Signaling events triggered by interferon (IFN) account for the molecular mechanisms of antiviral effect. JAK-STAT pathway plays a critical role in IFN signaling, and other pathways are also implicated in IFN-mediated antiviral effect. Changes in mitogen-activated protein kinase (MAPK) and STAT1 pathways were evaluated in human hepatoma cells Huh7 and HepG2 upon IFN alpha treatment.
Phosphorylation of ERK was significantly and specifically up-regulated, whereas enhanced phosphorylation of upstream kinase MEK was unobservable upon IFN alpha treatment. A mild increase in p38 MAPK, SAPK/JNK and downstream target ATF-2 phosphorylation was detectable after exposure to IFN alpha, indicating differential up-regulation of the MAPK signaling cascades. Moreover, STAT1 phosphorylation was strongly enhanced by IFN alpha.
IFN alpha up-regulates MAPK and STAT1 pathways in human hepatoma cells, and may provide useful information for understanding the IFN signaling.
Pulmonary fibrosis remains a significant public health burden with no proven therapies. The mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK)/extracellular signal–regulated kinase (ERK) signaling cascade is a major pathway controlling cellular processes associated with fibrogenesis, including growth, proliferation, and survival. Activation of the MAPK/ERK pathway is detected in the lungs of human fibrosis samples; however, the effect of modulating the pathway in vivo is unknown. Overexpression of transforming growth factor (TGF)-α in the lung epithelium of transgenic mice causes a progressive pulmonary fibrosis associated with increased MEK/ERK activation localized primarily in mesenchymal cells. To determine the role of the MEK pathway in the induction of TGF-α–induced lung fibrosis, TGF-α was overexpressed for 4 weeks while mice were simultaneously treated with the specific MEK inhibitor, ARRY-142886 (ARRY). Treatment with ARRY prevented increases in lung cell proliferation and total lung collagen, attenuated production of extracellular matrix genes, and protected mice from changes in lung function. ARRY administered as a rescue treatment after fibrosis was already established inhibited fibrosis progression, as assessed by lung histology, changes in body weights, extracellular matrix gene expression, and lung mechanics. These findings demonstrate that MEK inhibition prevents progression of established fibrosis in the TGF-α model, and provides proof of concept of targeting the MEK pathway in fibrotic lung disease.
pulmonary fibrosis; transforming growth factor-α; epidermal growth factor receptor; mitogen-activated protein kinase/mitogen-activated protein kinase kinase/extracellular signal–regulated kinase; ARRY-142886
Although in vitro studies have determined that the activation of mitogen-activated protein (MAP) kinases is crucial to the activation of transcription factors and regulation of the production of proinflammatory mediators, the roles of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in acute lung injury have not been elucidated.
Saline or lipopolysaccharide (LPS, 6 mg/kg of body weight) was administered intratracheally with a 1-hour pretreatment with SP600125 (a JNK inhibitor; 30 mg/kg, IO), or PD98059 (an MEK/ERK inhibitor; 30 mg/kg, IO). Rats were sacrificed 4 hours after LPS treatment.
SP600125 or PD98059 inhibited LPS-induced phosphorylation of JNK and ERK, total protein and LDH activity in BAL fluid, and neutrophil influx into the lungs. In addition, these MAP kinase inhibitors substantially reduced LPS-induced production of inflammatory mediators, such as CINC, MMP-9, and nitric oxide. Inhibition of JNK correlated with suppression of NF-κB activation through downregulation of phosphorylation and degradation of IκB-α, while ERK inhibition only slightly influenced the NF-κB pathway.
JNK and ERK play pivotal roles in LPS-induced acute lung injury. Therefore, inhibition of JNK or ERK activity has potential as an effective therapeutic strategy in interventions of inflammatory cascade-associated lung injury.
