Ethanol-induced mitogen activated protein kinase activity mediated through protein kinase C.

The aim of this study was to determine the pathway(s) by which ethanol activates mitogen-activated protein kinase (MAPK) signaling and to determine the role of Ca2+ in the signaling process. MAPK signaling was determined by assessing MAPK activity, measuring phosphorylated extracellular signaling-regulated kinase (pp 44 ERK-1 and pp 42 ERK-2) expression and ERK activity by measuring ERK-2-dependent phosphorylation of a synthetic peptide as a MAPK substrate in rat vascular smooth muscle cells. Ethanol activated extracellular signal-regulated kinase expression (ERK 1 and 2) could be observed when vascular smooth muscle cells (VSMCs) were stimulated for 5 min or less, but was inhibited when cells are treated for 10 min or more with 1-16 mM of ethanol. Maximum ethanol-induced MAPK activity was observed within 5 min with 4 or 8 mM. Ethanol stimulated MAPK activity was blocked by the protein kinase C (PKC) inhibitor (GF109203X) and epidermal growth factor (EGF) receptor antagonist (PD153035) by 41 +/- 24 and 34 +/- 12.3%, respectively. The calcium channel blocker, diltiazem and the chelating agent, BAPTA, reduced the activation of MAPK activity by ethanol, significantly. The data demonstrate that ethanol-stimulated MAPK expression is mediated partially through both the EGF-receptor and PKC intermediates and that activation through the PKC intermediate is calcium-dependent.     

Synthesis of robust tunable oscillators using mitogen activated protein kinase cascades

Mitogen activated protein kinase (MAPK) cascade is evolutionally preserved in all eukaryotic cells, and regulates various cellular activities such as gene expression, mitosis, differentiation, and apoptosis. Recently, Bashor et al. have shown that Ste5 scaffold protein can be used to reshape the MAPK cascade through engineered feedback loops, and have used heuristic tuning mechanisms to synthesize the feedback. A problem of interest is to determine whether information regarding the underlying biochemical reactions can be used to synthesize robust feedback that will ensure that the resultant circuit has the desired properties. In this paper, we consider the problem of engineering feedback in MAPK cascade to synthesize an oscillator of the desired frequency. Our approach builds on the MAPK cascade model derived by Chikarmane et al. who have exploited the existence of a Hopf bifurcation point in the Markevich model of the MAPK cascade to synthesize the exciting kinase as a function of the doubly phosphorylated protein. We show how the L₁\mathcal{L}_1-control theory can be used for a robust synthesis of the oscillator and present the simulation results.     

Comparative genomic analysis of mitogen activated protein kinase gene family in grapevine

Mitogen activated protein kinases (MAPKs) are important proteins involved in the signal transduction of extracellular information to intracellular targets, and play a crucial role in the response to biotic and abiotic stresses. Although Arabidopsis MAPKs are used for identification of the putative MAPKs in higher plants, no grapevine MAPK gene nomenclature has yet been appeared in the literature. In this study, we have identified 12 members of grapevine MAPK gene (VvMPK) family via In-silico analysis of current grapevine genome database. The structural comparison of 12 VvMPKs through the analysis of chromosome locations, sequence annotation and paralogous gene pair indicated that VvMPKs have evolved by segmental duplication, rather than by tandem amplification. Although further functional analysis of VvMPKs through in vivo and in vivo experiments will be required, our study provides the basis for future research on the diverse signaling pathways medicated by MAPKs in grapevine.     

New tumor suppressor CXXC finger protein 4 inactivates mitogen activated protein kinase signaling

As a well-characterized master player in epigenetic regulatory network, EZH2 is widely implicated in the development of many malignancies. We previously found that EZH2 promoted Wnt/β-catenin activation through downregulation of CXXC4 expression. In this report, we demonstrated that CXXC4 inhibited MAPK signaling through binding to ERK-1/2 and abrogating the interaction of ERK 1/2 with MEK1/2. L183, the critical residue in CXXC4 ERK D domain, was found to be essential for CXXC4–ERK 1/2 interaction and the growth inhibitory effect of CXXC4 in human cancer cells. In summary, CXXC4 directly disrupted MEK1/2–ERK 1/2 interaction to inactivate MAPK signaling. L183 site is indispensable for the binding of CXXC4 to ERK1/2 and growth inhibitory effect of CXXC4. Therefore, EZH2 can activate MAPK signaling by inhibiting CXXC4 expression.     

