Role Of Extracellular Signal-regulated Kinase Research Articles

Differential roles of extracellular signal-regulated kinase 1/2 and p38MAPK in mechanical load-induced procollagen alpha1(I) gene expression in cardiac fibroblasts.

We have previously demonstrated that mechanical loading of cardiac fibroblasts leads to increased synthesis and gene expression of the extracellular matrix protein collagen. We hypothesised that the upregulation of procollagen gene expression in cardiac fibroblasts, in response to cyclic mechanical load, is mediated by one or more members of the MAP kinase family. To test this hypothesis, the effect of mechanical load on the activation of extracellular signal-regulated kinase (ERK) 1/2, p46/54JNK, and p38MAPK was examined in rat cardiac fibroblasts. Peak phosphorylation of ERK 1/2, p38MAPK kinases, and p46/54JNK was observed following 10-20 min of continuous cyclic mechanical load. Mechanical load significantly increased procollagen alpha1(I) mRNA levels up to twofold above static controls after 24 h. This increase was completely abolished by the MEK 1/2 inhibitor U0126, with no effect on basal levels. In contrast, SB203580, a specific inhibitor of p38MAPK, enhanced both basal and stretch-stimulated levels of procollagen mRNA. Consistent with this finding, selective activation of the p38MAPK signalling pathway by expression of MKK6(Glu), a constitutive activator of p38MAPK, significantly reduced procollagen alpha1(I) promoter activity. SB203580-dependent increase in procollagen alpha1(I) was accompanied by ERK 1/2 activation, and inhibition of this pathway completely prevented SB203580-induced procollagen alpha1(I) expression. These results suggest that mechanical load-induced procollagen alpha1(I) gene expression requires ERK 1/2 activation and that the p38MAPK pathway negatively regulates gene expression in cardiac fibroblasts. These pathways are likely to be key in events leading to matrix deposition during heart growth and remodelling induced by mechanical load.

Extracellular signal-regulated kinase 1/2 is required for the induction of group I metabotropic glutamate receptor-mediated epileptiform discharges.

Transient stimulation of group I metabotropic glutamate receptors (mGluRs) induces persistent prolonged epileptiform discharges in hippocampal slices via a protein synthesis-dependent process. At present, the signaling process underlying the induction of these epileptiform discharges remains unknown. We examined the possible role of extracellular signal-regulated kinases (ERK1 and ERK2) because these kinases can be activated by group I mGluRs, and their activation may regulate gene expression and alter protein synthesis. The group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 50 microm) induced activation of ERK1/2 in hippocampal slices. 2-(2-Diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059) (50 microm) a specific inhibitor of mitogen-activated protein kinase kinase (MEK), suppressed ERK1/2 activation by DHPG. PD98059 or another MEK inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (10 microm), also prevented the induction of the prolonged epileptiform discharges by DHPG. In the presence of ionotropic glutamate receptor inhibitors and tetrodotoxin (blockers), DHPG-induced epileptiform discharges were suppressed, whereas ERK1/2 activation persisted. Protein kinase C inhibitors (2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide, 1 microm; or chelerythrine, 10 microm) did not prevent the generation of DHPG-induced epileptiform discharges, nor did they suppress the activation of ERK1/2 by DHPG in slices pretreated with the blockers. Genistein (30 microm), a broad-spectrum tyrosine kinase inhibitor, suppressed the DHPG-induced epileptiform discharges and the ERK1/2 activation in the presence of blockers. Induction of DHPG-mediated epileptiform discharges was also suppressed by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (10 microm), an Src-family tyrosine kinase inhibitor. The study shows that group I mGluRs activate ERK1/2 through a tyrosine kinase-dependent process and that this activation of ERK1/2 is necessary for the induction of prolonged epileptiform discharges in the hippocampus.

