The MEK/ERK cascade: from signaling specificity to diverse functions

The ERK signaling cascade is a central MAPK pathway that plays a role in the regulation of various cellular processes such as proliferation, differentiation, development, learning, survival and, under some conditions, also apoptosis. The ability of this cascade to regulate so many distinct, and even opposing, cellular processes, raises the question of signaling specificity determination by this cascade. Here we describe mechanisms that cooperate to direct MEK-ERK signals to their appropriate downstream destinations. These include duration and strength of the signals, interaction with specific scaffolds, changes in subcellular localization, crosstalk with other signaling pathways, and presence of multiple components with distinct functions in each tier of the cascade. Since many of the mechanisms do not function properly in cancer cells, understanding them may shed light not only on the regulation of normal cell proliferation, but also on mechanisms of oncogenic transformation.     

Histone deacetylase inhibitors facilitate dihydroartemisinin-induced apoptosis in liver cancer in vitro and in vivo

Liver cancer ranks in prevalence and mortality among top five cancers worldwide. Accumulating interests have been focused in developing new strategies for liver cancer treatment. We have previously showed that dihydroartemisinin (DHA) exhibited antitumor activity towards liver cancer. In this study, we demonstrated that histone deacetylase inhibitors (HDACi) significantly augmented the antineoplastic effect of DHA via increasing apoptosis in vitro and in vivo. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis, due to the fact that inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis. Compared with DHA alone, the combined treatment with DHA and HDACi reduced mitochondria membrane potential, released cytochrome c into cytoplasm, increased p53 and Bak, decreased Mcl-1 and p-ERK, activated caspase 3 and PARP, and induced apoptotic cells. Furthermore, we showed that HDACi pretreatment facilitated DHA-induced apoptosis. In Hep G2-xenograft carrying nude mice, the intraperitoneal injection of DHA and SAHA resulted in significant inhibition of xenograft tumors. Results of TUNEL and H&E staining showed more apoptosis induced by combined treatment. Immunohistochemistry data revealed the activation of PARP, and the decrease of Ki-67, p-ERK and Mcl-1. Taken together, our data suggest that the combination of HDACi and DHA offers an antitumor effect on liver cancer, and this combination treatment should be considered as a promising strategy for chemotherapy.     

RNA viruses and the mitogenic Raf/MEK/ERK signal transduction cascade

The Raf/MEK/ERK signal transduction cascade belongs to the mitogen-activated protein kinase (MAPK) cascades. Raf/MEK/ERK signaling leads to stimulus-specific changes in gene expression, alterations in cell metabolism or induction of programmed cell death (apoptosis), and thus controls cell differentiation and proliferation. It is induced by extracellular agents, including pathogens such as RNA viruses. Many DNA viruses are known to induce cellular signaling via this pathway. As these pathogens partly use the DNA synthesis machinery for their replication, they aim to drive cells into a proliferative state. In contrast, the consequences of RNA virus-induced Raf/MEK/ERK signaling were less clear for a long time, but since the turn of the century the number of publications on this topic has rapidly increased. Research on this virus/host-interaction will broaden our understanding of its relevance in viral replication. This important control center of cellular responses is differently employed to support the replication of several important human pathogenic RNA viruses including influenza, Ebola, hepatitis C and SARS corona viruses.     

Exogenous interleukin-1 beta promotes the proliferation and migration of HeLa cells via the MEK/ERK signaling pathway

Objective

Interleukin-1 beta (IL-1β) is a crucial cytokine that has been implicated in cancer and metastasis development. However, its possible mechanistic role in cervical cancer remains unclear. This study aimed to investigate the functions of exogenous IL-1β in cervical cancer cell proliferation and migration.

Methods

HeLa cell proliferation and migration were measured using MTT and Transwell assays. A lentivirus-mediated packaging system was used to construct an IL-1β overexpressing cell line. MEK/ERK signal transduction was inhibited by pretreatment with the MEK inhibitor PD98059. qRT–PCR and Western blotting were used to test the expression of relevant genes.

Results

Exogenous IL-1β promoted the proliferation and migration of HeLa cells. In addition, overexpression of IL-1β in HeLa cells promoted cell proliferation. Mechanistically, exogenous IL-1β increased the phosphorylated MEK and ERK levels in HeLa cells and the expression of JUN, RELB, and NF-κB2. Alternatively, blockade of MEK inhibited the promoting proliferation effects of IL-1β and the expression of JUN, RELB, and NF-κB2.

Conclusions

Our data suggest that exogenous IL-1β regulates HeLa cell functions by regulating the MEK/ERK signaling pathway and by targeting JUN, RELB, and NF-κB2. Our study uncovered a potential association across IL-1β, cervical tumor development, and cancer progression.

