Extracellular Signal-regulated Protein Kinase Pathway Research Articles

Sphingosine-1-phosphate decreases melanin synthesis via microphthalmia-associated transcription factor phosphorylation through the S1P3 receptor subtype

Previously, we reported that sphingosine-1-phosphate (S1P) reduced melanin synthesis. In this study we have investigated S1P receptor-mediated extracellular signal-regulated protein kinase (ERK) activation and microphthalmia-associated transcription factor (MITF) phosphorylation. To examine S1P-induced signalling pathways, electron and confocal microscopic studies, reverse transcription-polymerase chain reaction and Western blot analysis were performed. S1P phosphorylated MITF at Ser73, which may have resulted in a MITF mobility shift. Furthermore, 90 kDa ribosomal S6 kinase-1 (RSK-1) phosphorylation was observed after S1P treatment. In addition, PD98059 abrogated the S1P-induced MITF mobility shift and RSK-1 activation. In experiments with MITF mutants, it was shown that dual phosphorylation at Ser73 and Ser409 was indispensable for MITF degradation. We investigated further the actions of S1P on its specific receptors. The results showed that pertussis toxin completely abolished the hypopigmentary effects and ERK pathway activation by S1P, suggesting that S1P regulated melanogenesis via its receptor. The use of specific receptor antagonists indicated that the S1P(3) receptor was dominantly involved in S1P-induced ERK activation and hypopigmentation. The results suggested that S1P reduced melanin synthesis via S1P(3) receptor-mediated ERK and RSK-1 activation, and subsequent MITF dual phosphorylation and degradation.

On the Possible Role of ERK, p38 and CaMKII in the Regulation of CGRP Expression in Morphine-Tolerant Rats

BackgroundThe neuropeptide, calcitonin gene-related peptide (CGRP) has been proposed to be a regulator of the development of morphine analgesic tolerance and thereby could be a target to reduce the induction of this phenomenon under clinical conditions. However, the mechanisms of CGRP regulation are unclear. We investigated here the possible role of the extracellular signal-regulated protein kinase (ERK), p38 and calcium/calmodulin-dependent protein kinase II (CaMKII) in CGRP regulation following chronic morphine treatment.ResultsA 7-day treatment with morphine (15 μg/day) led to an increase in CGRP contents in the spinal cord dorsal horn (SCDH) and dorsal root ganglion (DRG) and this effect was prevented by the inhibition of the ERK, p38 or CaMKII pathway. The phosphorylation/activation of ERK, p38 and CaMKII was enhanced in the SCDH following chronic morphine while in DRG only the phosphorylation of CaMKII was increased. Moreover, our chronic morphine treatment up-regulated neuronal nitric oxide synthase (nNOS) levels in the SCDH, an effect blocked by the inhibition of the ERK, p38 or CaMKII pathway. The blockade of nNOS activity also suppressed chronic morphine-induced CGRP increases in the DRG and SCDH. Double immunofluorescence studies revealed that nNOS and CaMKII are co-localized in the SCDH and that CaMKII is activated in CGRP-expressing DRG neurons.ConclusionsThe activation of spinal ERK, p38 and CaMKII, alongside nNOS, is involved in chronic morphine-induced CGRP up-regulation in both the DRG and SCDH. Moreover, the stimulation of CaMKII in the DRG likely directly regulates the expression of CGRP associated with morphine analgesic tolerance.

Characterized Polysaccharides from Black Soybean Induce Granulocyte Colony-Stimulated Factor Gene Expression in a Phosphoinositide 3-Kinase-dependent Manner

Black soybean (Glycine max L. merr.) is an edible Chinese medicine for nourishment spleen. In the present study, effects of characterized polysaccharides from black soybean (PGM) on granulocyte colony-stimulated factor (G-CSF) production in human peripheral blood mononuclear cells (PBMC) were determined and their action mechanisms were examined. The results indicated that PGM concentration-dependently enhanced G-CSF production in PBMC through modulation of mRNA expression. Data from Western blotting showed that PGM significantly induced the extracellular signal-regulated protein kinase (ERK) activation in PBMC. The nuclear factor (NF)-κB activation in PBMC was increased with PGM by modulation of IκB degradation and PKC θ activation. The levels of G-CSF mRNA in PGM-treated PBMC could be reduced by ERK inhibitor U0126 and NF-κB inhibitor pyrrolidine dithiocarbamate, respectively. Furthermore, the data showed that PGM stimulated phosphoinositide 3-kinase (PI3K)-regulated Akt phosphorylation. The PI3K inhibitor, Ly294002, blocked ERK, NF-κB, and PKC θ activation and G-CSF mRNA expression in PBMC induced by PGM. Thus, we first proved that the enhancement mechanisms of PGM on G-CSF production, appeared to be mediated, at least in part, through activation of PI3K, ERK, PKC θ, and NF-κB signaling pathways in PBMC. We suggest that PGM from black soybean is a potential G-CSF stimulator.

