Phosphorylation Of ERK MAP Kinases Research Articles

MGluR5 activity is required for the induction of ethanol behavioral sensitization and associated changes in ERK MAP kinase phosphorylation in the nucleus accumbens shell and lateral habenula

Metabotropic glutamate receptor subtype-5 (mGluR5) activity regulates a variety of behavioral pathologies associated with alcohol addiction. The main goal of this study was to determine if mGluR5 regulates the induction of ethanol-induced locomotor sensitization, which is a model of experience-dependent plasticity following initial exposure to drugs of abuse. The extracellular signal-regulated kinase (ERK1/2) pathway is downstream of mGluR5 and implicated in alcohol addiction; however, its role in sensitization remains unexplored. We sought to determine if mGluR5-mediated changes in ethanol-induced sensitization are associated with changes in ERK1/2 phosphorylation (pERK1/2) in specific brain regions. Adult male DBA/2 J mice were tested for acute locomotor response to ethanol (0 or 2 g/kg, IP) followed by a 9-day induction period in which the mGluR5 antagonist MPEP (0 or 30 mg/kg, IP) was administered prior to ethanol (0 or 2.5 g/kg, IP). One day later, ethanol (2 g/kg) produced a robust within- and between-group increase in locomotor activity, indicating sensitization in mice that received MPEP (0 mg/kg) during induction. MPEP (30 mg/kg) treatment during induction resulted in locomotor response to ethanol (2 g/kg) challenge that was equivalent to an acute response, indicating full blockade of sensitization. Sensitization was associated with increased pERK1/2 immunoreactivity (IR) in nucleus accumbens shell (AcbSh) and a reduction in lateral habenula (LHb), both of which were blocked by MPEP treatment during induction. Sensitization was also associated with mGluR5-independent increases in pERK1/2 IR in the nucleus accumbens core and decreases in the dentate gyrus and lateral septum. These data indicate that mGluR5 activity is required for the induction of ethanol locomotor sensitization and associated changes in ERK1/2 phosphorylation in the AcbSh and LHb, which raises the hypothesis that mGluR5-mediated cell signaling in these brain regions may mediate the induction of sensitization. Elucidating mechanisms of sensitization may increase understanding of how ethanol hijacks behavioral functions during the development of addiction.

선학초(仙鶴草)추출물의 대식세포에서의 LPS-유도 염증반응에 대한 효능 연구

Objectives : The aerial parts of Agrimonia pilosa Ledeb (Agrimoniae Herba; AH) has been traditionally used as a Korean medicine to treatment of abdominal pain, sore throat, headaches, bloody discharge, parasitic infections and eczema. In this study, we investigated the effect of AH ethanol extract on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells. Methods : AH was extracted by 30% ethanol (AH-E). Raw264.7 cells were treated with AH-E extract at different concentrations for 30 min and then stimulated with LPS (1μg/ml) or without for indicated times. Cell viability was measured by MTT assay, and nitric oxide (NO) production was measured by Griess assay. The expression of inflammatory mediators, iNOS and COX-2 and inflammatory cytokines, TNF-α, IL-1β, and IL-6 was detected by RT-PCR, and the phosphorylation of ERK1/2, p38 and JNK MAP kinases (MAPKs) was analyzed by Western blot. Also, the expression of NF-κB in nuclear and cytosol was detected by Western blot. Results : AH-E extract significantly decreased LPS-induced NO production in RAW264.7 cells. AH-E extract inhibited the mRNA expression of iNOS, COX-2, TNF-α, IL-1β, and IL-6 in LPS-stimulated cells with a dose-dependent manner. In addition, the phosphorylation of ERK, p38 and JNK MAPKs was also inhibited by AH-E extract. AP-E extract inhibited the nuclear translocation of NF-κB in LPS-stimulated cells. Conclusions : Our results suggest that AH-E extract has an anti-inflammatory activity in macrophages-mediated inflammation.

