Transforming growth factor-beta stimulates articular chondrocyte cell growth through p44/42 MAP kinase (ERK) activation

Transforming growth factor-beta1 (TGF-beta1) stimulates articular chondrocyte cell proliferation and extracellular matrix formation. We reported previously that immediate and transient expression of c-fos mRNA through protein kinase C activation is required for the mitogenic effect of TGF-beta1 on cultured rat articular chondrocytes (CRAC). In gel kinase assays using myelin basic protein (MBP) showed that total cell lysates from cells treated with TGF-beta1 caused rapid phosphorylation of MBP, which suggests the involvement of mitogen-activated protein kinase (MAPK) activation. To identify specific MAPK pathways activated by TGF-beta1, we performed in vitro kinase assays using specific substrates. TGF-beta1 induced a rapid activation of extracellular signal regulated kinase (ERK) with a peak at 5 min, which decreased to basal levels within 240 min after TGF-beta1 stimulation. In contrast, the c-jun N-terminal kinase activity increased only about 2.5-fold after 240 min of stimulation and p38 MAPK activity did not change significantly. ERK activation by TGF-beta1 was also confirmed by in vivo phosphorylation assays of Elk1. However, a specific MEK1 inhibitor, PD98059, significantly decreased TGF-beta1 induced Elk1 phosphorylation in a dose-dependent manner. Furthermore, PD98059 reduced the TGF-beta1-induced cell growth by 40%. These results indicate that TGF-beta1 specifically activates MEK1 and subsequent ERK pathways in CRAC, and that the activation of this MAPK pathway plays a role in the mitogenic response to TGF-beta1.     

Both ERK1 and ERK2 Are Required for Enterovirus 71 (EV71) Efficient Replication

It has been demonstrated that MEK1, one of the two MEK isoforms in Raf-MEK-ERK1/2 pathway, is essential for successful EV71 propagation. However, the distinct function of ERK1 and ERK2 isoforms, the downstream kinases of MEKs, remains unclear in EV71 replication. In this study, specific ERK siRNAs and selective inhibitor U0126 were applied. Silencing specific ERK did not significantly impact on the EV71-caused biphasic activation of the other ERK isoform, suggesting the EV71-induced activations of ERK1 and ERK2 were non-discriminative and independent to one another. Knockdown of either ERK1 or ERK2 markedly impaired progeny EV71 propagation (both by more than 90%), progeny viral RNA amplification (either by about 30% to 40%) and protein synthesis (both by around 70%), indicating both ERK1 and ERK2 were critical and not interchangeable to EV71 propagation. Moreover, suppression of EV71 replication by inhibiting both early and late phases of ERK1/2 activation showed no significant difference from that of only blocking the late phase, supporting the late phase activation was more importantly responsible for EV71 life cycle. Taken together, this study for the first time identified both ERK1 and ERK2 were required for EV71 efficient replication and further verified the important role of MEK1-ERK1/2 in EV71 replication.     

Hepatitis C virus core protein activates the MAPK/ERK cascade synergistically with tumor promoter TPA, but not with epidermal growth factor or transforming growth factor alpha

Persistent hepatitis C virus (HCV) infection is associated with the development of human hepatocellular carcinoma (HCC), although the mechanism of HCV-related hepatocarcinogenesis remains unclear. Recently, however, the close relationships between the development of HCC and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) cascade have been described. In the present study, we investigated the effects of HCV core protein on this MAPK/ERK cascade. HCV core protein significantly activated the MAPK/ERK cascade, including Elk1. We also examined whether HCV core protein acted synergistically along with hepatocyte mitogen-mediated MAPK/ERK activation. Interestingly, Elk-1 activities were further enhanced by the tumor promoter, 12-O-tetradecanoyl phorbol 13-acetate (TPA), but not by hepatocyte mitogens (epidermal growth factor [EGF] and transforming growth factor alpha [TGF-alpha]) in NIH3T3 cells and HepG2 cells expressing HCV core protein. Moreover, the MAPK/ERK activation by HCV core protein was blocked in the presence of the specific MEK1 inhibitor, PD98059. These results indicate that ERK activation by HCV core protein may be independent of hepatocyte mitogen-mediated signaling but synergistic with TPA, and HCV core protein may function at MEK1 or farther upstream of that component.     