Mitogen-Activated Protein Kinase Signaling

The mechanism by which cells respond to extracellular stimuli involves a series of signal transduction events across the cell membrane and through the cytoplasm to the nucleus. Mitogen-activated protein (MAP) kinases are important mediators of signal transduction and play a key role in the regulation of many cellular processes, such as cell growth and proliferation, differentiation, and apoptosis. In mammalian cells, three major groups of MAP kinases have been identified: extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAP kinase. It is well documented that ERK is typically stimulated by growth-related signals, whereas the JNK and p38 MAP kinase cascades are activated by various stress stimuli. Studies have indicated that MAP kinases are expressed abundantly in the central nervous system (CNS) and that ERK is involved in long-lasting neuronal plasticity, including long-term potentiation and memory consolidation. While the role of ERK in neuronal plasticity and behavioral adaptation is beginning to emerge, the role of MAP kinase signal transduction cascades in major psychiatric disorders, including schizophrenia, is not well understood. This review outlines the intermediates of this signaling cascade and downstream transcription factor targets and recent evidence implicating MAP kinases to important biological functions in the CNS. Evidence from human post-mortem studies, as well as from the phencyclidine model of schizophrenia, that different MAP kinase cascades may be involved in the pathogenesis of schizophrenia, and potentially in other psychiatric disorders, is presented. Knowledge of MAP kinase signaling will aid greatly in our ability t o understand causal changes in disease process and may lead to new therapeutic approaches in controlling or treating schizophrenia.

Fas ligation induces IL-1β-dependent maturation and IL-1β-independent survival of dendritic cells: different roles of ERK and NF-κB signaling pathways

AbstractThe mechanisms that underpin the intriguing capacity of Fas ligation on dendritic cells (DCs) to induce maturation and activation, rather than apoptosis, remain unclear. In the present study we confirm that Fas signaling induces both phenotypic and functional maturation of murine DCs, and we demonstrate that phenotypic maturation is associated with phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, activation of caspase-1, and secretion of interleukin-β (IL-1β). Specific inhibition of ERK1/2 diminished Fas ligation-induced caspase-1 activation, IL-1β secretion, and ensuing up-regulation of developmental markers, whereas treatment with neutralizing anti-IL-1β antibody abrogated phenotypic and functional maturation, indicating that IL-1β mediates Fas ligation-induced DC maturation in an autocrine manner. NF-κB activation was responsible for maintaining DC viability after Fas ligation. Inhibiting NF-κB did not affect either IL-1β secretion or phenotypic maturation but rather sensitized DCs to Fas-mediated apoptosis. In conclusion, positive signals originating from Fas are transduced through at least 2 different intracellular pathways in DCs, promoting not only survival but also an increase in maturation that correlates with increased antigen-presentation capability. (Blood. 2003;102:4441-4447)

A neuroprotective role of extracellular signal-regulated kinase in n-acetyl- o-methyldopamine-treated hippocampal neurons after exposure to in vitro and in vivo ischemia

In response to cerebral ischemia, neurons activate survival/repair pathways in addition to death cascades. Activation of cyclic AMP-response-element-binding protein (CREB) is linked to neuroprotection in experimental animal models of stroke. However, a role of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MAPK/ERK or MEK), an upstream kinase for CREB, and its relation to CREB phosphorylation in neuroprotection in cerebral ischemia has not been delineated. Previously, we reported that N-acetyl- O-methyldopamine (NAMDA) significantly protected CA1 neurons after transient forebrain ischemia [J Neurosci 19 (1999b) 87.8]. The current study is to investigate whether NAMDA-induced neuroprotection occurs via the activation of ERK and its downstream effector, CREB. NAMDA induced ERK1/2 and CREB phosphorylation with increased survival of HC2S2 hippocampal neurons subjected to oxygen-glucose deprivation. These effects were reversed by U0126, a MEK kinase inhibitor. Similarly, animals treated with NAMDA following ischemia showed increased ERK and CREB phosphorylation in the CA1 subregion of the hippocampus during early reperfusion period with increased number of surviving neurons examined 7 days following ischemia. The NAMDA-induced neuroprotection was abolished by U0126 administered shortly after reperfusion. The results showed that the ERK-CREB signaling pathway might be involved in NAMDA-induced neuroprotection following transient global ischemia and imply that the activation of the pathway in neurons may be an effective therapeutic strategy to treat stroke or other neurological syndromes.

Redox regulation of reactive oxygen species-induced p38 MAP kinase activation and barrier dysfunction in lung microvascular endothelial cells.