Mechanical impact induces cartilage degradation via mitogen activated protein kinases

To determine the activation of Mitogen activated protein (MAP) kinases in and around cartilage subjected to mechanical damage and to determine the effects of their inhibitors on impaction-induced chondrocyte death and cartilage degeneration. The phosphorylation of MAP kinases was examined with confocal microscopy and immunoblotting. The effects of MAP kinase inhibitors on impaction-induced chondrocyte death and proteoglycan (PG) loss were determined with fluorescent microscopy and 1, 9-Dimethyl-Methylene Blue (DMMB) assay. The expression of catabolic genes at mRNA levels was examined with quantitative real-time PCR. Early p38 activation was detected at 20 min and 1h post-impaction. At 24h, enhanced phosphorylation of p38 and extracellular signal-regulated protein kinase (ERK)1/2 was visualized in chondrocytes from in and around impact sites. The phosphorylation of p38 was increased by 3.0-fold in impact sites and 3.3-fold in adjacent cartilage. The phosphorylation of ERK-1 was increased by 5.8-fold in impact zone and 5.4-fold in adjacent cartilage; the phosphorylation of ERK-2 increased by 4.0-fold in impacted zone and 3.6-fold in adjacent cartilage. Furthermore, the blocking of p38 pathway did not inhibit impaction-induced ERK activation. The inhibition of p38 or ERK pathway significantly reduced injury-related chondrocyte death and PG losses. Quantitative Real-time PCR analysis revealed that blunt impaction significantly up-regulated matrix metalloproteinase (MMP)-13, Tumor necrosis factor (TNF)-α, and ADAMTS-5 expression. These findings implicate p38 and ERK mitogen activated protein kinases (MAPKs) in the post-injury spread of cartilage degeneration and suggest that the risk of post-traumatic osteoarthritis (PTOA) following joint trauma could be decreased by blocking their activities, which might be involved in up-regulating expressions of MMP-13, ADAMTS-5, and TNF-α.

Calcitonin gene-related peptide as a regulator of neuronal CaMKII–CREB, microglial p38–NFκB and astroglial ERK–Stat1/3 cascades mediating the development of tolerance to morphine-induced analgesia

Tolerance to morphine-induced analgesia is an intractable phenomenon, often hindering its prolonged applications in the clinics. The enhanced pronociceptive actions of spinal pain-related molecules such as calcitonin gene-related peptide (CGRP) may underlie this phenomenon and could be a promising target for intervention. We demonstrate here how CGRP regulates the development of morphine analgesic tolerance at the spinal level. A 7-day treatment with morphine led to tolerance to its analgesic effects and enhanced expression of CGRP and its receptor subunits calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1). Activation of several cell-type-specific kinase transcription factor cascades is required to mediate this tolerance, including calcium/calmodulin-dependent protein kinase II (CaMKII) and cAMP response element-binding protein (CREB) in neurons, p38 and nuclear factor kappa B (NFκB) in microglia and extracellular signal-regulated protein kinase (ERK) and signal transducer and activator of transcription 1 and 3 (Stat1/3) in astrocytes, because inhibitors of CaMKII, p38 and ERK pathways correspondingly reduced the increases in phosphorylated CREB, acetylated-NFκB and phosphorylated Stat1/3 levels and attenuated the development of tolerance. Interestingly, these cascades were linked to the regulation of glutamatergic N-methyl- d-aspartate (NMDA) receptor expression. Chronic morphine-induced behavioural responses and biochemical events were all subjugated to modulation by disrupting CGRP receptor signaling. Together, these data suggest that CGRP contributes to the development of tolerance to morphine-induced analgesia by regulating the activation of the neuronal CaMKII–CREB, microglial p38–NFκB and astroglial ERK–Stat1/3 cascades. Targeting CGRP-associated signaling molecules may prolong or restore morphine’s analgesic properties upon a chronic exposure.

