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PubMed2002

MEK-ERK pathway is expressed but not activated in high proliferating, self-renewing cord blood hematopoietic progenitors.

INTRODUCTION: The expression, activity and functions of mitogen-activated protein (MAP) kinases in primary human hematopoietic progenitors (HP) have not yet been fully clarified. MATERIAL AND METHODS: To perform our experiments we used a stroma-free cell culture system in which the combination of FLT3 ligand (FL), stem cell factor (SCF) and thrombopoietin (TPO) induces massive expansion and proliferation of cord blood HP. The addition of IL-3 results in a rapid decrease of HP due to the prevalence of maturation and cell death. To detect extracellular regulated kinase (ERK) immunoenzymatic activity we recovered HP from FL, SCF and TPO stimulated long term cultures (LTC) after four weeks of culture. Some samples were recovered 16 h after addition of IL-3 to the LTC. We selectively immunoprecipitated p44/42 ERK kinase from 245 microg of cell lysates. We then analysed dual-phosphorylation of ERK-activating kinase-kinase (p45 MEK1/2) and of p44 ERK1 and p42 ERK2, and investigated MEK and ERK expression. RESULTS: ERK activity, MEK1, and p42 and p44 ERK dual-phosphorylation were undetectable in the expanding, greatly proliferating and self-renewing HP. However, after addition of IL-3 sustained (still detectable 16 h after the stimulus) and high levels of ERK activity and dual-phosphorylation of the kinases were seen. The levels of MEK and ERK expression were stable in the different phases. CONCLUSIONS: These findings add new information on the intracellular mechanisms of HP and help explain the very low levels of hematopoietic toxicity recently seen when treating cancer with down-modulators of ERK activity.

Antigens, CD34↗
PubMed2002

Group-I metabotropic glutamate receptors, mGlu1a and mGlu5a, couple to extracellular signal-regulated kinase (ERK) activation via distinct, but overlapping, signalling pathways.

The coupling of the group I metabotropic glutamate receptors, mGlu1a and mGlu5a, to the extracellular signal-regulated protein kinase (ERK) pathway has been studied in Chinese hamster ovary cell-lines where receptor expression is under inducible control. Both mGlu receptors stimulated comparable, robust and agonist concentration-dependent ERK activations in the CHO cell-lines. The mGlu1a receptor-mediated ERK response was almost completely attenuated by pertussis toxin (PTx) pretreatment, whereas the mGlu5a-ERK response, and the phosphoinositide response to activation of either receptor, was PTx-insensitive. mGlu1a and mGlu5a receptor coupling to ERK occurred via mechanisms independent of phosphoinositide 3-kinase activity and intracellular and/or extracellular Ca2+ concentration. While acute treatment with a protein kinase C (PKC) inhibitor did not attenuate agonist-stimulated ERK activation, down-regulation of PKCs by phorbol ester treatment for 24 h did attenuate both mGlu1a and mGlu5a receptor-mediated responses. Further, inhibition of Src non-receptor tyrosine kinase activity by PP1 attenuated the ERK response generated by both receptor subtypes, but only mGlu1a receptor-ERK activation was attenuated by PDGF receptor tyrosine kinase inhibitor AG1296. These findings demonstrate that, although expressed in a common cell background, these closely related mGlu receptors utilize different G proteins to cause ERK activation and may recruit different tyrosine kinases to facilitate this response.

Animals↗
PubMed2003

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys.