     

Growth arrest signaling of the Raf/MEK/ERK pathway in cancer

The Raf/MEK/extraceUular signal-regulated kinase （ERK） pathway has a pivotal role in facilitating cell proliferation, and its deregulated activation is a central signature of many epithelial cancers. However paradoxically, sustained activity of Raf/MEK/ERK can also result in growth arrest in many different cell types. This anti-proliferative Raf/MEK/ERK signaling also has physiological significance, as exemplified by its potential as a tumor suppressive mechanism. Therefore, significant questions include in which cell types and by what mechanisms this pathway can mediate such an opposing context of signaling. Particularly, our understating of the role of ERK1 and ERK2, the focal points of pathway signaling, in growth arrest signaling is still limited. This review discusses these aspects of Raf/MEK/ ERK-mediated growth arrest signaling.     

Insulin enhances growth hormone induction of the MEK/ERK signaling pathway

Growth hormone (GH) plays an important role in growth and metabolism by signaling via at least three major pathways, including STATs, ERK1/2, and phosphatidylinositol 3-kinase/Akt. Physiological concentrations of insulin promote growth probably by modulating liver GH receptor (GHR) levels in vivo, but the possible effects of insulin on GH-induced post-GHR signaling have yet to be studied. We hypothesized that short-term insulin, similar to the fluctuations that occur following feeding, affects GH-induced post-GHR signaling. Our present studies suggest that, in rat H4IIE hepatoma cells, insulin (4 h or less) selectively enhanced GH-induced phosphorylation of MEK1/2 and ERK1/2, but not GH-induced activation of STAT5 and Akt. Although insulin pretreatment altered GH-induced formation of Shc.Grb2.SOS complex, it did not significantly affect GH-induced activation of other signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1. Immunofluorescent staining indicated that insulin pretreatment facilitated GH-induced cell membrane translocation of MEK1/2. Insulin pretreatment also increased the amount of MEK association with its scaffolding protein, KSR. In summary, short-term insulin treatment of cultured, liver-derived cells selectively sensitized GH-induced MEK/ERK phosphorylation independent of JAK2, Ras, and Raf-1, but likely resulted from increased cell membrane translocation of MEK1/2. These findings suggest that insulin may be necessary for sensitization of cells to GH-induced ERK1/2 activation and provides a potential cellular mechanism by which insulin promotes growth.     

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.     

Cbl-b promotes chemotherapy-induced apoptosis in rat basophilic leukemia cells by suppressing PI3K/Akt activation and enhancing MEK/ERK activation

The ubiquitin ligase Cbl-b is a negative regulator of the PI3K/Akt pathway, the survival pathway implicated in chemotherapy resistance. However, it remains unclear whether Cbl-b can regulate chemosensitivity through modulating Akt activation. In this study, VP-16-induced RBL-2H3 cells apoptosis was accompanied by the activation of Akt and ERK. The PI3K inhibitor LY294002, not the ERK inhibitor PD98059, enhanced the apoptosis. In addition, down-regulation of Cbl-b was also detected. Over expression of Cbl-b significantly enhanced VP-16-induced cell apoptosis with inhibition of Akt activity, while a dominant negative (DN) RING Finger domain mutation completely abolished this enhancement. On the other hand, ERK activity was enhanced by Cbl-b, and the ERK inhibitor PD98059 reversed Cbl-b-enhanced apoptosis. The consistent results were also showed in the process of Ara-c treatment. These observations indicate that Cbl-b promotes RBL-2H3 apoptosis induced by VP-16 or Ara-c, probably through inhibition of Akt and activation of ERK.     

The role of phosphatidic acid in the regulation of the Ras/MEK/Erk signaling cascade

Phosphatidic acid (PA) is an important second messenger produced by the activation of numerous cell surface receptors. Recent data have suggested that PA regulates multiple cellular processes. This review addresses primarily the role of PA in the regulation of the Erk1/2 cascade pathway. A model for the regulation of Erk1/2 phosphorylation by cell surface receptors is presented. According to this model, agonists stimulate the binding of GTP to Ras and the activation of phospholipase D to generate phosphatidic acid. PA promotes the binding of cRaf-1 kinase to the membrane, where it interacts with Ras.GTP and other regulatory components of the pathway. Ras-Raf complexes remain bound to the surface of endosomes, where scaffolding complexes involving Ras, cRaf-1, MEK and Erk are formed. Complete activation and coupling of the cascade requires endocytosis, a process that is also modulated by PA.     

