Role Of Extracellular Signal-regulated Kinase Research Articles

Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinases pathways

We have previously demonstrated that transforming growth factor-β (TGF-β) in the presence of elevated levels of Smad3, its primary signaling protein, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The mechanism is partly through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluated the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TGF-β-induced VSMC proliferation. Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with small interfering RNA (siRNA). For in vivo experiments, male Sprague-Dawley rats underwent carotid balloon injury, followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK, and proliferating cell nuclear antigen. In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time-dependent and concentration-dependent manner. Overexpression of the signaling protein Smad3 enhanced TGF-β-induced activation of ERK MAPK, whereas inhibition of Smad3 with a siRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by proliferating cell nuclear antigen. Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that TGF-β enhances VSMC proliferation through a Smad3/ERK MAPK signaling pathway. These findings at least partly explain the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

Activation of the extracellular signal‐regulated kinase pathway contributes to the behavioral deficit of fragile x‐syndrome

Fragile X syndrome (FXS) is a developmental disorder caused by the loss of Fragile X Mental Retardation 1 (FMR1) gene function because of a CGG repeat expansion (> 200 repeats) in the gene. The molecular mechanism(s) linking loss of FMR1 function to the molecular pathology and cognitive/behavioral disability remain unclear. Given the critical role of extracellular signal-regulated kinase (ERK) in synaptic plasticity and neurodevelopment, a number of recent studies have investigated ERK phosphorylation under basal conditions or upon mGluR-induction using neuronal and peripheral tissues from Fmr1 knockout mice and peripheral tissues from FXS patients. However, these reports have presented conflicting results. The current study is the first to focus on the levels of ERK phosphorylation in brain tissue from human FXS patients. In both human brain tissue and brain tissue from Fmr1 knockout mice there was significantly increased phosphorylation of MEK1/2 and ERK. Indeed, treating Fmr1 knockout mice with the MEK1/2 inhibitor SL327 abrogated audiogenic seizure activity, a feature of the Fmr1 knockout mice that replicates the symptom in patients with FXS. These findings suggest that activation of the ERK pathway results in some cardinal cognitive and clinical features in FXS patients and likely have profound translational implications.

ERK1/2 and Akt pathway activated during (3R,6R)-bassiatin(1)-induced apoptosis in MCF-7 cells

(3R,6R)-bassiatin(1) was isolated from the endogenous fungus, Fusarium oxysporum J8-1-2. Previous studies showed that (3R,6R)-bassiatin(1) has anti-oestrogen properties which make it cytotoxic to ER (oestrogen receptor)-positive breast cancer cells (MCF-7) by cell cycle arrest and induction of apoptosis. (3R,6R)-bassiatin(1) suppresses mRNA and protein expression of the ERα and oestrogen responsive genes of cyclin D1 and PR. We have investigated the interaction between (3R,6R)-bassiatin(1) and ERα and followed the roles of ERK (extracellular-signal-regulated kinase), Akt and GSK3β (glycogen synthase kinase 3β) during (3R,6R)-bassiatin(1)-induced apoptosis of MCF-7 cells. (3R,6R)-bassiatin(1) competed with E2 (17β-estradiol) for ERα active sites to inhibit ERα activation. However, while ERK1/2 and Akt were activated, GSK3β was inactivated during (3R,6R)-bassiatin(1)-induced apoptosis, suggesting that this compound is indeed an anti-oestrogen agent that can also activate the survival signalling pathway. Apoptosis caused by (3R,6R)-bassiatin(1) may be related to activation of ERK.

The effects of metabotropic glutamate receptor 7 allosteric agonist N,N′-dibenzhydrylethane-1,2-diamine dihydrochloride on developmental sevoflurane neurotoxicity: role of extracellular signal-regulated kinase 1 and 2 mitogen-activated protein kinase signaling pathway