BACKGROUND: The proliferation of mural epithelial cells is a major cause of progressive cyst enlargement in autosomal-dominant polycystic kidney disease (ADPKD). Adenosine 3', 5' cyclic monophosphate (cAMP) stimulates the proliferation of cells from ADPKD cysts, but not cells from normal human kidney cortex (HKC), through the activation of protein kinase A (PKA), mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK/MAPK). In the current study, we examined the signaling pathway between PKA and MEK in ADPKD and HKC cells. METHODS: Primary cultures of human ADPKD and HKC cells were prepared from nephrectomy specimens. We determined the effects of cAMP and epidermal growth factor (EGF) on the activation of ERK, B-Raf and Raf-1 in ADPKD and HKC cells by immune kinase assay and Western blot. RESULTS: 8-Br-cAMP increased phosphorylated ERK (2.7- +/- 0.6-fold, N = 7), and B-Raf kinase activity (3.6- +/- 1.1-fold, N = 5) in cells from ADPKD kidneys; levels of phosphorylated Raf-1 were not changed. Inhibition of PKA by H89 strikingly decreased cAMP-stimulated phosphorylation of ERK and B-Raf, and MAPK inhibition by PD98059 blocked the effect of the nucleotide to activate ERK. By contrast, in HKC cells 8-Br-cAMP did not activate B-Raf and ERK. EGF stimulated the phosphorylation of ERK and Raf-1 in both ADPKD and HKC cells, but had no effect on B-Raf. 8-Br-cAMP and EGF conjointly increased ERK activation above that of either agonist alone in ADPKD cells, and this combined effect was abolished by PD98059, indicating that ERK was activated by EGF- and cAMP-responsive cascades that converge at MAPK. CONCLUSION: cAMP activates ERK and increases proliferation of ADPKD epithelial cells, but not cells from normal human kidney cortex, through the sequential phosphorylation of PKA, B-Raf and MAPK in a pathway separate from, but complementary to, the classical receptor tyrosine kinase cascade. Consequently, cAMP and EGF have great potential to accelerate the progressive enlargement of renal cysts.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2004

The role of oxytocin, protein kinase A, and ERK-related MAP-kinase in the control of porcine ovarian follicle functions.

The aim of our in vitro experiments was to study the role of oxytocin (OT), cAMP/protein kinase A (PKA), and mitogen-activated protein kinase (ERKs MAP-kinase) in the control of ovarian cell functions as well as the role of PKA and MAPK in mediating OT effects on these processes. The whole porcine ovarian follicles were cultured in the presence or absence of OT (1, 10, 100 ng/ml), PKA inhibitor Rp-cAMPS (10 nM), MAP-kinase inhibitor PD98059 (1 microg/ml), or their combination. The release of prostaglandins F (PGF) and E (PGE) were determined by RIA, PKA (alpha-cat subunit), the proliferation-associated peptide PCNA and ERK-1, -2 expression in cell lyzates were analysed by Western-blotting. OT stimulated the release of PGF and PGE, and accumulation of PKA, ERK-1/-2, and PCNA in cell lysate. PD98059 decreased the basal PGF and PGE output, as well as reduced both ERK-1 and ERK-2 accumulation in cell lysates. Rp-cAMPS decreased PKA accumulation in cell lysates. Rp-cAMPS prevented the OT-induced stimulation of PKA, ERK-1, ERK-2, PGF, and PGE, PD98059 did so for PKA, PGF, and PGE. However, PD98059 reduced either basal or OT-induced p-ERK level. OT-stimulated PCNA accumulation was only slightly modified by these blockers. These observations suggest that OT, PKA, and ERKs MAPK can be involved in the control of PGs release and proliferation of ovarian cells. The influence of OT on both PKA and MAPK, and the ability of PKA and MAPK blockers to prevent completely or partially OT effects suggest, that effects of OT on PGF and PGE can be mediated by both PKA and MAPK. The role of MAPK and PKA in mediating the proliferative effects of OT seems to be minor assuming the involvement of other intracellular messengers.

Animals↗
PubMed1998

Modes of action of aspirin-like drugs: salicylates inhibit erk activation and integrin-dependent neutrophil adhesion.

The anti-inflammatory effects of high-dose salicylates are well recognized, incompletely understood and unlikely due entirely to cyclooxygenase (COX) inhibition. We have previously reported a role for activation of the kinase Erk in CD11b/CD18 integrin-dependent adhesiveness of human neutrophils, a critical step in inflammation. We now report the effects of salicylates on neutrophil Erk and adhesion. Exposure of neutrophils to aspirin or sodium salicylate (poor COX inhibitor) inhibited Erk activity and adhesiveness of formylmethionyl-leucyl-phenylalanine- and arachidonic acid-stimulated neutrophils, consistent with anti-inflammation but not COX inhibition (IC50s = 1-8 mM). In contrast, indomethacin blocked neither Erk nor adhesion. Inhibition of Mek (proximal activator of Erk) also blocked stimulation of Erk and adhesion by formylmethionyl-leucyl-phenylalanineand arachidonic acid. Salicylate inhibition of Erk was independent of protein kinase A activation and generation of extracellular adenosine. These data are consistent with a role for Erk in stimulated neutrophil adhesion, and suggest that anti-inflammatory effects of salicylates may be mediated via inhibition of Erk signaling required for integrin-mediated responses.

