ERK MAP Kinase Signaling Research Articles

Circadian clock-controlled diurnal oscillation of Ras/ERK signaling in mouse liver

Accumulating evidence indicates that ERK MAP kinase signaling plays an important role in the regulation of the circadian clock, especially in the clock-resetting mechanism in the suprachiasmatic nucleus (SCN) in mammals. Previous studies have also shown that ERK phosphorylation exhibits diurnal variation in the SCN. However, little is known about circadian regulation of ERK signaling in peripheral tissues. Here we show that the activity of Ras/ERK signaling exhibits circadian rhythms in mouse liver. We demonstrate that Ras activation, MEK phosphorylation, and ERK phosphorylation oscillate in a circadian manner. As the oscillation of ERK phosphorylation is lost in Cry1/Cry2 double-knockout mice, Ras/ERK signaling should be under the control of the circadian clock. Furthermore, expression of MAP kinase phosphatase-1 (Mkp-1) shows diurnal changes in liver. These results indicate that Ras/ERK signaling is strictly regulated by the circadian clock in liver, and suggest that the circadian oscillation of the activities of Ras, MEK, and ERK may regulate diurnal variation of liver function and/or homeostasis.

A Genome-Wide RNAi Screen Reveals MAP Kinase Phosphatases as Key ERK Pathway Regulators during Embryonic Stem Cell Differentiation

Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.

Naringin attenuates EGF-induced MUC5AC secretion in A549 cells by suppressing the cooperative activities of MAPKs-AP-1 and IKKs-IκB-NF-κB signaling pathways

Naringenin, the aglycone of naringin, has been reported to attenuate MUC5AC secretion by inhibiting activity of nuclear factor kappa B (NF-κB) via EGFR-PI3K-Akt/ERK MAPKinase signaling pathways. However, previous studies demonstrated that the MUC5AC promoter was located in two different regions: an activator protein-1 (AP-1) binding site and a NF-κB binding site. The current study comprehensively determined the involvement of MAPKs/AP-1 and IKKs/IκB/NF-κB in epidermal growth factor (EGF)-induced A549 cells, and sought to ascertain the signaling pathways of naringin imparted in suppression of EGF-induced MUC5AC secretion. The results showed that naringin of 100μM not only significantly decreased EGF-induced overexpressions of both MUC5AC mucin and mRNA in A549 cells, but also suppressed the phosphorylation of EGF receptor, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK), as well as nucleus NF-κB p65 and AP-1. Moreover, any of three MAPKs inhibitors (PD98059, SB203580, and SP600125) significantly inhibited EGF-induced MUC5AC secretion. And as compared to MG132, the inhibitor κB (IκB) phosphorylation inhibitor of SN50 was more effective in reducing EGF-induced MUC5AC secretion because of suppression of nucleus AP-1. Meanwhile, as compared to naringin, both SP600125 and azithromycin were less effective in suppressing EGF-induced secretion of MUC5AC because of the unchanged nucleus NF-κB p65. These results indicated that naringin attenuates EGF-induced MUC5AC secretion in A549 cells by suppressing the cooperative activities of MAPKs/AP-1 and IKKs/IκB/NF-κB signaling pathways.

Discoidin domain receptor 1 expression in activated T cells is regulated by the ERK MAP kinase signaling pathway

The expression and function of discoidin domain receptor 1 (DDR1) in T cells are still poorly explored. We have recently shown that activation of primary human T cells via their T cell receptor leads to increased expression of DDR1, which promoted their migration in three-dimensional collagen. In the present study, we provide evidence that activated T cells bind collagen through DDR1. We found that the DDR1:Fc blocking molecule significantly reduced the ability of activated T cells to bind soluble biotinylated collagen. However, DDR1:Fc had no impact on the adhesion of activated T cells to collagen and overexpression of DDR1 in Jurkat T cells did not enhance their adhesion. Together, our results indicate that DDR1 can promote T cell migration without enhancing adhesion to collagen, suggesting that it can contribute to the previously described amoeboid movement of activated T cells in collagen matrices. Our results also show that CD28, in contrast to IL-2 expression, did not costimulate the expression of DDR1 in primary human T cells. Using specific inhibitors, we demonstrated that TCR-induced expression of DDR1 in T cells is regulated by the Ras/Raf/ERK MAP Kinase and PKC pathways but not by calcium/calcineurin signaling pathway or the JNK and P38 MAP Kinases. Thus, our study provides additional insights into the physiology of DDR1 in T cells and may therefore further our understanding of the regulatory mechanisms of T cell migration.