Positive regulation of the Egr-1/osteopontin positive feedback loop in rat vascular smooth muscle cells by TGF-β, ERK, JNK, and p38 MAPK signaling

Previous studies identified a positive feedback loop in rat vascular smooth muscle cells (VSMCs) in which early growth response factor-1 (Egr-1) binds to the osteopontin (OPN) promoter and upregulates OPN expression, and OPN upregulates Egr-1 expression via the extracellular signal-regulated protein kinase (ERK) signaling pathway. The current study examined whether transforming growth factor-β (TGF-β) activity contributes to Egr-1 binding to the OPN promoter, and whether other signaling pathways act downstream of OPN to regulate Egr-1 expression. ChIP assays using an anti-Egr-1 antibody showed that amplification of the OPN promoter sequence decreased in TGF-β DNA enzyme-transfected VSMCs relative to control VSMCs. Treatment of VSMCs with PD98059 (ERK inhibitor), SP600125 (JNK inhibitor), or SB203580 (p38 MAPK inhibitor) significantly inhibited OPN-induced Egr-1 expression, and PD98059 treatment was associated with the most significant decrease in Egr-1 expression. OPN-stimulated VSMC cell migration was inhibited by SP600125 or SB203580, but not by PD98059. Furthermore, MTT assays showed that OPN-mediated cell proliferation was inhibited by PD98059, but not by SP600125 or SB203580. Taken together, the results of the current study show that Egr-1 binding to the OPN promoter is positively regulated by TGF-β, and that the p38 MAPK, JNK, and ERK pathways are involved in OPN-mediated Egr-1 upregulation.

CNK1 Promotes Invasion of Cancer Cells through NF-κB–Dependent Signaling

Hallmarks of cancer cells are uncontrolled proliferation, evasion of apoptosis, angiogenesis, cell invasion, and metastasis, which are driven by oncogenic activation of signaling pathways. Herein, we identify the scaffold protein CNK1 as a mediator of oncogenic signaling that promotes invasion in human breast cancer and cervical cancer cells. Downregulation of CNK1 diminishes the invasiveness of cancer cells and correlates with reduced expression of matrix metalloproteinase 9 (MMP-9) and membrane-type 1 MMP (MT1-MMP). Ectopic expression of CNK1 elevates MT1-MMP promoter activity in a NF-kappaB-dependent manner. Moreover, CNK1 cooperates with the NF-kappaB pathway, but not with the extracellular signal-regulated protein kinase pathway, to promote cell invasion. Mechanistically, CNK1 regulates the alternative branch of the NF-kappaB pathway because knockdown of CNK1 interferes with processing of NF-kappaB2 p100 to p52 and its localization to the nucleus. In agreement with this, the invasion of CNK1-depleted cells is less sensitive to RelB downregulation compared with the invasion of control cells. Moreover, CNK1-dependent MT1-MMP promoter activation is blocked by RelB siRNA. Thus, CNK1 is an essential mediator of an oncogenic pathway involved in invasion of breast and cervical cancer cells and is therefore a putative target for cancer therapy.

Modified Si-Miao-San extract inhibits the release of inflammatory mediators from lipopolysaccharide-stimulated mouse macrophages

Ethnopharmacological relevance Modified Si-Miao-San (mSMS) is a prescription modified from Si-Miao-San which is an ancient Chinese prescription used to treat various ailments. Aim of the study Modified Si-Miao-San (mSMS) has been used for the treatment of infectious and inflammatory disorders in the clinic. This study was aimed to investigate its anti-inflammatory activity and underlying mechanism at cellular and molecular levels. Materials and methods We stimulated RAW264.7 cells with Lipopolysaccharide (LPS) and observed effects of mSMS on the release of inflammatory mediators such as: tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), NO, and relative gene expressions. Meanwhile, we also investigated the modulation of mSMS in inflammatory signal transduction mediated through extracellular signal-regulated protein kinase (ERK) and nuclear factor-κB (NF-κB) pathway. Results Our findings demonstrated that mSMS significantly inhibited the excessive production of NO, TNF-α and IL-6 and the over expression of relative genes in LPS-stimulated macrophages. In addition, mSMS suppressed LPS-induced ERK1/2-phosphorylation and inhibited the activation of NF-κB by attenuation of IκB-α degradation. Conclusions Our results suggest that the anti-inflammatory properties of mSMS might result from the inhibition of inflammatory mediators, such as NO, TNF-α and IL-6, via suppression of ERK and NF-κB dependent pathways.

