Constitutive activation of the MEK/ERK pathway mediates all effects of oncogenic H-ras expression in primary erythroid progenitors

Oncogenic mutations in ras genes frequently occur in patients with myeloid disorders, and in these patients erythropoiesis is often affected. Previously, we showed that expression of oncogenic H-ras in purified mouse primary fetal liver erythroid progenitors blocks terminal erythroid differentiation and supports erythropoietin (Epo)-independent proliferation. As a first step in understanding the underlying molecular mechanisms we examined the signaling pathways downstream of Ras in primary erythroid cells. We found that 3 major pathways are abnormally activated by oncogenic H-ras: Raf/ERK (extracellular signal-regulated kinase), phosphatidyl inositol 3 (PI3)-kinase/Akt, and RalGEF/RalA. However, only constitutive activation of the MEK (MAPK [mitogen-activated protein kinase]/ERK kinase)/ERK pathway alone could recapitulate all of the effects of oncogenic H-ras expression in blocking erythroid differentiation and inducing Epo-independent proliferation. Although expression of a constitutively active Akt kinase (ca.Akt) in erythroid progenitors does not significantly affect erythroid differentiation in the presence of Epo, coexpression of ca.Akt together with a constitutively active MEK causes prolonged Epo-independent proliferation of erythroid progenitors in addition to a block in differentiation. Moreover, the effects of oncogenic H-ras expression on primary erythroid cells are blocked by the addition of U0126, a specific inhibitor of MEK1 and MEK2, allowing normal terminal erythroid proliferation and differentiation. Our data suggest that the interruption of constitutive MEK/ERK signaling is a potential therapeutic strategy to correct impaired erythroid differentiation in patients with myeloid disorders.     

Constitutive degradation of PML/RARalpha through the proteasome pathway mediates retinoic acid resistance

PML/RARalpha is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RARalpha, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RARalpha protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RARalpha is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RARalpha protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RARalpha, but not RARalpha, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RARalpha protein may lead to RA resistance and that PML/RARalpha expression is crucial to convey RA sensitivity to APL cells.     

Angelica stimulates proliferation of murine bone marrow mononuclear cells by the MAPK pathway

Murine bone marrow mononuclear cells (MNC) were isolated and co-incubated with Angelica to investigate its effects on bone marrow cells and the underlying mechanism of action. Angelica stimulates MNC proliferation as determined by the 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Our results also suggest that the mechanism of action involves the phosphorylation of ERK1/2 and P38, two key proteins in the MAPK pathway. MAPK inhibitors, PD 98059 and SB 203580, block MNC proliferation caused by Angelica. Taken together, our results show that Angelica induces the proliferation of murine MNC by activating ERK1/2 and P38 MAPK proteins.     

NMDA receptor mediates tau-induced neurotoxicity by calpain and ERK/MAPK activation

The altered function and/or structure of tau protein is postulated to cause cell death in tauopathies and Alzheimer's disease. However, the mechanisms by which tau induces neuronal death remain unclear. Here we show that overexpression of human tau and of some of its N-terminal fragments in primary neuronal cultures leads to an N-methyl-D-aspartate receptor (NMDAR)-mediated and caspase-independent cell death. Death signaling likely originates from stimulation of extrasynaptic NR2B-subunit-containing NMDARs because it is accompanied by dephosphorylation of cAMP-response-element-binding protein (CREB) and it is inhibited by ifenprodil. Interestingly, activation of NMDAR leads to a crucial, sustained, and delayed phosphorylation of extracellular-regulated kinases 1 and 2, whose inhibition largely prevents tau-induced neuronal death. Moreover, NMDAR involvement causes the fatal activation of calpain, which, in turn, degrades tau protein into a 17-kDa peptide and possibly other highly toxic N-terminal peptides. Some of these peptides are hypothesized, on the basis of our in vitro experiments, to initiate a negative loop, ultimately leading to cell death. Thus, inhibition of calpain largely prevents tau degradation and cell death. Our findings unravel a cellular mechanism linking tau toxicity to NMDAR activation and might be relevant to Alzheimer's disease and tauopathies where NMDAR-mediated toxicity is postulated to play a pivotal role.     

p38MAPK信号通路与内皮细胞激活

内皮细胞激活是内皮细胞损伤的始动因素,是引起各种不同程度的炎症反应和细胞凋亡的前提条件 ;丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK) 是哺乳动物细胞中重要的信号转导通路,其中 p38MAPK 通路在细胞应激、细胞生长、凋亡和炎症等多种生理和病理过程中起重要作用,越来越引起业界的广泛关注,本文就 p38MAPK 信号通路及其与内皮细胞激活的相关性做一综述,旨在进一步对内皮细胞损伤引发心血管疾病研究提供参考资料。     