Soluble amyloid precursor protein: a novel proliferation factor of adult progenitor cells of ectodermal and mesodermal origin

IntroductionSoluble amyloid precursor protein α (sAPPα) is a proteolyte of APP cleavage by α-secretase. The significance of the cleavage and the physiological role of sAPPα are unknown. A crystal structure of a region of the amino terminal of sAPPα reveals a domain that is similar to cysteine-rich growth factors. While a previous study implicates sAPPα in the regulation of neural progenitor cell proliferation in the subventricular zone of adult mice, the ubiquitous expression of APP suggests that its role as a growth factor might be broader.MethodssAPPα and α-secretase activities were determined in neural progenitor cells (NPCs), mesenchymal stem cells (MSC) and human decidua parietalis placenta stem cells (hdPSC). Inhibition of α-secretase was achieved by treatment with the matrixmetalloproteinase inhibitor GM6001, and proliferation was determined using clonogenic and immunocytochemical analysis of cell-lineage markers. Recovery of proliferation was achieved by supplementing GM6001-treated cells with recombinant soluble APPα. Expression of APP and its cellular localization in the subventricular zone was determined by Western blot and immunohistochemical analyses of APP wild type and knockout tissue. Alterations in pERK and pAKT expression as a function of soluble APPα production and activity in NPCs were determined by Western blot analysis.ResultsHere we show that sAPPα is a proliferation factor of adult NPCs, MSCs and hdpPSC. Inhibition of α-secretase activity reduces proliferation of these stem cell populations in a dose-dependent manner. Stem cell proliferation can be recovered by the addition of sAPPα in a dose-dependent manner, but not of media depleted of sAPPα. Importantly, sAPPα operates independently of the prominent proliferation factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but in association with ERK signaling and MAP-kinase signaling pathways. Levels of sAPPα and putative α-secretase, ADAM10, are particularly high in the subventricular zone of adult mice, suggesting a role for sAPPα in regulation of NPCs in this microenvironment.ConclusionsThese results determine a physiological function for sAPPα and identify a new proliferation factor of progenitor cells of ectodermal and mesodermal origin. Further, our studies elucidate a potential pathway for sAPPα signaling through MAP kinase activation.

β-1,4-Galactosyltransferase I Promotes Tumor Necrosis Factor-α Autocrine via the Activation of MAP Kinase Signal Pathways in Schwann Cells

Recent studies have demonstrated that aberrant galactosylation is associated with some inflammation diseases. β-1,4-Galactosyltransferase-I (β-1,4-GalT I), which transferred galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a β-1,4-linkage, was considered to be the major galactosyltransferase among the seven members of the subfamily responsible for β4 galactosylation. To elucidate the expression and possible function of β-1,4-GalT I in the peripheral nervous system (PNS) inflammatory diseases, we performed a tumor necrosis factor-alpha (TNF-α) autocrine inflammatory model in Schwann cells (SCs). In this study, we found that silencing of β-1,4-GalT I suppressed TNF-α autocrine, while overexpression of β-1,4-GalT I promoted TNF-α autocrine in TNF-α-treated SCs. Meanwhile, anti-TNFR1 antibody suppressed the expression of β-1,4-GalT I, and TNF-α autocrine. β-1,4-GalT I conferred its effect by promoting ERK, JNK, and P38 MAP kinase signal pathways activation in TNF-α-induced SCs. Thus, the present data shows that during SCs activation, β-1,4-GalT I may play an important role in the release of inflammatory mediators.

Naringenin attenuates mucous hypersecretion by modulating reactive oxygen species production and inhibiting NF-κB activity via EGFR-PI3K-Akt/ERK MAPKinase signaling in human airway epithelial cells

