Acrylonitrile-induced extracellular signal-regulated kinase (ERK) activation via protein kinase C (PKC) in SK-N-SH neuroblastoma cells

Acrylonitrile (ACN) is classified by IARC as a probable carcinogen. Chronic exposure to ACN increases the incidence of tumors in various organs of test animals, including the brain and lung. ERK1/2 activation plays crucial roles in cell proliferation and is involved in many steps of tumor progression. Therefore, this study examined whether ACN altered the activation state of ERK1/2 in human neuroblastoma SK-N-SH cells. Treatment of these cells with ACN greatly increased phosphorylation of ERK1/2 in dose- and time-dependent manners. This effect was inhibited by PD 98059 and U 0126, specific inhibitors of MEK, indicating that MEK, an upstream activator of ERK1/2, was directly involved in ACN-induced ERK1/2 activation. Furthermore, the activation of ERK1/2 by ACN was attenuated by inhibition of PKC with GF 109203X, rottlerin and prolonged incubation with PMA (phorbol 12-myristate 13-acetate). This demonstrated the participation of PKC in the ACN-stimulated activation of ERK1/2. Taken together, our results indicate that ACN-induced ERK1/2 activation involves PKC through a MEK-dependent pathway.     

Involvement of the extracellular signal-regulated protein kinase (ERK) pathway in the induction of apoptosis by cadmium chloride in CCRF-CEM cells

When CCRF-CEM cells were incubated with 5–40 μM CdCl_2, apoptosis was observed most clearly at 10 μM. Prior to the development of apoptosis, mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, were activated with different sensitivity to CdCl₂ exposure. ERK and p38 MAPK were phosphorylated with incubation of 1 μM CdCl_2, but higher than 20 μM CdCl₂ was required for the clear phosphorylation of JNK. In the time–course study, ERK and p38 MAPK were phosphorylated earlier than JNK after CdCl₂ exposure. The in vitro activities of MAPKs also increased in response to CdCl₂ exposure. Pretreatment with an intracellular Ca²⁺ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), suppressed almost completely CdCl₂-induced phosphorylation of JNK and p38 MAPK, but not ERK phosphorylation, indicating that the activation of JNK and p38 MAPK depends on the intracellular Ca²⁺ but that of ERK does not. On the other hand, treatment with a MAPK/ERK kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), suppressed CdCl₂-induced ERK activation and the apoptosis as well. The inhibition of p38 MAPK activity with SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]1H-imidazole) did not protect cells from apoptosis. The present results showed that the activation of ERK, JNK, and p38 MAPK is differently regulated in CCRF-CEM cells exposed to CdCl_2, and that the ERK pathway seems to be responsible for the induction of apoptosis by CdCl₂ exposure in this human T cell line.     

Chlorpyrifos oxon potentiates diacylglycerol-induced extracellular signal-regulated kinase (ERK 44/42) activation, possibly by diacylglycerol lipase inhibition.

Chlorpyrifos oxon (CPO) activates extracellular signal-regulated kinase (ERK 44/42) in Chinese hamster ovary (CHOK1) cells but the mechanism is not defined. This study tests the hypothesis that diacylglycerol (DAG) is the secondary messenger responsible for CPO-induced ERK 44/42 activation. It is known that DAG is sequentially hydrolyzed by DAG lipase and monoacylglycerol (MAG) lipase, both of which are organophosphate sensitive. Inhibition of these enzymes might therefore lead to the accumulation of DAG and MAG, of which only DAG is a secondary messenger. The experiments show that treatment of CHOK1 cells with CPO significantly inhibits DAG/MAG lipase activity and elevates cellular DAG levels. Pretreatment of CHOK1 cells with CPO or a carbamate known to be a DAG lipase inhibitor, followed by treatment with a cell-permeable DAG (1,2-dihexanoyl-sn-glycerol), results in synergistic activation of ERK 44/42. CPO-potentiated DAG-induced ERK 44/42 activation is both time and concentration dependent. This activation is blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase, suggesting that these enzymes are important in CPO/DAG cellular signaling. Activation by a stable DAG analogue (phorbol ester) was not altered by CPO, suggesting that DAG metabolism is the probable target for CPO-potentiated DAG-induced ERK 44/42 activation. These observations support the hypothesis that CPO potentiates DAG signaling in CHOK1 cells by inhibiting a CPO-sensitive DAG lipase, thereby providing a potential mechanism of toxicity not associated with acetylcholinesterase inhibition.     