Acetaminophen↗
PubMed2000

Distinct roles of the adaptor protein Shc and focal adhesion kinase in integrin signaling to ERK.

It has been proposed that integrins activate ERK through the adaptor protein Shc independently of focal adhesion kinase (FAK) or through FAK acting on multiple target effectors, including Shc. We show that disruption of the actin cytoskeleton by cytochalasin D causes a complete inhibition of FAK but does not inhibit Shc signaling and activation of ERK. We have then generated primary fibroblasts carrying a targeted deletion of the segment of beta(1) subunit cytoplasmic domain required for activation of FAK. Analysis of these cells indicates that FAK is not necessary for efficient tyrosine phosphorylation of Shc, association of Shc with Grb2, and activation of ERK in response to matrix adhesion. In addition, integrin-mediated activation of FAK does not appear to be required for signaling to ERK following growth factor stimulation. To examine if FAK could contribute to the activation of ERK in a cell type-specific manner through the Rap1/B-Raf pathway, we have used Swiss-3T3 cells, which in contrast to primary fibroblasts express B-Raf. Dominant negative studies indicate that Shc mediates the early phase and peak, whereas FAK, p130(CAS), Crk, and Rap1 contribute to the late phase of integrin-dependent activation of ERK in these cells. In addition, introduction of B-Raf enhances and sustains integrin-mediated activation of ERK in wild-type primary fibroblasts but not in those carrying the targeted deletion of the beta(1) cytoplasmic domain. Thus, the Shc and FAK pathways are activated independently and function in a parallel fashion. Although not necessary for signaling to ERK in primary fibroblasts, FAK may enhance and prolong integrin-mediated activation of ERK through p130(CAS), Crk, and Rap1 in cells expressing B-Raf.

3T3 Cells↗
PubMed2001

Membrane proximal ERK signaling is required for M-calpain activation downstream of epidermal growth factor receptor signaling.

Localization of signaling is critical in directing cellular outcomes, especially in pleiotropic signaling pathways. The extracellular signal-regulated kinase (ERK)/microtubule-associated protein kinase, which promotes cell migration, proliferation, and differentiation is found in the nucleus and throughout the cytoplasm. Recently, it has been shown that nuclear translocation of ERK is required for transcriptional changes and cell proliferation. However, the cellular consequences, of cytoplasmic signaling have not been defined. We explored whether cytoplasmic, specifically membrane-proximal, ERK signaling is involved in growth factor-induced cell motility. We previously have demonstrated that increased M-calpain activity downstream of epidermal growth factor receptor (EGFR)-mediated ERK activation is necessary for epidermal growth factor (EGF)-induced motility. Calpain isoforms also have been found in nuclear, cytosolic, and plasma membrane-associated compartments in a variety of cell types. We now employ cell engineering approaches to control localization of the upstream EGFR and ERK activities to examine the spatial effect of upstream signal locale on downstream calpain activity. With differential ligand-induced internalization and trafficking-restricted receptor variants, we find that calpain activity is triggered only by plasma membrane-restricted activated EGFR, not by internalized (although still active) EGFR. Cells transfected with membrane-targeted ERK1 and ERK2, which sequester endogenous ERKs, exhibited normal EGF-induced calpain activity. Transfection of an inactive ERK phosphatase (MKP-3/Pyst1) that sequesters ERK in the cytoplasm prevented calpain activation as well as de-adhesion. These data strongly suggest that EGF-induced calpain activity can be enhanced near sites of membrane-proximal EGFR-mediated ERK signaling, providing insights about how calpain activity might be regulated and targeted to enhance its effects on adhesion-related substrates.

Animals↗
PubMed2002

Metalloproteinases stimulate ErbB-dependent ERK signaling in human skin organ culture.