Reactive oxygen species (ROS)-mediated compromise of endothelial barrier integrity has been implicated in a number of pulmonary disorders, including adult respiratory distress syndrome, pulmonary edema, and vasculitis. The mechanisms by which ROS increase endothelial permeability are unclear. We hypothesized that ROS-induced changes in cellular redox status (thiols) may contribute to endothelial barrier dysfunction. To test this hypothesis, we used N-acetylcysteine (NAC) and diamide to modulate intracellular levels of cellular glutathione (GSH) and investigated hydrogen peroxide (H(2)O(2))-mediated mitogen-activated protein kinase (MAPK) activation and transendothelial electrical resistance (TER). Exposure of bovine lung microvascular endothelial cells (BLMVECs) to H(2)O(2), in a dose- and time-dependent fashion, increased endothelial permeability. Pretreatment of BLMVECs with NAC (5 mM) for 1 h resulted in partial attenuation of H(2)O(2)-induced TER (a measure of increase in permeability) and GSH. Furthermore, treatment of BLMVECs with diamide, which is known to reduce the intracellular GSH, resulted in significant reduction in TER, which was prevented by NAC. To understand further the role of MAPKs in ROS-induced barrier dysfunction, we examined the role of extracellular signal-regulated kinase (ERK) and p38 MAPK on H(2)O(2)- and diamide-mediated permeability changes. Both H(2)O(2) and diamide, in a dose-dependent manner, activated ERK and p38 MAPK in BLMVECs. However, SB203580, an inhibitor of p38 MAPK, but not PD98059, blocked H(2)O(2)- and diamide-induced TER. Also, NAC prevented H(2)O(2)- and diamide-induced p38 MAPK, but not ERK activation. These results suggest a role for redox regulation of p38 MAPK in ROS-dependent endothelial barrier dysfunction.

Retinoic acid-induced neuritogenesis of human neuroblastoma SH-SY5Y cells is ERK independent and PKC dependent.

Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA-induced neuritogenesis, we used human neuroblastoma SH-SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal-regulated kinase (ERK) 1 and 2 in RA-induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA-induced neuritogenesis (percentage of neurite-bearing cells and neurite length) but significantly reduced cell survival. In addition, we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA-induced ERK phosphorylation does not follow the classic Raf kinase-dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI 3-K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3-K inhibitor wortmannin, we observed a significant dose-dependent reduction in ERK phosphorylation. RA-induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration-dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA-induced neuritogenesis.

Molecular Mechanisms of Renal Tissue Repair in Survival from Acute Renal Tubule Necrosis: Role of ERK1/2 Pathway

Our earlier studies with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) showed that prior administration of a low priming dose of 15 mg/kg, i.p. to mice, given 72 hours before administration of a normally lethal dose of DCVC (75 mg/kg, i.p.) led to renal tubule necrosis, however sustained renal tubule regeneration was observed and these mice recovered from renal failure and survived. The objective of the present study was to investigate the role of extracellular signal-regulated kinase (ERK) pathway in this autoprotection model. Following the priming dose of DCVC, IL-6 protein and mRNA increased markedly as early as 1 hour after dosing, peaking at 3 hours with a 1.5-fold increase in plasma. Immunocytochemistry on kidney sections using specific antibodies against TGF-alpha, HB-EGF, EGFr, IGF-1Rbeta, Grb-2, and phospho-p44/42 MAP kinase (ERK1/2) revealed a significantly higher staining of these molecules 3 to 72 hours after dosing, indicating up regulation of the ERK pathway. Following a lethal dose of DCVC (75 mg/kg) the early increase in these signaling molecules was not sustained, being markedly reduced 24 and 36 hours after dosing, leading to inhibition of S-phase DNA synthesis, cell division and renal tubule repair. In contrast, prior treatment with a low dose of DCVC, followed by a high dose led to a sustained stimulation of the renal ERK pathway, renal tubule regeneration and recovery from acute renal failure. These results suggest that a sustained activation of the ERK1/2 pathway may be a key factor in enabling a continued renal tubule repair and hence protection from the progressive phase of DCVC-induced acute renal tubular necrosis in the mouse.

Phosphorylation of extracellular signal-regulated kinases 1/2 is predominantly enhanced in the microglia of the rat spinal cord following dorsal root transection