Hepatitis C virus core protein enhances the activation of the transcription factor, Elk1, in response to mitogenic stimuli

Mitogen-activated protein kinase (MAPK) pathways play key roles in cell proliferation, transformation of mammalian cells, and the stress response. We and other investigators showed that hepatitis C virus (HCV) core protein has an oncogenic potential, but its mechanism has remained unknown. We previously demonstrated that the MAPK-extra-cellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway and its downstream target, the serum response element (SRE), is activated in BALB/3T3 cells producing HCV core protein. To elucidate the precise mechanism by which HCV core protein activates the MEK-ERK pathway, we transiently expressed HCV core protein in several cell lines and studied the signal transduction of the pathway, using Gal4-Elk1 luciferase assay, in vitro kinas assay of MAPK, and Western blotting analysis. We discovered that, in the presence of mitogenic signal, HCV core protein enhanced Elk1 activation working downstream of MEK without affecting ERK activity and Elk1 phosphorylation. Our data suggest that HCV core protein may activate Elk1 through a pathway alternative to the typical phosphorylation cascade. These findings might give new insights into the role of HCV in hepatocarcinogenesis.     

Activation of mitogen-activated protein kinase kinase (MEK)/extracellular signal regulated kinase (ERK) signaling pathway is involved in myeloid lineage commitment

Common lymphoid progenitors (CLPs) are lymphoid-lineage-committed progenitor cells. However, they maintain a latent myeloid differentiation potential that can be initiated by stimulation with interleukin-2 (IL-2) via ectopically expressed IL-2 receptors. Although CLPs express IL-7 receptors, which share the common gamma chain with IL-2 receptors, IL-7 cannot initiate lineage conversion in CLPs. In this study, we demonstrate that the critical signals for initiating lineage conversion in CLPs are delivered via IL-2 receptor beta (IL-2R beta) intracellular domains. Fusion of the A region of the IL-2R beta cytoplasmic tail to IL-7R alpha enables IL-7 to initiate myeloid differentiation in CLPs. We found that Shc, which associates with the A region, mediates lineage conversion signals through the mitogen activated protein kinase (MAPK) pathway. Because mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitors completely blocked IL-2-mediated lineage conversion, MAPK activation, specifically via the MEK/ERK pathway, is critically involved in the initiation of this event. Furthermore, formation of granulocyte/macrophage (GM) colonies by hematopoietic stem cells, but not by common myeloid progenitors (CMPs), was severely reduced in the presence of MEK/ERK inhibitors. These results demonstrate that activation of MEK/ERK plays an important role in GM lineage commitment.     

Discovering differential protein expression caused by CagA-induced ERK pathway activation in AGS cells using the SELDI-ProteinChip platform

AIM： To identify the protein expression differences related to the CagA-induced ERK pathway activation in AGS cells. METHODS： Human AGS cells transfected with cagA and blank vector were treated with specific mitogenactivated protein kinase kinase （MEK） inhibitor. Total cell proteins were combined by strong anion exchange （SAX2） and weak cation exchange （CM10） ProteinChip arrays and analyzed using surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry （SELDI-TOF- MS） proteomics technology. Protein expression profiles were compared with those of inhibitor-untreated cagA transfectants. SwissProt/TrEMBL database searching for differentially expressed proteins was carried out using the TagIdent tool with the pI and mass information. RESULTS： When a total of 16 proteins that showed expression differences in inhibitor-untreated cagA transfectants were compared with vector transfectants, three proteins with m/z 4229, 8162 and 9084 were found to have no expression differences after treatment with MEK inhibitor, while the other 13 maintained the same expression differences after inhibitor treatment. Seven pieces of meaningful matching information for the three proteins were obtained from database searching. CONCLUSION： Biomarkers with m/z 4229, 8162 and 9084 are ERKI/2 phosphorylation dependent, andtherefore are the downstream molecules of ERK1/2 in the ERK/MAPK signaling pathway. The three biomarkers may be important cancer-associated proteins according to SwissProt/TrEMBL database information.     

