre-type dependent manner. Slow-twitch (soleus) and fast-twitch (epitrochlearis, extensor digitorum longus) rat skeletal muscles were exposed to intermittent tetanic stimulation. Compared with the contralateral non-stimulated muscle, contractions increased ERK1/2 phosphorylation to the same extent in fast- and slow-twitch muscles. Significant increase in phosphorylation of p38 MAPK was observed in the fast-twitch muscles only. The total amount of ERK1/2 and p38 MAPK proteins was higher in the slow-twitch soleus muscle. In conclusion, MAP kinase signalling pathways are differentially activated and expressed in slow- and fast-twitch muscles. In addition, this activation is owing to muscle contraction per se and do not demand additional external influence.     

Redox paradox: effect of N-acetylcysteine and serum on oxidation reduction-sensitive mitogen-activated protein kinase signaling pathways

The thiol reducing agent N-acetylcysteine (NAC) is commonly used as an "antioxidant" in studies examining gene expression, signaling pathways, and outcome in acute and chronic models of lung injury. It is less widely appreciated that NAC can also undergo auto-oxidation and behave as an oxidant. We showed previously that NAC can have opposite effects on the activation of nuclear factor-kappaB depending on whether or not serum is present, and that the effects of NAC in the absence of serum are mimicked by various oxidants. Here we show that in a serum-depleted environment (0.1% fetal bovine serum), NAC substantially inhibited lipopolysaccharide (LPS) activation of the mitogen-activated protein kinases (MAPKs), namely extracellular signal-regulated kinase (ERK), p38mapk, and c-Jun NH2-terminal kinase (JNK). By contrast, in the presence of 10% serum, NAC had no effect on LPS activation of p42 and p44 ERK and in fact enhanced LPS induction of p38mapk and JNK phosphorylation. Because serum can significantly alter the redox state, these findings highlight the importance of the local redox milieu in signal transduction.     

MicroRNA-145通过丝裂原活化蛋白激酶和磷脂酰肌醇3激酶/蛋白质丝氨酸苏氨酸激酶通路调控肺癌A549细胞系转移及侵袭

目的 探讨microRNA-145(miR-145)对非小细胞肺癌A549细胞转移、侵袭及对丝裂原活化蛋白激酶(MAPK)和磷脂酰肌醇3激酶/蛋白质丝氨酸苏氨酸激酶（PI3K/AKT）通路的作用。 方法 将非小细胞肺癌A549细胞分成miR-145模拟物（mimics）组和negative-mimics组（miR-NC）以及antago miR-145组（抑制剂组）和antago miR control 组（antago-NC）,采用Transwell迁移实验及基质胶侵袭实验等检测miR-145对人非小细胞肺癌A549迁移、侵袭能力的影响;Western blotting方法分析miR-145对MAPK和PI3K/AKT通路的影响。此外,采用细胞外调节蛋白激酶（ERK）及AKT的通路抑制剂分别作用于A549细胞系,检测A549细胞迁移、侵袭能力的改变。 结果 miR-145 mimics组穿过细胞数（90.67±10.33）明显少于miR-NC组（175.33±23.67）,miR-145 mimics组穿过基质胶的细胞数（153.33±22.33）少于miR-NC组 (77.33±13.67）,P<0.05;antago-NC组通过小室的细胞数量以及穿过基质胶的细胞数量明显少于antago miR-145组（P<0.05）,结果说明,miR-145具有抑制非小细胞肺癌A549细胞迁移、侵袭的能力;miR-145 mimics转染可分别抑制A549细胞中90%、78%以及73%的ERK1/2、AKT的ser-473位点和thr-308位点的磷酸化,antago miR-145转染可促进A549细胞中ERK1/2、AKT的ser-473位点和thr-308位点的磷酸化,增加115%、125%以及129%,而当抑制MAPK通路及PI3K/AKT通路的激活后,A549细胞的转移及侵袭能力下降。 结论 miR-145通过MAPK和PI3K/AKT通路调控肺癌A549细胞转移及侵袭。     

HIV-1 gp120-induced TNF-{alpha} production by primary human macrophages is mediated by phosphatidylinositol-3 (PI-3) kinase and mitogen-activated protein (MAP) kinase pathways

Human immunodeficiency virus type 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein gp120 to CD4 followed by a chemokine receptor, but these interactions may also take place independently from infection. gp120 stimulation of primary human macrophages is known to trigger production of cytokines implicated in pathogenesis, particularly tumor necrosis factor alpha (TNF-alpha), but the mechanisms have not been determined. We sought to define the pathways responsible for TNF-alpha secretion by monocyte-derived macrophages (MDM) following HIV-1 gp120 stimulation. MDM exposure to recombinant macrophage-tropic (R5) gp120 led to dose- and donor-dependent release of TNF-alpha, which was cyclohexamide-sensitive and associated with up-regulated message. Pretreatment with specific inhibitors of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1/2 (ERK-1/2; PD98059, U0126) and p38 (SB202190, PD169316) inhibited the secretion of TNF-alpha. gp120-elicited TNF-alpha production was also blocked by phosphatidylinositol-3 kinase (PI-3K) inhibitors (wortmannin, LY294002). Moreover, PI-3K inhibition ablated gp120-induced phosphorylation of p38 and ERK-1/2. The response was inhibited by a CC chemokine receptor 5 (CCR5)-specific antagonist, indicating that CCR5 was in large part responsible. These results indicate that gp120-elicited TNF-alpha production by macrophages involves chemokine receptor-mediated PI-3K and MAPK activation, that PI-3K is an upstream regulator of MAPK in this pathway, and that p38 and ERK-1/2 independently regulate TNF-alpha production. These gp120-triggered signaling pathways may be responsible for inappropriate production of proinflammatory cytokines by macrophages, which are believed to play a role in immunopathogenesis and in neurological sequelae of AIDS.     

体外表达的Glypican-3的CTL表位肽诱导特异性免疫应答

目的 探讨体外表达的Glypican-3(GPC3)能否诱导出GPC3特异性的细胞免疫应答.方法 体外表达GPC3的CTL表位肽GPC3298-306及GPC3144-152偶联血蓝蛋白(KLH)后免疫Balb/c小鼠,检测免疫后小鼠脾淋巴细胞免疫活性,包括IFN-γ的分泌,以及该淋巴细胞对表达GPC3的鼠肿瘤细胞细胞毒活性.结果 经GPC3298-306及GPC3144-152免疫后,小鼠脾细胞中分泌IFN-γ的T淋巴细胞阳性率明显增高(P＜0.01);脾淋巴细胞中CD4+T/CD8+T比值亦明显升高(P＜0.05);脾淋巴细胞对表达GPC3的鼠肿瘤细胞有特异性的杀伤作用.结论 体外表达的GW3的CTL表位肽能诱导出GPC3特异性的细胞免疫,提示体外表达的GPC3的CTL表位肽GPC3298-306及GPC3144-152可能在在以GPC3为靶向的抗肿瘤免疫治疗中发挥重要作用.