The present study was designed to evaluate the possible neuroprotective effects of metabotropic glutamate receptor (mGluR7) allosteric agonist N,N′-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) on developmental sevoflurane neurotoxicity. To achieve the objective, hippocampal cultures (7 DIV, 7 day in vitro) were treated with different doses of l-(+)-2-Amino-4-phosphonobutyric acid (l-AP4, an agonist of group III mGluRs), (RS)-α-Methylserine-O-phosphate (MSOP, an antagonist of group III mGluRs), AMN082 or cis-2-[[(3,5-Dichlorophenyl)amino]carbonyl]cyclohexanecarboxylic acid (VU0155041, an agonist of mGluR4) before exposed to sevoflurane. Cell apoptosis were determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL)-staining. For in vivo study, rat pups (7 PND, 7 postnatal day) were injected with AMN082, l-AP4 or saline before sevoflurane exposure. Extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, caspase-3, Bcl-2, and Bax were detected by Western blot. The locomotor activity and cognitive functions were evaluated by open-field test and Morris water maze (MWM), respectively. We found that l-AP4 prevented sevoflurane-induced cell apoptosis, but MSOP promoted. Specially, application of AMN082 contributed to the relief of sevoflurane-induced apoptosis in vitro, whereas VU0155041 did not. In addition, sevoflurane treatment led to a decrease of Bcl-2 and an increase of caspase-3 and Bax, which were mitigated by AMNO82 in vivo. Moreover, we showed that sevoflurane treatment resulted in a remarkable suppression of phospho-ERK1/2, which was restored by AMN082. Application of U0126 (an inhibitor of MEK) abolished the neuroprotective effects of AMN082 on sevoflurane neurotoxicity both in vitro and in vivo. In addition, sevoflurane exposure also led to an increase of phospho-JNK, but SP600125 (an inhibitor of JNK) did not attenuate sevoflurane-induced apoptosis. The total and phosphorylated p38 remained unchanged in sevoflurane-treated rat pups. Finally, AMN082 improved the learning and memory defects caused by postnatal sevoflurane exposure without alternations in emotion or locomotor activity. These preliminary data indicate that AMN082 may protect immature brain against sevoflurane neurotoxicity, and the ERK1/2 MAP kinase signaling is likely to be involved. Further studies are needed to fully assess the neuroprotective role of mGluR7 agonist AMN082 in developmental anesthetic neurotoxicity.

ERK1/2 activation in quercetin-treated BEAS-2B cell plays a role in Nrf2-driven HO-1 expression

Quercetin, a member of the flavonoids, has been heralded as chemopreventive agent, generally because of its free radical-scavenging and antiproliferative activities. In this process, NF-E2-related factor-2 (Nrf2) plays a role in protecting cells from oxidative damage; however, the exact mechanisms in which bronchial epithelial cells respond to quercetin by activating Nrf2 are not fully understood. Here, we report the role of extracellular signal-regulated kinase (ERK) activation in response to quercetin. Quercetin treatment decreased the viability of human bronchial epithelial BEAS-2B cells in a dose- and time-dependent manner. ERK1/2 was activated within 10 min of quercetin addition and then remained increased above control level throughout 48 h, whereas AKT phosphorylation was suppressed from the first 1 h of quercetin treatment. Inhibition of Erk1/2 phosphorylation attenuated quercetin-induced loss of cell viability. Nrf2 levels in both nuclear and whole cell lysates increased after quercetin treatment and was accompanied by the increase of heme oxygenase-1 (HO-1) protein level, indicating that increased HO-1 expression is Nrf2-mediated. Activation of the Nrf2/HO-1 system after quercetin treatment was suppressed by the pretreatment with MEK inhibitor, PD98059. Also, silencing Nrf2 with siRNA decreased cell viability and augmented cytotoxic effect of anticancer drug LBH589. Overall, our results indicate that quercetin induces the up-regulation of Nrf2-mediated HO-1 expression, at least in part, via MEK/ERK1/2 signaling, as an adaptive mechanism which may provide advantages for cell proliferation and enhance survival despite endogenous and/or exogenous oxidative insults.