Requirement for ERK1/2 activation in the regulation of progesterone production in human granulosa-lutein cells is stimulus specific

This study was conducted to determine whether the ERK1/2 family of MAPKs can be modulated by physiological regulators of the human corpus luteum, and whether this activation is important for progesterone secretion in human granulosa-lutein (hGL) cells. Human LH (hLH), hCG, and agents that indirectly elevate cAMP [cholera toxin, forskolin, (Bu)(2)cAMP], time- and dose-dependently activated ERK1/2 in hGL cells. ERK1/2 activation was reduced by preincubation with PKA inhibitors, including myristoylated PKI, suggesting that cAMP mediates ERK1/2 activation. Two structurally distinct inhibitors of MAPK kinase (MEK), PD 98059 and U 0126, abrogated hLH/hCG-induced ERK1/2 activation, but had no effect on hLH-, hCG-, or 22R-hydroxycholesterol-stimulated progesterone secretion. In contrast, both inhibitors blocked cholera toxin-, forskolin-, and (Bu)(2)cAMP-induced ERK1/2 phosphorylation concomitant with a reduction in progesterone secretion. The known luteotropin, PGE(2), promoted MEK- and cAMP-dependent activation of ERK1/2, and inhibitors of either MEK or PKA decreased PGE(2)-induced progesterone synthesis. Our findings demonstrate that the requirement for ERK1/2 activation as a regulator of progesterone synthesis in hGL cells is stimulus dependent, and that the MEK inhibitor-sensitive step is distal to cAMP generation, but proximal to the conversion of cholesterol to pregnenolone.     

Signaling dynamics of the KSR1 scaffold complex

Scaffold proteins contribute to the spatiotemporal control of MAPK signaling and KSR1 is an ERK cascade scaffold that localizes to the plasma membrane in response to growth factor treatment. To better understand the molecular mechanisms of KSR1 function, we examined the interaction of KSR1 with each of the ERK cascade components, Raf, MEK, and ERK. Here, we identify a hydrophobic motif within the proline-rich sequence (PRS) of MEK1 and MEK2 that is required for constitutive binding to KSR1 and find that MEK binding and residues in the KSR1 CA1 region enable KSR1 to form a ternary complex with B-Raf and MEK following growth factor treatment that enhances MEK activation. We also find that docking of active ERK to the KSR1 scaffold allows ERK to phosphorylate KSR1 and B-Raf on feedback S/TP sites. Strikingly, feedback phosphorylation of KSR1 and B-Raf promote their dissociation and result in the release of KSR1 from the plasma membrane. Together, these findings provide unique insight into the signaling dynamics of the KSR1 scaffold and reveal that through regulated interactions with Raf and ERK, KSR1 acts to both potentiate and attenuate ERK cascade activation, thus regulating the intensity and duration of ERK cascade signaling emanating from the plasma membrane during growth factor signaling.     

Scaffolding mechanism of arrestin-2 in the cRaf/MEK1/ERK signaling cascade

Arrestins were initially identified for their role in homologous desensitization and internalization of G protein–coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange–mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.