JNK; ERK; LPS; acute lung injury; NF-κB
The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays a central role in intracellular signaling by many extracellular stimuli. One target of the ERK cascade is peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor that promotes differentiation and apoptosis. It was previously demonstrated that PPARγ activity is attenuated upon mitogenic stimulation due to phosphorylation of its Ser84 by ERKs. Here we show that stimulation by tetradecanoyl phorbol acetate (TPA) attenuates PPARγ's activity in a MEK-dependent manner, even when Ser84 is mutated to Ala. To elucidate the mechanism of attenuation, we found that PPARγ directly interacts with MEKs, which are the activators of ERKs, but not with ERKs themselves, both in vivo and in vitro. This interaction is facilitated by MEKs' phosphorylation and is mediated by the basic D domain of MEK1 and the AF2 domain of PPARγ. Immunofluorescence microscopy and subcellular fractionation revealed that MEK1 exports PPARγ from the nucleus, and this finding was supported by small interfering RNA knockdown of MEK1 and use of a cell-permeable interaction-blocking peptide, which prevented TPA-induced export of PPARγ from the nucleus. Thus, we show here a novel mode of downregulation of PPARγ by its MEK-dependent redistribution from the nucleus to the cytosol. This unanticipated role for the stimulation-induced nuclear shuttling of MEKs shows that MEKs can regulate additional signaling components besides the ERK cascade.
Cell shape plays a role in cell growth, differentiation, and death.
Herein, we used the hepatocyte, a normal, highly differentiated cell
characterized by a long G1 phase, to understand the mechanisms
that link cell shape to growth. First, evidence was provided that the
mitogen-activated protein kinase kinase (MEK)/extracellular
signal-regulated kinase (ERK) cascade is a key transduction pathway
controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1
phase did not lead to cell proliferation but induced cell shape
spreading and demonstration was provided that this MAPK-dependent
spreading was required for reaching G1/S transition and DNA
replication. Moreover, epidermal growth factor (EGF) was found to
control this morphogenic signal in addition to its mitogenic effect.
Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment
resulted in inhibition of EGF-dependent DNA replication. Our data led
us to assess the first third of G1, is exclusively devoted to the
growth factor-dependent morphogenic events, whereas the mitogenic
signal occured at only approximately mid-G1 phase. Moreover,
these two growth factor-related sequential signaling events involved
successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms.
In addition, we demonstrated that inhibition of extracellular matrix
receptor, such as integrin β1 subunit, leads to cell arrest
in G1, whereas EGF was found to up-regulated integrin β1 and
fibronectin in a MEK-ERK–dependent manner. This process in relation to
cytoskeletal reorganization could induce hepatocyte spreading, making
them permissive for DNA replication. Our results provide new insight
into the mechanisms by which a growth factor can temporally control
dual morphogenic and mitogenic signals during the G1 phase.
This paper focuses on the role that mitogen-activated protein kinases (MAPKs) play in drug-induced kidney injury. The MAPKs, of which there are four major classes (ERK, p38, JNK, and ERK5/BMK), are signalling cascades which have been found to be broadly conserved across a wide variety of organisms. MAPKs allow effective transmission of information from the cell surface to the cytosolic or nuclear compartments. Cross talk between the MAPKs themselves and with other signalling pathways allows the cell to modulate responses to a wide variety of external stimuli. The MAPKs have been shown to play key roles in both mediating and ameliorating cellular responses to stress including xenobiotic-induced toxicity. Therefore, this paper will discuss the specific role of the MAPKs in the kidney in response to injury by a variety of xenobiotics and the potential for therapeutic intervention at the level of MAPK signalling across different types of kidney disease.
Regulation of gene expression is an essential step during long-term memory formation. Recently, the involvement of DNA-binding transcription factors and chromatin remodeling in synaptic plasticity have been intensively studied. The process of learning was shown to be associated with chromatin remodeling through histone modifications such as acetylation and phosphorylation. We have previously shown that the MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase) regulatory cascade plays a key role in the food aversion conditioning in the mollusk Helix. Specifically, command neurons of withdrawal behavior exhibit a learning-dependent asymmetry (left–right) in MAPK/ERK activation. Here, we expanded our molecular studies by focusing on a potential MAPK/ERK target – histone H3. We studied whether there is a learning-induced MAPK/ERK-dependent acetylation of histone H3 in command neurons RPa(2/3) and LPa(2/3) of the right and left parietal ganglia and whether it is asymmetrical. We found a significant learning-dependent increase in histone H3 acetylation in RPa(2/3) neurons but not in LPa(2/3) neurons. Such an increase in right command neurons depended on MAPK/ERK activation and correlated with a lateralized avoidance movement to the right visible 48 h after training. The molecular changes found in a selective set of neurons could thus represent a lateralized memory process, which may lead to consistent turning in one direction when avoiding a food that has been paired with an aversive stimulus.
learning; epigenetics; histone H3 acetylation; MAPK/ERK; neuronal networks; lateralization; Helix; chromatin remodeling
Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1+ does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.