Effects of Agrimoniae Herba 30% ethanol extract on LPS-induced inflammatory responses in RAW264.7 macrophage cells

Objectives : The aerial parts of Agrimonia pilosa Ledeb (Agrimoniae Herba; AH) has been traditionally used as a Korean medicine to treatment of abdominal pain, sore throat, headaches, bloody discharge, parasitic infections and eczema. In this study, we investigated the effect of AH ethanol extract on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells. Methods : AH was extracted by 30% ethanol (AH-E). Raw264.7 cells were treated with AH-E extract at different concentrations for 30 min and then stimulated with LPS (1μg/ml) or without for indicated times. Cell viability was measured by MTT assay, and nitric oxide (NO) production was measured by Griess assay. The expression of inflammatory mediators, iNOS and COX-2 and inflammatory cytokines, TNF-α, IL-1β, and IL-6 was detected by RT-PCR, and the phosphorylation of ERK1/2, p38 and JNK MAP kinases (MAPKs) was analyzed by Western blot. Also, the expression of NF-κB in nuclear and cytosol was detected by Western blot. Results : AH-E extract significantly decreased LPS-induced NO production in RAW264.7 cells. AH-E extract inhibited the mRNA expression of iNOS, COX-2, TNF-α, IL-1β, and IL-6 in LPS-stimulated cells with a dose-dependent manner. In addition, the phosphorylation of ERK, p38 and JNK MAPKs was also inhibited by AH-E extract. AP-E extract inhibited the nuclear translocation of NF-κB in LPS-stimulated cells. Conclusions : Our results suggest that AH-E extract has an anti-inflammatory activity in macrophages-mediated inflammation.

Evidence for a regulatory role of Cullin-RING E3 ubiquitin ligase 7 in insulin signaling

Dysfunctional regulation of signaling pathways downstream of the insulin receptor plays a pivotal role in the pathogenesis of insulin resistance and type 2 diabetes. In this study we report both in vitro and in vivo experimental evidence for a role of Cullin-RING E3 ubiquitin ligase 7 (CRL7) in the regulation of insulin signaling and glucose homeostasis. We show that Cul7−/− mouse embryonic fibroblasts displayed enhanced AKT and Erk MAP kinase phosphorylation upon insulin stimulation. Depletion of CUL7 by RNA interference in C2C12 myotubes led to increased activation of insulin signaling pathways and cellular glucose uptake, as well as a reduced capacity of these cells to execute insulin-induced degradation of insulin receptor substrate 1 (IRS1). In vivo, heterozygosity of either Cul7 or Fbxw8, both key components of CRL7, resulted in elevated PI3 kinase/AKT activation in skeletal muscle tissue upon insulin stimulation when compared to wild-type controls. Finally, Cul7+/− or Fbxw8+/− mice exhibited enhanced insulin sensitivity and plasma glucose clearance. Collectively, our findings point to a yet unrecognized role of CRL7 in insulin-mediated control of glucose homeostasis by restraining PI3 kinase/AKT activities in skeletal muscle cells.

Lipoxin A4-mediated KATP potassium channel activation results in cystic fibrosis airway epithelial repair