Berberine chloride mediates its anti-leishmanial activity via differential regulation of the mitogen activated protein kinase pathway in macrophages

Background

A complex interplay between Leishmania and macrophages influences parasite survival and necessitates disruption of signaling molecules, eventually resulting in impairment of macrophage function. In this study, we demonstrate the immunomodulatory activity of Berberine chloride in Leishmania infected macrophages.

Principal Findings

The IC₅₀ of Berberine chloride, a quaternary isoquinoline alkaloid was tested in an amastigote macrophage model and its safety index measured by a cell viability assay. It eliminated intracellular amastigotes, the IC₅₀ being 2.8 fold lower than its IC₅₀ in promastigotes (7.10 µM vs. 2.54 µM) and showed a safety index >16. Levels of intracellular and extracellular nitric oxide (NO) as measured by flow cytometry and Griess assay respectively showed that Berberine chloride in Leishmania infected macrophages increased production of NO. Measurement of the mRNA expression of iNOS, IL-12 and IL-10 by RT-PCR along with levels of IL-12p40 and IL-10 by ELISA showed that in infected macrophages, Berberine chloride enhanced expression of iNOS and IL-12p40, concomitant with a downregulation of IL-10. The phosphorylation status of extracellular signal related kinase (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK) was studied by western blotting. In infected macrophages, Berberine chloride caused a time dependent activation of p38 MAPK along with deactivation of ERK1/2; addition of a p38 MAPK inhibitor SB203580 inhibited the increased generation of NO and IL-12p40 by Berberine chloride as also prevented its decrease of IL-10.

Conclusions

Berberine chloride modulated macrophage effector responses via the mitogen activated protein kinase (MAPK) pathway, highlighting the importance of MAPKs as an antiparasite target.     

P38 mitogen activated protein kinase expression and regulation by interleukin-4 in human B cell non-Hodgkin lymphomas

The prevalence and regulation of p38 mitogen activated protein kinase (MAPK) expression in human lymphomas have not been extensively studied. In order to elucidate the role of p38 MAPK in lymphomagenesis, we examined the expression of native and phosphorylated p38 (p-p38) MAPK in cell lines derived from human hematopoietic neoplasms including B cell lymphoma-derived cell lines using Western blot analysis. The p-p38 MAPK protein was also analyzed in 30 B cell non-Hodgkin lymphoma (NHL) tissue biopsies by immunohistochemistry. Our results show that the expression of p38 MAPK was up-regulated in most of the cell lines as compared with peripheral blood lymphocytes, while the expression of p-p38 MAPK was more variable. A subset of B cell NHL biopsies showed increased expression of p-p38 MAPK relative to reactive germinal center cells. Interleukin-4 (IL-4) induced a dose-dependent increase in the expression of p-p38 MAPK (1.6- to 2.8-fold) in cell lines derived from activated B cell-like diffuse large B cell lymphoma (DLBCL) but not those from germinal center-like DLBCL. No change was seen in native p38 MAPK. The in vitro kinase activity of p38 MAPK, however, was induced (1.6- to 3.2-fold) in all five cell lines by IL-4. Quantitative fluorescent RT-PCR demonstrated that all four isoforms of p38 MAPK gene were expressed in the lymphoma cell lines, with p38γ and p38β isoforms being predominant. IL-4 stimulation increased the expression of β, γ, and δ isoforms but not α isoform in two cell lines. In conclusion, there is constitutive expression and activation of p38 MAPK in a large number of B-lymphoma-derived cell lines and primary lymphoma tissues, supportive of its role in lymphomagenesis. The differential IL-4 regulation of p38 MAPK expression in cell lines derived from DLBCL may relate to the cellular origin of these neoplasms.

Electronic supplementary material

The online version of this article (doi:10.1007/s12308-009-0049-5) contains supplementary material, which is available to authorized users.     

Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway

Introduction

The c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis.

Methods

Three 2''-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis.

Results

MKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1β), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01).

Conclusions

MKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.     

Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway

Musculoskeletal pain is common across all populations and costly in terms of impact on the individual and, more generally, on society. In most health-care systems, the first person to see the patient with a musculoskeletal problem such as back pain is the general practitioner, and access to other professionals such as physiotherapists, chiropractors, or osteopaths is still either largely controlled by a traditional medical model of referral or left to self-referral by the patient. In this paper, we examine the arguments for the general practitioner-led model and consider the arguments, and underpinning evidence, for reconsidering who should take responsibility for the early assessment and treatment of patients with musculoskeletal problems.     