A physiological concentration of glucocorticoid inhibits the pro-inflammatory cytokine-induced proliferation of adult rat cardiac fibroblasts: Roles of extracellular signal-regulated kinase 1/2 and nuclear factor-κB

1. Inflammation-induced proliferation of cardiac fibroblasts plays an important role in cardiac remodelling. Pharmacological doses of exogenous glucocorticoids (GC) are the most effective therapy for inflammatory diseases. Similarly, physiological concentrations of endogenous GC have recently been shown to have anti-inflammatory effects. Therefore, the aim of the present study was to determine whether a physiological concentration of GC could inhibit pro-inflammatory cytokine-stimulated proliferation of cardiac fibroblasts and to explore the mechanisms involved. 2. Cardiac fibroblasts were isolated from adult male Sprague-Dawley rats and cell proliferation was measured using a CCK-8 kit. Western blotting was used to detect protein expression of extracellular-regulated kinase (ERK) 1/2 and nuclear factor (NF)-κB. 3. Cardiac fibroblast proliferation was significantly increased by tumour necrosis factor-α, interleukin (IL)-1β and angiotensin II and was accompanied by upregulated protein expression of ERK1/2 and NF-κB. A physiological concentration of hydrocortisone (127 ng/mL) not only inhibited the proliferation of cardiac fibroblasts, but also suppressed activation of ERK1/2 and NF-κB. These effects of hydrocortisone were abrogated by the glucocorticoid receptor (GR) antagonist RU-486 (100 nmol/L). Furthermore, inflammation-induced cardiac fibroblast proliferation was also blocked by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (100 nmol/L) and the NF-κB inhibitor pyrrolidine dithiocarbamate (1 μmol/L). Cytokine-induced ERK1/2 phosphorylation and cyclin D1 expression were attenuated by U0126, suggesting that the ERK1/2 and NF-κB signalling pathways were involved in cardiac fibroblast proliferation. 4. In conclusion, the results of the present study indicate that a physiological concentration of hydrocortisone can inhibit inflammation-induced proliferation of cardiac fibroblasts by preventing the activation of ERK1/2 and NF-κB.

The roles of extracellular signal‐regulated kinase 1/2 pathway in regulating osteogenic differentiation of murine preosteoblasts MC3T3‐E1 cells on roughened titanium surfaces

Surface roughness of titanium-based implants may enhance osteogenic differentiation of cells in vitro and bone-to-implant contact in vivo. Nevertheless, how surface roughness regulates the signaling pathway of osteoblasts is little understood. The study intended to investigate specifically the roles of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in regulating osteogenic differentiation of MC3T3-E1 murine preosteoblast cells on Ti surfaces. Substrates applied were two groups of titanium disks: (1) sand-blasted and acid-etched rough surfaces (SLA) and (2) smooth pretreated Ti surfaces (PT). Surface morphology of the two groups was examined by scanning electron microscope, and cell morphology cultured on Ti disks was observed by confocal microscope. The levels of alkaline phosphatase (ALP) activity and calcium deposition were measured and compared between the two groups. Real-time polymerase chain reaction was applied to detect the expression levels of osteogenic genes including runt related protein 2 (Runx2), osterix (OSX), osteocalcin (OCN) and osteoprotegerin (OPN) of the cells cultured on the two groups of substrates and on SLA surfaces treated with ERK1/2 inhibitor, PD98095. ERK1/2 activities in MC3T3-T1 cells were measured by Western-blotting on the two surfaces with or without PD98095. Cells cultured on rougher SLA surfaces displayed a more differentiated morphology. ALP activities at 7 days and 14 days and the calcium deposition at 28 days were significantly higher on SLA surfaces. The expression levels of Runx2, OSX, OPN and OCN were upregulated by the effect of surface roughness and PD98095 further upregulated the expression levels of these osteogenic genes on SLA surfaces. ERK1/2 phosphorylation was continuously inhibited by surface roughness at 2 days, 4 days and 6 days. In contrast, no marked alterations in ERK1/2 phosphorylation on PT surfaces were observed. PT surfaces treated with PD98095 (50 μM) and SLA surfaces without PD98095 both demonstrated reduced ERK1/2 phosphorylation of the cells, and the inhibitive effect of SLA surfaces was milder than that of PD98095. In conclusion, ERK1/2 pathway may be a negative regulator of cell differentiation in a dosage-dependent manner, and the enhancing effect of surface roughness on osteoblastic differentiation may be mediated through inhibiting ERK1/2 pathway.

Role of extracellular signal-regulated kinase 1/2 in cigarette smoke-induced mucus hypersecretion in a rat model.

