Extracellular Signal-regulated Kinase Protein Levels Research Articles

Transient Receptor Potential Ankyrin 1-dependent Activation of Extracellular Signal-regulated Kinase 2 in the Cerebral Cortices Contributes to Cortical Spreading Depolarization

Extracellular signal-regulated kinase (ERK) are serine/threonine-selective proteins and ERK1/2 can be phosphorylated in peripheral and central brain regions after cortical spreading depolarization (CSD) and calcitonin gene-related peptide; However, it remains unclear about whether and how ERK activity modulates CSD that correlates to migraine aura. Here, we determined the role of ERK in regulating CSD and explored the underlying mechanism involving transient receptor potential ankyrin 1 (TRPA1), a stress-sensing cation channel. CSD was recorded using intrinsic optical imaging in mouse brain slices, and electrophysiology in rats. Phosphorylated ERK (pERK1/2) and interleukin-1β (IL-1β) protein levels were detected using Western blot or enzyme-linked immunosorbent assay, respectively. IL-1β mRNA level was detected using qPCR. The results showed that an ERK inhibitor, SCH77298, markedly prolonged CSD latency and reduced propagation rate in mouse brain slices. Corresponding to this, CSD induction increased levels of cytosolic pERK1/2 in ipsilateral cerebral cortices of rats, the elevation of which correlated to the level of IL-1β mRNA. Mechanistic analysis showed that pre-treatment of an anti-TRPA1 antibody reduced the cytosolic pERK2 level but not pERK1 following CSD in cerebral cortices of rats and this level of pERK2 correlated with that of cerebral cortical IL-1β protein. Furthermore, an ERK activator, AES16-2M, but not its scrambled control, reversed the prolonged CSD latency by a TRPA1 inhibitor, HC-030031, in mouse brain slices. These data revealed a crucial role of ERK activity in regulating CSD, and elevation of pERK and IL-1β production induced by CSD is predominantly TRPA1 channel-dependent, thereby contributing to migraine pathogenesis.

Effect of Hydrogen Sulfide on the Mitogen-Activated Protein Kinase Signaling Pathway in Cultured Skin Macrophages of Burned Rats

BackgroundThis study aims to investigate the effect of hydrogen sulfide on the mitogen-activated protein kinases signaling pathway in in vitro cultured skin macrophages of burned rats. Materials and methodsThirteen healthy Sprague–Dawley rats were divided into five groups: normal control group, burned control group, sodium hydrogen sulfide group, glibenclamide group, and sodium hydrogen sulfide + glibenclamide group. The burned rats were made into a deep II° 5% total body surface area flame burn injury model. The skin basement macrophages were separated from the skin of normal rats and the wound skin of burned rats and cultured. At 1, 6, and 12 h after intervention, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 protein levels were detected by Western blot, and ERK, p38, and JNK messenger RNA (mRNA) levels were detected by reverse transcription polymerase chain reaction. ResultsDifferences in ERK, p38, and JNK mRNA and protein levels between the normal control group and burned control group were statistically significant (P < 0.05). At the same time point, the ERK, p38, and JNK mRNA and protein levels in the NaSH group were different from those in other groups, and the differences were statistically significant (P < 0.05). ConclusionsHydrogen sulfide has a regulatory effect on ERK, JNK, and p38 in the mitogen-activated protein kinases signaling pathway in macrophages of burned rats.

Effects of lncRNA BANCR on endometriosis through ERK/MAPK pathway.

