Deficiency of mitogen-activated protein kinase phosphatase-1 results in iNOS-mediated hypotension in response to low-dose endotoxin

2008 ◽  
Vol 294 (4) ◽  
pp. H1621-H1629 ◽  
Author(s):  
Thomas J. Calvert ◽  
Louis G. Chicoine ◽  
Yusen Liu ◽  
Leif D. Nelin
Keyword(s):  
Blood Pressure ◽  
Protein Kinase ◽  
Low Dose ◽  
Experimental Period ◽  
Mitogen Activated Protein Kinase ◽  
No Production ◽  
Wild Type ◽  
Proinflammatory Response ◽  
Arterial Blood ◽  
Mitogen Activated Protein

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential in limiting the proinflammatory response to lipopolysaccharide (LPS). We hypothesized that Mkp-1−/− mice would respond to low-dose LPS with a fall in blood pressure due to augmented expression of inducible nitric oxide (NO) synthase (iNOS). To test this hypothesis, Mkp-1−/− mice and their wild-type littermates were treated with 10 μg/kg iv LPS, and mean arterial blood pressure (MAP) and exhaled NO production (exNO) were measured. Tissues were harvested for an assessment of iNOS protein levels. Wild-type mice had no change in MAP or exNO during the experimental period, whereas Mkp-1−/− mice had a fall ( P < 0.005) in MAP [79 ± 5% of baseline (BL)] and an increase ( P < 0.01) in exNO (266 ± 50% of BL) after 150 min. The tissue levels of iNOS were greater in Mkp-1−/− than in wild-type mice. In additional experiments, 60 min after LPS treatment, Mkp-1−/− and wild-type mice were given Nω-nitro-l-arginine methyl ester (l-NAME) or aminoguanidine, and MAP and exNO were monitored for 90 min. Treatment with l-NAME prevented the LPS-induced increase in exNO and decrease in MAP but resulted in decreased exNO and elevated MAP in wild-type mice. Aminoguanidine prevented the increase in exNO and the fall in MAP caused by LPS in Mkp-1−/− mice, without significantly affecting MAP or exNO in wild-type mice. These results demonstrate that a deficiency of MKP-1 results in an exaggerated hypotensive response to LPS mediated by augmented iNOS expression. We speculate that defects in the Mkp-1 gene may increase susceptibility for the development of septic shock.

scholarly journals RICK Promotes Inflammation and Lethality after Gram-Negative Bacterial Infection in Mice Stimulated with Lipopolysaccharide

2009 ◽  
Vol 77 (4) ◽  
pp. 1569-1578 ◽  
Author(s):  
Jong-Hwan Park ◽  
Yun-Gi Kim ◽  
Gabriel Núñez
Keyword(s):  
Protein Kinase ◽  
Regulatory Protein ◽  
Mitogen Activated Protein Kinase ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Proinflammatory Response ◽  
Interacting Protein ◽  
Gram Negative ◽  
Mitogen Activated Protein

ABSTRACT RICK (receptor-interacting protein-like interacting caspase-like apoptosis regulatory protein kinase), a serine-threonine kinase, functions downstream of the pattern recognition receptors Nod1 and Nod2 to mediate NF-κB and mitogen-activated protein kinase (MAPK) activation in response to specific microbial stimuli. However, the function of RICK in the recognition and host defense of gram-negative bacteria remains poorly understood. We report here that infection of wild-type and RICK-deficient macrophages with Pseudomonas aeruginosa and Escherichia coli elicited comparable activation of NF-κB and MAPKs as well as secretion of proinflammatory cytokines. However, production of interleukin 6 (IL-6) and IL-1β induced by these gram-negative bacteria was impaired in RICK-deficient macrophages when the cells had previously been stimulated with lipopolysaccharide (LPS) or E. coli. The diminished proinflammatory response of RICK-deficient macrophages to bacteria was associated with reduced activation of NF-κB and MAPKs. Importantly, mutant mice deficient in RICK were less susceptible than wild-type mice to P. aeruginosa infection when the animals had previously been stimulated with LPS. The reduced lethality of RICK-deficient mice infected with P. aeruginosa was independent of pathogen clearance but was associated with diminished production of proinflammatory molecules in vivo. These results demonstrate that RICK contributes to the induction of proinflammatory responses and susceptibility to gram-negative bacteria after exposure to LPS, a condition that is associated with reduced Toll-like receptor signaling.

scholarly journals TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response to Toxoplasma gondii

2004 ◽  
Vol 72 (10) ◽  
pp. 5662-5667 ◽  
Author(s):  
Nicola J. Mason ◽  
Jim Fiore ◽  
Takashi Kobayashi ◽  
Katherine S. Masek ◽  
Yongwon Choi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Toxoplasma Gondii ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Interleukin 12 ◽  
Wild Type ◽  
Kinase Activation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-κB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasite Toxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-κB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-κB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6−/− mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6−/− mice failed to produce IL-12(p40) in response to STAg, and TRAF6−/− macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6−/− macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.