Naringenin (Nar) is a flavonoid derived from plant foods. It has been shown to have anti-inflammatory properties. Many studies have shown that overexpression of reactive oxygen species (ROS) and nuclear factor-κB (NF-κB) leads to increased mucin (MUC) 5AC expression in chronic inflammation of the airway. In addition, some studies have reported that naringenin inhibits NF-κB activity in a murine model of asthma. We speculated that naringenin might be associated with mucous hypersecretion, but the molecular mechanisms remain to be defined. Our study has also investigated whether naringenin could inhibit production of ROS and the activity of NF-κB on the inflammatory pulmonary diseases induced by human neutrophil elastase (HNE) and reduce the mRNA and protein levels of MUC5AC as shown by reverse transcriptase-polymerase chain reaction and real-time PCR (RT-PCR). Serum total MUC5AC protein was detected by enzyme-linked immunosorbent assay (ELISA), the protein morphological changes of MUC5AC were also observed by immunofluorescence and confocal laser technology. Hyperactivation of epidermal growth factor receptor (EGFR) signaling is commonly involved in the mucous hypersecretion process and initiates both the activation of extracellular signal-related kinases 1/2 (ERK1/2) and of phosphatidylinositol 3-kinase (PI3K) and Akt kinase. NF-κB is a key factor downstream of PI3K/Akt signaling, which induces overexpression of the MUC5AC gene. Our data revealed that naringenin inhibited the activation of EGFR resulting in the downregulation of the enzyme activities. Naringenin also reduced the protein expressions of p-EGFR, PI3K, p-Akt, p-ERK1/2, and NF-κB as shown by western blotting. Furthermore, naringenin significantly inhibited PI3K/Akt and ERK MAPKinase signaling with a concurrent reduction in production of ROS and NF-κB activities. These results suggest that naringenin may play a protective role by minimizing mucous production during airway inflammation by down-regulating ROS production and inhibiting the NF-κB activity via EGFR-PI3K-Akt/ERK MAPKinase signaling pathway.

Long-Term Potentiation in the CA1 Hippocampus Induced by NR2A Subunit-Containing NMDA Glutamate Receptors Is Mediated by Ras-GRF2/Erk Map Kinase Signaling

BackgroundNMDA-type glutamate receptors (NMDARs) are major contributors to long-term potentiation (LTP), a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood.Methodology/Principal FindingsIn this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules.Conclusions/SignificanceThis study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocamus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity.

W1726 ERK MAP Kinase Signalling and PEA3 are Synergistic in Predicting Adversity in Gastric Adenocarcinomas

Introduction The PEA3 sub family consists of 3 similar transcription factors, PEA3, ER81 and ERM. In gastric cancer cells PEA3 is activated by H pylori and it has been shown to regulate MMP-7 and invasion. PEA3 mRNA expression correlates with an adverse prognosis in gastric adenocarcinomas. We have previously shown that PEA3 and/or ER81 mRNA correlates with adverse the indicator, MMP-1 and MMP-7. Very high PEA3 protein expression in isolation is associated with tumour metastases. However tumours with PEA3 mRNA and lower protein expression did not correlate with prognostic markers. PEA3 is regulated by ERK MAP kinase signalling in oesophageal adenocarcinomas and we investigated if this pathway plays an important role in gastric adenocarcinomas. Methods Patients with gastric adenocarcinoma (37) and controls (11) were selected. Tissue microarrays were constructed for immunohistochemical studies of PEA3 and pERK. The t test and chi square test were used for statistical analysis. A p value Results In patients with gastric adenocarcinoma, active ERK signalling is present in 60%. From these, ERK signalling is associated with an advanced tumour stage (AJCC stage 1 67%, stage 2: 33%, stage 3: 21% stage 4: 80% p=0.04). Active signalling is present in stage 1 and 2 tumours (55%). Survival is significantly reduced in patients with active ERK signalling (41 vs 34 months P=0.05) on univariate analysis. Adenocarcinomas with PEA3 and pERK co-occurrence frequently have distant metastases 60% (T and N stage vs M stage p=0.048). Survival is worse in patients with combined pERK and PEA3 presence compared to PEA3 or pERK presence in isolation (12 vs 30 vs 63 months p=0.071). Conclusion Our results indicate that ERK MAP kinase signalling is an important marker of an advanced tumour stage and prognosis in gastric adenocarcinoma. The signalling pathway is frequently active in early stage (AJCC 1–3) tumours. Previous studies have shown poor results with MEK inhibition treatment in Pancreatic, Colon, Lung and Breast cancers. This may be due to the selection of heterogeneous tumours with an advanced disease stage or tumours with activation of alternative signalling pathways. Our results suggest consideration of trialling MEK inhibition treatment in patients with earlier stage disease in gastric adenocarcinoma. The more adverse outcome in patients with the combination of ERK MAP signalling and PEA3 indicates that a combined approach with inhibition of PEA3, could be considered in selected patients.