To investigate the role of ERK signaling in human skin responses to wounding, organ cultures of human skin were maintained for 0.5-24 h in the presence of various inhibitors, followed by measurement of ERK phosphorylation or mRNA levels. The MEK inhibitor PD98059 produced near-complete (97-98%) inhibition of ERK phosphorylation, whereas inhibition of c-Fos, c-Jun, HB-EGF, AR, and VEGF mRNA by this compound was incomplete (41-65%). PD98059 was significantly more effective than either PD158780 or BB2516 as an inhibitor of ERK phosphorylation and of the rapid rise in c-Fos and c-Jun mRNA expression. In contrast, all three compounds inhibited the more delayed rise in HB-EGF mRNA to the same extent. Exogenous epidermal growth factor abrogated the inhibition of ERK phosphorylation caused by BB2516. These data indicate that one or more metalloproteinases activate ErbB signaling in skin organ culture, that ErbB signaling plays an important but not exclusive role in the activation of ERK, and that non-ERK pathways contribute to gene expression in this system. Because metalloproteinase-mediated cleavage of the HB-EGF transmembrane precursor is known to be ERK-dependent, our data suggest that ERK activation resulting from initial trauma leads to metalloproteinase-mediated cleavage of HB-EGF, thereby triggering the ErbB signaling cascade.

Blotting, Western↗
PubMed2004

Transient versus sustained phosphorylation and nuclear accumulation of ERKs underlie anti-versus pro-apoptotic effects of estrogens.

Sex steroids exert anti-apoptotic effects on osteoblasts/osteocytes but exert pro-apoptotic effects on osteoclasts, in both cases requiring activation of the extracellular signal-regulated kinases (ERKs). To explain the mechanistic basis of this divergence, we searched for differences in the kinetics of phosphorylation and/or in the subcellular localization of ERKs in response to 17beta-estradiol in the two cell types. In contrast to its transient effect on ERK phosphorylation in osteocytic cells (return to base line by 30 min), 17beta-estradiol-induced ERK phosphorylation in osteoclasts was sustained for at least 24 h following exposure to the hormone. Conversion of sustained ERK phosphorylation to transient, by means of cholera toxin-induced activation of the adenylate cyclase/cAMP/protein kinase A pathway, abrogated the pro-apoptotic effect of 17beta-estradiol on osteoclasts. Conversely, prolongation of ERK activation in osteocytes, by means of leptomycin B-induced inhibition of ERK export from the nucleus or overexpression of a green fluorescent protein-ERK2 mutant that resides permanently in the nucleus, converted the anti-apoptotic effect of 17beta-estradiol to a pro-apoptotic one. These findings indicate that the kinetics of ERK phosphorylation and the length of time that phospho-ERKs are retained in the nucleus are responsible for pro-versus anti-apoptotic effects of estrogen on different cell types of bone and perhaps their many other target tissues.

Active Transport, Cell Nucleus↗
PubMed2004

Phosphorylation of ERK and CREB in cultured hippocampal neurons after haloperidol and risperidone administration.

The purpose of the present paper was to determine whether the brief exposure of neurons to antipsychotic drugs is associated with the activation of extracellular signal-regulated kinases (ERK) and cyclic adenosine 3',5'-monophosphate (cAMP) response element (CRE) binding protein (CREB). The activation of ERK-1/2 and CREB can be monitored by immunoblotting with antibodies that specifically recognize p-ERK-1/2 (phosphorylated on Thr-202 and Tyr-204) and p-CREB (phosphorylated on Ser-133). In hippocampal neuron cultures at 25 days in vitro (DIV), the levels of ERK and CREB phosphorylation significantly increased after treatment with haloperidol (50 nmol/L) and risperidone (50 nmol/L), except when risperidone was administered at the p-CREB level. However, risperidone also increased the p-CREB level at an insignificant rate in the same direction. At 10 DIV, none of the antipsychotic drugs significantly increased the level of ERK and CREB phosphorylation. The difference between levels of ERK and CREB phosphorylation in response to haloperidol and risperidone at 25 DIV was also observed. Risperidone significantly increased the level of ERK-1/2 phosphorylation, but not the level of CREB phosphorylation. Haloperidol, in contrast, had a different effect. These data indicate that neuronal maturation affects the phosphorylation of ERK and CREB in response to antipsychotic drugs. Furthermore, these results demonstrate that different antipsychotic drugs could lead to different profiles of ERK and CREB phosphorylation in neurons.