scholarly journals A single amino acid substitution (N297A) in the conserved NPXXY sequence of the human N-formyl peptide receptor results in inhibition of desensitization and endocytosis, and a dose-dependent shift in p42/44 mitogen-activated protein kinase activation and chemotaxis

2000 ◽  
Vol 352 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Jeannie M. GRIPENTROG ◽  
Algirdas J. JESAITIS ◽  
Heini M. MIETTINEN
Keyword(s):  
Protein Kinase ◽  
Transmembrane Domain ◽  
Mitogen Activated Protein Kinase ◽  
Formyl Peptide Receptor ◽  
Wild Type ◽  
Peptide Receptor ◽  
Formyl Peptide ◽  
Mitogen Activated Protein ◽  
Dose Dependent Increase ◽  
Dose Dependent

The formyl peptide receptor (FPR) is a G-protein-coupled receptor (GPCR) that mediates chemotaxis and stimulates the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase pathway. We have examined the functional effects of substitutions of a conserved aspartic acid residue in the second transmembrane domain (D71A) and of residues in the conserved NPXXY motif in the seventh transmembrane domain (N297A and Y301A). These mutated receptors, expressed in Chinese hamster ovary (CHO) cells, bind ligand with affinities similar to wild-type FPR, but the D71A mutant is uncoupled from G-protein [Miettinen, Mills, Gripentrog, Dratz, Granger and Jesaitis (1997) J. Immunol 159, 4045–4054]. In the present study, we show that both the D71A and N297A mutations resulted in defective endocytosis. The N297A substitution also prevented desensitization, as determined by intracellular calcium mobilization by sequential stimulation with ligand. In chemotaxis assays, the N297A mutation resulted in cell migration towards gradients of up to 100nM N-formyl-methionyl-leucyl-phenylalanine (fMLF), whereas cells expressing the wild-type FPR and the Y301A mutant were no longer chemotactically responsive at 10–100nM fMLF. Maximal activation of p42/44 MAPK occurred in CHO cells expressing wild-type FPR at 10nM–100nM fMLF, whereas cells expressing the N297A mutant showed a dose-dependent increase in the amount of phosphorylated p42/44 MAPK up to 1–10µM fMLF. Since the MAPK kinase inhibitor PD98059 blocked fMLF-induced chemotaxis, our results suggest that the dose-dependent increase in p42/44 MAPK activation may correlate with the increased chemotactic migration of N297A transfectants at 10nM–100nM fMLF.

scholarly journals Concerted Activity of Tyrosine Phosphatase SHP-2 and Focal Adhesion Kinase in Regulation of Cell Motility

1999 ◽  
Vol 19 (4) ◽  
pp. 3125-3135 ◽  
Author(s):  
Santos Mañes ◽  
Emilia Mira ◽  
Concepción Gómez-Mouton ◽  
Zhizuang Joe Zhao ◽  
Rosa Ana Lacalle ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Cell Motility ◽  
Focal Adhesion ◽  
Tyrosine Phosphatase ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Igf I ◽  
Mitogen Activated Protein ◽  
Mcf 7

ABSTRACT The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Using MCF-7 cells, we found that insulin-like growth factor I (IGF-I) induces the adhesion of MCF-7 to vitronectin and collagen in a dose- and time-dependent manner, suggesting that IGF-I triggers the activation of different integrins. On the other hand, IGF-I promotes the association of insulin receptor substrate 1 with the focal adhesion kinase (FAK), paxillin, and the tyrosine phosphatase SHP-2, resulting in FAK and paxillin dephosphorylation. Abrogation of SHP-2 catalytic activity with a dominant-negative mutant (SHP2-C>S) abolishes IGF-I-induced FAK dephosphorylation, and cells expressing SHP2-C>S show reduced IGF-I-stimulated chemotaxis compared with either mock- or SHP-2 wild-type-transfected cells. This impairment of cell migration is recovered by reintroduction of a catalytically active SHP-2. Interestingly, SHP-2-C>S cells show a larger number of focal adhesion contacts than wild-type cells, suggesting that SHP-2 activity participates in the integrin deactivation process. Although SHP-2 regulates mitogen-activated protein kinase activity, the mitogen-activated protein kinase kinase inhibitor PD-98059 has only a marginal effect on MCF-7 cell migration. The role of SHP-2 as a general regulator of cell chemotaxis induced by other chemotactic agents and integrins is discussed.