To investigate the regulatory role of long non-coding ribonucleic acid (lncRNA) BRAF-activated non-coding RNA (BANCR) in rats with endometriosis (EMs) and its mechanism of action. A total of 30 healthy, unmated, female Sprague-Dawley (SD) rats were selected and divided into sham-operation group, model group and lncRNA BANCR intervention group, and a rat model of EMs was established by means of autotransplantation. The volume of eutopic endometrium in each group of rats was measured, and hematoxylin and eosin (HE) staining was applied to detect the impacts on the pathological morphology of ectopic endometrial tissues in each group. The levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and MMP-9 in the rat serum were determined by virtue of enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) was performed to measure the messenger RNA (mRNA) levels of extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK) in the uterine tissues in each group of rats, and Western blotting assay was adopted to detect the levels of phosphorylated ERK and MAPK proteins in the rat uterine tissues in each group. Compared with those in sham-operation group, the volume of eutopic endometrium in the rats was increased markedly, the pathological morphology was poorer, and the content of VEGF, MMP-2 and MMP-9 in the serum, the mRNA levels of ERK and MAPK in the uterine tissues, and the levels of phosphorylated ERK and MAPK proteins were elevated notably in model group. The rats in lncRNA BANCR intervention group had evidently decreased volume of eutopic endometrium, improved pathological morphology and significantly declined content of serum VEGF, MMP-2 and MMP-9, ERK and MAPK mRNA levels, and phosphorylated ERK and MAPK protein levels in the uterine tissues than those in model group. LncRNA BANCR inhibitor can repress the development of ectopic endometrial tissues by inhibiting the generation of angiogenic factors in the EMs focus, and its mechanism may be related to the inhibition on the ERK/MAPK signaling pathway.

Research on cardioprotective effect of irbesartan in rats with myocardial ischemia-reperfusion injury through MAPK-ERK signaling pathway.

The aim of this study was to explore whether the mechanism of Irbesartan (IRB) in the treatment of rats with myocardial ischemia-reperfusion injury (MIRI) was related to the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) signaling pathway. The rat model of MIRI was first successfully established. All rats were randomly divided into 5 groups, including the Sham group (sham operation control group, ligation only), Model group (MIRI rat model group), IRB12.5 group [low-dose IRB (12.5 mg/kg/d) group], IRB50.0 group [medium-dose IRB (50.0 mg/kg/d) group] and IRB200.0 group [high-dose IRB (200.0 mg/kg/d) group]. After treatment of IRB in MIRI rats, the activities of four myocardial enzyme indexes, including creatine kinase-MB (CK-MB), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and cardiac troponin T (cTnT), were detected via enzyme-linked immunosorbent assay (ELISA). The effect of IRB on myocardial apoptosis in MIRI rats was detected via Annexin V-fluorescein isothiocyanate/Propidium Iodide (FITC/PI) double staining. Meanwhile, the messenger ribonucleic acid (mRNA) levels of ERK, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in myocardial cells after treatment of IRB were detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Furthermore, the protein levels of ERK and p-ERK were detected via Western blotting. Different concentrations of IRB could protect myocardium from MIRI. IRB at doses of 50.0 mg/kg/d and 200.0 mg/kg/d could significantly downregulate myocardial enzyme indexes in MIRI (p<0.01). Meanwhile, both the doses could markedly inhibit myocardial apoptosis in MIRI rat model by regulating the expressions of apoptosis-related genes (Bcl-2 and Bax) (p<0.01), eventually improving myocardial pathological damage. At the same time, it could also significantly decrease the mRNA and protein levels of ERK in the MAPK-ERK signaling pathway (p<0.05). The cardioprotective mechanism of IRB in MIRI rats may be related to the inhibition of the activation of the MAPK-ERK signaling pathway.

RA promotes proliferation of primordial germ cell-like cells differentiated from porcine skin-derived stem cells.

Previous studies have shown that primordial germ cell-like cells (PGCLCs) can be obtained from human, porcine and mouse skin-derived stem cells (SDSCs). In this paper, we found retinoic acid (RA), the active derivative of vitamin A, accelerated the growth of porcine primordial germ cells (pPGCs) and porcine PGCLCs (pPGCLCs) which were derived from porcine SDSCs (pSDSCs). Moreover, flow cytometry results revealed that the proliferation promoting effect of RA was attenuated by U0126, a specific inhibitor of extracellular signal-regulated kinase (ERK). Western blot analysis showed the protein level of ERK, phosphorylated ERK, cyclin D1 (CCND1), and cyclin-dependent kinase 2 (CDK2) increased after stimulation with RA, and this effect could also be abolished by U0126. Our data revealed that ablation of ERK expression by U0126 should significantly decrease proliferation of pPGCLCS. This reduction was because CCND1 and CDK2 proteins level decrease and subsequently the pPGCLCs were arrested in the G0/G1 phase. In addition, we also confirmed RA indeed promoted the proliferation of pPGCs isolated from porcine fetal genital ridges in vitro. Furthermore, our data indicated that DNA methylation pattern were changed in pPGCLCs and this pattern were more similar to pPGCs.