Artemisinin attenuated ischemic stroke induced cell apoptosis through activation of ERK1/2/CREB/BCL-2 signaling pathway in vitro and in vivo

Ischemic stroke is characterized by the presence of both brain ischemic and reperfusion-induced injuries in the brain, leading to neuronal dysfunction and death. Artemisinin, an FDA-approved antimalarial drug, has been reported to have neuroprotective properties. However, the effect of artemisinin on ischemic stroke is not known. In the present study, we investigated the effect of artemisinin on ischemic stroke using an oxygen-glucose deprivation/reperfusion (OGD/RP) cellular model and a mouse middle cerebral artery occlusion (MCAO) animal model and examined the underlying mechanisms. The obtained results revealed that a subclinical antimalarial concentration of artemisinin increased cell viability and decreased LDH release and cell apoptosis. Artemisinin also attenuated the production of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (Δψm). Importantly, artemisinin attenuated the infarction volume and the brain water content in the MCAO animal model. Artemisinin also improved neurological and behavioural outcomes and restored grasp strength and the recovery of motor function in MCAO animals. Furthermore, artemisinin treatment significantly inhibited the molecular indices of apoptosis, oxidative stress and neuroinflammation and activated the ERK1/2/CREB/BCL-2 signaling pathway. Further validation of the involved signaling pathway by the ERK1/2 inhibitor PD98059 revealed that inhibiting the ERK1/2 signaling pathway or silencing ERK1/2 reversed the neuroprotective effects of artemisinin. These results indicate that artemisinin provides neuroprotection against ischemic stroke via the ERK1/2/CREB/BCL-2 signaling pathway. Our study suggests that artemisinin may play an important role in the prevention and treatment of stroke.     

SHP-2 promotes the maturation of oligodendrocyte precursor cells through Akt and ERK1/2 signaling in vitro

Background

Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs), which are responsible for myelination. Myelin is essential for saltatory nerve conduction in the vertebrate nervous system. However, the molecular mechanisms of maturation and myelination by oligodendrocytes remain elusive.

Methods and Findings

In the present study, we showed that maturation of oligodendrocytes was attenuated by sodium orthovanadate (a comprehensive inhibitor of tyrosine phosphatases) and PTPi IV (a specific inhibitor of SHP-2). It is also found that SHP-2 was persistently expressed during maturation process of OPCs. Down-regulation of endogenous SHP-2 led to impairment of oligodendrocytes maturation and this effect was triiodo-L-thyronine (T3) dependent. Furthermore, over-expression of SHP-2 was shown to promote maturation of oligodendrocytes. Finally, it has been identified that SHP-2 was involved in activation of Akt and extracellular-regulated kinases 1 and 2 (ERK1/2) induced by T3 in oligodendrocytes.

Conclusions

SHP-2 promotes oligodendrocytes maturation via Akt and ERK1/2 signaling in vitro.     

An enzyme-linked immunosorbent assay for the Raf/MEK1/MAPK signaling cascade.

The Ras-MAPK signaling cascade transmits mitogenic stimuli from growth factor receptors and activated Ras to the cell nucleus. Inappropriate Ras activation is associated with approximately 30% of all human cancers. The kinase components of the Ras-MAPK signaling cascade are attractive targets for pharmaceutical intervention. Therefore, we have developed a high-throughput, nonradioactive ELISA method to monitor Raf and MEK1 kinase activity. In this assay system activated Raf phosphorylates and activates MEK1, which in turn phosphorylates MAPK. Antibodies that specifically detect phosphorylated MAPK (vs. nonphosphorylated MAPK) made enzyme-linked immunosorbent assay (ELISA) development possible. This assay detects inhibitors of Raf and/or MEK1 and has been used to screen large numbers of random compounds. The specific target of inhibition in the Raf/MEK1/MAPK ELISA can be subsequently identified by secondary assays which directly measure Raf phosphorylation of MEK1 or MEK1 phosphorylation of MAPK.     

电磁辐射对大鼠海马Raf／MEK／ERK信号通路的影响

目的：研究电磁辐射后大鼠海马Raf／MEK／ERK通路相关信号分子的表达变化规律。探讨辐射损伤机制。方法：分别采用X波段高功率微波（X-HPM）、S波段高功率微波（S-HPM）及电磁脉冲（EMP）模拟源辐射大鼠,建立电磁辐射动物模型。通过Western blot检测海马Raf-1、磷酸化Raf-1和磷酸化ERK的表达。结果：三种电磁辐射后6h-14d,Raf-1表达均下调,以7d最为显著,至28d基本恢复,辐射组间未见明显差异。辐射后6h和7d,磷酸化Raf-1和磷酸化ERK表达均上调,6h较为明显,磷酸化ERK的变化以两微波组更为显著。S-HPM辐射后6h～14d,磷酸化Raf-1表达持续上调,磷酸化ERK的变化呈波浪状,以6h和3d为高峰。结论：Raf／MEK／ERK信号通路参与了电磁辐射所致海马损伤;ERK通路过度活化导致神经元凋亡与坏死可能是电磁辐射致认知功能障碍的重要机制。     

MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal

MEK/ERK signaling plays a crucial role in a diverse set of cellular functions including cell proliferation, differentiation and survival, and recently has been reported to negatively regulate mouse embryonic stem cell (mESC) self-renewal by antagonizing STAT3 activity. However, its role in human ESCs (hESCs) remains unclear. Here we investigated the functions of MEK/ERK in controlling hESC activity. We demonstrated that MEK/ERK kinases were targets of fibroblast growth factor (FGF) pathway in hESCs. Surprisingly, we found that, in contrast to mESCs, high basal MEK/ERK activity was required for maintaining hESCs in an undifferentiated state. Inhibition of MEK/ERK activity by specific MEK inhibitors PD98059 and U0126, or by RNA interference, rapidly caused the loss of self-renewal capacity. We also showed that MEK/ERK signaling cooperated with phosphoinositide 3-kinase (PI3K)/AKT signaling in maintaining hESC pluripotency. However, MEK/ERK signaling had little or no effect on regulating hESC proliferation and survival, in contrast to PI3K/AKT signaling. Taken together, these findings reveal the unique and crucial role of MEK/ERK signaling in the determination of hESC cell fate and expand our understanding of the molecular mechanisms behind the FGF pathway maintenance of hESC pluripotency. Importantly, these data make evident the striking differences in the control of self-renewal between hESCs and mESCs.     

Proliferative signaling by ERBB proteins and RAF/MEK/ERK effectors in polycystic kidney disease

A primary pathological feature of polycystic kidney disease (PKD) is the hyperproliferation of epithelial cells in renal tubules, resulting in formation of fluid-filled cysts. The proliferative aspects of the two major forms of PKD—autosomal dominant PKD (ADPKD), which arises from mutations in the polycystins PKD1 and PKD2, and autosomal recessive PKD (ARPKD), which arises from mutations in PKHD1—has encouraged investigation into protein components of the core cell proliferative machinery as potential drivers of PKD pathogenesis. In this review, we examine the role of signaling by ERBB proteins and their effectors, with a primary focus on ADPKD. The ERBB family of receptor tyrosine kinases (EGFR/ERBB1, HER2/ERBB2, ERBB3, and ERBB4) are activated by extracellular ligands, inducing multiple pro-growth signaling cascades; among these, activation of signaling through the RAS GTPase, and the RAF, MEK1/2, and ERK1/2 kinases enhance cell proliferation and restrict apoptosis during renal tubuloepithelial cyst formation. Characteristics of PKD include overexpression and mislocalization of the ERBB receptors and ligands, leading to enhanced activation and increased activity of downstream signaling proteins. The altered regulation of ERBBs and their effectors in PKD is influenced by enhanced activity of SRC kinase, which is promoted by the loss of cytoplasmic Ca²⁺ and an increase in cAMP-dependent PKA kinase activity that stimulates CFTR, driving the secretory phenotype of ADPKD. We discuss the interplay between ERBB/SRC signaling, and polycystins and their depending signaling, with emphasis on thes changes that affect cell proliferation in cyst expansion, as well as the inflammation-associated fibrogenesis, which characterizes progressive disease. We summarize the current progress of preclinical and clinical trials directed at inhibiting this signaling axis, and discuss potential future strategies that may be productive for controlling PKD.     

Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells

The mitogen-activated protein kinase cascade operates downstream of Ras to convey cell-surface signals to the nucleus via nuclear translocation of ERK1 and ERK2. We and others have recently demonstrated that activation of ERK1/2 by growth factors is required for proliferation of intestinal epithelial crypt cells. However, it remained to be established whether ERK1/2 activation alone was sufficient to trigger intestinal epithelial cell (IEC) proliferation. To this aim, retrovirus encoding the hemagglutinin-tagged MAPK/ERK kinase (MEK)1 wild type (wtMEK), the upstream activator of ERK1/2, or a constitutively active mutant of MEK1 (MEK1-S218D/S222D; caMEK) were used to infect nonimmortalized human normal intestinal epithelial crypt cell cultures [human intestinal epithelial cells (HIEC)] and rodent immortalized intestinal crypt cells (IEC-6). Stable expression of caMEK but not wtMEK in HIEC led to the irreversible arrest of cellular proliferation (premature senescence). Concomitant with the onset of cell-cycle arrest was the induction of the cyclin-dependent kinase inhibitors p21(Cip), p53, and p16(INK4A). By contrast, overexpression of caMEK in IEC-6 cells induced growth factor relaxation for DNA synthesis, promoted morphological transformation and growth in soft agar, and did not affect expression of p21(Cip), p53, and p16(INK4A). We provided evidences that ERK1b, an alternatively spliced isoform of ERK1, is activated and may contribute to the deregulation of contact inhibition cell growth and transformation of these cells. Constitutive activation of MEK in IECs can produce either premature senescence or forced mitogenesis depending on the integrity of a senescence program controlled by the cell cycle inhibitors p53, p16(INK4A), and p21(CIP).