Interferon (IFN)-α affects the growth, differentiation and function of various cell types by transducing regulatory signals through the Janus tyrosine kinase/signal transducers of activation and transcription (Jak/STAT) pathway. The signalling pathways employing the mitogen-activated ERK-activating kinase (MEK) and the extracellular-regulated kinase (ERK) are critical in growth factors signalling. Engagement of the receptors, and subsequent stimulation of Ras and Raf, initiates a phosphorylative cascade leading to activation of several proteins among which MEK and ERK play a central role in routing signals critical in controlling cell development, activation and proliferation. We demonstrate here that 24–48 h following treatment of transformed T- and monocytoid cell lines with recombinant human IFN-α2b both the phosphorylation and activity of MEK1 and its substrates ERK1/2 were reduced. In contrast, the activities of the upstream molecules Ras and Raf -1 were not affected. No effect on MEK/ERK activity was observed upon short-term exposure (1–30 min) to IFN. The anti-proliferative effect of IFN-α was increased by the addition in the culture medium of a specific inhibitor of MEK, namely PD98059. In conclusion, our results indicate that IFN-α regulates the activity of the MEK/ERK pathway and consequently modulates cellular proliferation through a Ras / Raf -independent mechanism. Targeting the MEK/ERK pathway may strengthen the IFN-mediated anti-cancer effect. © 2000 Cancer Research Campaign
IFN-α; cellular proliferation; MEK/ERK pathway
ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879–29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17Gag, and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.
The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo.
Anopheles gambiae is the primary mosquito vector of human malaria parasites in sub-Saharan Africa. To date, three innate immune signaling pathways, including the nuclear factor (NF)-kappaB-dependent Toll and immune deficient (IMD) pathways and the Janus kinase/signal transducers and activators of transcription (Jak-STAT) pathway, have been extensively characterized in An. gambiae. However, in addition to NF-kappaB-dependent signaling, three mitogen-activated protein kinase (MAPK) pathways regulated by JNK, ERK and p38 MAPK are critical mediators of innate immunity in other invertebrates and in mammals. Our understanding of the roles of the MAPK signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted.
In this study, we present the orthologs and phylogeny of 17 An. gambiae MAPKs, two of which were previously unknown and two others that were incompletely annotated. We also provide detailed temporal activation profiles for ERK, JNK, and p38 MAPK in An. gambiae cells in vitro to immune signals that are relevant to malaria parasite infection (human insulin, human transforming growth factor-beta1, hydrogen peroxide) and to bacterial lipopolysaccharide. These activation profiles and possible upstream regulatory pathways are interpreted in light of known MAPK signaling cascades.
The establishment of a MAPK "road map" based on the most advanced mosquito genome annotation can accelerate our understanding of host-pathogen interactions and broader physiology of An. gambiae and other mosquito species. Further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the MAP kinase cascades and other networked pathways will facilitate identification of the "master signaling regulators" in biomedically important mosquito species.
Cell migration is critical for normal development and for pathological processes including cancer cell metastasis. Dynamic remodeling of focal adhesions and the actin cytoskeleton are crucial determinants of cell motility. The Rho family and the mitogen-activated protein kinase (MAPK) module consisting of MEK-extracellular signal-regulated kinase (ERK) are important regulators of these processes, but mechanisms for the integration of these signals during spreading and motility are incompletely understood. Here we show that ERK activity is required for fibronectin-stimulated Rho-GTP loading, Rho-kinase function, and the maturation of focal adhesions in spreading cells. We identify p190A RhoGAP as a major target for ERK signaling in adhesion assembly and identify roles for ERK phosphorylation of the C terminus in p190A localization and activity. These observations reveal a novel role for ERK signaling in adhesion assembly in addition to its established role in adhesion disassembly.