Baicalein inhibits the proliferation and invasion of oral squamous cell carcinoma and its possible signaling pathway

To investigate the relationship between the anti-proliferation effect of baicalein and extracellular signal-regulated kinase and focal adhesion kinase(ERK-FAK) signal pathway in oral squamous cell carcinoma (OSCC). The study included two parts and each part contained 4 groups, including control, 20 μmol/L BAI, 40 μmol/L BAI, 80 μmol/L BAI or control, 40 μmol/L BAI, MEK inhibitor(0.33 nmol/L),MEK inhibitor(0.33 nmol/L)+40 μmol/L BAI.Each group was treated in triplicate for 24 hours and 48 hours.Cell counting kit-8 (CCK8) was used to detect the inhibitory effect of baicalein; Polymerase chain reaction(PCR) and Western blot were used to analysis the effect of Baicalein on E-cadherin and Vimentin. The expressions of extracellular signal-regulated kinase(ERK), phosphorylated (p-ERK), focal adhesion kinase (FAK) and phosphorylated focal adhesion kinase(p-FAK) were detected by Western blot. The regulatory effect of MEK inhibitor(U0126) on Baicalein was tested by Western blot assay. The survival rate of cells treated with BAI is much lower than that of control group(P<0.01); the mRNA and protein levels of E-cadherin were obviously higher than those of control group, while the mRNA and protein levels of Vimentin were lower than those of control group(P<0.01).The protein levels of p-ERK and p-FAK treated with BAI were much lower than those of control group(P<0.01), but the total ERK and FAK had no obvious changes (P<0.05).The protein level of E-cadherin treated with MEK inhibitor was higher than that of control group(P<0.01) and the protein levels of Vimentin, p-ERK and p-FAK were lower than those of control group (P<0.01), while the total protein levels of ERK and FAK were the same(P<0.05). Baicalein can inhibit the proliferation and invasiveness of OSCC, which may be mediated by ERK-FAK signal pathway.

Effects of microRNA-126 on cell proliferation, apoptosis and tumor angiogenesis via the down-regulating ERK signaling pathway by targeting EGFL7 in hepatocellular carcinoma.

This study intends to explore the effects of microRNA-126 (miR-126) on cell proliferation, apoptosis, and tumor angiogenesis in hepatocellular carcinoma (HCC) by regulating epidermal growth factor-like domain 7 (EGFL7) through extracellular signal-regulated kinase (ERK) signaling. HCC tissues and adjacent normal tissues were obtained from 184 HCC patients. HCC cells were separately transfected with recombinant plasmids. Western blotting and qRT-PCR were applied to detect miR-126 and EGFL7, ERK, Fas/FasL, Bcl-2, Caspase mRNA and protein levels. CCK8 and TUNEL were performed to determinate cell proliferation and apoptosis. Flow cytometry was used to analyze cell cycle distribution. Rats model of HCC was constructed, and tumor weight and the number of new blood vessels were recorded after 3 weeks of tumor transplantation. Compared with the adjacent normal tissues, HCC tissues exhibited lower miR-126 expression, and higher EGFL7, and ERK mRNA and protein levels. Overexpression of miR-126 in HCC cell lines suppressed EGFL7, ERK, Bcl-2, and P-ERK, and increased apoptotic-associated proteins Fas/FasL and Caspase-3, and it inhibited cell proliferation and induced cell apoptosis. Overexpression of miR-126 in nude mice resulted in reduced tumor weight and less new blood vessels in tumors. The inhibition of miR-126 decreased cell apoptosis, and enhanced cell proliferation and tumor angiogenesis. This study demonstrates that miR-126 might decrease cell proliferation, induce apoptosis, and inhibit tumor angiogenesis in HCC by inhibiting EGFL7 via down-regulating the ERK signaling pathway.