The present study was initiated to investigate the role of extracellular signal-regulated kinases (ERK) 1/2 signaling pathway in the early response of spinal cord and associated dorsal root ganglion (DRG) to rhizotomy by using Western blotting and immunohistochemical techniques in a rat model of L3 and L4 dorsal root transection. The results showed that there were a considerable amount of total and phosphorylated ERK 1/2 protein in both spinal cord and DRG in normal animals killed under pentobarbital anesthesia. The total ERK 1/2 distributed in both glia and neurons, while phosphorylated ERK 1/2 dominantly existed in the latter in the gray matter of spinal cord, as demonstrated with double immunofluorescent staining. Twenty-four and forty-eight hours after axotomy, the phosphorylation level of ERK 1/2 in the operation side of dorsal spinal cord was much higher than that in the contralateral side, while the total ERK 1/2 level seemed unchanged. The increased expression of Fos protein was also seen in the dorsal spinal cord at lesion side twelve and twenty-four hours after axotomy. Double fluorescent staining proved that the phosphorylated ERK 1/2 positive cells in the ipsilateral dorsal spinal cord after axotomy predominantly were microglia and small portion was oligodendrocytes, whereas the Fos expression was mainly in neurons. In normal DRG, most neurons, especially the medium and small-sized ones, and the satellite cells contained total ERK 1/2-like immunoreactivity, whereas only a small portion of neurons and satellite cells contained phosphorylated ERK 1/2. After unilateral dorsal rhizotomy, there were no detectable changes for the phosphorylation of ERK 1/2 in either neurons or satellite cells in DRG. Collectively, the present results suggest that both ERK and Fos signal pathways involve the cellular activation in the spinal cord following dorsal rhizotomy, with ERK mainly in microglia and Fos in neurons. The increase of phosphorylation of ERK 1/2 in microglia of spinal cord after rhizotomy implicates that ERK signaling pathway involves intracellular activity of microglia responding to the experimental injury.

Extracellular Signal-regulated Kinase 1/2 is Involved in the Activation of NADPH Oxidase Induced by FMLP Receptor but not by Complement Receptor 3 in Rat Neutrophils

This experiment was performed to clarify the role of extracellular signal-regulated kinase, ERK1/2, in NADPH oxidase-dependent O2- production in rat peritoneal neutrophils. When neutrophils were exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) to stimulate an N-formyl peptide receptor, not only the production of O2- but also the activation of ERK1/2 was observed. The translocation of an NADPH oxidase component, p47phox, from cytosol to membrane also occurred in neutrophils stimulated with fMLP. U0126, an ERK1/2 kinase inhibitor, inhibited both the production of O2- and the translocation of p47phox elicited by fMLP. On the other hand, when complement receptor 3 of neutrophils was stimulated with opsonized zymosan (OZ), weaker activation of ERK1/2 than that by fMLP was observed. In this case, U0126 showed no inhibition against the production of O2- and slight inhibition against the translocation of p47phox. Large inhibition against the OZ-induced production of O2- was only observed in neutrophils treated with GF109203X, a PKC inhibitor. The present study indicates that receptor dependence exists in the ERK1/2 signaling pathway leading to the activation of NADPH oxidase.

Inhibition of calcineurin facilitates the induction of memory for sensitization in Aplysia: requirement of mitogen-activated protein kinase.

The induction of both synaptic plasticity and memory is thought to depend on the balance between opposing molecular regulatory factors, such as protein kinases and phosphatases. Here we show that inhibition of protein phosphatase 2B (calcineurin, CaN) facilitates the induction of intermediate-term memory (ITM) and long-term memory (LTM) for tail shock-induced sensitization in Aplysia without any effect on short-term memory. To identify the molecular cascade underlying the improvement of memory by inhibition of CaN, we examined the role of extracellular signal-regulated kinase 1/2/mitogen-activated protein kinase (MAPK). Molecular experiments revealed that one pulse of serotonin, which by itself does not activate MAPK, leads to significant MAPK activation in the sensory neurons of the pleural ganglia when CaN is inhibited. Extending these observations, behavioral experiments showed that the facilitated induction of ITM and LTM produced by CaN inhibition depends on MAPK activity. These results demonstrate: (i) that CaN acts as an inhibitory constraint in the formation of long-lasting phases of memory, and (ii) that facilitated induction of ITM and LTM by CaN inhibition requires MAPK activity.

Epidermal growth factor induces c-fos and c-jun mRNA via Raf-1/MEK1/ERK-dependent and -independent pathways in bovine luteal cells