Airway mucus hypersecretion is an important pathophysiological feature of chronic obstructive pulmonary disease, which is closely associated with cigarette smoking. However, the signal transduction pathway from the cell surface to the nucleus through which cigarette smoke causes upregulation of mucin gene expression is not well known. This study was designed to investigate the role of extracellular signal-regulated Kinase 1/2 (ERK 1/2) in airway mucus hypersecretion induced by cigarette smoke in rats. A rat model of airway mucus hypersecretion was induced by exposure to cigarette smoke for 4 weeks.Rats exposed to inhalation of cigarette smoke or normal saline were given an intraperitoneal injection of U0126, a specific MEK1 kinase inhibitor, at doses of 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg for 14 days. Expression of MUC5AC mRNA and protein, ERK 1/2 and phosphorylated-ERK 1/2 (p-ERK 1/2) were detected by RT-PCR, immunohistochemistry and Western blotting. Cigarette smoke significantly increased airway goblet cells metaplasia, induced the overexpression of MUC5AC mRNA and protein in bronchial epithelia, and increased the ratio of p-ERK 1/2 and ERK 1/2. U0126 significantly attentuated the expression of MUC5AC mRNA and protein induced by cigarette smoke (P < 0.05). Moreover, there was a significant positive correlation between the ratio of p-ERK1/2 to ERK1/2 and the expression of MUC5AC mRNA and protein (P < 0.05). Inhibition of ERK 1/2 by U0126 decreased the ratio of p-ERK 1/2 to ERK 1/2 and expression of MUC5AC mRNA and protein. ERK 1/2 may play an essential role in cigarette smoke-induced mucus hypersecretion in vivo.

Effect of opioid receptor blockade on acetaldehyde self-administration and ERK phosphorylation in the rat nucleus accumbens

We have previously shown that acetaldehyde (ACD), the first metabolite of ethanol, regulates its motivational properties and possesses reinforcing effects by itself. A large and still growing body of evidence indicates that the endogenous opioidergic system plays a critical role in the motivational effects of ethanol and suggests a role for extracellular signal-regulated kinase (ERK) in these effects of both ethanol and ACD. The present study was undertaken to examine if opioid-mediated mechanisms are involved in the reinforcing properties of ACD and in ACD-elicited ERK activation. To this end, Wistar rats were trained to orally self-administer ACD (0.2%) by nose poking. Responses on active nose poke caused delivery of ACD solution, whereas responses on inactive nose poke had no consequences. The effect of pretreatment with a nonselective opioid receptor antagonist, naltrexone (NTX), was evaluated during (1) maintenance of ACD self-administration, (2) deprivation effect after ACD extinction, and (3) ACD self-administration under a progressive-ratio schedule of reinforcement. Additionally, we tested the effect of NTX on saccharin (0.05%) reinforcement, as assessed by oral self-administration, and on ACD-elicited ERK phosphorylation in the nucleus accumbens (Acb), as assessed by immunohistochemistry. Finally, we examined the effect of a μ 1-selective opioid receptor antagonist, naloxonazine (NLZ), on the maintenance phase of ACD and saccharin self-administration. The results indicate that NTX (0.4–0.8 mg/kg) reduced the maintenance, the deprivation effect, and the break points of ACD self-administration without suppressing saccharin self-administration. Moreover, NTX decreased ACD-elicited ERK activation in the Acb shell and core. NLZ (10–15 mg/kg) reduced the maintenance phase of ACD self-administration without interfering with saccharin self-administration, whereas both NTX and NLZ failed to modify responses on inactive nose poke indicating the lack of a nonspecific behavioral activation. Overall, these results indicate that the opioid system is implicated in the reinforcing properties of ACD and suggest an involvement of ERK. The finding that NTX and NLZ reduce ACD but not saccharin self-administration indicates that these effects are specific to ACD.