Mechanisms of transforming growth factor β1 /Smad signalling mediated by mitogen-activated protein kinase pathways in keloid fibroblasts

Keloids are recognized as benign tumours characterized by fibroblastic proliferation and accumulation of extracellular matrix, especially collagen deposition. The transforming growth factor (TGF)-beta(1)/Smad pathway plays an important role in keloid pathogenesis; however the underlying mechanisms are not fully understood. To define further the mechanisms of TGF-beta(1)/Smad signal transduction mediated by mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 pathways, in keloid fibroblasts. In the absence or presence of three MAPK (ERK, JNK and p38)-specific inhibitors, keloid fibroblasts were stimulated with exogenous TGF-beta(1) to activate Smad signalling. Smad protein expression was measured by immunoprecipitation/immunoblotting and immunofluorescence; plasminogen activator inhibitor (PAI)-1 transcriptional activity was measured by real-time reverse transcriptase-polymerase chain reaction analysis. TGF-beta(1) induced Smad2/3 phosphorylation at both the C-terminal and the linker region, thus promoting formation of the Smad2/3/4 complex and nuclear translocation, and PAI-1 mRNA expression in keloid fibroblasts; in addition, TGF-beta(1) decreased inhibitory Smad7 expression. Meanwhile, the p38 inhibitor significantly inhibited Smad2/3 phosphorylation, especially at the linker region, and furthermore blocked Smad2/3/4 complex formation, and thus decreased PAI-1 mRNA expression; decreased Smad7 expression induced by TGF-beta(1) was also reversed by the p38 inhibitor. The ERK and JNK inhibitors interrupted Smad2/3/4 complex translocation into the nucleus and consequently decreased PAI-1 mRNA expression. These results suggested that the ERK, JNK and p38 pathways mediate TGF-beta(1)/Smad signal transduction and might be considered as specific targets of drug therapy for keloids.

Regulation of calpain B from Drosophila melanogaster by phosphorylation

Calpain B is one of the two catalytically competent calpain (calcium-activated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombinant protein by protein kinase A as well as by the extracellular signal-regulated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca2+ binding region of an EF-hand motif, and Ser240 close to the autocatalytic activation site of calpain B, as being the residues phosphorylated by protein kinase A. In the transducer region of the protease, Thr747 was shown to be the target of the ERK phosphorylation. Based on the results of three different assays, we concluded that the treatment of calpain B with protein kinase A and ERK1 and ERK2 kinases increases the rate of the autoproteolytic activation of the enzyme, together with the rate of the digestion of external peptide or protein substrates. Phosphorylation also elevates the Ca2+ sensitivity of the protease. The kinetic analysis of phosphorylation mimicking Thr747Glu and Ser845Glu calpain B mutants confirmed the above conclusions. Out of the three phosphorylation events tested in vitro, we verified the in vivo phosphorylation of Thr747 in epidermal growth factor-stimulated Drosophila S2 cells. The data obtained suggest that the activation of the ERK pathway by extracellular signals results in the phosphorylation and activation of calpain B in fruit flies.

Happyhour, a Ste20 Family Kinase, Implicates EGFR Signaling in Ethanol-Induced Behaviors

The consequences of alcohol use disorders (AUDs) are devastating to individuals and society, yet few treatments are currently available. To identify genes regulating the behavioral effects of ethanol, we conducted a genetic screen in Drosophila and identified a mutant, happyhour (hppy), due to its increased resistance to the sedative effects of ethanol. Hppy protein shows strong homology to mammalian Ste20 family kinases of the GCK-1 subfamily. Genetic and biochemical experiments revealed that the epidermal growth factor (EGF)-signaling pathway regulates ethanol sensitivity in Drosophila and that Hppy functions as an inhibitor of the pathway. Acute pharmacological inhibition of the EGF receptor (EGFR) in adult animals altered acute ethanol sensitivity in both flies and mice and reduced ethanol consumption in a preclinical rat model of alcoholism. Inhibitors of the EGFR or components of its signaling pathway are thus potential pharmacotherapies for AUDs.

