Involvement of staphylococcal protein A and cytoskeletal actin in Staphylococcus aureus invasion of cultured human oral epithelial cells

Following the coincidental discovery that beta-actin isolated from renal epithelial cells was precipitated by staphylococcal protein A (SPA), the possibility that SPA and cytoskeletal actin filaments may be involved in Staphylococcus aureus infection of epithelial cells was considered. Therefore, to clarify the potential role of SPA and actin filaments in S. aureus infection, the invasion efficiency of S. aureus was determined quantitatively by measuring the number of cfu of viable organisms recovered from cultured KB cells. S. aureus invasion was found to be time dependent (0-60 min) and increased linearly when increasing numbers of bacteria were added (10(4)-10(6) cfu/ml). However, significant variation in the level of invasion was noted in protein A-deficient S. aureus Wood 46. Cytochalasin B inhibited the invasion efficiency of S. aureus in a dose-dependent manner. The present study suggests that interaction of staphylococcal protein A and cytoskeletal actin filaments is involved in the S. aureus invasion of cultured KB cells, and this process may contribute, in part, to the intracellular movement, cell-to-cell spread and dissemination of S. aureus within human oral epithelial cells in vivo.     

Dysregulation of mitogen-activated protein kinase signaling pathways in simian immunodeficiency virus encephalitis

Central nervous system (CNS) disease is a frequent complication of human immunodeficiency virus (HIV)-1 infection. Identification of cellular mechanisms that control virus replication and that mediate development of HIV-associated neuropathology will provide novel strategies for therapeutic intervention. The milieu of the CNS during HIV infection is extraordinarily complex because of infiltration of inflammatory cells and production of chemokines, cytokines, and neurotoxic molecules. Cells in the CNS must integrate signaling pathways activated simultaneously by products of virus replication and infiltrating immune cells. In this study, we examined activation of mitogen-activated protein kinases (MAPKs) in the CNS of simian immunodeficiency virus-infected macaques during acute, asymptomatic, and terminal infection. We demonstrate that significantly increased (P < 0.02) activation of ERK MAPK, typically associated with anti-apoptotic and neuroprotective pathways, occurs predominantly in astrocytes and immediately precedes suppression of virus replication and macrophage activation that occur after acute infection. In contrast, significantly increased activation of proapoptotic, neurodegenerative MAPKs JNK (P = 0.03; predominantly in macrophages/microglia), and p38 (P = 0.03; predominantly in neurons and astrocytes) after acute infection correlates with subsequent resurgent virus replication and development of neurological lesions. This shift from classically neuroprotective to neurodegenerative MAPK pathways suggests that agents that inhibit activation of JNK/p38 may be protective against HIV-associated CNS disease.     

Rho-kinase regulates thrombin-stimulated interleukin-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

We have previously reported that thrombin stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of thrombin in the thrombin-stimulated IL-6 synthesis and the involvement of Rho-kinase in MC3T3-E1 cells. Thrombin time-dependently induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and myosin phosphatase targeting subunit-1 (MYPT-1), a Rho-kinase substrate. While SP600125, an inhibitor of SAPK/JNK, failed to reduce IL-6 synthesis, PD98059, a specific inhibitor of MEK, and SB203580 and BIRB0796, potent inhibitors of p38 MAP kinase, suppressed the IL-6 synthesis induced by thrombin. Y27632, a specific Rho-kinase inhibitor, significantly reduced thrombin-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed thrombin-stimulated IL-6 synthesis. Y27632 and fasudil failed to affect thrombin-induced phosphorylation of p44/p42 MAP kinase. Y27632 as well as fasudil attenuated thrombin-induced phosphorylation of p38 MAP kinase. These results strongly suggest that Rho-kinase regulates thrombin-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

Involvement of the ERK mitogen-activated protein kinase in cell resistance to complement-mediated lysis

Sublytic doses of complement desensitize cells and make them resistant to lytic complement doses. This process, named complement-induced protection, requires calcium ion influx, protein kinase C activation and protein synthesis. The involvement of the extracellular signal-regulated kinase, ERK, in cell desensitization by sublytic complement was examined in erythroleukaemia K562 cells and in COS-7 cells. As shown here, ERK is activated in K562 and COS-7 cells within 10 min of sublytic immune attack and then shows a decline and a second peak of activation at 20 min. C7- and C8-deficient human sera have a small effect on ERK activity. However, a significant increase in ERK activation is observed when C7 or C8, respectively, is added back to these sera. Complement-induced ERK activation was blocked in cells treated with GF109203X or Go6976, two selective PKC inhibitors, as well as by treatment with PD098059, an inhibitor of MEK1, the ERK kinase. PD098059 treatment also sensitized K562 cells to complement-mediated lysis and prevented complement-induced protection. COS-7 cells transfected with a dominant-negative MEK plasmid were incapable of undergoing the process of complement-induced protection. In conclusion, cell desensitization by sublytic doses of the complement membrane attack complex involves a signalling cascade that includes PKC-mediated ERK activation.     