The mitogen-activated protein kinase (MAPK) signaling cascade is an intracellular signaling pathway that is activated by diverse external stresses and regulates various cellular functions such as differentiation and proliferation (1). MAPK activation cascades consist of three components: MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. MAP3K phosphorylates and activates MAP2K, which in turn phosphorylates and activates MAPK (1, 2). In mammals, there are four distinct MAPK groups, ERKs (ERK1 and 2), JNKs (JNK1, 2, and 3), p38 (p38α through δ), and ERK5, each of which has its own upstream MAP3Ks and MAP2Ks (2, 3).A long-standing question in biochemistry is how diverse input signals can generate a specific MAPK signaling cascade. In other words, it is unknown how MAPK signaling modules dynamically interact in a spatiotemporal manner. To accomplish signaling fidelity, scaffolding proteins play an important role in colocalizing MAPK signaling modules. MAPK scaffolding proteins facilitate activation, regulate subcellular localization, and/or modulate the negative feedback of a specific MAPK signaling, which helps to organize a specific MAPK module to link the input signaling to proper biological outcomes (4, 5). Owing to the diverse roles of MAPK scaffolding proteins, they have been considered as potential therapeutic targets (6, 7).In mammals, a number of MAPK scaffolding proteins, including JIP, KSR, paxillin, MORG1, JSAP1, and arrestins, have been identified (8, 9). The functional role of MAPK scaffolding and the molecular and structural mechanisms of how the MAPK scaffolding proteins interact with MAPK signaling components vary depending on the scaffolding proteins (4). Therefore, it is important to study the details of the scaffolding mechanism of each MAPK scaffolding protein to gain a better understanding of the MAPK signaling mechanism and to precisely regulate a specific MAPK signaling pathway for therapeutic purposes.Arrestins were first discovered as proteins that play a key role in homologous desensitization and internalization of G protein–coupled receptors (GPCRs) (10). Four arrestins (arrestin-1 through -4) have been identified in humans: arrestin-1 and -4 (visual and cone arrestins, respectively) are expressed exclusively in the visual system, whereas arrestin-2 and -3 (also known as β-arrestin1 and 2, respectively) are ubiquitously expressed (10). Agonist-activated GPCRs, after coupling with G proteins, are phosphorylated by GPCR kinases. Arrestins bind active phosphorylated GPCRs, precluding receptor coupling to G proteins and facilitating receptor internalization (10, 11). In GPCR internalization, arrestins act as scaffolding proteins, linking the receptor with components of internalization machinery, such as clathrin and AP-2 (11–15). Arrestins have also been reported to interact with numerous signaling proteins, including MAPKs, and perform multiple functions (16, 17). The interaction with these signaling proteins occurs either in the basal or GPCR-induced active state of arrestins (11, 15, 18).Arrestins are the only known scaffolding proteins for MAPKs that are regulated by GPCRs. The interaction between MAPKs and arrestins has been extensively studied (11, 15, 19–21). Arrestins regulate GPCR-mediated ERK1/2 activation by scaffolding cRaf-1, MEK1, and ERK1/2 (22, 23) and regulate GPCR-dependent or -independent JNK3 signaling by scaffolding ASK1, MKK4/7, and JNK3 (24–26). Although the cellular and physiological effects of arrestins in MAPK signaling cascades have been studied extensively, the structural mechanisms governing arrestin/MAPK interactions have not been fully elucidated. Only a few studies have suggested a scaffolding mechanism of arrestins for ERK1/2 and JNK3 signaling components. A molecular simulation approach has suggested the binding interfaces of GPCR, arrestin-2, cSrc, cRaf, MEK1, and ERK1 (27), and mutation or truncation studies have investigated the interaction sites between arrestin and ERK1/2 signaling components (22, 28). Recent studies suggested an arrestin-3-mediated scaffolding and signal amplification mechanism of the JNK3 cascade (26, 29).Here, we studied the interaction of ERK1/2 signaling cascade components (cRaf, MEK1, and ERK2) with arrestin-2 using a combination of hydrogen/deuterium exchange–mass spectrometry (HDX-MS), Trp-induced bimane fluorescence quenching, and NMR spectroscopy. HDX-MS measures the exchange rate between the hydrogen atoms of amides in the protein backbone and the deuterium atoms in the solvent, which can provide an insight into protein–protein binding interfaces (30). NMR and Trp-induced bimane fluorescence quenching enable the detailed mapping of interaction interfaces within proteins (29, 31).     