Blockade of Cannabinoid CB1 receptor attenuates the acquisition of morphine-induced conditioned place preference along with a downregulation of ERK, CREB phosphorylation, and BDNF expression in the nucleus accumbens and hippocampus

Cannabinoid CB1 receptor (CB1R) is highly expressed in the mesocorticolimbic system and associated with drug craving and relapse. Clinical trials suggest that CB1R antagonists may represent new therapies for drug addiction. However, the downstream signaling of CB1R is not fully elucidated. In the present study, we investigated the relationship between CB1R and the extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF) signaling in the nucleus accumbens (NAc) and hippocampus in morphine-induced conditioned place preference (CPP), which is used to assess the morphine-induced reward memory. The protein level of CB1R, ERK, CREB, and BDNF were detected by western blotting. Additionally, a CB1R antagonist, AM251, was used to study whether blockade of CB1R altered the CPP and above-mentioned molecules. We found an increase of CB1R expression in the NAc and hippocampus of the mice following morphine CPP, but not those after repeated morphine in home cage without context exposure (NO-CPP). Both morphine CPP and NO-CPP induced an upregulation of ERK, CREB phosphorylation and BDNF expression. Furthermore, pretreatment with AM251 before morphine attenuated the CPP acquisition and CB1R expression as well as the activation of ERK-CREB-BDNF cascade. Collectively, these findings demonstrate that (1) Repeated morphine with context exposures but not merely the pharmacological effects of morphine increased CB1R expression both in the NAc and hippocampus. (2) CB1R antagonist mediated blockade of ERK-CREB-BDNF signaling activation in the NAc and hippocampus may be an important mechanism underlying the attenuation of morphine CPP.

Hypoxia Activates Extracellular Signal‐Regulated Kinase (ERK) via EGFR Activation in Human Pulmonary Microvascular Endothelial Cells

We have previously shown that hypoxia‐induced proliferation of human pulmonary microvascular endothelial cells (hPMVEC) depends on epidermal growth factor receptor (EGFR)‐mediated arginase II induction. We hypothesized that hypoxia‐induced EGFR activation leads to phosphorylation of extracellular signal‐regulated kinase (ERK) which then leads to increased arginase II expression in hPMVEC. Cells were incubated in hypoxia (5% CO2, 1% O2) for 0, 2, or 24 hours and western blotting was performed for phospho‐ERK and total ERK. We found that hypoxia resulted in greater levels of phospho‐ERK protein with little effect on total ERK protein levels at both 2 and 24 hours. hPMVECs were transfected with an siRNA against EGFR or scramble for 24 hours, allowed to recover for 24 hours, and then incubated in hypoxia or normoxia (5% CO2, 20% O2) for 24 hours. Cells incubated in hypoxia and treated with EGFR siRNA had lower phospho‐ERK levels than hypoxic cells treated with scramble. Furthermore, hypoxic hPMVECs treated with the EGFR siRNA had lower levels of arginase II protein than did hypoxic cells treated with scramble. In the normoxia exposed cells there were lower levels of arginase II protein than in hypoxia exposed cells and there was little effect of the EGFR siRNA on arginase II protein levels. Our findings suggest that in hPMVEC hypoxia activates EGFR which in turn phosphorylates ERK eventually leading to increased levels of arginase II. We speculate that ERK may be a potential therapeutic target to prevent or reverse the vascular remodeling seen in pulmonary hypertension associated with hypoxia.

Eucalyptol suppresses matrix metalloproteinase-9 expression through an extracellular signal-regulated kinase-dependent nuclear factor-kappa B pathway to exert anti-inflammatory effects in an acute lung inflammation model.