Animals↗
PubMed2004

Modulation of extracellular signal-regulated kinase (ERK) activity by acute and chronic opioid treatment in neuronal and glial cell lines.

Acute mu opioid application has been shown to activate extracellular signal-related kinases (ERKs) in various non-neural cell lines. However, ERK activation in neuronal cells following acute morphine treatment is more questionable. Moreover, the ERK activation phenomenon observed in vivo after withdrawal of chronic opioids has never been demonstrated in vitro. The goal of this study was to determine if mu agonist treatment induced ERK activation acutely or after withdrawal of chronic opioids in one glial and three neuronal cell lines. We found that acute application of opioids was not able to activate ERK in neuronal cell lines but was able to activate ERK in a glial cell line. In another set of experiments, cells were chronically treated with escalating doses of a mu opioid agonist. After 8 days, the agonist was removed from the media and naloxone applied. Acute ERK activation was not seen in any tested cell line after agonist removal. These findings suggest that opioids may acutely activate ERK in non-neuronal cells, and that the acute ERK activation observed in some brain regions during opioid withdrawal in vivo might be mediated by indirect effects on neuronal cells.

Animals↗
PubMed2004

Emerging role for ERK as a key regulator of neuronal apoptosis.

Extracellular signal-regulated kinases (ERKs) are traditionally viewed as a survival factor in the mitogen-activated protein kinase (MAPK) family. On the other hand, some recent reports have suggested that ERK can also be responsible for neuronal cell death in various neurodegeneration models. In-depth studies on the action of ERK in apoptosis, however, have not been done. A recent study has revealed that ERK is a key apoptotic factor in potassium deprivation-induced neuronal cell death by showing that ERK inhibitors protect neurons from low potassium conditions, whereas constitutively activated ERK activates cell death. Most important, this study shows how ERK can promote neuronal cell death by causing plasma membrane and DNA damage that is independent of caspase-3 activity. Further studies on the mechanism of ERK in neuronal cell death will shed light on the possibility of using ERK as a therapeutic target in treating neurodegeneration.

Animals↗
PubMed2002

Mitochondrial K(ATP) channel openers activate the ERK kinase by an oxidant-dependent mechanism.

Extracellular signal-regulated kinases (ERKs) are key regulatory proteins that mediate cell survival, proliferation, and differentiation. Reactive oxygen species (ROS) may play a role in activation of the ERK pathway. Because mitochondria are a major source of ROS, we investigated whether mitochondria-derived ROS play a role in ERK activation. Diazoxide, a potent mitochondrial ATP-sensitive K+ (K(ATP)) channel opener, is known to depolarize the mitochondrial membrane potential and cause a reversible oxidation of respiratory chain flavoproteins, thus increasing mitochondrial ROS production. Using THP-1 cells as a model, we postulated that opening mitochondrial K(ATP) channels would increase production of ROS and, thereby, regulate the activity of the ERK kinase. We found that opening mitochondrial K(ATP) channels by diazoxide induced production of ROS as determined by an increased rate of dihydroethidium and dichlorofluorescein fluorescence. This increased production of ROS was associated with increased phosphorylation of ERK kinase in a time-dependent fashion. The MEK inhibitors PD-98059 and U-0126 blocked ERK activation mediated by diazoxide. N-acetylcysteine, but not diphenyleneiodonium, attenuated ERK activation mediated by diazoxide. Adenovirus-mediated overexpression of manganese superoxide dismutase, which is expressed in mitochondria, decreased the rate of dihydroethidium oxidation as well as ERK activation. We conclude that mitochondrial K(ATP) channel openers trigger ERK activation via mitochondria-derived ROS.

Adenosine Triphosphate↗
PubMed2002

VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide.