Abstract 1564: Fluvastatin and atorvastatin inhibit human leukemic natural killer cell function

Abstract Clinically relevant doses of statins were investigated for their in vitro inhibitory effects on proliferation, cell cycle progression, cytotoxicity, and ERK1 and ERK2 MAP kinase activation in the YT-INDY human natural killer cell leukemia. YT-INDY is a model for studying new therapies against natural killer cell leukemias. Statins interrupt the formation of cholesterol by inhibiting HMG-CoA reductase, which is responsible for controlling the rate of cholesterol synthesis. Distinct from their effects on LDL-cholesterol, statins have been shown to inhibit the growth and induced apoptosis of various leukemia cell lines. Compared to controls, a seven day incubation with 10 uM and 5 uM fluvastatin inhibited YT-INDY proliferation 24% and 13%, respectively. Under the same conditions, atorvastatin inhibited proliferation 21% and 9%, respectively. Mevalonate (1 mM) fully reversed the effects of the statins, indicating that the compounds were acting through inhibition of HMG-CoA reductase. When cell cycle analysis was performed, it was determined that, following seven days of incubation with 10 uM fluvastatin or atorvastatin, there was an increased percentage of cells in the G0/G1 phase and a reduction in the S and G2/M phases. While 5 uM statin concentrations showed a modest effect on cell cycle progression, the results were not statistically significant. These data indicated that the statins altered YT-INDY cell cycle progression, thus reducing cell proliferation. A two-day incubation of 5 uM fluvastatin or atorvastatin was sufficient to cause nearly complete abrogation of natural killer cell cytotoxicity, which could be reversed with 1 mM mevalonate or 10 uM geranylgeranyl pyrophosphate. This is significant because natural killer cell cytotoxicity is thought to produce the pulmonary damage seen in some natural killer cell leukemia patients. Since both proliferation and cytotoxicity are dependent upon the ERK MAP kinase signal transduction pathway, we investigated the effect of low dose statins on ERK1 and ERK2 activation. We showed that as little as 5 uM fluvastatin or atorvastatin significantly inhibited the activation of ERK1 and ERK2, which are constitutively expressed in YT-INDY. Finally, flow cytometric analysis of YT-INDY incubated for seven days with 5 uM or 10 uM fluvastatin or atorvastatin decreased the forward scatter and side scatter parameters. This indicated that YT-INDY cell size and granularity were reduced. From these investigations, we concluded that 1) clinically relevant concentrations of fluvastatin and atorvastatin inhibit the proliferation of YT-INDY, 2) inhibitory effects of the statins appear to be dependent on decreasing cell cycle progression, and 3) ERK1 and ERK2 MAP kinase activation is severely reduced. Given the typical poor prognosis for natural killer cell leukemia patients, these results may point the way to better therapeutic strategies for this devastating cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1564.

OC-051 Erk MAP kinase signalling and PEA3 are synergistic in predicting adversity in gastric adenocarcinomas

<h3>Introduction</h3> The PEA3 sub family consists of 3 similar transcription factors, PEA3, ER81 and ERM. In gastric cancer cells PEA3 is activated by H pylori and it has been shown to regulate MMP-7 and invasion. PEA3 mRNA expression correlates with an adverse prognosis in gastric adenocarcinomas. We have previously shown that PEA3 and/or ER81 mRNA correlates with adverse the indicator, MMP-1 and MMP-7. Very high PEA3 protein expression in isolation is associated with tumour metastases. However tumours with PEA3 mRNA and lower protein expression did not correlate with prognostic markers. PEA3 is regulated by ERK MAP kinase signalling in oesophageal adenocarcinomas and we investigated if this pathway plays an important role in gastric adenocarcinomas. <h3>Methods</h3> Patients with gastric adenocarcinoma (37) and controls (11) were selected. Tissue microarrays were constructed for immunohistochemical studies of PEA3 and pERK. The t test and chi square test were used for statistical analysis. A p value &lt;0.05 was judged to be significant. <h3>Results</h3> In patients with gastric adenocarcinoma, active ERK signalling is present in 60%. From these, ERK signalling is associated with an advanced tumour stage (AJCC stage 1 67%, stage 2: 33%, stage 3: 21% stage 4: 80% p=0.04). Active signalling is present in stage 1 and 2 tumours (55%). Survival is significantly reduced in patients with active ERK signalling (41 vs 34 months P=0.05) on univariate analysis. Adenocarcinomas with PEA3 and pERK co-occurrence frequently have distant metastases 60% (T and N stage vs M stage p=0.048). Survival is worse in patients with combined pERK and PEA3 presence compared to PEA3 or pERK presence in isolation (12 vs 30 vs 63 months p=0.071). <h3>Conclusion</h3> Our results indicate that ERK MAP kinase signalling is an important marker of an advanced tumour stage and prognosis in gastric adenocarcinoma. The signalling pathway is frequently active in early stage (AJCC 1–3) tumours. Previous studies have shown poor results with MEK inhibition treatment in Pancreatic, Colon, Lung and Breast cancers. This may be due to the selection of heterogeneous tumours with an advanced disease stage or tumours with activation of alternative signalling pathways. Our results suggest consideration of trialling MEK inhibition treatment in patients with earlier stage disease in gastric adenocarcinoma. The more adverse outcome in patients with the combination of ERK MAP signalling and PEA3 indicates that a combined approach with inhibition of PEA3, could be considered in selected patients.