胰高糖素样肽-1受体激活对肝星状细胞p38MAPK信号通路的影响

目的 探讨肝星状细胞(HSC)上胰高糖素样肽-1(GLP-1)受体激活对胞内p38MAPK信号通路的影响. 方法 体外培养人HSC并进行形态学鉴定,随机选取样本用Western blot法检测其GLP-1受体蛋白的表达情况;设立对照组和实验组HSC进行体外培养,实验组予以GLP-1受体激动剂-利拉鲁肽,对照组予以等量等渗盐水,细胞培养120 h后用RT-PCR检测各样本的p38MAPK mRNA表达水平,用Western blot检测各样本磷酸化p38MAPK蛋白表达水平.对数据进行两独立样本均数t检验分析. 结果 人HSC上存在GLP-1受体蛋白的表达.实验组和对照组相比,磷酸化p38MAPK蛋白相对表达水平下降,差异具有统计学意义(18.0±2.8与21.2±2.5,t=3.814,P＜0.01);p38MAPK mRNA相对表达水平亦下降,差异亦具有统计学意义(37.9±2.4与43.3±4.7,t=4.478,P＜0.01). 结论 HSC上GLP-1受体激活后能够下调HSC内p38MAPK mRNA的表达,也能够降低胞内磷酸化的p38MAPK蛋白水平,起到抑制p38MAPK信号通路的作用.     

Mitochondrial PKCepsilon and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKCepsilon-MAPK interactions and differential MAPK activation in PKCepsilon-induced cardioprotection

Although activation of protein kinase C (PKC) epsilon and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKCepsilon signaling modules in na?ve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKCepsilon and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKCepsilon, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKCepsilon greatly increased mitochondrial PKCepsilon expression and activity, which was concomitant with increased mitochondrial interaction of PKCepsilon with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKCepsilon activity, as the interactions were also observed in mice expressing inactive PKCepsilon. However, although both active and inactive PKCepsilon bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKCepsilon but not in mice expressing inactive PKCepsilon. Examination of potential downstream targets of mitochondrial PKCepsilon-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKCepsilon forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKCepsilon-ERK modules appears to play a role in PKCepsilon-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.     

CD44-mediated phagocytosis induces inside-out activation of complement receptor-3 in murine macrophages

Diverse receptors, including Fcgamma receptors and beta(2) integrins (complement receptor-3 [CR3], CD11b/CD18), have been implicated in phagocytosis, but their distinct roles and interactions with other receptors in particle engulfment are not well defined. CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, may have additional functions in regulation of inflammation and phagocytosis. We have recently reported that CD44 is a fully competent phagocytic receptor that is able to trigger ingestion of large particles by macrophages. Here, we investigated the role of coreceptors and intracellular signaling pathways in modulation of CD44-mediated phagocytosis. Using biotinylated erythrocytes coated with specific antibodies (anti-CD44-coated erythrocytes [Ebabs]) as the phagocytic prey, we determined that CD44-mediated phagocytosis is reduced by 45% by a blocking CD11b antibody. Further, CD44-mediated phagocytosis was substantially (42%) reduced in CD18-null mice. Immunofluorescence microscopy revealed that CD11b is recruited to the phagocytic cup. The mechanism of integrin activation and mobilization involved activation of the GTPase Rap1. CD44-mediated phagocytosis was also sensitive to the extracellular concentration of the divalent cation Mg(2+) but not Ca(2+). In addition, buffering of intracellular Ca(2+) did not affect CD44-mediated phagocytosis. Taken together, these data suggest that CD44 stimulation induces inside-out activation of CR3 through the GTPase Rap1.     