Aberrant extracellular signal-regulated kinase (ERK) 5 signaling in hippocampus of suicide subjects.

Extracellular signal-regulated kinase 5 (ERK5), the newest member of the mitogen-activated protein (MAP) kinase family, is regulated differently than the other MAP kinases. Emerging evidence suggest the role of ERK5 signaling in promoting cell proliferation, differentiation, neuronal survival, and neuroprotection. The present study investigates whether suicide brain is associated with alterations in components of the ERK5 signaling cascade. In the prefrontal cortex (PFC) and hippocampus of suicide subjects (n=28) and nonpsychiatric control subjects (n=21), we examined the catalytic activities and protein levels of ERK5 and upstream MAP kinase kinase MEK5 in various subcellular fractions; mRNA levels of ERK5 in total RNA; and DNA-binding activity of myocyte enhancer factor (MEF)2C, a substrate of ERK5. In the hippocampus of suicide subjects, we observed that catalytic activity of ERK5 was decreased in cytosolic and nuclear fractions, whereas catalytic activity of MEK5 was decreased in the total fraction. Further, decreased mRNA and protein levels of ERK5, but no change in protein level of MEK5 were noted. A decrease in MEF2C-DNA-binding activity in the nuclear fraction was also observed. No significant alterations were noted in the PFC of suicide subjects. The observed changes were not related to a specific psychiatric diagnosis. Our findings of reduced activation and/or expression of ERK5 and MEK5, and reduced MEF2C-DNA-binding activity demonstrate abnormalities in ERK5 signaling in hippocampus of suicide subjects and suggest possible involvement of this aberrant signaling in pathogenic mechanisms of suicide.     

alpha(1)-Adrenergic receptor stimulation of cell motility requires phospholipase D-mediated extracellular signal-regulated kinase activation

Phospholipase D is suspected to play a role in tumorigenesis, and the inhibition of phospholipase D has been associated with changes in several cellular events including invasion and migration. We report here that the specific alpha(1)-adrenergic receptor agonist, phenylepherine, signals to a growth factor pathway in a manner that requires phospholipase D activity in CCL39 fibroblasts. Phenylepherine increased extracellular signal-regulated kinase phosphorylation eightfold and promoted stress fiber formation threefold. Stress fiber formation was blocked when extracellular signal-regulated kinase activation was inhibited. Stimulation of CCL39 fibroblasts by phenylepherine increased the rate of wound healing fourfold in a wounding assay, while treatment with the MEK inhibitor, PD98059 reduced the closure of phenylepherine-induced wound healing to control levels. Addition of 1-butanol but not 2-butanol inhibited extracellular signal-regulated kinase activation by phenylepherine, presumably by blocking the formation of phosphatidic acid. Exogenously added cell permeable phosphatidic acid increased extracellular signal-regulated kinase activation in a time- and dose-dependent manner as well as stimulated the formation of stress fibers. 1-butanol also significantly inhibited the ability of phenylepherine to stimulate stress fiber formation and wound healing. Taken together, these results indicate a novel role for phospholipase D in the activation of the extracellular signal-regulated kinase growth factor pathway to stimulate early cellular events induced by phenylepherine.     

Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation

There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders.     