FGFR3 signaling induces a reversible senescence phenotype in chondrocytes similar to oncogene-induced premature senescence

Oncogenic activation of the RAS–ERK MAP kinase signaling pathway can lead to uncontrolled proliferation but can also result in apoptosis or premature cellular senescence, both regarded as natural protective barriers to cell immortalization and transformation. In FGFR3-related skeletal dyplasias, oncogenic mutations in the FGFR3 receptor tyrosine kinase cause profound inhibition of cartilage growth resulting in severe dwarfism, although many of the precise mechanisms of FGFR3 action remain unclear. Mutated FGFR3 induces constitutive activation of the ERK pathway in chondrocytes and, remarkably, can also cause both increased proliferation and apoptosis in growing cartilage, depending on the gestational age. Here, we demonstrate that FGFR3 signaling is also capable of inducing premature senescence in chondrocytes, manifested as reversible, ERK-dependent growth arrest accompanied by alteration of cellular shape, loss of the extracellular matrix, upregulation of senescence markers (α-GLUCOSIDASE, FIBRONECTIN, CAVEOLIN 1, LAMIN A, SM22α and TIMP 1), and induction of senescence-associated β-GALACTOSIDASE activity. Our data support a model whereby FGFR3 signaling inhibits cartilage growth via exploiting cellular responses originally designed to eliminate cells harboring activated oncogenes.

Role of ERK map kinase and CRM1 in IL‐1β‐stimulated release of HMGB1 from cortical astrocytes

Reactive astrocytes are traditionally thought to impede brain plasticity after stroke. However, we previously showed that reactive astrocytes may also contribute to stroke recovery, partly via the release of a nuclear protein called high-mobility group box 1 (HMGB1). Here, we investigate the mechanisms that allow stimulated astrocytes to release HMGB1. Exposure of rat primary astrocytes to IL-1beta for 24 h elicited a dose-dependent HMGB1 response. Immunostaining and western blots of cell lysates showed increased intracellular levels of HMGB1. Western blots confirmed that IL-1beta induced a release of HMGB1 into astrocyte conditioned media. MAP kinase signaling was involved. Levels of phospho-ERK were increased by IL-1beta, and the MEK/ERK inhibitor U0126 decreased HMGB1 upregulation in the stimulated astrocytes. Since HMGB1 is a nuclear protein, the role of the nuclear protein exporter, chromosome region maintenance 1 (CRM1), was assessed as a candidate mechanism for linking MAP kinase signaling to HMGB1 release. IL-1beta increased CRM1 expression in concert with a translocation of HMGB1 from nucleus into cytoplasm. Blockade of IL-1beta-stimulated HMGB1 release with the ERK inhibitor U0126 was accompanied by a downregulation of CRM1. Our findings reveal that IL-1beta stimulates the release of HMGB1 from activated astrocytes via ERK MAP kinase and CRM1 signaling. These data suggest a novel pathway by which inflammatory cytokines may enhance the ability of reactive astrocytes to release prorecovery mediators after stroke.