Epidermal growth factor (EGF) modulates the actions of gonadotropins in the corpus luteum. The membrane-associated EGF receptors undergo rapid tyrosine phosphorylation and internalization upon ligand binding in ovarian cells, including luteal cells. However, little is known about the post-receptor signaling events induced by EGF that lead to the transcriptional regulation of EGF-responsive genes in the ovary. The present study was designed to examine in bovine luteal cells (1) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling cascade (Raf/MEK/ERK) by EGF; (2) mRNA expression of AP-1 transcription factors, i.e. c-fos and c-jun, in response to EGF; and (3) the role of ERK in EGF-induced expression of c-fos and c-jun mRNA. Raf-1 and B-Raf, but not A-Raf, were activated by EGF (10 ng/ml) and the pharmacological protein kinase C (PKC) activator phorbol myristate acetate (PMA, 20 nM). Activation of Raf resulted in the phosphorylation and activation of MAPK kinase (MEK1) which subsequently activated ERKs. Treatment with EGF-induced the phosphorylation of both ERK2 and ERK1 in a time and concentration dependent manner. Additionally, activated ERK was found in the nucleus of the cells following treatment with EGF (10 ng/ml) and PMA (PMA, 20 nM) for 5 min. Depletion of PKC by chronic PMA treatment (2.5 μM, 24 h) only partially inhibited the stimulatory effects of EGF on Raf-1, ERK2 and ERK1. These data demonstrate that PKC-dependent and independent-mechanisms are involved in EGF activation of the Raf/MEK/ERK signaling cascade in bovine luteal cells. EGF rapidly and transiently stimulated the expression of c-fos and c-jun mRNA in bovine luteal cells. Maximal induction of c-fos and c-jun mRNA by EGF occurred within 30 min of treatment with 10 ng/ml EGF. Treatment with the MEK1 inhibitor PD098059 (50 μM) abolished EGF-induced ERK activation. However, blocking EGF-induced ERK activation by pretreatment with PD098059 only partially attenuated EGF-induced c-fos and c-jun mRNA expression. Thus, additional pathways are implicated in the regulation of c-fos and c-jun mRNA expression by EGF in bovine luteal cells.

Nitric oxide as a modulator of gastric mucin synthesis: role of ERK and p38 mitogen-activated protein kinase activation.

Nitric oxide (NO) is an important biological messenger in the regulation of tissue homeostasis and pathophysiological processes. Here, we investigated the effect of NO on gastric mucus glycoprotein (mucin) synthesis, apoptotic processes, and the involvement of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Exposure of gastric mucosal cells to NO donor led to a dose-dependent decrease (up to 48%) in mucin synthesis, accompanied by a marked increase in caspase-3 activity and apoptosis. Inhibition of ERK with PD98059 accelerated (up to 23.8%) the NO-induced decrease in mucin synthesis, and cause further enhancement in caspase-3 activity and apoptosis. Blockade of p38 kinase with SB203580 produced reversal in the NO-induced reduction in mucin synthesis, and substantially countered the induced increase in caspase-3 activity and apoptosis. Moreover, caspase-3 inhibitor not only blocked the NO-induced increase in caspase-3 activity but also produced an increase in mucin synthesis. Thus, the detrimental influence of NO on mucin synthesis is closely linked to caspase-3 activation and apoptosis, and involves ERK and p38 kinase participation. Activation of p38 kinase leads to the upregulation of proapoptotic signal, while ERK activation stimulates the anti-apoptotic pathway.

Regulation of gonadotropin alpha subunit gene expression by dopamine D(2) receptor agonist in clonal mouse gonadotroph alphaT3-1 cells.

Pituitary prolactin biosynthesis is negatively regulated by hypothalamic dopamine through D(2) receptors in pituitary lactotrophs, but little is known about the direct effect of dopamine on gonadotrophs. In this study, the clonal gonadotroph-derived cell line, alphaT3-1, was used to examine whether gene expression of the pituitary gonadotropin alpha subunit, stimulated with GnRH or pituitary adenylate cyclase-activating polypeptide (PACAP), was controlled by dopamine D(2) receptor. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated the presence of dopamine D(2) receptors in alphaT3-1 cells. Both GnRH and PACAP increased alpha subunit gene expression. GnRH-induced alpha subunit gene expression was not affected by quinpirol, a specific dopamine D(2) receptor agonist. In contrast, PACAP-induced gene expression was significantly lower in the presence of quinpirol. The roles of extracellular signal-regulated kinase (ERK) and cAMP in the expression of the alpha subunit gene were examined. GnRH activated ERK, but PACAP did not, and the activation was not inhibited by quinpirol. GnRH-induced alpha subunit gene expression was completely inhibited by an ERK inhibitor, PD098059. Cyclic AMP accumulation in alphaT3-1 cells was increased by treatment with PACAP, and quinpirol inhibited this effect. GnRH did not affect cAMP production in these cells. These results suggest that in alphaT3-1 cells, dopamine D(2) receptors negatively regulate pituitary alpha subunit gene expression in association with the cAMP-dependent pathway, but not with the ERK pathway.