We tested the hypothesis that VEGF regulates endothelial hyperpermeability to macromolecules by activating the ERK-1/2 MAPK pathway. We also tested whether PKC and nitric oxide (NO) mediate VEGF-induced increases in permeability via the ERK-1/2 pathway. FITC-Dextran 70 flux across human umbilical vein endothelial cell monolayers served as an index of permeability, whereas Western blots assessed the phosphorylation of ERK-1/2. VEGF-induced hyperpermeability was inhibited by antisense DNA oligonucleotides directed against ERK-1/2 and by blockade of MEK and Raf-1 activities (20 microM PD-98059 and 5 microM GW-5074). These blocking agents also reduced ERK-1/2 phosphorylation. The PKC inhibitor bisindolylmaleimide I (10 microM) blocked both VEGF-induced ERK-1/2 activation and hyperpermeability. The NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (200 microM) and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidiazoline-1-oxyl-3-oxide (100 microM) abolished VEGF-induced hyperpermeability but did not block ERK-1/2 phosphorylation. These observations demonstrate VEGF-induced hyperpermeability involves activation of PKC and NOS as well as Raf-1, MEK, and ERK-1/2. Furthermore, our data suggest that ERK-1/2 and NOS are elements of different signaling pathways in VEGF-induced hyperpermeability.

Capillary Permeability↗
PubMed2004

Translocation of caveolin regulates stretch-induced ERK activity in vascular smooth muscle cells.

Mechanical stress contributes to vascular disease related to hypertension. Activation of ERK is key to mediating cellular proliferation and vascular remodeling in response to stretch stress. However, the mechanism by which stretch mediates ERK activation in the vascular tissue is still unclear. Caveolin, a major component of a flasklike invaginated caveolae, acts as an adaptor protein for an integrin-mediated signaling pathway. We found that cyclic stretch transiently induced translocation of caveolin from caveolae to noncaveolar membrane sites in vascular smooth muscle cells (VSMCs). This translocation of caveolin was determined by detergent solubility, sucrose gradient fractionation, and immunocytochemistry. Cyclic stretch induced ERK activation; the activity peaked at 5 min (the early phase), decreased gradually, but persisted up to 120 min (the late phase). Disruption of caveolae by methyl-beta-cyclodextrin, decreasing the caveolar caveolin and accumulating the noncaveolar caveolin, enhanced ERK activation in both the early and late phases. When endogenous caveolins were downregulated, however, the late-phase ERK activation was subsided completely. Caveolin, which was translocated to noncaveolar sites in response to stretch, is associated with beta1-integrins as well as with Fyn and Shc, components required for ERK activation. Taken together, caveolin in caveolae may keep ERK inactive, but when caveolin is translocated to noncaveolar sites in response to stretch stress, caveolin mediates stretch-induced ERK activation through an association with beta1-integrins/Fyn/Shc. We suggest that stretch-induced translocation of caveolin to noncaveolar sites plays an important role in mediating stretch-induced ERK activation in VSMCs.

Actin Cytoskeleton↗
PubMed2004

ERK-mediated uterine artery contraction: role of thick and thin filament regulatory pathways.

We have demonstrated that extracellular signal-regulated kinase (ERK) plays an important role in the regulation of uterine artery contraction. The present study tested the hypothesis that ERK regulates thick and thin filament regulatory pathways in the uterine artery. Isometric tension, intracellular free Ca2+ concentration ([Ca2+]i), and 20-kDa myosin light chain (LC20) phosphorylation were measured simultaneously in uterine arteries isolated from near-term (140 days gestation) pregnant sheep. Phenylephrine produced time-dependent increases in [Ca2+]i and LC20 phosphorylation that preceded the contraction, which were inhibited by the MEK (ERK) inhibitor PD-098059. In addition, PD-098059 decreased the intercept of the regression line of LC20 phosphorylation vs. [Ca2+]i but increased the rate of tension development vs. LC20 phosphorylation. In contrast to phenylephrine, phorbol 12,13-bibutyrate (PDBu) produced contractions without changing [Ca2+]i or LC20 phosphorylation. PD-098059 potentiated PDBu-induced contractions without affecting [Ca2+]i and LC20 phosphorylation. PDBu produced time-dependent increases in phosphorylation of p42 and p44 ERK and ERK-dependent phosphorylation of caldesmon at Ser789 in the uterine artery. PD-098059 blocked PDBu-mediated phosphorylation of p42 and p44 ERK and caldesmon. The results indicate that ERK may regulate force by a dual regulation of thick and thin filaments in uterine artery smooth muscle. ERK potentiates the thick filament regulatory pathway by enhancing LC20 phosphorylation via increases in [Ca2+]i and Ca2+ sensitivity of LC20 phosphorylation. In contrast, ERK attenuates the thin filament regulatory pathway and suppresses contractions independent of changes in LC20 phosphorylation in the uterine artery.