Fluoride-induced cyclooxygenase-2 expression and prostaglandin E2 production in A549 human pulmonary epithelial cells

To clarify the mechanisms of fluoride-induced airway diseases, we examined the expression of cyclooxygenase-2 (COX-2), an important mediator of airway inflammation, in A549 human pulmonary epithelial cells treated with sodium fluoride (NaF). Following exposure to 5mM NaF, COX-2 protein and COX-2 transcript increased markedly. However, no change was observed in COX-1 expression. NaF-induced accumulation of COX-2 transcript was abolished by actinomycin D, but not cycloheximide. The level of prostaglandin E(2), a major product of COX enzymes, increased in response to NaF exposure, and its production was abolished by the selective COX-2 inhibitor NS-398. Phosphorylated forms of mitogen-activated protein kinases (MAPKs)-including extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase, and p38-increased after NaF exposure, while treatment with the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 markedly suppressed COX-2 expression. Furthermore, NaF-induced COX-2 expression was markedly suppressed by the Src family kinase (SFK) inhibitor PP2, but only partially suppressed by the epidermal growth factor receptor (EGFR) inhibitor PD153035. These results suggest that NaF induces COX-2 expression by transcriptional up-regulation via p38 and ERK pathways, at least in part, and that SFKs may be upstream tyrosine kinases responsible for NaF-induced COX-2 expression in A549 cells.

5-Hydroxytryptophan Acts on the Mitogen-Activated Protein Kinase Extracellular-Signal Regulated Protein Kinase Pathway to Modulate Cyclooxygenase-2 and Inducible Nitric Oxide Synthase Expression in RAW 264.7 Cells

In this study, we evaluated the effect of 5-hydroxytryptophan on anti-inflammatory and analgesic activity in RAW 264.7 cells. Cells were treated with different concentrations of 5-hydroxytryptophan for either 1 h or for 24 h. The anti-inflammatory effect was then analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting and reverse transcription polymerase chain reaction (RT-PCR). In addition, the analgesic activity was evaluated by measuring the acetic acid-induced writhing response. We found that 5-hydroxytryptophan significantly reduced the acetic acid-induced writhing response. Moreover we evaluated the effects of 5-hydroxytryptophan on the release of several inflammatory mediators including nitric oxide (NO) and interleukin-6 (IL-6). Our results demonstrated that 5-hydroxytryptophan inhibited the lipopolysaccharide (LPS)-induced expression of NO and IL-6. Furthermore, we found that 5-hydroxytryptophan played a role in LPS induced inducible nitric oxide synthase (iNOS), cyclo oxygenase-2 (COX-2) and extracellular-signal regulated protein kinase (ERK) activation. Taken together, these results indicate that 5-hydroxytryptophan has the potential for use in the treatment of inflammatory disease and as an analgesic.

ERK Regulates Renal Cell Proliferation and Renal Cyst Expansion in inv Mutant Mice

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage kidney disease in children and young adults. Inv mice are a model for human nephronophthisis type 2 (NPHP2) and characterized by multiple renal cysts and situs inversus. Renal epithelial cells in inv cystic kidneys show increased cell proliferation. We studied the ERK pathway to understand the mechanisms that induce cell proliferation and renal cyst progression in inv kidneys. We studied the effects of ERK suppression by administering PD184352, an oral mitogen-activated protein kinase kinase (MEK) inhibitor on renal cyst expansion, extracellular signal-regulated protein kinase (ERK) activity, bromo-deoxyuridine (BrdU) incorporation and expression of cell-cycle regulators in invΔC kidneys. Phosphorylated ERK (p-ERK) level increased along with renal cyst enlargement. Cell-cycle regulators showed a high level of expression in invΔC kidneys. PD184352 successfully decreased p-ERK level and inhibited renal cyst enlargement. The inhibitor also decreased expression of cell-cycle regulators and BrdU incorporation in renal epithelial cells. The present results showed that ERK regulated renal cell proliferation and cyst expansion in inv mutants.