Effects of nicotine on apoptosis in human gingival fibroblasts

AimCigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants and it is a world widely known problem to health. Nicotine is the major compound of tobacco and known as the cause of gingivitis and periodontitis. It induces intracellular oxidative stress recognized as the important agent in the damage of biological molecules. The aim of this study is to clarify the cytotoxic pathway of nicotine in human gingival fibroblasts (HGFs). MethodsHuman gingival fibroblasts stimulated by nicotine were used as an in vitro model. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability and reactive oxygen species (ROS) generation was assessed with 2,7-dichlorofluoroscein diacetate (DCF-DA). Morphological change was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay, stained with 4,6-diamidino-2-phenylindole (DAPI). To delineate the roles of extracellular signal-regulated kinase (ERK), P38 and c-Jun N-terminal kinase (JNK), Western blot and caspase-3 (CASP3) activity assay were performed. ResultsExposure of the human gingival fibroblasts to nicotine reduced cell viability by time and dose dependent and increased the generation of ROS. It also showed morphological evidence of increased apoptosis, resulted in transient activation of JNK and ERK concomitant with activation of P38, and stimulated apoptosis as evidenced by CASP3 activation and Poly ADP ribose polymerase (PARP) cleavage. ConclusionThese results suggest that nicotine induces apoptosis through the ROS generation and CASP3 dependent pathways in HGFs.

Erk in Kidney Diseases

Acute or chronic kidney injury results from various insults and pathological conditions, and is accompanied by activation of compensatory repair mechanisms. Both insults and repair mechanisms are initiated by circulating factors, whose cellular effects are mediated by activation selective signal transduction pathways. Two main signal transduction pathways are activated during these processes, the phosphatidylinositol 3′ kinase (PI-3K)/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK) cascades. This review will focus on the latter, and more specifically on the role of extracellular signal-regulated kinase (ERK) cascade in kidney injury and repair.

Role of Extracellular Signal-Regulated Kinase in Synaptic Transmission and Plasticity of a Nociceptive Input on Capsular Central Amygdaloid Neurons in Normal and Acid-Induced Muscle Pain Mice

Application of phorbol 12,13-diacetate (PDA) caused marked enhancement of synaptic transmission of nociceptive parabrachio-amygdaloid (PBA) input onto neurons of the capsular central amygdaloid (CeAC) nucleus. The potentiation of PBA-CeAC EPSCs by PDA involved a presynaptic protein kinase C (PKC)-dependent component and a postsynaptic PKC-extracellular-regulated kinase (ERK)-dependent component. NMDA glutamatergic receptor (NMDAR)-dependent long-term potentiation (LTP) of PBA-CeAC EPSCs, which was also dependent on the PKC-ERK signaling pathway, was induced by tetanus stimulation at 100 Hz. In slices from mice subjected to acid-induced muscle pain (AIMP), phosphorylated ERK levels in the CeAC increased, and PBA-CeAC synaptic transmission was postsynaptically enhanced. The enhanced PBA-CeAC synaptic transmission in AIMP mice shared common mechanisms with the postsynaptic potentiation effect of PDA and induction of NMDAR-dependent LTP by high-frequency stimulation in normal slices, both of which required ERK activation. Since the CeAC plays an important role in the emotionality of pain, enhanced synaptic function of nociceptive (PBA) inputs onto CeAC neurons might partially account for the supraspinal mechanisms underlying central sensitization.

The Role of ERK1/2 Signaling Pathway in Coronary Microembolization-Induced Rat Myocardial Inflammation and Injury

Aim: Inflammation plays an important role in coronary microembolization (CME)-induced myocardial injury. The present study was designed to investigate the role of extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathway in regulating myocardial inflammation and cardiac function in a rat model of CME. Methods: Sprague-Dawley rats were randomly divided into three groups: sham-operated group (sham group), CME group and PD98059 group (15 animals per group). CME was produced by injection of 42-µm microspheres into the left ventricle with occlusion of the ascending aorta. Rats in the PD98059 group were injected with PD98059, a specific ERK1/2 inhibitor, 30 min before the CME operation. Western blotting and immunohistochemistry analysis were used to determine the activation of ERK1/2. Echocardiography was employed to evaluate cardiac function. Hematoxylin-eosin staining was performed to assay myocardial inflammation. Expression of TNF-α mRNA was determined by RT-PCR analysis, and activity of NF-ĸB was assessed by electrophoretic mobility shift assay. Results: CME dramatically induced cardiac dysfunction (left ventricular ejection fraction, LVEF, was 72.97 ± 3.20% in the CME vs. 82.69 ± 3.50% in the sham group, p < 0.05) and local myocardial inflammatory response, both of which were ameliorated significantly by PD98059 (LVEF was 76.46 ± 4.46 and p < 0.05 vs. CME group). When compared to the CME group, PD98059 markedly attenuated the increased phosphorylation of ERK1/2 (0.48 ± 0.11 vs. 0.92 ± 0.10, p < 0.05), expression of TNF-α mRNA (0.42 ± 0.06 vs. 0.94 ± 0.04, p < 0.05) and activity of NF-ĸB (104.83 ± 13.65 vs. 540.79 ± 24.95, p < 0.05) in CME rat myocardium. Conclusions: The present study demonstrates a novel role of the ERK1/2 signaling pathway in promoting myocardium inflammation and dysfunction in CME, and suggests that ERK1/2 is a novel potential therapeutic target for CME.