The acute lung injury (ALI) model is characterised by a severe acute inflammatory response in the lungs that represents the pathogenesis of acute respiratory distress syndrome (ARDS). In this study, we sought to elucidate the anti-inflammatory mechanism of eucalyptol in relation to tissue remodelling in acute lung inflammation. BALB/C mice were intraperitoneally injected with eucalyptol (100, 200 or 400 mg/kg) or dexamethasone (1 mg/kg) 1 h before intratracheal challenge with lipopolysaccharide (LPS; 1.5 mg/kg) and sacrificed after 4 h. The anti-inflammatory effects of eucalyptol were assessed by determining cell counts, measuring cytokine and nitric oxide production and performing Western blotting and histological analyses. Eucalyptol attenuated inflammation-associated increases in cell numbers, matrix metalloproteinase-9 (MMP-9) expression, production of cytokines (tumour necrosis factor-α and interleukin-6) and nitric oxide, and nuclear factor-kappa B (NF-κB) and phosphorylated extracellular signal-regulated kinase protein levels induced by LPS in bronchoalveolar lavage fluid from ALI mice. Furthermore, pretreatment with 400 mg/kg eucalyptol prevented LPS-induced histopathological changes. Collectively, these results indicate that eucalyptol acts through a mechanism involving decreased MMP-9 expression and an extracellular signal-regulated kinase-dependent NF-κB pathway to exert anti-inflammatory actions in acute lung inflammation. Thus, eucalyptol may be a potentially important agent in the treatment of pulmonary inflammation.

Change of extracellular signal-regulated kinase expression in pulmonary arteries from smokers with and without chronic obstructive pulmonary disease

ABSTRACTBackground: Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease (COPD) by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell (PASMC) proliferation. However, the molecular mechanism underlying this process remains poorly understood. Objectives: The purpose of this study was to investigate the role of extracellular signal-regulated kinase (ERK) in pulmonary arteries from smokers with normal lung function and smokers with mild to moderate COPD. Methods: The peripheral lung tissues were obtained from 14 nonsmokers with normal lung function, 18 smokers with normal lung function, and 16 smokers with mild to moderate COPD. The morphological changes of pulmonary arteries were observed by hematoxylin-eosin (HE) staining. Primary cultured human pulmonary artery smooth muscle cells (HPASMCs) were exposed to cigarette smoke extract (CSE). Cell proliferation was determined by cell counting and Methyl thiazolyl tetrazolium assay. Protein expression was analyzed by western blotting. Results: Morphometrical analysis showed that the pulmonary vessel wall thickness in smoker group and COPD group was significantly greater than that in nonsmoker group (P < .01). The protein level of ERK was significantly increased in smoker group and COPD group as compared with nonsmoker group (P < .01). The expression of ERK was significantly increased in HPASMCs at protein levels when HPASMCs were treated with 5% CSE (P < .01), which significantly promoted the proliferation of HPASMCs (P < .01). Conclusions: Increased expression of ERK might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with and without COPD.

Stable Knockdown of Clusterin by Vector-Based RNA Interference in a Human Breast Cancer Cell Line Inhibits Tumour Cell Invasion and Metastasis

Overexpression of the clusterin (CLU) gene occurs in breast cancer and is associated with lymph node metastasis. The present study explored the effect of CLU silencing on invasion and metastasis, and the relationship between CLU expression and the extracellular signal-regulated kinase (ERK) / matrix metalloproteinase-9 (MMP) signalling pathway in human breast cancer cells. A pcDNA3.1-based RNA interference approach was used to knockdown the CLU gene in MDA-231 cells (MDA-231-CLUi); control MDA-231 cells were transfected with an empty vector (MDA-231-Vec). Reverse transcription-polymerase chain reaction was used to assess CLU and MMP-9 mRNA levels, and Western blotting was used to analyse CLU, MMP-9 and ERK protein levels. Metastatic potential was evaluated using in vitro and in vivo models of invasion and metastasis. Compared with MDA-231-Vec cells, the MDA-231-CLUi cells demonstrated reduced migration and invasion in vitro and decreased metastatic potential in vivo. Reintroduction and reexpression of the CLU gene into the MDA-231-CLUi cells restored the invasive phenotype. MMP-9 mRNA and protein levels were reduced in MDA-231-CLUi cells, and there was a correlation between activated ERK and CLU and MMP-9 protein levels. CLU may regulate the aggressive behaviour of human breast cancer cells through modulation of ERK signalling and MMP9 expression.