The role of extracellular signal-regulated kinase in cognitive and motor deficits following experimental traumatic brain injury

Traumatic brain injury (TBI) causes neuronal death and alters the plasticity (e.g. morphology) of surviving neurons. Both of these events contribute to TBI-associated neurological deficits, such as memory dysfunction. Although a majority of current research is directed towards identifying biochemical cascades responsible for cell death, little is known about mechanisms of altered neuronal plasticity following TBI. Extracellular signal-regulated kinases (Erk1 and 2) play a critical role in growth and have been implicated in long-lasting neuronal plasticity and memory storage. The activation of Erk following TBI was investigated utilizing an antibody that specifically binds to dually phosphorylated Erk. Using this antibody, we report that lateral cortical impact injury in rats increases Erk phosphorylation both in the cortex and the hippocampus as early as 10 min post-injury. Double immunostaining experiments using either a neuron-specific or an astroglial-specific marker show that the active Erk is localized almost exclusively in neuronal cells. Furthermore, the increase in phospho-Erk immunoreactivity was initially localized to axons and at later time points was observed to be predominantly in the cell soma. This suggests that Erk redistributed over time and may play a role in retrograde signaling. Administration of inhibitors of the Erk cascade worsened retrograde amnesia, impaired performances in hippocampus- and amygdala-dependent memory tasks, and exacerbated motor deficits following TBI. Furthermore, inhibition of this cascade did not have any overt effects on cell survival, but altered neuronal morphology as detected by a dendritic-specific marker. These findings suggest that the Erk cascade plays an essential role for the maintenance of neuronal function and plasticity following TBI.

Control of mycobacterial replication in human macrophages: roles of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways.

Intracellular persistence of mycobacteria may result from an intricate balance between bacterial replication and signaling events leading to antimicrobial macrophage activities. Using human monocyte-derived macrophages, we investigated the relevance of mitogen-activated protein kinase activation for the growth control of Mycobacterium avium isolates differing in their abilities to multiply intracellularly. The highly replicative smooth transparent morphotype of M. avium strain 2151 induced significantly less p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation than the smooth opaque morphotype of the same strain, which was gradually eliminated from macrophage cultures. Inhibition of the p38 pathway by highly specific inhibitors did not significantly affect mycobacterial replication within macrophages, regardless of the in vitro virulence of the M. avium strain. However, repression of the ERK1/2 pathway further enhanced intracellular growth of highly replicative M. avium strains, although it did not increase survival of the poorly replicating M. avium isolate. Inhibition of the ERK1/2 pathway resulted in decreased tumor necrosis alpha (TNF-alpha) secretion irrespective of the virulence of the M. avium isolate used for infection, revealing that TNF-alpha could have been only partially responsible for the control of intracellular M. avium growth. In conclusion, ERK1/2- and TNF-alpha-independent pathways are sufficient to limit intramacrophage growth of less-virulent M. avium strains, but early ERK1/2 activation in infected macrophages is critically involved in controlling the growth of highly replicative M. avium strains.

MAPK activation is involved in posttranscriptional regulation of RSV-induced RANTES gene expression.

Airway epithelial cells represent the primary cell target of respiratory syncytial virus (RSV) infection. They actively participate in the lung immune/inflammatory response that follows RSV infection by expressing chemokines, small chemotactic cytokines that recruit and activate leukocytes. Regulated on activation, normal T cell expressed, and presumably secreted (RANTES) is a member of the CC chemokine subfamily and is strongly chemotactic for T lymphocytes, monocytes, basophils, and eosinophils, cell types that are present or activated in the inflammatory infiltrate that follows RSV infection of the lung. RSV infection of airway epithelial cells induces RANTES expression by increasing gene transcription and stabilizing RNA transcripts. The signaling pathway regulating RANTES gene expression after RSV infection has not been determined. In this study, we examined the role of extracellular signal-regulated kinase (ERK) and p38, members of the mitogen-activated protein (MAP) kinase (MAPK) family, in RSV-induced RANTES production. RSV infection of alveolar epithelial cells induced increased phosphorylation and catalytic activity of ERK and the upstream kinases Raf-1 and MAP ERK kinase. Induction of the MAP signaling cascade required a replication-competent virus. RSV infection of alveolar epithelial cells also induced activation of p38 MAPK. Inhibition of ERK and p38 activation significantly reduced RSV-induced RANTES mRNA and protein secretion without affecting RANTES gene transcription or transcription factor activation. These results indicate that the MAPK signaling cascade regulates RANTES production in alveolar epithelial cells through a posttranscriptional mechanism.