Lack of involvement of extracellular signal-regulated kinase (ERK) in the agonist-induced endothelial nitric oxide synthesis

In a recent paper, it was shown that stimulation of endothelial cells with bradykinin (BK) leads to phosphorylation of endothelial nitric oxide synthase (eNOS) mediated by extracellular signal-regulated kinase (ERK) (J. Biol. Chem. 275 (2000) 30707). Since in vitro phosphorylation by ERK reduced the catalytic activity of eNOS, it was suggested that this mechanism may be an important determinant of nitric oxide signalling in endothelial cells. To explore the physiological role of ERK as regulator of nitric oxide synthesis in intact cells, we measured the effects of the kinase inhibitor PD 98059 on BK- and ATP-induced nitric oxide formation in cultured endothelial cells and isolated vascular smooth muscle strips. PD 98059 completely inhibited ERK activation by BK and ATP in porcine aortic endothelial cells without affecting eNOS activation. Moreover, PD 98059 did not potentiate relaxation of isolated porcine pulmonary arteries to BK or ATP, indicating that ERK-catalysed eNOS phosphorylation does not contribute to the regulation of nitric oxide formation in intact cells or tissues.     

An anticancer agent icaritin induces sustained activation of the extracellular signal-regulated kinase (ERK) pathway and inhibits growth of breast cancer cells

Icaritin, a prenylflavonoid derivative from Epimedium Genus, regulates many cellular processes. However, the function and the underlying mechanisms of icaritin in breast cancer cell growth have not been well established. Here, we report that icaritin strongly inhibited the growth of breast cancer MDA-MB-453 and MCF7 cells. At concentrations of 2-3 μM, icaritin induced cell cycle arrest at the G(2)/M phase accompanied by a down-regulation of the expression levels of the G(2)/M regulatory proteins such as cyclinB, cdc2 and cdc25C. Icaritin at concentrations of 4-5 μM, however, induced apoptotic cell death characterized by the accumulation of the annexin V- and propidium iodide-positive cells, cleavage of poly ADP-ribose polymerase (PARP) and down-regulation of the Bcl-2 expression. In addition, icaritin also induced a sustained phosphorylation of extracellular signal-regulated kinase (ERK) in these breast cancer cells. U0126, a specific ERK activation inhibitor, abrogated icaritin-induced G2/M cell cycle arrest and cell apoptosis. Icaritin more potently inhibited growth of the breast cancer stem/progenitor cells compared to anti-estrogen tamoxifen. Our results indicate that icaritin is a potent growth inhibitor for breast cancer cells and provide a rationale for preclinical and clinical evaluations of icaritin for breast cancer therapy.     

Honokiol-induced neurite outgrowth promotion depends on activation of extracellular signal-regulated kinases (ERK1/2)

We have found that honokiol [4-allyl-2-(3-allyl-4-hydroxy-phenyl)-phenol] can promote neurite outgrowth and mobilize intracellular Ca2+ store in primary cultured rat cortical neurons. In this study, we examined the effects of honokiol on extracellular signal-regulated kinases (ERK1/2) and Akt, and their possible relationship to neurite outgrowth and Ca2+ mobilization. Honokiol-induced neurite outgrowth in the cultured rat cortical neurons was significantly reduced by PD98059, a mitogen-activated protein kinase kinase (MAPKK, MAPK/ERK kinase MEK, direct upstream of ERK1/2) inhibitor, but not by LY294002, a phosphoinositide 3-kinase (PI3K, upstream of Akt) inhibitor. Honokiol also significantly enhanced the phosphorylation of ERK1/2 in a concentration-dependent manner, whereas the effect of honokiol on Akt phosphorylation was characterized by transient enhancement in 10 min and lasting inhibition after 30 min. The phosphorylation of ERK1/2 enhanced by honokiol was inhibited by PD98059 as well as by KN93, a Ca2+/calmodulin-dependent kinase II (CaMK II) inhibitor. Moreover, the products of the phosphoinositide specific phospholipase C (PLC)-derived inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG) were measured after honokiol treatment. Together with our previous findings, these results suggest that the signal transduction from PLC, IP3, Ca2+, and CaMK II to ERK1/2 is involved in honokiol-induced neurite outgrowth.     