Direct Activation of TACE-Mediated Ectodomain Shedding by p38 MAP Kinase Regulates EGF Receptor-Dependent Cell Proliferation

Inflammatory stimuli activate ectodomain shedding of TNF-alpha, L-selectin, and other transmembrane proteins. We show that p38 MAP kinase, which is activated in response to inflammatory or stress signals, directly activates TACE, a membrane-associated metalloprotease that is also known as ADAM17 and effects shedding in response to growth factors and Erk MAP kinase activation. p38alpha MAP kinase interacts with the cytoplasmic domain of TACE and phosphorylates it on Thr(735), which is required for TACE-mediated ectodomain shedding. Activation of TACE by p38 MAP kinase results in the release of TGF-alpha family ligands, which activate EGF receptor signaling, leading to enhanced cell proliferation. Conversely, depletion of p38alpha MAP kinase activity suppresses EGF receptor signaling and downstream Erk MAP kinase signaling, as well as autocrine EGF receptor-dependent proliferation. Autocrine EGF receptor activation through TACE-mediated ectodomain shedding intimately links inflammation and cancer progression and may play a role in stress and conditions that relate to p38 MAP kinase activation.

The Extracellular Signal‐Regulated Kinase Is Involved in the Effects of Sildenafil on Pulmonary Vascular Remodeling

Pulmonary hypertension is a group of diseases comprising vascular constriction and obstructive changes of the pulmonary vasculature. Phosphodiesterase type 5 inhibitors, for example, sildenafil, can alleviate vascular remodeling in the monocrotaline pulmonary hypertension model in rats. We investigate the mechanisms of sildenafil on the pulmonary vascular remodeling of pulmonary hypertension induced by monocrotaline (MCT) in rats. Thirty Sprague-Dawley rats (weighing 200-220 g) were administered with MCT abdominal cavity injection or equivalent volume of normal saline (NS) (which were treated as C group n = 10) to induce pulmonary hypertension model. Fourteen days later, 20 MCT treated rats were randomly fed with sildenafil (25mg/kg/day) or placebo as S, P group (10 rats for each group), respectively. Another 6 weeks later, mean pulmonary artery pressure (mPAP), index of right ventricular hypertrophy (RV/LV+S) of all animals were measured under general anesthesia. Pulmonary tissue was collected to investigate pathological features of pulmonary arteries and to measure protein expression of ERK(1)/ERK(2) and MKP1. After 6 weeks, there were significant elevated mPAP and RV/LV+S in both P and S groups. The ratio of wall thickness to vessel diameter in pulmonary arteries with diameters <200 microm were increased in both P and S groups. But the ratio of wall thickness to vessel diameter was smaller in S group than that in P group. The phosphorylation level of ERK(1)/ERK(2) were elevated in both P and S groups, but the level of phosphorlation ERK(1)/ERK(2) were lower in S group than that in P group. Intriguingly, the expression level of MKP1 was significantly increased in both S and P groups, while it was higher in S group than that in P group. The sildenafil can decrease mPAP and inhibit the progress of pulmonary vascular remodeling in pulmonary hypertension rats. The ERK-MAP kinase signaling pathway might play a role during this process.

Differential regulation of MAP kinase activation by a novel splice variant of human MAP kinase phosphatase-2

MAP kinase phosphatase-2 (MKP-2) is a member of the family of dual specificity phosphatases that functions to inactivate the ERK and JNK MAP kinase signalling pathways. Here, we identify a novel human MKP-2 variant (MKP-2-S) lacking the MAP kinase binding site but retaining the phosphatase catalytic domain. Endogenous MKP-2-S transcripts and proteins were found in PC3 prostate and MDA-MB-231 breast cancer cells and also human prostate biopsies. Cellular transfection of MKP-2-S gave rise to a nuclear protein of 33kDa which displayed phosphatase activity comparable to the formerly described long form of MKP-2 (MKP-2-L). Due to its lack of a kinase interacting motif (KIM), MKP-2-S did not bind to JNK or ERK; MKP-2-L bound ERK and to a lesser extent JNK. Protein turnover of adenoviral expressed MKP-2-S was accelerated relative to MKP-2-L, with a greater susceptibility to proteosomal-mediated degradation. MKP-2-S retained its ability to deactivate JNK in a similar manner as MKP-2-L and was an effective inhibitor of LPS-stimulated COX-2 induction. However, unlike MKP-2-L, MKP-2-S was unable to reverse serum-induced ERK activation or significantly inhibit endothelial cell proliferation. These findings reveal the occurrence of a novel splice variant of MKP-2 which is unable to bind ERK and may be significant in the dysregulation of MAP kinase activity in certain disease states, particularly in breast and prostate cancers.