Therapeutic Effect Of Exercise Training On Cardiac Fibrotic Pathway In Obesity

Background. No information regarding transforming growth factor beta (TGFb) and connective tissue growth factor (CTGF) in obese heart as well as no information regarding the effects of exercise training on cardiac fibrosis in obesity was available. The purpose of this study was to evaluate TGF-beta-CTGF-related fibrotic pathway in obese heart and the effects of exercise training on cardiac fibrosis. METHODS: Twelve lean Zucker rats (LZR) and twelve obese Zucker rats (OZR) at 5-6 months of age were served as sedentary lean group and sedentary obese group as well as twelve obese rats underwent swimming exercise 3 hour daily for 1 month (OZR-EX). After exercise training or sedentary status, the excised hearts were measured by Hematoxylin and Eosin (H&E) staining, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Masson trichrome staining and Western Blotting. RESULTS: Myocardial architecture abnormality, more cardiac TUNEL-positive apoptotic cells, and minor cardiac fibrosis were found in OZR, compared to LZR. The protein levels of TGF-beta, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), CTGF, matrix metalloproteinase (MMP)-9, and MMP2 were significantly increased in OZR relative to LZR. Exercise training decreased OZR-induced myocardial architecture abnormality, cardiac TUNEL-positive apoptotic cells, and cardiac fibrosis as well as exercise training decreased OZR-induced fibrosis-related protein levels of TGF-beta, MEK, ERK, CTGF, MMP-9, and MMP2. CONCLUSIONS: Exercise training prevented cardiac fibrosis and cardiac TGF-beta-CTGF fibrotic pathway in obese models. Our findings imply that exercise therapy could be one of possible therapeutic approaches to prevent delirious cardiac fibrosis in obesity.

Naringenin-7- O-glucoside protects against doxorubicin-induced toxicity in H9c2 cardiomyocytes by induction of endogenous antioxidant enzymes

Doxorubicin, a widely used chemotherapeutic agent, can give rise to severe cardiotoxicity that limits its clinical use by generation of reactive oxygen species (ROS) and apoptosis. Protection or alleviation of doxorubicin cardiotoxicity can be achieved by administration of natural phenolic compounds via activating endogenous defense systems and antiapoptosis. Naringenin-7- O-glucoside (NARG), isolated from Dracocephalum rupestre Hance, has been demonstrated to protect against cardiomyocyte apoptosis. In the present study, we investigated the effects of NARG on endogenous antioxidant enzymes against doxorubicin toxicity and the potential role of extracellular signal-regulated kinase (ERK) in regulation of NARG-induced Nrf2-dependent gene expression in H9c2 cardiomyocytes. The mRNA expression of glutamate-cysteine ligase modifier subunit (GCLM) and glutamate-cysteine ligase catalytic subunit (GCLC) was upregulated by NARG as detected by RT-PCR. NARG (10, 20, and 40 μM) pretreatment increased NAD (P) H: quinone oxidoreductase (NQO1), ERK, and Nrf2 protein levels in cardiomyocytes as detected by Western blotting. These results suggest that NARG could prevent cardiomyocytes from doxorubicin-induced toxicity by induction of endogenous antioxidant enzymes via phosphorylation of ERK1/2 and nuclear translocation of Nrf2.

Effect of extracellular signal-regulated kinase pathway on nitric oxide release by human umbilical vein endothelial cell induced by placental growth factor-1