The main cause of morbidity and mortality in cystic fibrosis (CF) is progressive lung destruction as a result of persistent bacterial infection and inflammation, coupled with reduced capacity for epithelial repair. Levels of the anti-inflammatory mediator lipoxin A₄ (LXA₄) have been reported to be reduced in bronchoalveolar lavages of patients with CF. We investigated the ability of LXA₄ to trigger epithelial repair through the initiation of proliferation and migration in non-CF (NuLi-1) and CF (CuFi-1) airway epithelia. Spontaneous repair and cell migration were significantly slower in CF epithelial cultures (CuFi-1) compared with controls (NuLi-1). LXA₄ triggered an increase in migration, proliferation, and wound repair of non-CF and CF airway epithelia. These responses to LXA₄ were completely abolished by the ALX/FPR2 receptor antagonist, Boc2 and ALX/FPR2 siRNA. The KATP channel opener pinacidil mimicked the LXA₄ effect on migration, proliferation, and epithelial repair, whereas the KATP channel inhibitor, glibenclamide, blocked the responses to LXA₄. LXA₄ did not affect potassium channel expression but significantly upregulated glibenclamide-sensitive (KATP) currents through the basolateral membrane of NuLi-1 and CuFi-1 cells. MAP kinase (ERK1/2) inhibitor, PD98059, also inhibited the LXA₄-induced proliferation of NuLi-1 and CuFi-1 cells. Finally, both LXA₄ and pinacidil stimulated ERK-MAP kinase phosphorylation, whereas the effect of LXA₄ on ERK phosphorylation was inhibited by glibenclamide. Taken together, our results provided evidence for a role of LXA₄ in triggering epithelial repair through stimulation of the ALX/FPR2 receptor, KATP potassium channel activation, and ERK phosphorylation. This work suggests exogenous delivery of LXA₄, restoring levels in patients with CF, perhaps as a potential therapeutic strategy.

A Quantitative Model of ERK MAP Kinase Phosphorylation in Crowded Media

Cytoplasm contains a large number of macromolecules at extremely high densities. How this striking nature of intracellular milieu called macromolecular crowding affects intracellular signaling remains uncharacterized. Here, we examined the effect of macromolecular crowding on ERK MAPK phosphorylation by MEK MAPKK. Addition of polyethylene glycol-6000 (PEG-6000) as a crowder to mimic intracellular environments, elicited a biphasic response to the overall ERK phosphorylation rate. Furthermore, probability of processive phosphorylation (processivity) of tyrosine and threonine residues within the activation loop on ERK increased non-linearly for increasing PEG-6000 concentration. Based on the experimental data, we developed for the first time a mathematical model integrating all of the effects of thermodynamic activity, viscosity, and processivity in crowded media, and found that ERK phosphorylation is transition-state-limited reaction. The mathematical model allows accurate estimation of the effects of macromolecular crowding on a wide range of reaction kinetics, from transition-state-limited to diffusion-limited reactions.

Increased sensitivity to alcohol induced changes in ERK Map kinase phosphorylation and memory disruption in adolescent as compared to adult C57BL/6J mice

Adolescence is a critical period of brain development that is accompanied by increased probability of risky behavior, such as alcohol use. Emerging research indicates that adolescents are differentially sensitive to the behavioral effects of acute ethanol as compared to adults but the neurobiological mechanisms of this effect remain to be fully elucidated. This study was designed to evaluate effects of acute ethanol on extracellular signal-regulated kinase phosphorylation (p-ERK1/2) in mesocorticolimbic brain regions. We also sought to determine if age-specific effects of ethanol on p-ERK1/2 are associated with ethanol-induced behavioral deficits on acquisition of the hippocampal-dependent novel object recognition (NOR) test. Adolescent and adult C57BL/6J mice were administered acute ethanol (0 0.5, 1, or 3g/kg, i.p.). Brains were removed 30-min post injection and processed for analysis of p-ERK1/2 immunoreactivity (IR). Additional groups of mice were administered ethanol (0 or 1g/kg) prior to the NOR test. Analysis of p-ERK1/2 IR showed that untreated adolescent mice had significantly higher levels of p-ERK1/2 IR in the nucleus accumbens shell, basolateral amygdala (BLA), central amygdala (CeA), and medial prefrontal cortex (mPFC) as compared to adults. Ethanol (1g/kg) selectively reduced p-ERK1/2 IR in the dentate gyrus and increased p-ERK1/2 IR in the BLA only in adolescent mice. Ethanol (3g/kg) produced the same effects on p-ERK1/2 IR in both age groups with increases in CeA and mPFC, but a decrease in the dentate gyrus, as compared to age-matched saline controls. Pretreatment with ethanol (1g/kg) disrupted performance on the NOR test specifically in adolescents, which corresponds with the ethanol-induced inhibition of p-ERK1/2 IR in the hippocampus. These data show that adolescent mice have differential expression of basal p-ERK1/2 IR in mesocorticolimbic brain regions. Acute ethanol produces a unique set of changes in ERK1/2 phosphorylation in the adolescent brain that are associated with disruption of hippocampal-dependent memory acquisition.