Extracellular Signal-Regulated Kinases 1 and 2 Regulate the Balance Between Eccentric and Concentric Cardiac Growth

An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, whereas volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood. To determine the role of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in regulating the cardiac hypertrophic response. Here, we used mice lacking all ERK1/2 protein in the heart (Erk1(-/-) Erk2(fl/fl-Cre)) and mice expressing activated mitogen-activated protein kinase kinase (Mek)1 in the heart to induce ERK1/2 signaling, as well as mechanistic experiments in cultured myocytes to assess cellular growth characteristics associated with this signaling pathway. Although genetic deletion of all ERK1/2 from the mouse heart did not block the cardiac hypertrophic response per se, meaning that the heart still increased in weight with both aging and pathological stress stimulation, it did dramatically alter how the heart grew. For example, adult myocytes from hearts of Erk1(-/-) Erk2(fl/fl-Cre) mice showed preferential eccentric growth (lengthening), whereas myocytes from Mek1 transgenic hearts showed concentric growth (width increase). Isolated adult myocytes acutely inhibited for ERK1/2 signaling by adenoviral gene transfer showed spontaneous lengthening, whereas infection with an activated Mek1 adenovirus promoted constitutive ERK1/2 signaling and increased myocyte thickness. A similar effect was observed in engineered heart tissue under cyclic stretching, where ERK1/2 inhibition led to preferential lengthening. Taken together, these data demonstrate that the ERK1/2 signaling pathway uniquely regulates the balance between eccentric and concentric growth of the heart.

Angiotensin II induces interleukin-6 synthesis in osteoblasts through ERK1/2 pathway via AT1 receptor

Background Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteoblast secretion of IL-6 plays an important role in the regulation of bone metabolism. Angiotensin II (Ang II) has been shown to regulate the expression of potent inflammatory factors, including MCP-1 and IL-6, by stimulating endothelia cells, vascular smooth muscle cells (VSMC) and monocytes. However, of the mechanism by which Ang II regulates IL-6 expression in osteoblasts is unknown. Aims The present study was designed to investigate the effect of Ang II on IL-6 expression in osteoblasts isolated from mice. The receptor(s) required and the potential role of extracellular signal-regulated kinase 1/2 (ERK1/2) activation in Ang II-induced IL-6 synthesis was also examined in these cells. Methods The osteoblasts were isolated from the calvaria of mice and cultured in α-MEM medium. IL-6 mRNA expression and protein synthesis was determined by qPCR and ELISA analyses. ERK1/2 kinase activation was determined by western blot. Results The results indicate that Ang II induced IL-6 mRNA expression and protein synthesis in cultured osteoblasts. However, these effects were abolished by pre-treatment with Ang II type 1 (AT1) receptor antagonist, losartan, and the ERK1/2 inhibitor, U0126, inhibited Ang II-mediated IL-6 expression and the phosphorylation of ERK1/2. Conclusion Ang II induces the synthesis of IL-6 in osteoblasts through activation of the ERK1/2 pathway via the AT1 receptor.

Extracellular signal-regulated kinase (ERK) is involved in LPS-induced disturbances in intestinal motility