Eph receptors and their ligands (ephrins) play an important role in axonal guidance, topographic mapping, and angiogenesis. The signaling pathways mediating these activities are starting to emerge and are highly cell- and receptor-type specific. Here we demonstrate that activated EphB1 recruits the adaptor proteins Grb2 and p52Shc and promotes p52Shc and c-Src tyrosine phosphorylation as well as MAPK/extracellular signal–regulated kinase (ERK) activation. EphB1-mediated increase of cell migration was abrogated by the MEK inhibitor PD98059 and Src inhibitor PP2. In contrast, cell adhesion, which we previously showed to be c-jun NH2-terminal kinase (JNK) dependent, was unaffected by ERK1/2 and Src inhibition. Expression of dominant-negative c-Src significantly reduced EphB1-dependent ERK1/2 activation and chemotaxis. Site-directed mutagenesis experiments demonstrate that tyrosines 600 and 778 of EphB1 are required for its interaction with c-Src and p52Shc. Furthermore, phosphorylation of p52Shc by c-Src is essential for its recruitment to EphB1 signaling complexes through its phosphotyrosine binding domain. Together these findings highlight a new aspect of EphB1 signaling, whereby the concerted action of c-Src and p52Shc activates MAPK/ERK and regulates events involved in cell motility.
Eph receptor; MAPK; Src kinase; p52Shc; chemotaxis
Peroxisome proliferator–activated receptor (PPAR)-γ is a ligand-activated transcription factor and regulates inflammation. Posttranslational modifications regulate the function of PPARγ, potentially affecting inflammation. PPARγ contains a mitogen-activated protein kinase (MAPK) site, and phosphorylation by extracellular signal-regulated kinase (ERK)-1/2 leads to inhibition of PPARγ. This study investigated the kinetics of PPARγ expression and activation in parenchymal and immune cells in sepsis using the MAPK/ERK kinase (MEK)-1 inhibitor, an upstream kinase of ERK1/2. Adult male Sprague Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture. Rats received intraperitoneal injection of vehicle or the MEK1 inhibitor PD98059 (5 mg/kg) 30 min before cecal ligation and puncture. Rats were euthanized at 0, 1, 3, 6 and 18 h after cecal ligation and puncture. Control animals used were animals at time 0 h. Lung, plasma and peripheral blood mononuclear cells (PBMCs) were collected for biochemical assays. In vehicle-treated rats, polymicrobial sepsis resulted in significant lung injury. In the lung and PBMCs, nuclear levels of PPARγ were decreased and associated with an increase in phosphorylated PPARγ and phosphorylated ERK1/2 levels. Treatment with the MEK1 inhibitor increased the antiinflammatory plasma adipokine adiponectin, restored PPARγ expression in PBMCs and lung, and decreased lung injury. The inflammatory effects of sepsis cause changes in PPARγ expression and activation, in part, because of phosphorylation of PPARγ by ERK1/2. This phosphorylation can be reversed by ERK1/2 inhibition, thereby improving lung injury.
The response of the postnatal heart to growth and stress stimuli includes activation of a network of signal transduction cascades, including the stress activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK) and the extracellular signal-regulated kinase (ERK1/2) pathways. In response to increased workload, the mitogen-activated protein kinase kinase (MAPKK) MEK1 has been shown to be active. Studies embarking on mitogen-activated protein kinase (MAPK) signaling cascades in the heart have indicated peroxisome-proliferators activated-receptors (PPARs) as downstream effectors that can be regulated by this signaling cascade. Despite the importance of PPARα in controlling cardiac metabolism, little is known about the relationship between MAPK signaling and cardiac PPARα signaling.
Using co-immunoprecipitation and immunofluorescence approaches we show a complex formation of PPARα with MEK1 and not with ERK1/2. Binding of PPARα to MEK1 is mediated via a LXXLL motif and results in translocation from the nucleus towards the cytoplasm, hereby disabling the transcriptional activity of PPARα. Mice subjected to voluntary running-wheel exercise showed increased cardiac MEK1 activation and complex formation with PPARα, subsequently resulting in reduced PPARα activity. Inhibition of MEK1, using U0126, blunted this effect.