MEK/ERK pathway is aberrantly active in Hodgkin disease: a signaling pathway shared by CD30, CD40, and RANK that regulates cell proliferation and survival

The mitogen-activated protein kinase (MAPK) (also called extracellular signal-regulated kinase [ERK]) pathway has been implicated in malignant transformation and in the regulation of cellular growth and proliferation of several tumor types, but its expression and function in Hodgkin disease (HD) are unknown. We report here that the active phosphorylated form of MAPK/ERK is aberrantly expressed in cultured and primary HD cells. Inhibition of the upstream MAPK kinase (also called MEK) by the small molecule UO126 inhibited the phosphorylation of ERK and demonstrated a dose- and time-dependent antiproliferative activity in HD cell lines. UO126 modulated the levels of several intracellular proteins including B-cell lymphoma protein 2 (Bcl-2), myeloid cell leukemia-1 (Mcl-1) and caspase 8 homolog FLICE-inhibitory protein (cFLIP), and induced G2M cell-cycle arrest or apoptosis. Furthermore, UO126 potentiated the activity of apoliprotein 2/tumor necrosis factor-related apoptosis-inducing ligand (APO2L/TRAIL) and chemotherapy-induced cell death. Activation of CD30, CD40, and receptor activator of nuclear kappabeta (RANK) receptors in HD cells by their respective ligands increased ERK phosphorylation above the basal level and promoted HD cell survival. UO126 inhibited basal and ligand-induced ERK phosphorylation, and inhibited ligand-induced cell survival of HD cell lines. These findings provide a proof-of-principle that inhibition of the MEK/ERK pathway may have therapeutic value in HD.     

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 microM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.     

Raf-1 is not required for megakaryocytopoiesis or TPO-induced ERK phosphorylation

Thrombopoietin stimulates extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in megakaryocytes, and the classic mitogen-activated protein (MAP) kinase (Raf/mitogen-induced extracellular kinase [MEK]/ERK) pathway has been implicated directly and indirectly to play a critical role in megakaryocytopoiesis. However, the involvement of specific Raf family members in megakaryocytopoiesis is unknown. raf-1(-/-) mice were therefore used to directly determine the role of Raf-1 in megakaryocytopoiesis. Surprisingly, raf-1(-/-) mice have a modestly higher platelet count than their raf-1(+/+) littermates. Nonetheless, the absence of Raf-1 does not alter thrombopoietin-induced expansion of primary megakaryocyte-lineage cells, the development of apoptotic megakaryocytes in the presence or absence of thrombopoietin, or the development of megakaryocyte DNA ploidy distribution. Moreover, raf-1(-/-) megakaryocytes do not have a compensatory increase in A-Raf or B-Raf expression, and thrombopoietin-induced ERK1/2 phosphorylation is similar in raf-1(-/-) and raf-1(+/+) megakaryocytes. These unexpected findings demonstrate that Raf-1 is dispensable for megakaryocytopoiesis, and for thrombopoietin-induced ERK1/2 activation in primary megakaryocyte-lineage cells.     

Human neutrophils utilize a Rac/Cdc42-dependent MAPK pathway to direct intracellular granule mobilization toward ingested microbial pathogens

Elevated levels of mitogen-activated protein kinase/extracellular regulatory kinase (MAPK/ERK) activity are frequently found in some cancer cells. In efforts to reduce tumor growth, attempts have been made to develop cancer therapeutic agents targeting the MAPK. Here, by use of biologic, biochemical, and gene manipulation methods in human polymorphonuclear neutrophils (PMNs), we have identified a key pathway important in normal cell function involving MAPK/ERK in PMNs for growth inhibition of Candida albicans. Contact with C albicans triggered MAPK/ERK activation in PMNs within 5 minutes, and blocking of MAPK/ERK activation, either by the pharmacologic reagent PD098059 or by dominant-negative MAPK kinase (MEK) expression via vaccinia viral delivery, suppressed antimicrobial activity. Rac and Cdc42, but not Ras or Rho, were responsible for this MAPK/ERK activation. Expression of dominant-negative Rac (N17Rac) or Cdc42 (N17Cdc42) eliminated not only C albicans- mediated ERK phosphorylation but also phagocytosis and granule migration toward the ingested microbes, whereas dominant-negative Ras (N17Ras) and Rho (N19Rho) did not. PAK1 (p21-activated kinase 1) activation is induced by C albicans, suggesting that PAK1 may also be involved in the Rac1 activation of MAPK/ERK. We conclude from these data that Rac/Cdc42-dependent activation of MAPK/ERK is a critical event in the immediate phagocytic response of PMNs to microbial challenge. Therefore, use of MAPK pharmacologic inhibitors for the treatment of cancer may result in the interruption of normal neutrophil function. A balance between therapeutic outcome and undesirable side effects must be attained to achieve successful and safe anticancer therapy.     