Left ventricular mitogen activated protein kinase signaling following polymicrobial sepsis during streptozotocin-induced hyperglycemia

We hypothesized that sepsis during hyperglycemia would activate left ventricular (LV) mitogen activated protein kinase (MAPK) signaling mechanisms and modulate generation of endothelin-1 (ET-1) and nitric oxide (NO) that can contribute to the progression of LV dysfunction. A single injection of streptozotocin (STZ, 60 mg/kg, via tail vein) was used to produce type 2 diabetes in male SD rats. Polymicrobial sepsis and sham-sepsis were induced using single i.p. injection of cecal inoculum and sterile 5% dextrose water, respectively, on the 13th and 27th day following STZ injection. Both 2-week (2-wk) and 4-wk diabetes groups were associated with hyperglycemia and weight loss. LV end diastolic pressure (LVEDP) was significantly increased in 4-wk diabetes but not in 2-wk diabetes group. Plasma concentration of tumor necrosis factor-alpha (TNF-alpha) was significantly increased in 4-wk diabetes+sepsis group as compared to sham, 2-wk diabetes+sepsis and sepsis groups. Elevated plasma and LV ET-1 and NO byproducts (NOx) along with LV preproET-1 and inducible nitric oxide synthase (iNOS) protein expression were observed in 4-wk but not in 2-wk diabetes group. Sepsis further elevated LV iNOS and preproET-1 in 4-wk diabetes group. Up-regulated phosphorylation of LV p38-MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2) and heat shock protein-27 (Hsp27) was observed in 4-wk diabetes group. Sepsis caused a factorial increase in LV p38-MAPK and Hsp27 phosphorylation and iNOS up-regulation but not ERK1/2 following progression from 2-wk to 4-wk diabetes. The study provides evidence that sepsis up-regulated LV iNOS, p38-MAPK phosphorylation and elevated LVEDP during 4-wk diabetes. We concluded that sepsis contributes in the development of LVEDP dysfunction and alteration in signaling mechanisms depending upon the progression from 2-wk to 4-wk diabetes in the rat.     

Oxidized low density lipoproteins stimulate galactosyltransferase activity, ras activation, p44 mitogen activated protein kinase and c-fos expression in aortic smooth muscle cells

Previously, our laboratory has shown that oxidized low densitylipoproteins (Ox-LDL) can exert a concentration-dependent stimulationin the proliferation of aortic smooth muscle cells, "a hallmarkin the pathogenesis of atherosclerosis" (Chatterjeey,S. (1992)Mol Cell Biochem., 111, 143–147). Here we report a novelaspect of Ox-LDL-mediated signal transduction. We demonstratethat in aortic smooth muscle cells, Ox-LDL stimulates the activityof a UDP-galactose:glucosylceramide ß1     

The relation between mitogen activated protein kinase (MAPK) pathway and different genes expression in patients with beta Thalassemia

Backgroundβ-thalassemia is an inherited hemoglobinopathy resulting in quantitative changes in the β-globin chain. Understanding the molecular basis of that disorder requires studying the expression of genes controlling the pathways that affect the erythropoietic homeostasis especially the MAPK pathway. The MAPKs are a family of serine/threonine kinases that play an essential role in connecting cell-surface receptors to DNA in the nucleus of the cell.Aimto study the effect of expression of GNAI2, DUSP5 and ARRB1 genes on MAPK signaling pathway in pediatric patients with beta thalassemia.MethodsForty children with beta thalassemia major (TM), forty children with beta thalassemia intermedia (TI) and forty age and gender matched healthy controls were enrolled in this study. Detection of GNAI2, DUSP5 and ARRB1 mRNA expression was done by real time polymerase chain reaction (RT-PCR).Resultsrevealed increased expression of ARRB1 (Arrestin Beta 1) gene, and decreased expression of both GNAI2 (Guanine nucleotide-binding protein G (i) subunit alpha-2) and DUSP5 (Dual specificity protein phosphatase 5) genes in both patient groups than control groups respectively.ConclusionsChange in the rate of expression of ARRB1, GNAI2 and DUSP5 may have a role in the pathogenesis of abnormal hematopoiesis in cases of β thalassemia through affecting the MAPK pathway.     

OS-2 mitogen activated protein kinase regulates the clock-controlled gene ccg-1 in Neurospora crassa

OS-2 MAP kinase is involved in osmoadaptation in Neurospora crassa. Clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an OS-2 dependent manner. In contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. These results suggest that OS-2 participates in the regulation of certain circadian-clock output genes.     