Involvement of YC-1 in extracellular signal-regulated kinase action in rat cremasteric muscle

Objectives The nitric oxide (NO)–soluble guanylate cyclase (sGC) signalling pathway is attributed to the prevention of ischaemia–reperfusion (I/R)‐induced leucocyte–endothelium adhesive interactions. YC‐1 (3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole), a NO‐independent sGC activator, has been shown to exert cardiovascular benefits, but its action on leucocyte–endothelium interactions remains unknown. In this study, the direct effect and the underlying mechanism of the anti‐adhesive action of YC‐1 have been examined in cremasteric microcirculation. Methods Rat cremaster muscle was subjected to 4 h pudic‐epigastric artery ischaemia followed by 2 h reperfusion and intravital microscopy was used to observe leucocyte–endothelium interaction and to quantify functional capillaries in rat cremaster muscle flaps. Key findings The values for leucocyte rolling, adhering and transmigrating were 5.5‐, 6.9‐ and 8.8‐fold greater, respectively, in I/R than in sham‐control animals. YC‐1 treatment rescued functional capillary density and reduced leucocyte rolling, adhering and transmigrating in I/R injured cremaster muscles to levels observed in sham‐controls. Interestingly, these effects were completely blocked by the MEK (extracellular signal‐regulated kinase (ERK) kinase) inhibitor (PD98059) but not by sGC or protein kinase C inhibitors. Cotreatment of PD98059 with YC‐1 caused a 3.3‐, 7.5‐ and 8.3‐fold increase in the values for leucocyte rolling, adhering and transmigrating, respectively, in postcapillary venules of I/R‐injured cremaster muscle. Conclusions This study has indicated that the anti‐adhesive and functional capillary density rescue properties of YC‐1 were mediated predominantly by the activation of ERK but not sGC, although YC‐1 was identified to be a sGC activator. A better understanding of the action of YC‐1 on the microvasculature may help shed light on its therapeutic potential for cardiovascular disease.     

Cell-specific extracellular signal-regulated kinase activation by multiple G protein-coupled receptor families in hippocampus

Several families of G protein-coupled receptors (GPCR) have been shown to activate extracellular signal-regulated kinase (ERK) in transfected cells and non-neuronal systems. However, little is known about GPCR activation of ERK in brain. Because ERK is an important component in the regulation of synaptic plasticity, in this study we examined ERK activation by three families of GPCR that respond to major neuromodulatory neurotransmitters in the hippocampus. We used an immunocytochemical approach to examine ERK activation by muscarinic acetylcholine (mAChR), metabotropic glutamate (mGluR), and beta-adrenergic (beta-AR) receptors in CA1 neurons of mouse hippocampal slices. Because these GPCR families comprise receptors coupling to each of the major heterotrimeric G proteins, we examined whether ERK activation differs according to G-protein coupling. By using immunocytochemistry, we were able to examine not only whether each family of receptors activates ERK, but also the cellular populations and subcellular distributions of activated ERK. We demonstrated that M1 mAChRs and group I mGluRs, both of which are Gq-coupled receptors, activate ERK in CA1 pyramidal neurons, although activation in response to mAChR is more robust. The G(i/o)-coupled group II mGluRs activate ERK in glia scattered throughout CA1, and Gs-coupled beta-AR receptors activate ERK in scattered interneurons. Thus, we demonstrated that GPCR coupling to Gq, G(i/o), and Gs all activate ERK in the hippocampus, although each does so with unique properties and distributions.     