Thrombin induces striatal neurotoxicity depending on mitogen-activated protein kinase pathways in vivo

Intracerebral hemorrhage represents stroke characterized by formation and expansion of hematoma within brain parenchyma. Blood-derived factors released from hematoma are considered to be involved in poor prognosis of this disorder. We previously reported that thrombin, a blood-derived serine protease, induced cytotoxicity in the cerebral cortex and the striatum in organotypic slice cultures, which depended on mitogen-activated protein kinase (MAPK) pathways. Here we investigated the mechanisms of thrombin cytotoxicity in the striatum in vivo. Thrombin microinjected into the striatum of adult rats induced neuronal death and microglial activation around the injection site. Neuronal loss without any sign of nuclear fragmentation was observed as early as 4 h after thrombin injection, which was followed by gradual neuronal death exhibiting nuclear fragmentation. Thrombin-induced damage assessed at 72 h after injection was partially but significantly reduced by concomitant administration of inhibitors of MAPK pathways. Activation of extracellular signal-regulated kinase (ERK) and p38 MAPK in response to thrombin was verified by Western blot analysis. Moreover, phosphorylated ERK and p38 MAPK were localized prominently in reactive microglia, and inhibition of microglial activation by minocycline attenuated thrombin-induced damage, suggesting that reactive microglia were responsible for thrombin-induced neuronal death. Thus, MAPK pathways and microglial activation may serve as therapeutic targets of pathogenic conditions associated with hemorrhagic stroke.     

Involvement of p38-mitogen-activated protein kinase in Staphylococcus aureus-induced neutrophil apoptosis

Apoptosis occurred in human neutrophils within an hour of exposure to viable serum-opsonized Staphylococcus aureus, as indicated by appearance of cells with condensed nuclei, fragmented DNA, and increased phosphatidylserine exposure. In contrast, serum-opsonized, heat-killed S. aureus did not induce apoptosis. This discrepancy could not be explained by differences in bacterial uptake or total NADPH-oxidase activity. Suppressing phagocytosis by pretreating the neutrophils with cytochalasin b or by using nonopsonized bacteria did not prevent apoptosis. A supernatant from bacteria grown for 2 h in nutrient broth had a strong proapoptotic influence that was abrogated by heat treatment. Exposure to viable S. aureus or supernatant also led to activation of p38-mitogen-activated protein kinase in the neutrophils. Inhibition of this kinase with SB203580 reduced the apoptosis-inducing capacity of both bacteria and supernatant. We conclude that S. aureus activates p38-mitogen-activated protein kinase in neutrophils and induces apoptosis, probably mediated by a bacteria-derived soluble factor(s).     

Involvement of protein kinase C signalling and mitogen-activated protein kinase in the amebocyte-producing organ of Biomphalaria glabrata (Mollusca)

Mechanisms that regulate hemocyte production in molluscs, at either the organismal or cellular levels, are not well understood. In the present study, 24-h saline cultures of the amebocyte-producing organ (APO) of the schistosome-transmitting snail Biomphalaria glabrata were used to test for the potential involvement of protein kinase C (PKC) signalling in hematopoiesis. Exposure to phorbol myristate acetate (PMA), an activator of PKC, resulted in an increase in the number of dividing hematopoietic cells in APOs from schistosome-resistant Salvador snails. PMA-induced cell division was blocked by treatment with U0126, an inhibitor of the mitogen-activated protein kinase kinase, MEK1/2. These results suggest that PKC-induced activation of the mitogen-activated protein kinase, ERK1/2, is involved in cell division in the APO.     

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).     

Treponema denticola activates mitogen-activated protein kinase signal pathways through Toll-like receptor 2

Treponema denticola, a spirochete indigenous to the oral cavity, is associated with host inflammatory responses to anaerobic polymicrobial infections of the root canal, periodontium, and alveolar bone. However, the cellular mechanisms responsible for the recognition of T. denticola by the innate immune system and the underlying cell signaling pathways that regulate the inflammatory response to T. denticola are currently unresolved. In this study, we demonstrate that T. denticola induces innate immune responses via the utilization of Toll-like receptor 2 (TLR2) but not TLR4. Assessment of TLR2/1 and TLR2/6 heterodimers revealed that T. denticola predominantly utilizes TLR2/6 for the induction of cellular responses. Analysis of the mitogen-activated protein kinase (MAPK) signaling pathway in T. denticola-stimulated monocytes identified a prolonged up-regulation of the MAPK extracellular signal-related kinase 1/2 (ERK1/2) and p38, while no discernible increase in phospho-c-Jun N-terminal kinase 1/2 (JNK1/2) levels was observed. With the aid of pharmacological inhibitors selectively targeting ERK1/2 via the mitogen-activated protein kinase/extracellular signal-related kinase 1/2 kinase and p38, we further demonstrate that ERK1/2 and p38 play a major role in T. denticola-mediated pro- and anti-inflammatory cytokine production.     