Animals↗
PubMed2001

Role of reactive oxygen species in IL-1 beta-stimulated sustained ERK activation and MMP-9 induction.

We have recently demonstrated that interleukin-1 beta (IL-1 beta) stimulates matrix metalloproteinase-9 (MMP-9) induction. In this study we have investigated the roles of superoxide and extracellular signal-regulated kinase (ERK) activation in MMP-9 induction following exposure to IL-1 beta. IL-1 beta stimulated biphasic ERK activation in vascular smooth muscle (VSM) cells, a transient activation that reached a maximum at 15 min and declined to baseline levels within 1 h, and a second phase of sustained ERK activation lasting up to 8 h. To determine the role of ERK in IL-1 beta-stimulated MMP-9 induction, we treated cells with the specific ERK pathway inhibitor PD-98059 at different time intervals after IL-1 beta stimulation. Addition of PD-98059 up to 4 h after IL-1 beta stimulation significantly inhibited MMP-9 induction, suggesting a role for sustained ERK activation in MMP-9 induction. IL-1 beta treatment stimulated superoxide production in VSM cells that was inhibited by pretreatment of cells with the superoxide scavenger N-acetyl-L-cysteine (NAC) and also by overexpression of the human manganese superoxide dismutase (MnSOD) gene. Treatment of VSM cells with NAC selectively inhibited the sustained phase of ERK activation without influencing the transient phase, suggesting a role for reactive oxygen species in sustained ERK activation. In addition, both NAC treatment and MnSOD overexpression significantly inhibited IL-1 beta-stimulated MMP-9 induction (P < 0.05). The results demonstrate that IL-1 beta-dependent MMP-9 induction is mediated by superoxide-stimulated ERK activation.

Acetylcysteine↗
PubMed2003

Role of osteoclast extracellular signal-regulated kinase (ERK) in cell survival and maintenance of cell polarity.

UNLABELLED: Morphological changes of osteoclasts by a MEK1 inhibitor, PD98059, were investigated to clarify a role of ERK. PD98059 promoted apoptosis of osteoclasts and the loss of ruffled borders. This study supports the importance of ERK in survival and polarity of osteoclasts. INTRODUCTION: Extracellular signal-regulated kinase (ERK) is a mitogen activated protein kinase (MAPK) that has been reported to play a role in the survival and apoptosis of osteoclasts. However, the precise signal transduction mechanism is not fully understood. The aim of this study was to clarify the role of ERK in osteoclasts by histological analysis. MATERIALS AND METHODS: Using a rat calvarial organ culture system, the inhibition of ERK phosphorylation by PD98059, a MAPK/ERK kinase 1 (MEK1) inhibitor, was assayed by immunoblotting. Morphological changes in osteoclasts induced by PD98059 were elucidated by light and electron microscopy. The cellular localization of ERK was also determined by immunoelectron microscopy. RESULTS: PD98059 inhibited phosphorylated ERK after a 1-h incubation. Ultrastructural study demonstrated that PD98059 induced the accumulation of vesicles and vacuoles in osteoclasts and the loss of ruffled border at 1 h. At 3 h, some osteoclasts showed apoptosis with nuclear condensation, and at 6 h after PD98059 treatment, many osteoclasts were detached from the bone surface and had lost their cell polarity. Electron microscopic immunohistochemistry revealed that ERK was mainly localized in the cytoplasm of clear zones in control osteoclasts, but apoptotic osteoclasts also showed immunoreactivity in clear zone-like structures in contact with osteoblast-lineage cells. CONCLUSION: These findings indicate that ERK in osteoclasts is involved in their survival and may be involved in the formation of a ruffled border and the maintenance of cell polarity.

Animals↗