ERK1/2 signaling pathway in mast cell activation‐induced sensitization of esophageal nodose C‐fiber neurons

Sensitization of esophageal nociceptive afferents by inflammatory mediators plays an important role in esophageal inflammatory nociception. Our previous studies demonstrated that esophageal mast cell activation increases the excitability of esophageal nodose C‐fibers. But the intracellular mechanism of this sensitization process is still less clear. We hypothesize that extracellular signal‐regulated kinases 1 and 2 (ERK1/2) signaling pathway plays an important role in mast cell activation‐induced sensitization of esophageal nodose C‐fiber neurons. Mast cell activation and in vivo esophageal distension‐induced phosphorylations of ERK1/2 were studied by immuno‐staining and Western blot in esophageal nodose neurons. Extracellular recordings were performed from nodose neurons using ex vivo esophageal‐vagal preparations with intact nerve endings in the esophagus. Nerve excitabilities were compared by action potentials evoked by esophageal distensions before and after mast cell activations with/without pretreatment of mitogen‐activated protein kinases (MAPK)/ERK kinase inhibitor U0126. The expressions of phospho‐ERK1/2 (p‐ERK1/2) in the same nodose ganglia were then studied by Western blot. Mast cell activation enhances in vivo esophageal distension‐induced phosphorylation of ERK1/2 in nodose neurons. This can be prevented by pretreatment with mast cell stabilizer cromolyn. In ex vivo esophageal‐vagal preparations, both mast cell activation and proteinase‐activated receptor 2 (PAR2)‐activating peptide perfusion increases esophageal distension‐induced mechano‐excitability of esophageal nodose C‐fibers and phosphorylation of ERK1/2 in nodose neurons. Pretreatment with MAPK/ERK kinase inhibitor U0126 prevents these potentiation effects. Collectively, our data demonstrated that mast cell activation enhances esophageal distension‐induced mechano‐excitability and phosphorylation of ERK1/2 in esophageal nodose C‐fiber neurons. This reveals a new intracellular pathway of esophageal peripheral sensitization and inflammatory nociception.     

The chemotactic and mitogenic responses of vascular smooth muscle cells to insulin-like growth factor-I require the activation of ERK1/2

Insulin-like growth factors (IGFs) play an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. IGFs exert these biological actions through the activation of the IGF-I receptor and its downstream signaling network. While the involvement of the IRS-PI3 kinase-Akt pathway in mediating the chemotactic and mitogenic actions of IGFs is clear, the role of the mitogen-activated protein kinase (MAPK) signaling pathway is still under debate. In this study, the role of ERK1 and 2 in mediating the chemotactic and mitogenic actions of IGF-I in cultured porcine VSMCs was investigated. IGF-I treatment caused a significant increase in the phosphorylation state, as well as the kinase activity, of ERK1 and 2. Compared to the strong and sustained MAPK activation induced by platelet-derived growth factor-BB, the IGF-I-induced MAPK activation was weaker and more transient. Specific inhibition of the MAPK activation by PD98059 or U0126, two selective MEK inhibitors, significantly inhibited IGF-I-stimulated cell proliferation, and reduced the number of cells that migrated towards IGF-I. The p38 MAPK inhibitor SB203580 had no such effect. Likewise, depletion of ERK1/2 using antisense oligonucleotides abolished the IGF-I-induced VSMC migration and proliferation. These results suggest that the chemotactic and mitogenic responses of VSMCs to IGF-I require the activation of ERK1 and 2.     

Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.