Lipopolysaccharide (LPS) is a causative agent of sepsis. A relationship has been described between LPS, free radicals, and cyclooxygenase-2 (COX-2). Here, we investigate the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPK) in the effect of LPS on intestinal motility, oxidative stress status, and COX-2 expression. Rabbits were injected with (i) saline, (ii) LPS, (iii) U0126, an ERK MAPK inhibitor, or (iv) U0126+LPS. Duodenal contractility was studied in an organ bath with acetylcholine, prostaglandin E(2), and KCl added. Neuromuscular function was assessed by electrical field stimulation (EFS). Neurotransmitter blockers were used to study the EFS-elicited contractile response. The formation of products of oxidative damage to proteins (carbonyls), lipids, [malondialdehyde (MDA), and 4-hydroxyalkenals (4-HDA)] was quantified in plasma and intestine. The protein expression of phospho-ERK (p-ERK), total ERK, and COX-2 in the intestine was measured by western blot, and p-ERK was localized by immunohistochemistry. Acetylcholine, prostaglandin E(2), and KCl-induced contractions decreased with LPS. Electrical field stimulation induced a neurogenic contraction that was reduced by LPS. Lipopolysaccharide increased p-ERK and COX-2 expression and the levels of carbonyls and MDA+4-HDA. U0126 blocked the effect of LPS on acetylcholine, prostaglandin E(2), KCl, and EFS-induced contractions, the levels of carbonyls and MDA+4-HDA and p-ERK and COX-2 expression. Phospho-ERK was detected mostly in the neurons of the myenteric and submucosal ganglia. We can suggest that ERK is involved in the mechanism of action of LPS in the intestine.

Lactobacillus plantarum surface layer adhesive protein protects intestinal epithelial cells against tight junction injury induced by enteropathogenic Escherichia coli

Lactobacillus plantarum (LP) has previously been used for the treatment and prevention of intestinal disorders and disease. However, the role of the LP surface layer adhesive protein (SLAP) in inhibition of epithelial cell disruption is not fully understood. The aim of the present study was to investigate the protective effects of purified SLAP on Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). The role of ERK in LP-mediated inhibition of tight junction (TJ) injury was also evaluated in order to determine the molecular mechanisms underlying the protective effects of LP in epithelial cells. SLAP was extracted and purified from LP cells using a porcine stomach mucin-Sepharose 4B column. SLAP-mediated inhibition of bacterial adhesion was measured using a competition-based adhesion assay. Expression of TJ-associated proteins, maintenance of TJ structure, and levels of extracellular signal regulated kinase (ERK) and ERK phosphorylation were assessed in SLAP-treated cells by a combination of real-time PCR, western blotting, and immunofluorescence microscopy. Cell permeability was analyzed by measurement of trans-epithelial electrical resistance (TER) and dextran permeability. The effect of SLAP on levels of apoptosis in epithelial cells was assessed by flow cytometry. Results from these experiments revealed that treatment with SLAP decreased the level of adhesion of EPEC to Caco-2 cells. SLAP treatment also enhanced expression of TJ proteins at both the mRNA and protein levels and affected F-actin distribution. Although ERK levels remained unchanged, ERK phosphorylation was increased by SLAP treatment. Caco-2 cells treated with SLAP exhibited increased TER and decreased macromolecular permeability, which was accompanied by a decrease in the level of apoptosis. Together, these results suggest that LP-produced SLAP protects intestinal epithelial cells from EPEC-induced injury, likely through a mechanism involving ERK activation.

A role for extracellular signal-regulated kinases 1 and 2 in the maintenance of persistent mechanical hyperalgesia in ovariectomized mice

Mitogen-activated protein kinases (MAPKs) are important signaling factors in many cellular processes including cell proliferation and survival during development and synaptic plasticity induced by acute nociception in the adult. There is extensive evidence for the involvement of members of the MAPK family, the extracellular signal-regulated kinases 1 and 2 (ERKs 1/2), in the development of acute inflammatory somatic and visceral pain, but their role in the maintenance of chronic pain states is unknown. We have previously shown that ovariectomy of adult mice (OVX) generates a persistent and estrogen-dependent abdominal hyperalgesic state that lasts for several months and is not related to a persistent nociceptive afferent input. Here we have used OVX mice to study a possible role of ERK 1/2 in the spinal processing of this form of chronic abdominal hyperalgesia. Eight weeks after OVX the mice showed a robust abdominal hyperalgesia and a significant increase in the activation of ERK1/2 in the lumbosacral spinal cord. This enhanced activation was not seen in control and sham-operated mice or in regions of the cord other than lumbosacral in OVX mice. Also, the increased activation of ERK 1/2 observed in OVX mice matched the time course of the hyperalgesic state as no activation was observed at week 1 after OVX when the hyperalgesic state had not yet developed. Administration of slow-release pellets containing 17β-estradiol at week 5 post OVX reversed both the development of the hyperalgesia and the enhanced activation of ERK 1/2, suggesting that this activation, like the hyperalgesic state, was estrogen-dependent. Intrathecal injections of the ERK 1/2 inhibitor U0126 successfully rescued the mice from the abdominal hyperalgesia for up to 24 h after the injection and also reversed the enhanced expression of ERK 1/2. Our study shows, for the first time, activation of ERK 1/2 in the spinal cord matching the time course of an estrogen-dependent chronic hyperalgesic state.