Here we show that activation of the MEK1-ERK1/2 pathway leads to specific inhibition of PPARα transcriptional activity. Furthermore we show that this inhibitory effect is mediated by MEK1, and not by its downstream effector kinase ERK1/2, through a mechanism involving direct binding to PPARα and subsequent stimulation of PPARα export from the nucleus.
Mitogen-activated protein kinases (p42/p44 MAPK, also called Erk2 and Erk1) are key mediators of signal transduction from the cell surface to the nucleus. We have previously shown that the activation of p42/p44 MAPK required for transduction of mitogenic signaling is associated with a rapid nuclear translocation of these kinases. However, the means by which p42 and p44 MAPK translocate into the nucleus after cytoplasmic activation is still not understood and cannot simply be deduced from their protein sequences. In this study, we have demonstrated that activation of the p42/ p44 MAPK pathway was necessary and sufficient for triggering nuclear translocation of p42 and p44 MAPK. First, addition of the MEK inhibitor PD 98059, which blocks activation of the p42/p44 MAPK pathway, impedes the nuclear accumulation, whereas direct activation of the p42/p44 MAPK pathway by the chimera ΔRaf-1:ER is sufficient to promote nuclear accumulation of p42/p44 MAPK. In addition, we have shown that this nuclear accumulation of p42/p44 MAPK required the neosynthesis of short-lived proteins. Indeed, inhibitors of protein synthesis abrogate nuclear accumulation in response to serum and accelerate p42/p44 MAPK nuclear efflux under conditions of persistent p42/p44 MAPK activation. In contrast, inhibition of targeted proteolysis by the proteasome synergistically potentiated p42/p44 MAPK nuclear localization by nonmitogenic agonists and markedly prolonged nuclear localization of p42/p44 MAPK after mitogenic stimulation. We therefore conclude that the MAPK nuclear translocation requires both activation of the p42/p44 MAPK module and neosynthesis of short-lived proteins that we postulate to be nuclear anchors.
growth factors; signal transduction; protein kinases; cell compartmentation; cell nucleus
Long-term potentiation (LTP) in the anterior cingulate cortex (ACC) is believed to be critical for higher brain functions including emotion, learning, memory and chronic pain. N-methyl-D-aspartate (NMDA) receptor-dependent LTP is well studied and is thought to be important for learning and memory in mammalian brains. As the downstream target of NMDA receptors, the extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinase (MAPK) cascade has been extensively studied for its involvement in synaptic plasticity, learning and memory in hippocampus. By contrast, the role of ERK in cingulate LTP has not been investigated. In this study, we examined whether LTP in ACC requires the activation of ERK. We found that P42/P44 MAPK inhibitors, PD98059 and U0126, suppressed the induction of cingulate LTP that was induced by presynaptic stimulation with postsynaptic depolarization (the pairing protocol). We also showed that cingulate LTP induced by two other different protocols was also blocked by PD98059. Moreover, we found that these two inhibitors had no effect on the maintenance of cingulate LTP. Inhibitors of c-Jun N-terminal kinase (JNK) and p38, other members of MAPK family, SP600125 and SB203850, suppressed the induction of cingulate LTP generated by the pairing protocol. Thus, our study suggests that the MAPK signaling pathway is involved in the induction of cingulate LTP and plays a critical role in physiological conditions.
Intracellular mitogen-activated protein kinase (MAPK) signaling cascades likely play an important role in the pathogenesis of cardiac and vascular disease. A substantial amount of basic science research has defined many of the details of MAPK pathway organization and activation, but the role of individual signaling proteins in the pathogenesis of various cardiovascular diseases is still being elucidated. In this review, the role of the MAPKs extracellular signal-regulated kinase (ERK), C-jun N-terminal kinase (JNK) and p38 MAPK in cardiac hypertrophy, cardiac remodeling after myocardial infarction, atherosclerosis and vascular restenosis will be examined with attention paid to genetically-modified murine model systems and to the use of pharmacologic inhibitors of protein kinases. Despite the complexities of this field of research, attractive targets for pharmacological therapy are emerging.
Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) promotes the progression of Kaposi's sarcoma (KS), a dominant malignancy in patients with AIDS. While 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced KSHV reactivation from latency is mediated by the protein kinase C δ and MEK/ERK mitogen-activated protein kinase (MAPK) pathways, we have recently shown that the MEK/ERK, JNK and p38 MAPK pathways modulate KSHV lytic replication during productive primary infection of human umbilical vein endothelial cells (Pan, H.Y., Xie, J.P., Ye, F.C., Gao, S.-J., 2006. J. Virol. 80, 5371-82). Here, we report that, besides the MEK/ERK pathway, the JNK and p38 MAPK pathways also mediate TPA-induced KSHV reactivation from latency. The MEK/ERK, JNK and p38 MAPK pathways were constitutively activated in latent KSHV-infected BCBL-1 cells. TPA treatment enhanced the levels of activated ERK and p38 but not those of activated JNK. Inhibitors of all three MAPK pathways reduced TPA-induced production of KSHV infectious virions in BCBL-1 cells in a dose-dependent fashion. The inhibitors blocked KSHV lytic replication at the early stage(s) of reactivation, and reduced the expression of viral lytic genes including RTA, a key immediate-early transactivator of viral lytic replication. Activation of MAPK pathways was necessary and sufficient for activating the promoter of RTA. Furthermore, we showed that the activation of RTA promoter by MAPK pathways was mediated by their downstream target AP-1. Together, these findings suggest that MAPK pathways might have general roles in regulating the life cycle of KSHV by mediating both viral infection and switch from viral latency to lytic replication.
Kaposi's sarcoma; KSHV; reactivation; MAPK pathways; AP-1; RTA; TPA
MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.
MEK1, a member of the mitogen-activated protein kinase (MAPK) cascade that directly activates extracellular signal-regulated kinase (ERK), induces cardiac hypertrophy in transgenic mice. Calcineurin is a calcium-regulated protein phosphatase that also functions as a positive regulator of cardiac hypertrophic growth through a direct mechanism involving activation of nuclear factor of activated T-cell (NFAT) transcription factors. Here we determined that calcineurin-NFAT and MEK1-ERK1/2 signaling pathways are interdependent in cardiomyocytes, where they directly coregulate the hypertrophic growth response. For example, genetic deletion of the calcineurin Aβ gene reduced the hypertrophic response elicited by an activated MEK1 transgene in the heart, while inhibition of calcineurin or NFAT in cultured neonatal cardiomyocytes also blunted the hypertrophic response driven by activated MEK1. Conversely, targeted inhibition of MEK1-ERK1/2 signaling in cultured cardiomyocytes attenuated the hypertrophic growth response directed by activated calcineurin. However, targeted inhibition of MEK1-ERK1/2 signaling did not directly affect calcineurin-NFAT activation, nor was MEK1-ERK1/2 activation altered by targeted inhibition of calcineurin-NFAT. Mechanistically, we show that MEK1-ERK1/2 signaling augments NFAT transcriptional activity independent of calcineurin, independent of changes in NFAT nuclear localization, and independent of alterations in NFAT transactivation potential. In contrast, MEK1-ERK1/2 signaling enhances NFAT-dependent gene expression through an indirect mechanism involving induction of cardiac AP-1 activity, which functions as a necessary NFAT-interacting partner. As a second mechanism, MEK1-ERK1/2 and calcineurin-NFAT proteins form a complex in cardiac myocytes, resulting in direct phosphorylation of NFATc3 within its C terminus. MEK1-ERK1/2-mediated phosphorylation of NFATc3 directly augmented its DNA binding activity, while inhibition of MEK1-ERK1/2 signaling reduced NFATc3 DNA binding activity. Collectively, these results indicate that calcineurin-NFAT and MEK1-ERK1/2 pathways constitute a codependent signaling module in cardiomyocytes that coordinately regulates the growth response through two distinct mechanisms.
Mitogen-activated protein kinase (MAPK) pathways provide a critical connection between extrinsic and intrinsic signals to cardiac hypertrophy. Extracellular signal-regulated protein kinase 5 (ERK5), an atypical MAP kinase is activated in the heart by pressure overload. However, the role of ERK5 plays in regulating hypertrophic growth and hypertrophy-induced apoptosis is not completely understood.