Effects of Molecular Crowding on Processive Phosphorylation of ERK MAP Kinase

Effects of Molecular Crowding on Processive Phosphorylation of ERK MAP Kinase

Increased sensitivity to alcohol induced changes in ERK Map kinase phosphorylation and memory disruption in adolescent as compared to adult C57BL/6J mice

Increased sensitivity to alcohol induced changes in ERK Map kinase phosphorylation and memory disruption in adolescent as compared to adult C57BL/6J mice

Identification of New Substrates of the Protein-tyrosine Phosphatase PTP1B by Bayesian Integration of Proteome Evidence

There is growing evidence that tyrosine phosphatases display an intrinsic enzymatic preference for the sequence context flanking the target phosphotyrosines. On the other hand, substrate selection in vivo is decisively guided by the enzyme-substrate connectivity in the protein interaction network. We describe here a system wide strategy to infer physiological substrates of protein-tyrosine phosphatases. Here we integrate, by a Bayesian model, proteome wide evidence about in vitro substrate preference, as determined by a novel high-density peptide chip technology, and “closeness” in the protein interaction network. This allows to rank candidate substrates of the human PTP1B phosphatase. Ultimately a variety of in vitro and in vivo approaches were used to verify the prediction that the tyrosine phosphorylation levels of five high-ranking substrates, PLC-γ1, Gab1, SHP2, EGFR, and SHP1, are indeed specifically modulated by PTP1B. In addition, we demonstrate that the PTP1B-mediated dephosphorylation of Gab1 negatively affects its EGF-induced association with the phosphatase SHP2. The dissociation of this signaling complex is accompanied by a decrease of ERK MAP kinase phosphorylation and activation.

Trans-Zeatin inhibits UVB-induced matrix metalloproteinase-1 expression via MAP kinase signaling in human skin fibroblasts

Ultraviolet (UV) irradiation induces the expression of matrix metalloproteinases (MMPs), disturbing the metabolism of extracellular matrix (ECM), and causes the characteristic changes of photoaging in skin. Inhibition of induction of MMPs is suggested to alleviate photoaging induced by UV irradiation. Zeatin, purified from Zea mays, is a member of the cytokinin group of plant growth factors, the activity of which is attributed to its more stable trans form. In this study, we investigated the effect of trans-Zeatin on UVB-induced matrix metalloproteinase-1 (MMP-1) expression in cultured human skin fibroblasts (HSFs) and studied the mechanisms of its actions. We found that pretreatment with trans-Zeatin significantly inhibits UVB-induced MMP-1 expression and c-Jun activation in a dose-dependent manner. We also observed that trans-Zeatin inhibits UVB-induced phosphorylation of ERK, JNK and p38 MAP kinases (MAPKs) dose-dependently. As expected, PD98059, an ERK inhibitor, SP600125, a JNK inhibitor and SB203580, a p38 MAPK inhibitor effectively inhibit UVB-induced phosphorylation of ERK, JNK and p38 MAPKs, respectively. Moreover, the inhibitory mechanism of trans-Zeatin was further demonstrated in MMP-1 secretion using MAPK-specific inhibitors. PD98059, SP600125 and SB203580 suppressed UVB-induced MMP-1 secretion, which is consistent with the above results. Collectively, our results suggest that trans-Zeatin inhibits UVB-induced MMP-1 expression, which may be mediated by inhibition of ERK, JNK and p38 MAPKs signaling pathways in HSFs. Trans-Zeatin is a potential agent for the management of skin photoaging.

Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology

BackgroundLysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP) cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development.ResultsOur results demonstrate that Lysophosphatidic Acid (LPA) and Sphingosine-1-phosphate (S1P) receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR)- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK.ConclusionThus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

Epigallocatechin-3-gallate inhibits interleukin-1β-induced MUC5AC gene expression and MUC5AC secretion in normal human nasal epithelial cells

It has been reported that the proinflammatory cytokine interleukin-1β (IL-1β) induces mucus hypersecretion in normal human nasal epithelial (NHNE) cells and that the MAP kinase pathway may be an important signal pathway in IL-1β-induced MUC5AC gene expression. Green tea ( Camellia sinensis) polyphenols are potent anti-inflammatory agents and have been shown to inhibit inflammation in tumor cell lines and cultured respiratory epithelial cells. In this study, we examined the effect of (−)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, on IL-1β-induced MUC5AC gene expression and secretion in NHNE cells. After cells had been treated with IL-1β (10 ng/ml) and pretreated with EGCG (10, 50 and 100 μM), mRNA expression of MUC5AC was determined by real-time polymerase chain reaction. The suppression of each signal pathway protein was determined by Western blot analysis after treatment with IL-1β and EGCG, respectively. IL-1β increased MUC5AC gene expression and MUC5AC secretion. EGCG markedly suppressed IL-1β-induced MUC5AC gene expression and MUC5AC secretion via suppression of the phosphorylation of ERK MAP kinase, MSK1, and transcription factor, cAMP response element-binding protein. IL-1β increased the number of cells staining positive with MUC5AC antibodies, and EGCG treatment decreased this number. Our data suggest that EGCG may be an effective inhibitor of IL-1β-induced mucus hypersecretion.

Integrin alpha9beta1 promotes cell migration via Src kinase mediated nitric oxide synthesis

Beta-1 integrins are important mediators of cell migration but may do so through different signaling mechanisms. However, c-src-activation seems to be a key signaling intermediate. Nitric oxide (NO) synthesis is another novel molecule that modulates cell migration, which may be utilized during integrin mediated cell migration. We used α9 expressing SW480 colon carcinoma cells, to explore a role for src and NO in α9β1 mediated-cell migration. We found that α9β1-ligation leads to c-Src phosphorylation (Tyr-416) and Erk MAP kinase phosphorylation; phospho-c-src was maximum at 5 min, and phospho-Erk maxima was at 20min. Pre-treated cells with src and Erk kinase inhibitors, PP1 and PD98059, inhibited α9-dependent cell motility. c-src appeared to signal upstream of Erk since inhibition of c-Src-kinase blocked Erk phosphorylation. Furthermore, α9β1-ligation enhanced NO synthesis through activation of inducible Nitric Oxide Synthase (iNOS), which was constitutively expressed in SW480 cells. Using the NO specific inhibitor L-NAME, α9β1-dependent cell migration was blocked. Also, enhanced iNOS phosphorylation and NO production were src kinase (but not Erk) dependent. These results suggest a novel signaling pathway for α9β1-induced cell migration through c-src kinase activation of iNOS and subsequent NO production.

Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cells

5alpha,8alpha-Epidioxy-22E-ergosta-6, 22-dien-3beta-ol (ergosterol peroxide) is a major antitumour sterol produced by edible or medicinal mushrooms. However, its molecular mechanism of action has yet to be determined. Here, we examine the anticancer and anti-inflammatory effects of ergosterol peroxide. After treating RAW264.7 macrophages with LPS and purified ergosterol peroxide or ergosterol, we determined LPS-induced inflammatory cytokines, nuclear DNA binding activity of transcription factors and phosphorylation of MAP kinases (MAPKs). HT29 colorectal adenocarcinoma cells were treated with ergosterol peroxide for 5 days. To investigate the antitumour properties of ergosterol peroxide, we performed DNA microarray and RT-PCR analyses and determined the reactive oxygen species (ROS) in HT29 cells. Ergosterol peroxide suppressed LPS-induced TNF-alpha secretion and IL-1alpha/beta expression in RAW264.7 cells. Ergosterol peroxide and ergosterol suppressed LPS-induced DNA binding activity of NF-kappaB and C/EBPbeta, and inhibited the phosphorylation of p38, JNK and ERK MAPKs. Ergosterol peroxide down-regulated the expression of low-density lipoprotein receptor (LDLR) regulated by C/EBP, and HMG-CoA reductase (HMGCR) in RAW264.7 cells. In addition, ergosterol peroxide showed cytostatic effects on HT29 cells and increased intracellular ROS. Furthermore, ergosterol peroxide induced the expression of oxidative stress-inducible genes, and the cyclin-dependent kinase inhibitor CDKN1A, and suppressed STAT1 and interferon-inducible genes. Our results suggest that ergosterol peroxide and ergosterol suppress LPS-induced inflammatory responses through inhibition of NF-kappaB and C/EBPbeta transcriptional activity, and phosphorylation of MAPKs. Moreover, ergosterol peroxide appears to suppress cell growth and STAT1 mediated inflammatory responses by altering the redox state in HT29 cells.

(±)-2-Chloropropionic acid elevates reactive oxygen species formation in human neutrophil granulocytes

(±)-2-Chloropropionic acid (2-CPA) is a neurotoxic compound which kills cerebellar granule cells in vivo, and makes cerebellar granule cells in vitro produce reactive oxygen species (ROS). We have studied the effect of 2-CPA on ROS formation in human neutrophil granulocytes in vitro. We found an increased formation of ROS after 2-CPA exposure using three different methods; the fluorescent probe DCFH-DA and the chemiluminescent probes lucigenin and luminol. Four different inhibitors of ROS formation were tested on the cells in combination with 2-CPA to characterize the signalling pathways. The spin-trap s-PBN, the ERK1/2 inhibitor U0126 and the antioxidant Vitamin E inhibited the 2-CPA-induced ROS formation completely, while the mitochondrial transition permeability pore blocker cyclosporine A inhibited the ROS formation partly. We also found that 2-CPA induced an increased nitric oxide production in the cells by using the Griess reagent. The level of reduced glutathione, measured with the DTNB assay, was decreased after exposure to high concentrations of 2-CPA. Western blotting analysis showed that 2-CPA exposure led to an elevated phosphorylation of ERK MAP kinase. This phosphorylation was inhibited by U0126. Based on these experiments it seems like the mechanisms for 2-CPA induced toxicity involves ROS formation and is similar in neutrophil granulocytes as earlier shown in cerebellar granule cells. This also implies that 2-CPA may be immunotoxic.

Human lactoferrin upregulates expression of KDR/Flk-1 and stimulates VEGF-A-mediated endothelial cell proliferation and migration

Lactoferrin (LF) is a multifunctional iron-binding glycoprotein, which plays a variety of biological processes including immunity. In this study, we demonstrate that human LF upregulates KDR/Flk-1 mRNA and protein levels in HUVECs at an optimal concentration of 5 μg/ml, which subsequently promotes the VEGF-induced proliferation and migration of the endothelial cells. Exposure of HUVECs to LF significantly increased VEGF-induced ERK MAP kinase phosphorylation. The maximal stimulation of KDR/Flk-1 expression by LF was correlated with LF-induced increase in cell proliferation and migration. These findings suggest that LF may stimulate in vivo angiogenesis via upregulation of KDR/Flk-1 expression in endothelial cells.