4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2)

A class of inhibitors of mitogen activated protein kinase-activated kinase 2 (MK2) was discovered via high-throughput screening. This compound class demonstrates activity against the enzyme with sub-μM IC₅₀ values, and suppresses LPS-induced TNFα levels in THP-1 cells. MK2 inhibition kinetic measurements indicated mixed binding approaching non-ATP competitive inhibition.     

Inhibition of mitogen activated protein kinase signaling affects gastrulation and spiculogenesis in the sea urchin embryo

The mitogen activated protein (MAP) kinase signaling cascade has been implicated in a wide variety of events during early embryonic development. We investigated the profile of MAP kinase activity during early development in the sea urchin, Strongylocentrotus purpuratus, and tested if disruption of the MAP kinase signaling cascade has any effect on developmental events. MAP kinase undergoes a rapid, transient activation at the early blastula stage. After returning to basal levels, the activity again peaks at early gastrula stage and remains high through the pluteus stage. Immunostaining of early blastula stage embryos using antibodies revealed that a small subset of cells forming a ring at the vegetal plate exhibited active MAP kinase. In gastrula stage embryos, no specific subset of cells expressed enhanced levels of active enzyme. If the signaling cascade was inhibited at any time between the one cell and early blastula stage, gastrulation was delayed, and a significant percentage of embryos underwent exogastrulation. In embryos treated with MAP kinase signaling inhibitors after the blastula stage, gastrulation was normal but spiculogenesis was affected. The data suggest that MAP kinase signaling plays a role in gastrulation and spiculogenesis in sea urchin embryos.     

Activation of mitogen activated protein kinase (MAPK) during D-galactosamine intoxication in the rat liver

A significant increase in plasma glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase was observed 6 h after intraperitoneal administration of D-galactosamine (D-Galn). Three hours after administration of D-Galn, the vitamin C concentration in the liver decreased significantly compared to that in a control group and thereafter the hepatic vitamin C concentration remained at a significantly lower level. Phosphorylated JNK (c-Jun NH2-terminal kinase) and phosphorylated ERK (extracellular signal-regulated kinase) started increasing 3 h after D-Galn treatment and remained at a high level for 6-12 h after the treatment, while phosphorylated p38 MAPK increased significantly 6 h after D-Galn administration. These results indicated that oxidative stress and the activation of JNK and ERK took place almost simultaneously, followed by the activation of p38 MAPK.     

A computer-based model for the regulation of mitogen activated protein kinase (MAPK) activation

Computer simulations and mathematical modeling of biological processes are becoming increasingly popular, and yet the complexity of the biochemical systems or the differences between experimental setups make it very difficult to establish a standard formula for these modeling projects. Before we can start using computer-based models for predictions or targeted experiment designs, it is very important to establish a reliable model on which those predictions can be based and experimentally tested. Here we attempt to present a computer model for the mitogen-activated protein kinase (MAPK) signaling cascade which is consistent with previously published experimental results. In this study we have focused our attention to a generic MAPK ERK (extracellular signal-regulated kinase) pathway activated by epidermal growth factor (EGF) in an attempt to understand how receptors may achieve different activation kinetics of the MAPK signaling. We successfully show that the level of receptor expression is one key determinant in this regulation, and that the binding affinity of the active receptor to adaptor proteins can have a small but albeit direct effect on the downstream activation.     

Stimulation of mitogen activated protein kinase by LDL and oxLDL in human U-937 macrophage-like cells

Mitogen activated protein kinase in extracts of U-937 macrophage-like cells was stimulated by LDL and oxLDL. A maximum value (161% of the basal phosphotransferase activity) was obtained after 6 min exposure to oxidized LDL (27 μ/ml) using APRTPGGRR peptide substrate. The activatory effect was more pronounced (LDL 181%, oxLDL 201%) when MAPK of stimulated cells was immunoprecipitated with anti-p42^MAPK antibodies and phosphotransferase activity was assayed in immune complexes. Stimulation produced by oxLDL was inhibited by poly I, fucoidan, dextran sulfate and by the MAPKK inhibitor PD 098059 but not by PMA-mediated depletion of PKC or by pre-treatment with chloroquine or with pertussis toxin. These results suggest a direct mitogenic effect of LDL which, in the case of oxLDL, is dependent on scavenger receptor ligation but not on G-protein mediated or PKC-dependent signal transduction.