Selective inhibition of mitogen-activated protein kinase phosphatases by zinc accounts for extracellular signal-regulated kinase 1/2-dependent oxidative neuronal cell death

Oxidative stress induced by glutathione depletion in the mouse HT22 neuroblastoma cell line and embryonic rat immature cortical neurons causes a delayed, sustained activation of extracellular signal-regulated kinase (ERK) 1/2, which is required for cell death. This sustained activation of ERK1/2 is mediated primarily by a selective inhibition of distinct ERK1/2-directed phosphatases either by enhanced degradation (i.e., for mitogen-activated protein kinase phosphatase-1) or as shown here by reductions in enzymatic activity (i.e., for protein phosphatase type 2A). The inhibition of ERK1/2 phosphatases in HT22 cells and immature neurons subjected to glutathione depletion results from oxidative stress because phosphatase activity is restored in cells treated with the antioxidant butylated hydroxyanisole. This leads to reduced ERK1/2 activation and neuroprotection. Furthermore, an increase in free intracellular zinc that accompanies glutathione-induced oxidative stress in HT22 cells and immature neurons contributes to selective inhibition of ERK1/2 phosphatase activity and cell death. Finally, ERK1/2 also functions to maintain elevated levels of zinc. Thus, the elevation of intracellular zinc within neurons subjected to oxidative stress can trigger a robust positive feedback loop operating through activated ERK1/2 that rapidly sets into motion a zinc-dependent pathway of cell death.     

The effect of active and passive intravenous cocaine administration on the extracellular signal-regulated kinase (ERK) activity in the rat brain

According to a current hypothesis of learning processes, recent papers pointed out to an important role of the extracellular signal-regulated kinase (ERK), in drug addiction. We employed the Western blotting techniques to examine the ERK activity immediately after cocaine iv self-administration and in different drug-free withdrawal periods in rats. To distinguish motivational vs. pharmacological effects of the psychostimulant intake, a “yoked” procedure was used. Animals were decapitated after 14 daily cocaine self-administration sessions or on the 1st, 3rd or 10th extinction days. At each time point the activity of the ERK was assessed in several brain structures, including the prefrontal cortex, hippocampus, dorsal striatum and nucleus accumbens.Passive, repeated iv cocaine administration resulted in a 45% increase in ERK phosphorylation in the hippocampus while cocaine self-administration did not change brain ERK activity. On the 1st day of extinction, the activity of the ERK in the prefrontal cortex was decreased in rats with a history of cocaine chronic intake: by 66% for “active” cocaine group and by 35% for “yoked” cocaine group. On the 3rd day the reduction in the ERK activity (25–34%) was observed in the hippocampus for both cocaine-treated groups, and also in the nucleus accumbens for “yoked” cocaine group (40%). On the 10th day of extinction there was no significant alteration in ERK activity in any group of rats.Our findings suggest that cortical ERK is involved in cocaine seeking behavior in rats. They also indicate the time and regional adaptations in this enzyme activity after cocaine withdrawal.     

Inhibition by pentoxifylline of extracellular signal-regulated kinase activation by platelet-derived growth factor in hepatic stellate cells.

1. It has been proposed that pentoxifylline (PTF) acts an antifibrogenic agent by reducing the synthesis of extracellular matrix components, and this possibility has been confirmed in animal models of hepatic fibrosis. In this study the effects of PTF on the proliferation of extracellular matrix producing cells induced by platelet-derived growth factor (PDGF) were evaluated. The study was performed on hepatic stellate cells, currently indicated as the major source of extracellular matrix in fibrotic liver. 2. PTF caused a dose-dependent reduction of PDGF-induced mitogenesis with an IC50 of 170 microM, identical to the EC50 for the increase in intracellular cyclic AMP levels. Preincubation with PTF did not affect either PDGF-receptor autophosphorylation or phosphotidylinositol 3-kinase activity, whereas it markedly reduced PDGF-stimulated extracellular signal-regulated kinase (ERK) activity and ERK isoform phosphorylation. PTF also reduced PDGF-induced c-fos mRNA expression, which is dependent on activation of the RAS/ERK pathway. In addition, the PDGF-induced increase in cytsolic-free calcium was almost completely prevented by pretreating the cells with PTF. 3. The results of the present study indicate that PTF, in addition to its effect on collagen deposition and degradation, may exert an antifibrogenic effect by reducing the PDGF-induced proliferation of extracellular matrix producing cells. This effect appears to be mediated by a reduction of PDGF-stimulated ERK activity as well as of other intracellular signalling pathways such as the PDGF-induced elevation of cytosolic-free calcium.     