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。     

Mechanisms of internalization of Staphylococcus aureus by cultured human osteoblasts

Staphylococcus aureus is an important bone pathogen, and evidence shows that this organism is internalized by chick osteoblasts. Here we report that S. aureus is internalized by human osteoblasts. Internalization was inhibited by monodansylcadaverine and cytochalasin D and to a lesser extent by ouabain, monensin, colchicine, and nocodazole. We propose that internalization occurs via a receptor-mediated pathway, requiring the participation of cytoskeletal elements, principally actin.     

Involvement of mitogen-activated protein kinase in peroxynitrite-induced cell death of human neuroblastoma SH-SY5Y cells

We have investigated the activation of mitogen-activated protein kinase (MAP kinase) in relation to cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Exposure of the cells to peroxynitrite caused transient increase in MAP kinase activity, and resulted in cell death. PD98059, a selective inhibitor of MAP kinase kinase, reduced peroxynitrite-induced cell death. These results suggest that the activation of MAP kinase may be involved in cell death induced by peroxynitrite.     

Atractylenolide I inhibits lipopolysaccharide-induced inflammatory responses via mitogen-activated protein kinase pathways in RAW264.7 cells

Atractylenolide I (ATL-I) is a bioactive component of Rhizoma Atractylodis macrocephalae. Although increasing evidence shows that ATL-I has an anti-inflammatory effect, the anti-inflammatory molecular mechanism of ATL-I is still unknown. In this study, we investigated the effect of ATL-I on cell viability by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and the level of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) by enzyme-linked immunosorbent assay (ELISA) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Further, we examined the effect of ATL-I on the activation of nuclear factor-kappaB (NF-κB) and phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) by Western blot. We also investigated the effect of ATL-I on the expression of myeloid differentiation protein-2 (MD-2), CD14, complement receptor 3 (CR3), scavenger receptor class A (SR-A), toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). We found that ATL-I showed no inhibitory effect on cell viability at concentrations ranging from 1?µM to 100?µM and markedly reduced the release of IL-6 and TNF-α at a concentrate-dependent manner. In addition, ATL-I suppressed the activity of nuclear NF-κB and the phosphorylation of ERK1/2 and p38 in LPS-treated RAW264.7 cells. Further analysis showed that ATL-I inhibited the expression of MD-2, CD14, SR-A, TLR4 and MyD88, but the expression of CR3 was unaffected. These data suggest that ATL-I shows an anti-inflammatory effect by inhibiting TNF-α and IL-6 production. The anti-inflammatory effects of ATL-I may be associated with the inhibition of the NF-κB, ERK1/2 and p38 signaling pathways.     

Staphylococcus aureus host cell invasion and post-invasion events

Staphylococcus aureus is now recognized as a facultative intracellular pathogen. The aim of this review is to discuss novel data regarding the invasion mechanism and post-invasion events with a focus on the fate of the infected phagosome in non-professional phagocytes and the role of S. aureus α-toxin.     

Activation of p38 mitogen-activated protein kinase during normal mitosis in the developing retina

The p38 member of the mitogen-activated protein kinase superfamily is engaged by phosphorylation in response to environmental stress signals, and may have either permissive or inhibitor roles upon cell proliferation. The cell cycle in the proliferative zone of the retina is tightly controlled and proceeds in synchrony with interkinetic migration of the neuroblast nuclei. We examined the association of p38 kinase activity with the cell cycle in the normal, non-stressed retina of the developing rat, maintained either in vivo or in vitro. Using immunohistochemistry, we show that mitotic profiles in the developing retina are highly enriched for phosphorylated p38. Blockade of p38 activity with the chemical inhibitor SB203580 for 4 h transiently arrested cells at the metaphase-anaphase transition and induced cell death after 20 h. p38 inhibition induced an aberrant mitotic profile, with chromosomes arranged in one side of the cell.The data show that p38 is active during normal mitosis and we suggest that p38 is required for the proper cell cycle progression during metaphase-anaphase transition in retinal neuroblasts.     

The sigma B regulon influences internalization of Staphylococcus aureus by osteoblasts

Individual strains of Staphylococcus aureus have different capacities to become internalized by osteoblasts. Here we report that the levels of sigma(B) expressed by S. aureus correlate with the capacity of this bacterium to be internalized by osteoblasts. However, sigma(B) is not essential for internalization and does not necessarily account for the differences in the capacities of strains to be internalized.     

丝裂原活化蛋白激酶信号途径在心肌肥厚中的作用进展

Myocardial hypertrophy is an independent risk factor for cardiac events. Mitogen-activated protein kinases(MAPK), including extracellular signal-regulated kinases, C-jun N-terminal kinases and P38-MAPK, are the common intracellular pathway of transducing hypertrophic signs. All three MAPK subfamilies play an important role in development of myocardial hypertrophy.