Role of mitogen-activated protein kinase-mediated cytosolic phospholipase A2 activation in arachidonic acid metabolism in human eosinophils.

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A(2) (PLA(2)) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA(2) activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC(50) = 8.5 nM)- and time-dependent (t(1/2) = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C(4) (LTC(4)) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA(2) (cPLA(2)) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A(2) by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca(2+)-independent phospholipase A(2) inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC(4) release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1-5 min, and declined thereafter. fMLP stimulation also increased cPLA(2) activity in eosinophils, which was inhibited completely by 30 microM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA(2) activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA(2) activation causes AA hydrolysis and LTC(4) secretion. We also find that cPLA(2) activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA(2) isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC(4).     

Activation of Transient Receptor Potential Vanilloid 4 Promotes the Proliferation of Stem Cells in the Adult Hippocampal Dentate Gyrus

Neurogenesis plays an important role in adult hippocampal function, and this process can be modulated by intracellular calcium. The activation of transient receptor potential vanilloid 4 (TRPV4) induces an increase in intracellular calcium concentration, but whether neurogenesis can be modulated by TRPV4 activation remains unclear. Here, we report that intracerebroventricular injection of the TRPV4 agonist GSK1016790A for 5 days enhanced the proliferation of stem cells in the hippocampal dentate gyrus (DG) of adult mice without affecting neurite growth, differentiation, or survival of newborn cells. GSK1016790A induced increases in the hippocampal protein levels of cyclin-dependent kinase (CDK) 6, CDK2, cyclin E1, and cyclin A2 but did not affect CDK4 and cyclin D1 expression. The phosphorylation of retinoblastoma protein (Rb) in hippocampi was enhanced in GSK1016790A-injected mice compared with control mice. Moreover, hippocampal protein levels of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation were enhanced by GSK1016790A. Finally, GSK1016790A-enhanced proliferation was markedly blocked by a MAPK/ERK kinase or p38 MAPK antagonist (U0126 or SB203580, respectively). The increased protein levels of CDK2 and CDK6, as well as those of cyclin E1 and cyclin A2, in GSK1016790A-injected mice were substantially reduced by co-injection of U0126 or SB203580. We conclude that TRPV4 activation results in the proliferation of stem cells in the adult hippocampal DG, which is likely mediated through ERK1/2 and p38 MAPK signaling to increase the expression of CDKs (CDK6 and CDK2) and cyclins (cyclin E1 and A2), phosphorylate Rb consequently, and accelerate the cell cycle ultimately.     

Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model

Mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in the activation of inflammatory cells. Recent findings revealed that the activity of p42/44 MAPK (also known as extracellular signal-regulated kinase (ERK)) in the lungs was significantly higher in asthmatic mice than in normal controls. We hypothesized that inhibition of ERK activity may have anti-inflammatory effects in allergic asthma. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of VCAM-1 expression, and airway hyperresponsiveness. Intraperitoneal administration of U0126, a specific MAPK/ERK kinase inhibitor, significantly (p < 0.05) inhibited OVA-induced increases in total cell counts, eosinophil counts, and IL-4, IL-5, IL-13, and eotaxin levels recovered in bronchoalveolar lavage fluid in a dose-dependent manner. U0126 also substantially (p < 0.05) reduced the serum levels of total IgE and OVA-specific IgE and IgG1. Histological studies show that U0126 dramatically inhibited OVA-induced lung tissue eosinophilia, airway mucus production, and expression of VCAM-1 in lung tissues. In addition, U0126 significantly (p < 0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine in a dose-dependent manner. Western blot analysis of whole lung lysates shows that U0126 markedly attenuated OVA-induced tyrosine phosphorylation of ERK1/2. Taken together, our findings implicate that inhibition of ERK signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.     