Inhibition of extracellular signal-regulated kinase (ERK1/2) activity reverses endotoxin-induced hypotension via decreased nitric oxide production in rats.

Overproduction of reactive oxygen and nitrogen species leads to oxidative stress and decreased total antioxidant capacity, which is responsible for high mortality from several inflammatory diseases such as endotoxic shock. Among reactive nitrogen species, nitric oxide (NO) produced by inducible NO synthase (iNOS) during endotoxemia is the major cause of vascular hyporeactivity, hypotension and multiple organ failure. This study was conducted to determine if mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK1/2) contributes to endotoxin-induced hypotension as well as vascular inflammation and oxidative stress via NO production. In conscious male Wistar rats, injection of endotoxin (10 mg kg(-1), i.p.) caused a decrease in mean arterial pressure (MAP) for 4h and increased levels of nitrite in serum, aorta and mesenteric artery. These effects of endotoxin were prevented by selective inhibition of ERK1/2 phosphorylation by MAPK kinase (MEK1/2) with U0126 (5 mg kg(-1), i.p.; 1h after endotoxin). Endotoxin also caused an increase in protein levels of phosphorylated ERK1/2 in aorta which was abolished by U0126. Selective inhibition of iNOS with phenylene-1,3-bis[ethane-2-isothiourea] dihydrobromide (1,3-PBIT) (10 mg kg(-1), i.p.; 1h after endotoxin) did not change the endotoxin-induced increase in ERK1/2 activity. Myeloperoxidase activity was increased in aorta and decreased in mesenteric artery by endotoxin, which was reversed by U0126. Endotoxin-induced decrease in one of the products of lipid peroxidation, malonedialdehyde (MDA) was prevented by U0126 in mesenteric artery; however, U0126 caused a further decrease in the levels of MDA in aorta. These data suggest that increased phosphorylation of ERK1/2 by MEK1/2 contributes to the endotoxin-induced hypotension via NO production rat aorta and mesenteric artery. It is likely that ERK1/2 mediates the effect of endotoxin on MPO activity in a different degree in the tissues suggesting possible involvement of any mediator and/or mechanism which also causes neutrophil infiltration during inflammatory response at least in mesenteric artery. Moreover, ERK1/2 seems to be involved in the endotoxin-induced increase in total antioxidant capacity in mesenteric artery.     

ERK/MAPK信号转导途径在急性髓系白血病发病机制中的作用

目的:研究急性髓系白血病细胞外信号调节激酶(ERK)/丝裂原活化蛋白激酶(M APK)的表达水平及其意义。方法:以急性髓系白血病细胞株HL-60细胞,31例初治急性髓系白血病(AM L)、14例AM L完全缓解(CR)和15例正常供髓者骨髓细胞为研究对象,应用流式细胞术检测磷酸化ERK 1/2信号分子的表达。结果:HL-60细胞,初治AM L、AM L-CR及正常骨髓细胞均表达磷酸化ERK 1/2,但表达水平不同。HL-60、初治AM L细胞与AM L-CR、正常细胞相比,磷酸化ERK 1/2表达水平均显著增高(P<0.05),高表达磷酸化ERK 1/2的初治AM L比例为80.6%(25/31)。结论:ERK/M APK信号途径的构成性激活在急性髓系白血病的发病中起重要作用。