Expression of β‐1,4‐galactosyltransferase I in rat Schwann cells

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of beta-1,4-galactosylation is performed by a family of beta-1,4-galactosyltransferases (beta-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. In the present study, real-time PCR revealed that the beta-1,4-GalT I mRNA reached peaks at 2 weeks after sciatic nerve crush and 3 days after sciatic nerve transection. Combined in situ hybridization for beta-1,4-GalT I mRNA and immunohistochemistry for S100 showed that beta-1,4-GalT I mRNAs were mainly located in Schwann cells after sciatic nerve injury. In conclusion, beta-1,4-GalT I might play important roles in Schwann cells during the regeneration and degeneration of the injured sciatic nerve. In other pathology, such as inflammation, we found that LPS administration affected beta-1,4-GalT I mRNA expression in sciatic nerve in a time- and dose-dependent manner, and beta-1,4-GalT I mRNA is expressed mainly in Schwann cells. These results indicated that beta-1,4-GalT I plays an important role in the inflammation reaction induced by intraperitoneal injection of LPS. Similarly, we found that beta-1,4-GalT I in Schwann cells in vitro was affected in a time- and concentration-dependent manner in response to LPS stimulation. All these results suggest that beta-1,4-GalT I play an important role in Schwann cells in vivo and vitro during pathology. In addition, beta-1,4-GalT I production was drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that Schwann cells which regulated beta-1,4-GalT I expression after LPS stimulation were via ERK, SAPK/JNK, and P38 MAP kinase signal pathways.

Building the perfect beast: complex mouse models teach surprisingly simple melanoma lessons

Building the perfect beast: complex mouse models teach surprisingly simple melanoma lessons

MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts

MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target messenger RNAs. Dysregulation of microRNAs by several mechanisms has been described in various disease states including cardiac disease. Whereas previous studies of cardiac disease have focused on microRNAs that are primarily expressed in cardiomyocytes, the role of microRNAs expressed in other cell types of the heart is unclear. Here we show that microRNA-21 (miR-21, also known as Mirn21) regulates the ERK-MAP kinase signalling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function. miR-21 levels are increased selectively in fibroblasts of the failing heart, augmenting ERK-MAP kinase activity through inhibition of sprouty homologue 1 (Spry1). This mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specific antagomir in a mouse pressure-overload-induced disease model reduces cardiac ERK-MAP kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction. These findings reveal that microRNAs can contribute to myocardial disease by an effect in cardiac fibroblasts. Our results validate miR-21 as a disease target in heart failure and establish the therapeutic efficacy of microRNA therapeutic intervention in a cardiovascular disease setting.

Autocrine Actions of Matrix Metalloproteinase (MMP)-2 Counter the Effects of MMP-9 to Promote Survival and Prevent Terminal Differentiation of Cultured Human Keratinocytes

Cultured human keratinocytes produce matrix metalloproteinase (MMP)-2 and MMP-9. In this study, using small interfering RNA (siRNA) for MMP-2 or MMP-9, we investigated the functions of these two gelatinases in the regulation of survival by measuring growth, differentiation, apoptosis, and migration of cultured keratinocytes. MMP-2 siRNA treatment significantly decreased keratinocyte growth and migration, and stimulated apoptosis fourfold. In addition, MMP-2 siRNA caused a 70% reduction in keratin-14 (K14) and a fourfold increase in K10. In contrast, MMP-9 siRNA treatment exerted opposite effects on cell growth, apoptosis, and K10 expression. MMP-2 appears to act through the ERK MAP kinase and caspase-3 signaling pathways as evidenced by the 53% reduction in the level of phosphorylated ERK1/2 and threefold increase in phosphorylated p38 and stronger staining for active caspase-3 in response to MMP-2 siRNA. Dual fluorescent staining revealed that almost all cultured cells stained positive for MMP-2, with only a few scattered cells being positive for MMP-9. There were considerably more BrdU-positive cells following MMP-9 siRNA treatment, indicating that MMP-9 inhibited proliferation. In conclusion, MMP-2 stimulates keratinocyte survival whereas MMP-9 promotes terminal differentiation.