(−)-Epigallocatechin-3-gallate induces up-regulation of Th1 and Th2 cytokine genes in Jurkat T cells

In the present study, we found that (−)-epigallocatechin-3-gallate (EGCG) significantly up-regulated the mRNA expression of the Th1/Th2 cytokines including IL-2, IFN-γ, IL-5 and IL-13 in Jurkat T cells. The EGCG-induced mRNA up-regulation of IL-2 and IL-5 was predominantly affected by the extracellular signal-regulated protein kinase (ERK) signalling, whereas IL-13 gene expression, the most responsive to the EGCG treatment, was dependent on neither ERK nor c-jun NH 2-terminal kinase (JNK) signalling. IFN-γ gene expression was partially mitigated by both inhibitors of the ERK and JNK pathways. Furthermore, catalase significantly attenuated the intracellular peroxide production, phosphorylation of ERK and JNK, and all cytokine gene expressions induced by EGCG. In addition, physiologically relevant concentrations of both EGCG and H 2O 2-induced up-regulation of IL-5 gene expression. Our findings provide biological evidence that EGCG induces Th1/Th2 cytokine mRNA expression via H 2O 2 production followed by activation of ERK or JNK in Jurkat T cells.

Role of extracellular signal-regulated protein kinase (ERK) in 17β-estradiol-mediated attenuation of lung injury after trauma-hemorrhage

Extracellular signal-regulated protein kinase (ERK) is known to be involved in pro-inflammatory and chemotactic events in response to injury. The aim of this study is to elucidate whether ERK plays any role in 17beta-estradiol (E2)-mediated attenuation of lung injury and pro-inflammatory mediators after trauma-hemorrhage. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure approximately 40 mm Hg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle (cyclodextrin), E2 (1 mg/kg body weight [BW]), or the ERK inhibitor PD98059 (2 mg/kg BW). At 2 h after sham operation or trauma-hemorrhage, various parameters were measured. Trauma-hemorrhage led to a significant increase in lung ERK phosphorylation, which was associated with increased lung myeloperoxidase activity, wet-to-dry weight ratio, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, and macrophage inflammatory protein-2 levels. Circulatory IL-6, TNF-alpha, and lactate levels were also increased after trauma-hemorrhage compared with shams. Administration of E2 or ERK inhibitor PD98059 after trauma-hemorrhage attenuated the trauma-hemorrhage-induced increase in lung injury markers, ERK phosphorylation and cytokines/chemokines, ICAM-1 production, as well as circulatory cytokines and lactate levels. These results collectively suggest that the salutary effects of E2 on the lung after trauma-hemorrhage are mediated via an ERK pathway and subsequent downregulation of pro-inflammatory mediator production.

Ethanol Attenuates the HFS‐Induced, ERK‐Mediated LTP in a Dose‐Dependent Manner in Rat Striatum

The striatum has been implicated to play a role in the control of voluntary behavior, and striatal synaptic plasticity is involved in instrumental learning. Ethanol is known to alter synaptic plasticity, in turn altering the behavior of human and animals. However, it remains unclear whether the striatum plays a role in the effects of ethanol on the central nervous system. The objective of this investigation was to study the effects of acute perfusion of ethanol on long-term potentiation (LTP) to elucidate the mechanisms of addictive drugs in the striatum. In addition, we investigated the contribution of intracellular extracellular signal regulated protein kinase (ERK) signaling pathway to corticostriatal LTP induction. The stimulation evoked population spikes (PS) were recorded from the dorsomedial striatum (DMS) slices of rat using the extracellular recording technique. The LTP in DMS slices was induced by high-frequency stimulation (HFS). The ERK level of the DMS was assessed with the Western blot technique. U0126, the inhibitor of ERK, eliminated or significantly attenuated the LTP induced by HFS of the PS in the DMS. MK801 and APV, N-methyl-d-aspartic acid receptor (NMDAR) antagonists, inhibited the induction of striatal LTP, and HFS-induced ERK activation decreased in the slices treated with MK801 in the DMS. Clinically relevant concentrations of ethanol (22 to 88 mM) dose-dependently attenuated the HFS-induced striatal LTP and ERK activation in this brain region. The LTP of the PS in the DMS is, at least partly, mediated by the ERK pathway coupling to NMDARs. Ethanol attenuated the HFS-induced, ERK-mediated LTP in a dose-dependent manner in this brain region. These results indicate that ethanol may change the synaptic plasticity of corticostriatal circuits underlying the learning of goal-directed instrumental actions, which is mediated by an intracellular ERK signaling pathway associated with NMDARs.