Herein, we investigate the in vivo role and signaling mechanism whereby ERK5 regulates cardiac hypertrophy and hypertrophy-induced apoptosis.
Methods and Results
We generated and examined the phenotypes of mice with cardiomyocyte-specific deletion of the erk5 gene (ERK5cko). In response to hypertrophic stress, ERK5cko mice developed less hypertrophic growth and fibrosis than controls. However, increased apoptosis together with upregulated expression levels of p53 and Bad were observed in the mutant hearts. Consistently, we found that silencing ERK5 expression or specific inhibition of its kinase activity using BIX02189 in neonatal rat cardiomyocytes (NRCMs) reduced myocyte enhancer factor 2 (MEF2) transcriptional activity and blunted hypertrophic responses. Furthermore, the inhibition of MEF2 activity in NRCMs using a non-DNA binding mutant form of MEF2 was found to attenuate the ERK5-regulated hypertrophic response.
These results reveal an important function of ERK5 in cardiac hypertrophic remodeling and cardiomyocyte survival. The role of ERK5 in hypertrophic remodeling is likely to be mediated via the regulation of MEF2 activity.
Cardiac hypertrophy; signal transduction; genetically modified mice
Extracellular signal-regulated protein kinase (ERK1/2) is a member of the mitogen-activated protein kinase (MAPK) signaling pathway and a key molecular target for ethanol (EtOH) and other drugs of abuse.
The aim of the study was to assess the role of two MAPK pathways, ERK1/2 and c-Jun N-terminal kinase (JNK), on the modulation of EtOH and sucrose self-administration.
Materials and methods
C57BL/6J mice were trained to lever press on a fixed-ratio 4 schedule with 9% EtOH/2% sucrose, or 2% sucrose, as the reinforcer. In experiments 1 and 2, mice were injected with the MEK1/2 inhibitor SL 327 (0–100 mg/kg) and the JNK inhibitor AS 6012452 (0–56 mg/kg) prior to self-administration. In experiment 3, SL 327 (0–100 mg/kg) was administered prior to performance on a progressive ratio (PR) schedule of EtOH reinforcement. In experiment 4, SL 327 and AS 601245 were injected 2 h before a locomotor test.
SL 327 (30 mg/kg) significantly increased EtOH self-administration without affecting locomotion. Higher doses of SL 327 and AS 601245 reduced EtOH-reinforced responding and locomotor activity. Reductions of both ligands on sucrose self-administration were due to decreases in motor activity. SL 327 pretreatment had no effect on PR responding.
ERK1/2 activity is more directly involved in modulating the reinforcing properties of EtOH than JNK activity due to its selective potentiation of EtOH-reinforced responding. The specificity of this effect to EtOH self-administration, rather than sucrose self-administration, suggests that the mechanism by which ERK1/2 increases EtOH-reinforced responding does not generalize to all reinforcing solutions and is not due to increased motivation to consume EtOH.
Alcohol drinking; ERK/MAPK; SL 327; AS 601245; Self-administration; Progressive ratio; Motor activity; Reinforcement; Protein kinase; Operant conditioning
Mechanical stretch rapidly activates multiple signaling cascades, including phospholipases and kinases, to stimulate protein synthesis and growth. The purpose of this study was to determine whether PLA2 activation contributes to stretch-induced phosphorylation of ERK2 in skeletal muscle myotubes. Myotubes derived from neonatal C57 mice were cultured on silicone membranes and subjected to brief cyclic stretch. Inhibition of PLA2 prevented ERK2 phosphorylation, while inhibition of prostaglandin or leukotriene synthesis did not. ERK2 phosphorylation was also blocked by genistein and PD98059, implicating the canonical raf-MEK-ERK cassette. It appears that PLA2, but not further metabolism of arachidonic acid, is required for stretch-induced activation of ERK2. Exposure to exogenous arachidonic acid had no effect on ERK2 phosphorylation, but exposure to lysophosphatidylcholine, the other metabolite of PLA2, caused a dose-dependent increase in ERK2 phosphorylation. These results suggest that stretch-induced activation of ERK2 may result from an interaction between PLA2 derived lysophosphatidylcholine and membrane receptors.
mechanotransduction; MAP kinase; phospholipase a2