scholarly journals The Phosphotyrosine Phosphatase SHP-2 Participates in a Multimeric Signaling Complex and Regulates T Cell Receptor (TCR) coupling to the Ras/Mitogen-activated Protein Kinase (MAPK) Pathway in Jurkat T Cells

1998 ◽  
Vol 187 (9) ◽  
pp. 1417-1426 ◽  
Author(s):  
Julie A. Frearson ◽  
Denis R. Alexander
Keyword(s):  
T Cells ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
T Cell Receptor ◽  
Mapk Pathway ◽  
Cell Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Jurkat T Cells ◽  
Mitogen Activated Protein

Src homology 2 (SH2) domain–containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase–mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-ζ chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2–associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3′-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3′-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process.

scholarly journals Stress-Activated Protein Kinase Pathway Functions To Support Protein Synthesis and Translational Adaptation in Response to Environmental Stress in Fission Yeast

2005 ◽  
Vol 4 (11) ◽  
pp. 1785-1793 ◽  
Author(s):  
Isabelle Dunand-Sauthier ◽  
Carol A. Walker ◽  
Jana Narasimhan ◽  
Amanda K. Pearce ◽  
Ronald C. Wek ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Osmotic Stress ◽  
Environmental Stress ◽  
Fission Yeast ◽  
Translation Initiation ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Eif2α Phosphorylation ◽  
Mitogen Activated Protein

ABSTRACT The stress-activated protein kinase (SAPK) pathway plays a central role in coordinating gene expression in response to diverse environmental stress stimuli. We examined the role of this pathway in the translational response to stress in Schizosaccharomyces pombe. Exposing wild-type cells to osmotic stress (KCl) resulted in a rapid but transient reduction in protein synthesis. Protein synthesis was further reduced in mutants disrupting the SAPK pathway, including the mitogen-activated protein kinase Wis1 or the mitogen-activated protein kinase Spc1/Sty1, suggesting a role for these stress response factors in this translational control. Further polysome analyses revealed a role for Spc1 in supporting translation initiation during osmotic stress, and additionally in facilitating translational adaptation. Exposure to oxidative stress (H2O2) resulted in a striking reduction in translation initiation in wild-type cells, which was further reduced in spc1 − cells. Reduced translation initiation correlated with phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) in wild-type cells. Disruption of Wis1 or Spc1 kinase or the downstream bZip transcription factors Atf1 and Pap1 resulted in a marked increase in eIF2α phosphorylation which was dependent on the eIF2α kinases Hri2 and Gcn2. These findings suggest a role for the SAPK pathway in supporting translation initiation and facilitating adaptation to environmental stress in part through reducing eIF2α phosphorylation in fission yeast.

scholarly journals Phosphoprotein Enriched in Astrocytes-15 kDa Expression Inhibits Astrocyte Migration by a Protein Kinase Cδ-dependent Mechanism

2006 ◽  
Vol 17 (12) ◽  
pp. 5141-5152 ◽  
Author(s):  
François Renault-Mihara ◽  
Frédéric Beuvon ◽  
Xavier Iturrioz ◽  
Brigitte Canton ◽  
Sophie De Bouard ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Bryostatin 1 ◽  
Calcium Calmodulin Dependent ◽  
Mitogen Activated Protein ◽  
Green Fluorescent

Phosphoprotein enriched in astrocytes-15 kDa (PEA-15), a phosphoprotein enriched in astrocytes, inhibits both apoptosis and proliferation in normal and cancerous cells. Here, analysis of PEA-15 expression in glioblastoma organotypic cultures revealed low levels of PEA-15 in tumor cells migrating away from the explants, regardless of the expression levels in the originating explants. Because glioblastomas are highly invasive primary brain tumors that can originate from astrocytes, we explored the involvement of PEA-15 in the control of astrocyte migration. PEA-15−/− astrocytes presented an enhanced motility in vitro compared with their wild-type counterparts. Accordingly, NIH-3T3 cells transfected by green fluorescent protein-PEA-15 displayed a reduced migration. Reexpression of PEA-15 restored PEA-15−/− astrocyte motility to wild-type levels. Pharmacological manipulations excluded a participation of extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and calcium/calmodulin-dependent protein kinase II in this effect of PEA-15. In contrast, treatment by bisindolylmaleimide, Gö6976, and rottlerin, and chronic application of phorbol 12-myristate 13-acetate and/or bryostatin-1 indicated that PKCδ mediated PEA-15 inhibition of astrocyte migration. PEA-15−/− astrocytes constitutively expressed a 40-kDa form of PKCδ that was down-regulated upon PEA-15 reexpression. Together, these data reveal a new function for PEA-15 in the inhibitory control of astrocyte motility through a PKCδ-dependent pathway involving the constitutive expression of a catalytic fragment of PKCδ.

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