Prolonged Nuclear Retention of Activated Extracellular Signal-regulated Kinase 1/2 Is Required for Hepatocyte Growth Factor-induced Cell Motility

We examined the signaling pathway by which hepatocyte growth factor (HGF) induces cell motility, with special focus on the role of extracellular signal-regulated kinase (ERK) in the nucleus. We used Madin-Darby canine kidney cells overexpressing ERK2 because of their prominent motility response to HGF. HGF stimulation of the cells induces not only a rapid, marked, and sustained activation and rapid nuclear accumulation of ERK1/2, but also a prolonged nuclear retention of the activated ERK1/2. Interruption of the ERK1/2 activation by PD98059 treatment of the cells 30 min after HGF stimulation abolishes the HGF-induced cell motility. Enforced cytoplasmic retention of the activated ERK1/2 by the expression of an inactive form of MKP-3 cytoplasmic phosphatase inhibits the cell motility response. Although epidermal growth factor stimulation of the cells induces the activation and nuclear accumulation of ERK1/2, it does not induce the prolonged nuclear retention of the activated ERK1/2, and fails to induce cell motility. In the nucleus, activated ERK1/2 continuously phosphorylate Elk-1, leading to the prolonged expression of c-fos, which results in the expression of several genes such as matrix metalloproteinase (mmp)-9; MMP-9 activity is required for the induction of the cell motility response. Our results indicate that the sustained activity of ERK1/2 in the nucleus is required for the induction of HGF-induced cell motility.

MAP kinases contribute to IL-8 secretion by intestinal epithelial cells via a posttranscriptional mechanism.

The intracellular pathways that regulate intestinal epithelial gene expression are poorly understood. In this study we examined the roles of extracellular signal-regulated kinase (ERK) and p38 in the expression of interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) using the human intestinal cell line HT-29. HT-29 cells were treated with tumor necrosis factor-alpha (TNF-alpha) in the presence or absence of ERK and p38 pathway inhibitors. TNF-alpha treatment resulted in increased IL-8 and ICAM-1 protein and mRNA synthesis, increased ERK and p38 activity, and activation of the transcription factors activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB). Inhibition of the ERK and p38 pathways attenuated IL-8 secretion but did not alter ICAM-1 expression. Furthermore, AP-1 and NF-kappaB DNA binding was not affected by ERK and p38 inhibition. In contrast, ERK and p38 inhibition resulted in the accelerated degradation of the IL-8 mRNA, suggesting that in HT-29 cells, p38 and ERK contribute to TNF-alpha-stimulated IL-8 secretion by intestinal epithelial cells via a posttranscriptional mechanism that involves stabilization of the IL-8 transcript.

Extracellular signal-regulated kinases phosphorylate 5-lipoxygenase and stimulate 5-lipoxygenase product formation in leukocytes.

5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. Here, we demonstrate that extracellular signal-regulated kinases (ERKs) can phosphorylate 5-LO in vitro. Efficient phosphorylation required the presence of unsaturated fatty acids and was abolished when Ser-663 was mutated to alanine. In intact HeLa cells stimulated with arachidonic acid (AA), impaired 5-LO product formation was evident in cells expressing the S663A-5-LO mutant compared with cells expressing wild-type 5-LO. For Mono Mac 6 cells, priming with phorbol myristate acetate (PMA) before stimulation with ionophore was required for ERK1/2 activation and efficient 5-LO phosphorylation, in parallel with substantial AA release and 5-LO product formation. Inhibition of PKC by GF109203x or MEK1/2 by U0126 (or PD98059) abolished the 5-LO up-regulation effects of PMA. In contrast, these inhibitors failed to suppress 5-LO product formation induced by stimuli such as AA plus ionophore, which apparently do not involve the ERK1/2 pathway. Based on inhibitor studies, ERKs are also involved in AA-stimulated 5-LO product formation in PMNL, whereas a role for ERKs is not apparent in 5-LO activation induced by ionophore or cell stress. Finally, the data suggest that ERKs and p38 MAPK-regulated MAPKAPKs can act in conjunction to stimulate 5-LO by phosphorylation.