Organ culture of the trigeminal ganglion induces enhanced expression of calcitonin gene-related peptide via activation of extracellular signal-regulated protein kinase 1/2

Clinical and experimental studies have revealed a central role of calcitonin gene-related peptide (CGRP) in primary headaches. The role of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in neuronal and glial cell expression of CGRP- immunoreactivity (-ir) in rat trigeminal ganglia was studied with an organ culture method. Sections of adult rat trigeminal ganglia were cultured for up to 48 hours, examined with immunohistochemistry and quantitative real-time polymerase chain reaction (PCR) assay. Specific antibodies against CGRP, phosphorylated ERK1/2 (pERK1/2), total ERK1/2 (tERK1/2), phosphorylated p38 (pp38), phosphorylated C-Jun-N-terminal protein kinase (pJNK), pro-calcitonin (pro-CT), CGRP receptor activity modifying protein 1 (RAMP1), glutamine synthetase (GS) and pro-CT were used. To explore molecular mechanisms involved in the organ culture-induced CGRP-ir in neurons and glial cells, the effects of the MEK/ERK1/2 inhibitor U0126, its inactive analogue U0124, the p38 inhibitor SB203580 and the JNK inhibitor SP600125 were studied. In fresh ganglia, small- and medium-sized neurons were CGRP-ir while some larger neurons displayed RAMP1-ir. Glial cells were negative to both. After organ culture, neurons showed enhanced CGRP- and RAMP1-ir. In addition, some glial cells were RAMP1- and CGRP-ir. Isolated glial cells and neurons were found to contain CGRP mRNA, and showed pro-CT-ir, suggestive of local formation of CGRP. Neurons and glial cells showed enhanced pERK1/2-ir already after two hours of organ culture and this remained elevated for 48 hours. There was transient pJNK-ir in neurons at two hours, while pp38-ir was not altered. U0126 reduced the enhanced pERK1/2-ir, while U0124 had no such effect; the CGRP-ir in neurons and glial cells was reduced at 48 hours and in parallel the CGRP mRNA expression was lower at 24 hours. We suggest that in conditions of elevated CGRP expression, inhibition of ERK1/2 might be an option for novel treatment.

ERK1/2 is involved in cyclic compressive force-induced IL-6 secretion in MLO-Y4 cells

We previously reported that cyclic compressive force (CCF) induced interleukin-6 mRNA expression in osteocyte-like MLO-Y4 cells. But little is known about how the stimuli are converted into the biochemical signals in MLO-Y4 cells. The aim of this research was to study the effect of CCF on the IL-6 secretion and the role of extracellular signal-regulated kinases 1/2 (ERK1/2) in this process. The cells were exposed to CCF with different magnitudes (1000, 2000 and 4000 μstrain), frequencies (0.5, 1.0 and 2.0 Hz) and durations (10 min, 30 min, 1 h, 3 h and 6 h) by a four-point bending system. The IL-6 secretion and ERK1/2 phosphorylation of the cells were determined by ELISA and Western blotting, respectively. The results showed that IL-6 protein secretion was significantly up-regulated in response to CCF in a magnitude-, frequency- and duration-dependent fashion. The phosphorylation of ERK1/2 also increased in all cases but not depended on the magnitude, frequency or duration of CCF. Furthermore, the inhibition of the ERK1/2 pathway by its specific inhibitor PD098059 decreased but not completely abrogated the IL-6 secretion from stressed MLO-Y4 cells. These findings demonstrate that CCF-induced IL-6 secretion occurs via a mechanism that involves ERK1/2 signaling pathway and suggest that modulation of this event contributes to the pathogenesis of osteoporosis and stress-induced pathological bone resorption as well.