4-Aminopyridine-induced epileptogenesis depends on activation of mitogen-activated protein kinase ERK

Extracellular signal-regulated kinases such as ERK1 [p44 mitogen-activated protein kinase (MAPK)] and ERK2 (p42 MAPK) are activated in the CNS under physiological and pathological conditions such as ischemia and epilepsy. Here, we studied the activation state of ERK1/2 in rat hippocampal slices during application of the K(+) channel blocker 4-aminopyridine (4AP, 50 micro m), a procedure that enhances synaptic transmission and leads to the appearance of epileptiform activity. Hippocampal slices superfused with 4AP-containing medium exhibited a marked activation of ERK1/2 phosphorylation that peaked within about 20 min. These effects were not accompanied by changes in the activation state of c-Jun N-terminal kinase (JNK), another member of the MAP kinase superfamily. 4AP-induced ERK1/2 activation was inhibited by the voltage-gated Na(+) channel blocker tetrodotoxin (1 micro m). We also found that application of the ERK pathway inhibitors U0126 (50 micro m) or PD98059 (100 micro m) markedly reduced 4AP-induced epileptiform synchronization, thus abolishing ictal discharges in the CA3 area. The effects induced by U0126 or PD98059 were not associated with changes in the amplitude and latency of the field potentials recorded in the CA3 area following electrical stimuli delivered in the dentate hylus. These data demonstrate that activation of ERK1/2 accompanies the appearance of epileptiform activity induced by 4AP and suggest a cause-effect relationship between the ERK pathway and epileptiform synchronization.     

Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

Coxsackievirus B3 (CVB3) is the most common human pathogen for viral myocarditis. We have previously shown that the signaling protein p21(ras) GTPase-activating protein (RasGAP) is cleaved and that mitogen-activated protein kinases (MAPKs) ERK1/2 are activated in the late phase of CVB3 infection. However, the role of intracellular signaling pathways in CVB3-mediated myocarditis and the relative advantages of such pathways to host or virus remain largely unclear. In this study we extended our prior studies by examining the interaction between CVB3 replication and intracellular signaling pathways in HeLa cells. We observed that CVB3 infection induced a biphasic activation of ERK1/2, early transient activation versus late sustained activation, which were regulated by different mechanisms. Infection by UV-irradiated, inactivated virus capable of receptor binding and endocytosis triggered early ERK1/2 activation, but was insufficient to trigger late ERK1/2 activation. By using a general caspase inhibitor (zVAD.fmk) we further demonstrated that late ERK1/2 activation was not a result of CVB3-mediated caspase cleavage. Treatment of cells with U0126, a selective inhibitor of MAPK kinase (MEK), significantly inhibited CVB3 progeny release and decreased virus protein production. Furthermore, inhibition of ERK1/2 activation circumvented CVB3-induced apoptosis and viral protease-mediated RasGAP cleavage. Taken together, these data suggest that ERK1/2 activation is important for CVB3 replication and contributes to virus-mediated changes in host cells. Our findings demonstrate coxsackievirus takeover of a particular host signaling mechanism and uncover a prospective approach to stymie virus spread and preserve myocardial integrity.     

Multiple signaling cascades are differentially involved in gene induction by double stranded RNA in interferon-alpha-primed cells.

Priming with interfon (IFN)alpha enhanced the ability of the synthetic double-stranded RNA polyriboinosinic acid: polyribocytidilic acid (pI:C), but not interleukin-1 beta, to activate both p38 mitogen-activated kinase (MAPK) and extracellular signal-regulated kinase (ERK) signaling cascades. Activation by pI:C in IFN alpha-primed cells was delayed compared to activation with interleukin-1 beta, and this delay was followed by high, sustained activation of p38 MAPK and a modest elevation of ERK activation. Pharmacologic inhibition of either the ERK or the p38 MAPK pathway, using U0126 and SB203580, respectively, reduced interleukin-6 protein induction by at least 70%, and combined inhibition of both pathways fully blocked interleukin-6 protein expression and reduced interleukin-6 mRNA induction by more than 80%. In contrast, induction of double-stranded RNA-activated protein kinase (PKR) mRNA and protein by IFN alpha and/or pI:C was minimally affected by either inhibitor. Induction of interferon-regulatory factor-1 (IRF-1) by pI:C in IFN alpha primed cells was profoundly inhibited by U0126 but not by SB203580. Thus, IFN alpha priming enhances activation of p38 MAPK and ERK pathways by pI:C but not by interleukin-1 beta, thereby enhancing the expression of some, but not all, genes that are induced by pI:C.     