Stimulation of Ebola virus production from persistent infection through activation of the Ras/MAPK pathway

Human infections with Ebola virus (EBOV) result in a deadly viral disease known as Ebola hemorrhagic fever. Up to 90% of infected patients die, and there is no available treatment or vaccine. The sporadic human outbreaks are believed to result when EBOV "jumps" from an infected animal to a person and is subsequently transmitted between persons by direct contact with infected blood or body fluids. This study was undertaken to investigate the mechanism by which EBOV can persistently infect and then escape from model cell and animal reservoir systems. We report a model system in which infection of mouse and bat cell lines with EBOV leads to persistence, which can be broken with low levels of lipopolysaccharide or phorbol-12-myristate-13-acetate (PMA). This reactivation depends on the Ras/MAPK pathway through inhibition of RNA-dependent protein kinase and eukaryotic initiation factor 2alpha phosphorylation and occurs at the level of protein synthesis. EBOV also can be evoked from mice 7 days after infection by PMA treatment, indicating that a similar mechanism occurs in vivo. Our findings suggest that EBOV may persist in nature through subclinical infection of a reservoir species, such as bats, and that appropriate physiological stimulation may result in increased replication and transmission to new hosts. Identification of a presumptive mechanism responsible for EBOV emergence from its reservoir underscores the "hit-and-run" nature of the initiation of human and/or nonhuman primate EBOV outbreaks and may provide insight into possible countermeasures to interfere with transmission.     

Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in endomitosis.

Thrombopoietin (TPO) plays a critical role in megakaryocyte proliferation and differentiation. Using various cultured cell lines, several recent studies have implicated the mitogen-activated protein kinase (MAPK) pathway in megakaryocyte differentiation. In the study reported here, we examined the role played by thrombopoietin-induced MAPK activity in a cytokine-dependent cell line (BAF3/Mpl) and in primary murine megakaryocytes. In both systems, extracellular signal-regulated protein kinase (ERK) 1 and 2 MAPK phosphorylation was rapidly induced by TPO stimulation. To identify the Mpl domain responsible for MAPK activation, BAF3 cells expressing truncated forms of the Mpl receptor were studied. Phosphorylation of ERKs did not require elements of the cytoplasmic signaling domain distal to Box 2 and was not dependent on phosphorylation of the adapter protein Shc. ERK activation in murine megakaryocytes was maximal at 10 minutes and was markedly decreased over the subsequent 3 hours. Next, the physiologic consequences of MAPK inhibition were studied. Using the MAPK kinase (MEK) inhibitor, PD 98059, blockade of MAPK activity substantially reduced TPO-dependent proliferation in BAF3/Mpl cells and markedly decreased mean megakaryocyte ploidy in cultures. To exclude an indirect effect of MAPK inhibition on stromal cells in whole bone marrow, CD41(+) cells were selected and then cultured in TPO. The number of polyploid megakaryocytes derived from the CD41-selected cells was also significantly reduced by MEK inhibition, as was their geometric mean ploidy. These studies show an important role for MAPK in TPO-induced endomitosis and underscore the value of primary cells when studying the physiologic effects of signaling pathways.     

Rhodiola crenulata reduces ventricular arrhythmia through mitigating the activation of IL-17 and inhibiting the MAPK signaling pathway

Cardiovascular Drugs and Therapy - Ventricular arrhythmia (VA) is related to inflammatory activity. Rhodiola crenulate (RC) and its main active component, salidroside, have been reported as...     

Methionine-induced hyperhomocysteinemia reverts fibrinolytic pathway activation in a murine model of acute promyelocytic leukemia

Increased fibrinolysis is an important component of acute promyelocytic leukemia (APL) bleeding diathesis. APL blasts overexpress annexin II (ANXII), a receptor for tissue plasminogen activator (tPA), and plasminogen, thereby increasing plasmin generation. Previous studies suggested that ANXII plays a pivotal role in APL coagulopathy. ANXII binding to tPA can be inhibited by homocysteine and hyperhomocysteinemia can be induced by L-methionine supplementation. In the present study, we used an APL mouse model to study ANXII function and the effects of hyperhomocysteinemia in vivo. Leukemic cells expressed higher ANXII and tPA plasma levels (11.95 ng/mL in leukemic vs 10.74 ng/mL in wild-type; P = .004). In leukemic mice, administration of L-methionine significantly increased homocysteine levels (49.0 μmol/mL and < 6.0 μmol/mL in the treated and nontreated groups, respectively) and reduced tPA levels to baseline concentrations. The latter were also decreased after infusion of the LCKLSL peptide, a competitor for the ANXII tPA-binding site (11.07 ng/mL; P = .001). We also expressed and purified the p36 component of ANXII in Pichia methanolica. The infusion of p36 in wild-type mice increased tPA and thrombin-antithrombin levels, and the latter was reversed by L-methionine administration. The results of the present study demonstrate the relevance of ANXII in vivo and suggest that methionine-induced hyperhomocysteinemia may reverse hyperfibrinolysis in APL.