To investigate the effect of extracellular signal-regulated kinase (ERK) pathway on nitric oxide (NO) release by human umbilical vein endothelial cell (HUVEC) induced by placental growth factor-1 (PLGF-1). During January to April 2006, 50 samples of umbilical vein blood were collected from newborns delivered by cesarean section due to intrauterine distress and abnormal fetal position. HUVEC were primarily cultured by trypsin digestion. Then the following procedures were performed: (1) Cells were identified using the morphology and VIII factor immunohistochemistry methods if the culture was satisfactory. (2) Cells were collected, and fms-like tyrosin kinase (Flt-1) protein and its mRNA expression were detected with immunoprints and RT-PCR methods. (3) The protein was extracted after cells were treated with PLGF-1 (cells were collected before the treatment and 2.5, 5, 10, 20 min after the treatment). The protein levels of ERK were determined by immunoprints. (4) The cells were cultured with serum-free culture medium containing PLGF-1 only (culture media were collected 20, 40, 160, 360, 480, 720 and 1440 min after the treatment). The quantity of NO was detected with nitrate reductase method. (5) The cells were cultured with serum-free culture medium containing PD98059, the inhibitor of mitogen-activated protein kinase (MAPK)/MEK for 60 min. Then the cells were cultured continuously with the serum-free culture medium containing PLGF-1 for 60 min. The culture media were collected. The quantity of NO was detected by nitrate reductase method. The samples were divided into treatment group and control group. Control group was exactly the same in treatment time, culture condition, and time to collect the cells as the treatment group, except that it was not treated with PLGF-1 or PD 98059. (1) By morphology and VIII factor immunohistochemistry the cultured cells were identified to be HUVEC. (2) Flt-1 mRNA and protein were expressed in HUVEC. (3) Expression of ERK protein started to increase at 2.5 min after treatment of HUVEC with PLGF-1, reaching the peak at 5 min, and decreased at 10 min. (4) In comparison with the control group, NO started to increase at 20 min after treatment of HUVEC with PLGF-1 (6.96 +/- 0.34) micromol/L, significantly increased at 40 min (9.45 +/- 0.59) micromol/L, and arrived at the peak at 480 min (15.82 +/- 0.69) micromol/L Comparison between the two groups showed a significant difference (P<0.05). (5) Release of NO from the cells treated with PD98059 for 1 hour and PLGF was significantly inhibited, compared with the cells treated with PLGF-1 only. ERK pathways play an important role in NO release by HUVEC induced by PLGF.

Involvement of Ras/Raf‐1/ERK actions in the magnolol‐induced upregulation of p21 and cell‐cycle arrest in colon cancer cells

Previously, we showed that magnolol induces cell-cycle arrest in cultured colon and liver cancer cells through an upregulation of the p21 protein. The aim of this study was to delineate the molecular mechanism underlying this magnolol-induced increase of p21 protein. Thus our RT-PCR analysis demonstrated that the mRNA levels of p21 were increased at 1 h after magnolol treatment and sustained for at least 24 h. The p21 promoter activity was also increased by magnolol treatment. Western blot analysis demonstrated that treatment of COLO-205 cells with magnolol increased the levels of phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment of the cells with PD98059 abolished the magnolol-induced upregulation of p21 protein, suggesting the involvement of an ERK pathway in the magnolol-induced upregulation of p21 in COLO-205 cells. Ras inhibitor peptide abolished the magnolol-induced increase of phosphorylated ERK protein levels, increase of p21 protein, and decrease of thymidine incorporation. Moreover, treatment of COLO-205 with magnolol increased the phosphorylated Raf-1 protein (the Ras target molecule). Pretreatment of the cells with Raf-1 inhibitor reversed the magnolol-induced decrease in thymidine incorporation. Treatment of the cells with CaM kinase inhibitor, but not protein kinase A (PKA) inhibitor or phosphatidylinosital 3-kinase (PI3K) inhibitor, abolished the magnolol-induced activation of ERK and decrease of thymidine incorporation. Taken together, our results suggest that magnolol activates ERK phosphorylation through a Ras/Raf-1-mediated pathway. Subsequently, p21 expression is increased, and finally thymidine incorporation is decreased.

Pregnancy up‐regulates protein and mRNA levels of ERK in ovine uterine artery

Our recent studies demonstrated that extracellular signal-regulated kinase (ERK) plays an important role in regulation of vascular contractility in ovine uterine artery. The present study was designed to test the hypotheses that pregnancy up-regulates ERK activity by increase in the ERK phosphorylation, protein and mRNA levels in uterine arteries. Uterine arteries were isolated from nonpregnant and near-term (~ 140 days) pregnant ewes. The phosphorylation and protein levels of ERK1/2 were detected by western blot. The mRNA levels of ERK1/2 were determined by RT-PCR. The phosphorylation and protein levels of ERK were significantly higher in the pregnant uterine arteries than those in the nonpregnant uterine arteries. Furthermore, the mRNA levels of ERK1/2 were also significantly higher in the pregnant uterine arteries than those in the nonpregnant uterine arteries. The results suggest that pregnancy up-regulates ERK1/2 protein expression levels in the uterine arteries. The increased protein levels, which are up regulated through transcription levels, lead to increase in ERK phosphorylation levels and its activities.