MAP kinases in lung endothelial permeability induced by microtubule disassembly

Lung endothelial barrier function is regulated by multiple signaling pathways, including mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK) 1/2 and p38. We have recently shown involvement of microtubule (MT) disassembly in endothelial cell (EC) barrier failure. In this study, we examined potential involvement of ERK1/2 and p38 MAPK in lung EC barrier dysfunction associated with MT disassembly. MT inhibitors nocodazole (0.2 microM) and vinblastine (0.1 microM) induced sustained activation of Ras-Raf-MEK1/2-ERK1/2 and MKK3/6-p38-MAPKAPK2 MAPK cascades in human and bovine pulmonary EC, as detected by phosphospecific antibodies and in MAPK activation assays. These effects were linked to increased permeability assessed by measurements of transendothelial electrical resistance and cytoskeletal remodeling analyzed by morphometric analysis of EC monolayers. MT stabilization by taxol (5 microM, 1 h) attenuated nocodazole-induced ERK1/2 and p38 MAPK activation and phosphorylation of p38 MAPK substrate 27-kDa heat shock protein and regulatory myosin light chains, the proteins involved in actin polymerization and actomyosin contraction. Importantly, only pharmacological inhibition of p38 MAPK by SB-203580 (20 microM, 1 h) attenuated nocodazole-induced MT depolymerization, actin remodeling, and EC barrier dysfunction, whereas the MEK/ERK1/2 inhibitor U0126 (5 microM, 1 h) exhibited no effect. These data suggest a direct link between p38 MAPK activation, remodeling of MT network, and EC barrier regulation.     

Mitogen-activated protein kinase signalling pathways triggered by the hepatitis C virus envelope protein E2: implications for the prevention of infection

OBJECTIVE: Hepatitis C virus (HCV) is a major pathogenic factor of liver diseases. During HCV infection, interaction of the envelope protein E2 of the virion, with target cells, is a crucial process for viral penetration into the cell and its propagation. We speculate that such interaction may trigger early signalling events required for HCV infection. MATERIALS AND METHODS: Human liver cell line L-02 was treated with HCV E2. The kinase phosphorylation levels of mitogen-activated protein kinase (MAPK) signalling pathways in the treated cells were analyzed by Western blotting. The proliferation of the E2-treated cells was evaluated by MTT assay. RESULTS: HCV E2 was shown to be an efficient activator for MAPK pathways. Levels of phosphorylation of upstream kinases Raf-1 and MEK1/2 were seen to be elevated following E2 treatment and similarly, phosphorylation levels of downstream kinases MAPK/ERK and p38 MAPK also increased in response to E2 treatment, and specificity of kinase activation by E2 was confirmed. E2-induced MAPK/ERK activation was inhibited by the MEK1/2 inhibitor U0126 in a concentration-dependent manner. Blockage of relevant cellular receptors reduced activation of Raf-1, MEK1/2, MAPK/ERK and p38 MAPK by E2, indicating efflux of the E2 signal from extracellular to the intracellular spaces. Thus, kinase cascades of MAPK pathways were continuously affected by E2 presence. Moreover, enhancement of cell proliferation by E2 appeared to be associated with the dynamic phosphorylation of MAPK/ERK and p38 MAPK. CONCLUSION: These results suggest that MAPK signalling pathways triggered by E2 may be a potential target for prevention of HCV infection.     

Murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells

Analyses of mitogen-activated protein kinases (MAPKs) in a mouse hepatitis virus (MHV)-infected macrophage-derived J774.1 cell line showed activation of two MAPKs, p38 MAPK and c-Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase (ERK). Activation of MAPKs was evident by 6 h postinfection. However, UV-irradiated MHV failed to activate MAPKs, which demonstrated that MHV replication was necessary for their activation. Several other MHV-permissive cell lines also showed activation of both p38 MAPK and JNK, which indicated that the MHV-induced stress-kinase activation was not restricted to any particular cell type. The upstream kinase responsible for activating MHV-induced p38 MAPK was the MAPK kinase 3. Experiments with a specific inhibitor of p38 MAPK, SB 203580, demonstrated that MHV-induced p38 MAPK activation resulted in the accumulation of interleukin-6 (IL-6) mRNAs and an increase in the production of IL-6, regardless of MHV-induced general host protein synthesis inhibition. Furthermore, MHV production was suppressed in SB 203580-treated cells, demonstrating that activated p38 MAPK played a role in MHV replication. The reduced MHV production in SB 203580-treated cells was, at least in part, due to a decrease in virus-specific protein synthesis and virus-specific mRNA accumulation. Interestingly, there was a transient increase in the amount of phosphorylation of the translation initiation factor 4E (eIF4E) in infected cells, and this eIF4E phosphorylation was p38 MAPK dependent; it is known that phosphorylated eIF4E enhances translation rates of cap-containing mRNAs. Furthermore, the upstream kinase responsible for eIF4E phosphorylation, MAPK-interacting kinase 1, was also phosphorylated and activated in response to MHV infection. Our data suggested that host cells, in response to MHV replication, activated p38 MAPK, which subsequently phosphorylated eIF4E to efficiently translate certain host proteins, including IL-6, during virus-induced severe host protein synthesis inhibition. MHV utilized this p38 MAPK-dependent increase in eIF4E phosphorylation to promote virus-specific protein synthesis and subsequent progeny virus production. Enhancement of virus-specific protein synthesis through virus-induced eIF4E activation has not been reported in any other viruses.     

MAP kinase dependent cyclinE/cdk2 activity promotes DNA replication in early sea urchin embryos

Sea urchins provide an excellent model for studying cell cycle control mechanisms governing DNA replication in vivo. Fertilization and cell cycle progression are tightly coordinated by Ca²⁺ signals, but the mechanisms underlying the onset of DNA replication after fertilization remain less clear. In this study we demonstrate that calcium-dependent activation of ERK1 promotes accumulation of cyclinE/cdk2 into the male and female pronucleus and entry into first S-phase. We show that cdk2 activity rises quickly after fertilization to a maximum at 4 min, corresponding in timing to the early ERK1 activity peak. Abolishing MAP kinase activity after fertilization with MEK inhibitor, U0126, substantially reduces the early peak of cdk2 activity and prevents cyclinE and cdk2 accumulation in both sperm pronucleus and zygote nucleus in vivo. Both p27^kip1 and roscovitine, cdk2 inhibitors, prevented DNA replication suggesting cdk2 involvement in this process in sea urchin. Inhibition of cdk2 activity using p27^kip1 had no effect on the phosphorylation of MBP by ERK, but completely abolished phosphorylation of retinoblastoma protein, a cdk2 substrate, indicating that cdk2 activity is downstream of ERK1 activation. This pattern of regulation of DNA synthesis conforms to the pattern observed in mammalian somatic cells.     

Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade

Influenza A viruses are important worldwide pathogens in humans and different animal species. The functions of most of the ten different viral proteins of this negative-strand RNA virus have been well elucidated. However, little is known about the virus-induced intracellular signalling events that support viral replication. The Raf/MEK/ERK cascade is the prototype of mitogen-activated protein (MAP) kinase cascades and has an important role in cell growth, differentiation and survival. Investigation of the function of this pathway has been facilitated by the identification of specific inhibitors such as U0126, which blocks the cascade at the level of MAPK/ERK kinase (MEK). Here we show that infection of cells with influenza A virus leads to biphasic activation of the Raf/MEK/ERK cascade. Inhibition of Raf signalling results in nuclear retention of viral ribonucleoprotein complexes (RNPs), impaired function of the nuclear-export protein (NEP/NS2) and concomitant inhibition of virus production. Thus, signalling through the mitogenic cascade seems to be essential for virus production and RNP export from the nucleus during the viral life cycle.     

The structure of SENP1-SUMO-2 complex suggests a structural basis for discrimination between SUMO paralogues during processing

eNOS (endothelial nitric oxide synthase) activity is post-translationally regulated in a complex fashion by acylation, protein-protein interactions, intracellular trafficking and phosphorylation, among others. Signalling pathways that regulate eNOS activity include phosphoinositide 3-kinase/Akt, cyclic nucleotide-dependent kinases [PKA (protein kinase A) and PKG], PKC, as well as ERKs (extracellular-signal-regulated kinases). The role of ERKs in eNOS activation remains controversial. In the present study, we have examined the role of ERK1/2 in eNOS activation in HUVEC-CS [transformed HUVEC (human umbilical-vein endothelial cells)] as well as a widely used model for eNOS study, transiently transfected COS-7 cells. U0126 pretreatment of HUVEC-CS potentiated ATP-stimulated eNOS activity, independent of changes in intracellular Ca2+ concentration ([Ca2+]i). In COS-7 cells transiently expressing ovine eNOS, U0126 potentiated A23187-stimulated eNOS activity, but inhibited ATP-stimulated activity. Compensatory changes in phosphorylation of five key eNOS residues did not account for changes in A23187-stimulated activity. However, in the case of ATP, altered phosphorylation and changes in [Ca2+]i may partially contribute to U0126 inhibition of activity. Finally, seven eNOS alanine mutants of putative ERK1/2 targets were generated and the effects of U0126 pretreatment on eNOS activity were gauged with A23187 and ATP treatment. T97A-eNOS was the only construct significantly different from wild-type after U0126 pretreatment and ATP stimulation of eNOS activation. In the present study, eNOS activity was either potentiated or inhibited in COS-7 cells, suggesting agonist dependence for MEK/ERK1/2 signalling [where MEK is MAPK (mitogen-activated protein kinase)/ERK kinase] to eNOS and a complex mechanism including [Ca2+]i, phosphorylation and, possibly, intracellular trafficking.     

LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.     

Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells

Neurotensin (NT) and epidermal growth factor (EGF) induced rapid extracellular-regulated protein kinase (ERK) activation through different signaling pathways in the K-Ras mutated human pancreatic carcinoma cell lines PANC-1 and MIA PaCa-2. NT stimulated ERK activation via a protein kinase C (PKC)-dependent (but EGF receptor-independent) pathway in PANC-1 and MIA PaCa-2 cells, whereas EGF promoted ERK activation through a PKC-independent pathway in these cells. Concomitant stimulation of these cells with NT and EGF induced a striking increase in the duration of ERK pathway activation as compared with that obtained in cells treated with each agonist alone. Stimulation with NT + EGF promoted synergistic stimulation of DNA synthesis and anchorage-independent growth. Addition of the MEK inhibitor U0126, either prior to stimulation with NT + EGF or 2 h after stimulation with NT + EGF prevented the synergistic increase in DNA synthesis and suppressed the sustained phase of ERK activation. Furthermore, treatment with the selective PKC inhibitor GF-1 converted the sustained ERK activation in response to NT and EGF into a transient signal and also abrogated the synergistic increase in DNA synthesis. Collectively, our results suggest that the sustained phase of ERK signaling mediates the synergistic effects of NT and EGF on DNA synthesis in pancreatic cancer cells.