Identification and Characterization of ERK MAP Kinase Phosphorylation Sites in Smad3

Smad3 is phosphorylated by ERK MAP kinase upon EGF treatment. We have mapped the ERK phosphorylation sites to Ser 207, Ser 203, and Thr 178 in Smad3. We show that, upon EGF treatment, Smad3 is rapidly phosphorylated in these sites, peaking at approximately 15-30 min and that MEK1 inhibitors PD98059 and U0216 inhibit Smad3 phosphorylation induced by EGF. Ser 207 is the best ERK site in Smad3. Its phosphorylation shows the highest EGF induction in Smad3. It is also a very sensitive site to EGF treatment, significantly responding to low concentrations of EGF. These three sites are also phosphorylated by recombinant ERK2 in vitro. We have compared the kinetic parameters of Smad3 with those of ELK1 and MBP for ERK2. We further show that mutation of the ERK phosphorylation sites increases the ability of Smad3 to stimulate a Smad target gene, suggesting that ERK phosphorylation inhibits Smad3 activity.

Human skeletal muscle cell differentiation is associated with changes in myogenic markers and enhanced insulin-mediated MAPK and PKB phosphorylation.

We hypothesized that myogenic differentiation of HSMC would yield a more insulin responsive phenotype. We assessed expression of several proteins involved in insulin action or myogenesis during differentiation of primary human skeletal muscle cultures (HSMC). Differentiation increased creatine kinase activity and expression of desmin and myocyte enhancer factor (MEF)2C. No change in expression was observed for big mitogen-activated protein kinase (BMK1/ERK5), MEF2A, insulin receptor (IR), hexokinase II, and IR substrates 1 and 2, while expression of glycogen synthase, extracellular signal-regulated kinase 1 and 2 (ERK1/2 MAP kinase) and the insulin responsive aminopeptidase increased after differentiation. In contrast to protein kinase B (PKB)a, expression of (PKB)b increased, with differentiation. Both basal and insulin-stimulated PI 3-kinase activity increased with differentiation. Insulin-mediated phosphorylation of PKB and ERK1/2 MAP kinase increased after differentiation. Components of the insulin-signalling machinery are expressed in myoblast and myotube HSMC; however, insulin responsiveness to PKB and ERK MAP kinase phosphorylation increases with differentiation.

Cultured gastrointestinal smooth muscle cells: cell response to contractile agonists depends on their phenotypic state.

In the digestive tract, the transit of ingested food induces a local contraction-relaxation reflex of which the smooth muscle cell (SMC) represents the functional unit. Although freshly isolated SMCs have been extensively used for in vitro studies, in specific cases cultured cells appear necessary. Because conventionally cultured SMCs lose their contractile properties, we have developed: (1) differentiated, contractile rabbit gastric SMCs (D-stim cells), cultured in a medium supplemented with insulin, and (2) proliferative, dedifferentiated rabbit gastric SMCs (P-stim cells), cultured in a medium supplemented with insulin, fetal serum, EGF and b-FGF. The proliferative index was 5 +/- 4% and 82 +/- 10%, respectively, for D-stim and P-stim cells. Expression of SM-myosin heavy chain was observed in 90% of D-stim cells, whereas it was progressively lost in P-stim cells. Carbachol (1-100 nM), glicentin (2 nM) and gastrin-17 (100 nM) induced contraction of D-stim cells cultured for 3 or 6 days, whereas they did not induce the contraction of P-stim cells; in contrast, gastrin-17 (10 nM) was able to stimulate DNA synthesis (1.86 +/- 0.09-fold increase) in P-stim cells. The coupling of muscarinic receptors to intracellular transduction pathways was evaluated in D-stim cells: at day 3, carbachol (100 nM) induced a twofold increase in the production of inositol tri-tetra-phosphates; in parallel, a phosphorylation of ERK MAP kinases occurred within 1 min of carbachol stimulation. In conclusion, cultured functional myocytes derived from mature tissue may be used for long-term studies concerning the events coupled either to proliferation or to motility regulation of differentiated SMCs due to the activation of G-protein-coupled receptors.