Partial cloning of the coding sequence of ERK2 gene in sheep uterine artery

Our recent studies demonstrated that extracellular signal-regulated kinase (ERK) played an important role in the regulation of vascular contractility of ovine uterine arteries. In addition, pregnancy significantly increased ERK protein and mRNA levels in the uterine artery. In an effort of attempting to study the regulation of ERK2 gene expression in the uterine artery, we designed primers based on known ERK2 mRNA sequences from human, rat, mouse and bovine. A 539 bp segment of ERK2 gene coding sequence was successfully amplified in the sheep uterine artery by RT-PCR. Homology analysis revealed that nucleotide sequence of the segments between sheep and human are 94% identical, which translate into 2 amino acids alteration, and between sheep and cow, 97% DNA sequence are identical, which also translate into 2 differences in amino acids sequence, suggesting that the ERK2 gene is highly conserved among species, reflex its important regulatory function in cellular processes.

The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin

Alterations in specific signal transduction pathways may explain the hyperproliferation and abnormal differentiation of the keratinocytes as well as the increased expression of inflammatory cytokines seen in psoriasis. Major signalling pathways used by eukaryotic cells to transduce extracellular signals into cellular responses impinge on the mitogen-activated protein kinases (MAPKs). To investigate the expression of the MAPK p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in psoriatic skin. Keratome biopsies were taken from patients with plaque-type psoriasis. Western blot analysis was used to determine p38, ERK and JNK activity and protein levels, whereas kinase assays were used to examine the kinase activity of p38. We demonstrated increased levels of the phosphorylated forms of p38 and ERK1/2 in lesional psoriatic skin compared with nonlesional psoriatic skin. No abnormality was found in the activation and expression of JNK1/2. Ex vivo kinase assays confirmed the increased activation of p38, and furthermore demonstrated increased kinase activity of the p38 isoforms p38alpha, p38beta and p38delta in lesional compared with nonlesional psoriatic skin. p38gamma was not detected in the psoriatic skin. Clearance of the psoriatic lesions, induced by climatotherapy at the Dead Sea for 4 weeks, led to a normalization in the activity of both p38 and ERK1/2. Taken together, our results demonstrate that the activity of the MAPKs p38alpha, p38beta and p38delta and ERK1/2 are increased in lesional psoriatic skin compared with nonlesional psoriatic skin, and that clearance of psoriasis normalizes the p38 and ERK1/2 activity. Thus, p38 and ERK1/2 might be potential targets in the treatment of psoriasis.

Platelet-derived Growth Factor Inhibits Smooth Muscle Cell Adhesion to Fibronectin by ERK-dependent and ERK-independent Pathways

The adhesion of cells to the extracellular matrix plays a major role in cell migration. Pretreatment with platelet-derived growth factor (PDGF) inhibited the adhesion of smooth muscle cells to fibronectin by 80%. This inhibition decreased as concentrations of fibronectin increased. In the presence of 200 microm GRGDS peptide, only 45% of PDGF-treated cells adhered to fibronectin compared with 80% of control cells. This indicates that a decrease in integrin avidity was induced by PDGF. Cell adhesion was partially restored when the activation of the extracellular signal-regulated kinase (ERK) was inhibited with PD98059. The remaining inhibition of adhesion (50%) was independent of the fibronectin concentration, suggesting that the ERK pathway is involved in the decrease in integrin avidity. This was confirmed by depleting ERK protein levels by treatment with ERK antisense oligonucleotide. The adhesion of ERK control oligonucleotide-treated cells decreased by 41% when the concentration of GRGDS peptide was increased from 50 to 200 microm but only decreased by 11% in ERK antisense oligonucleotide-treated cells. Treatment with PDGF also delayed focal complex assembly and inhibited stress fiber formation. Consistent with a delay in tyrosine phosphorylation of paxillin, PDGF treatment caused a lag in focal complex formation, although this was not associated with any change in Src family tyrosine kinase activity. Our results indicate that PDGF inhibits smooth muscle cells adhesion by two pathways. The first involves an ERK-dependent decrease in integrin avidity; the second involves the ERK-independent inhibition of focal complex assembly.