Downregulation of cyclin D1‐CDK4 protein in human embryonic lung fibroblasts (HELF) induced by silica is mediated through the ERK and JNK pathway

Silica is a factor in the induction of acute injury and chronic pulmonary fibrosis. In 1996, silica was also listed as a human carcinogen by the International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved in its pathologic effects are not well understood. We found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica for 2h decreased cyclin D1 and cyclin-dependent kinase 4 (CDK4) expression levels. Extracellular signal-regulated protein kinase (ERKs), c-Jun NH2-terminal amino kinase (JNKs), and p38 kinase, as well as their downstream transcription factor, AP-1, had different effects on the regulation of expression levels of cyclin D1 and CDK4 alterations induced by silica. Silica activates multiple signal transduction pathways involved in coordinating cellular responses to stress. We established the requirements for ERK and JNK, members of the mitogen-activated protein kinase (MAPK) family, in mediating G1 phase arrest of HELF induced by silica. Silica treatment activated ERK in a dose-dependent manner. AG126 (a chemical inhibitor of the ERK signaling pathway) and the dominant negative mutant of ERK2 (a molecular inhibitor of ERK2) prevented decreases in cyclin D1 and CDK4 expression levels. A chemical inhibitor of JNK, SP600125, prevented the decreased expression of both cyclin D1 and CDK4, whereas SB203580, a chemical inhibitor of p38, did not. Interestingly, curcumin prevented the decrease in DK4 expression, but not in cyclin D1. These results demonstrate that ERKs and JNKs are responsible for the decrease of cyclin D1 and CDK4 expression levels in HELF induced by silica. Activator protein-1 (AP-1) was responsible for the decrease of CDK4 expression level, but not that of cyclin D1. The findings help to explain the mechanisms of diseases induced by silica.

BDNF induces late-phase LTP of C-fiber evoked field potentials in rat spinal dorsal horn

Several lines of evidence have shown that in some brain regions brain-derived neurotrophic factor (BDNF) is important for long-term potentiation (LTP), a synaptic model of memory storage. In the present work we evaluate the role of BDNF in LTP of C-fiber evoked field potentials in spinal dorsal horn, a synaptic model of pain memory. We found that spinal application of BDNF-induced LTP of C-fiber evoked field potentials with a long latency, lasting for > 8 h, and the effect was blocked by either tyrosine kinase inhibitor (K252a) or BNDF scavenger (TrkB-Fc). The potentiation produced by BDNF was occluded by late-phase LTP (L-LTP) but not by early-phase LTP (E-LTP) induced by electrical stimulation. Pretreatment of K252a or TrkB-Fc selectively blocked spinal L-LTP induced by low-frequency stimulation (LFS) but not E-LTP. BDNF-induced LTP was completely abolished by the protein synthesis inhibitor (anisomycin), by N-methyl-D-aspartate (NMDA) receptor blocker (MK-801), by extracellular signal-regulated protein kinase (ERK) inhibitor (PD98059) or by p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) but not by c-Jun N-terminal kinase (JNK) inhibitor (SP600125). Nuclear factor-kappaB (NF-κB) inhibitor (PDTC) also suppressed spinal BDNF-LTP. The results suggest that BDNF play a crucial role in protein synthesis-dependent L-LTP in spinal dorsal horn via activation of ERK, p38 MAPK and NF-κB signal pathways.

Taurine inhibits osteoblastic differentiation of vascular smooth muscle cells via the ERK pathway

Vascular calcification develops within atherosclerotic lesions and results from a process similar to osteogenesis. Taurine is a free beta-amino acid and plays an important physiological role in mammals. We have recently demonstrated that vascular smooth muscle cells (VSMCs) express a functional taurine transporter. To evaluate the possible role of taurine in vascular calcification, we assessed its effects on osteoblastic differentiation of VSMCs in vitro. The results showed that taurine inhibited the beta-glycerophosphate-induced osteoblastic differentiation of VSMCs as evidenced by both the decreasing alkaline phosphate (ALP) activity and expression of the core binding factor alpha1 (Cbfalpha1). Taurine also activated the extracellular signal-regulated protein kinase (ERK) pathway. Inhibition of ERK pathway reversed the effect of taurine on ALP activity and Cbfalpha1 expression. These results suggested that taurine inhibited osteoblastic differentiation of vascular cells via the ERK pathway.