Cooperation of calcineurin and ERK for UTP-induced IL-6 production in HaCaT keratinocytes

UTP causes IL-6 production in HaCaT keratinocytes, which is partially inhibited by PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, suggesting that a pathway other than the extracellular signal-regulated kinase (ERK) pathway is involved in the production. In the present study, we examined the involvement of calcineurin in the UTP-induced interleukin (IL)-6 production in HaCaT keratinocytes. FK506 and cyclosporine A, calcineurin inhibitors, partially inhibited UTP-induced IL-6 mRNA expression and protein production. In addition, combined application of FK506 and PD98059 synergistically inhibited the UTP-induced IL-6 production. These results suggest that ERK and calcineurin are cooperatively involved in UTP-induced IL-6 production.     

Iron-induced interleukin-6 gene expression: possible mediation through the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways

Increased iron store in the body may increase the risk of many diseases such as cancer and inflammation. However, the precise pathogenic mechanism of iron has not yet been elucidated. In the present study, the early biological responses of cells to iron treatment were investigated in AP-1 luciferase reporter stably transfected mouse epidermal JB6 cells and primary rat hepatocytes. It was shown that water-soluble iron compounds, such as FeSO4 and Fe2(SO4)3, were more active in inducing AP-1 in JB6 cells than water-insoluble iron compounds, such as Fe2O3 and FeS. Iron stimulated mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-jun NH2 terminal kinases (JNKs), both in JB6 cells and in primary rat hepatocytes, as determined by the phosphorylation assay. Interestingly, the increase in AP-1 luciferase activity by iron was inhibited by the pretreatment of the cells with PD98059, a specific MEK1 inhibitor, and SB202190, a p38 kinase inhibitor. Levels of interleukin-6 (IL-6), a pro-inflammatory cytokine, were increased in JB6 cells by iron in a dose-dependent manner. The increase in IL-6 and its mRNA by iron was also eliminated by the pretreatment of the cells with PD98059 and SB202190. Since the IL-6 promoter contains an AP-1 binding site, our studies indicate that the iron-induced IL-6 gene expression may be mediated through ERKs and p38 MAPK pathways, possibly one of the important mechanisms for the pathogenesis of iron overload.     

Targeting the extracellular signal-regulated kinase pathway in cancer therapy

The extracellular signal-regulated kinase (ERK) pathway is a major determinant in the control of diverse cellular processes such as proliferation, survival, and motility. This pathway is often upregulated in human cancers and as such represents an attractive target for mechanism-based approaches to cancer treatment. However, specific blockade of the ERK pathway alone induces mostly cytostatic rather than proapoptotic effects, resulting in limited therapeutic efficacy. Blockade of the constitutively activated ERK pathway by an ERK kinase (MEK) inhibitor sensitizes tumor cells to apoptotic cell death induced by several cytotoxic anticancer agents including microtubule-destabilizing agents and histone deacetylase inhibitors, not only in vitro but also in tumor zenografts in vivo. Thus, low concentrations of these anticancer drugs that by themselves show little cytotoxicity effectively kill tumor cells in which the ERK pathway is constitutively activated when co-administrated with a MEK inhibitor. The combination of a cytostatic signaling pathway inhibitor (MEK inhibitors) and conventional anticancer drugs (microtubule-destabilizing agents or histone deacetylase inhibitors) provides an excellent basis for the development of safer anticancer chemotherapies with enhanced efficacy through lowering the required dose of the latter cytotoxic drugs.     

Nickel-induced proteins in human HaCaT keratinocytes: annexin II and phosphoglycerate kinase

It has been established in previous in vitro experiments with human HaCaT keratinocytes that nickel becomes cytotoxic at concentrations higher than 100 microM and that it is accumulated mainly in the cytosolic fraction (Ermolli et al., 2000). The aim of this work was to search possible biomarkers of metal insult, i.e. nickel-binding proteins or proteins differentially expressed in the cytosolic fraction of nickel-exposed cells (up to 1 mM nickel) as compared to controls. Cytosolic proteins were studied by isoelectric focusing (IEF) and two-dimensional gel electrophoresis (2-DE). Separation by IEF revealed nickel-induced changes in the abundance of cytosolic proteins as visualised with nickel-nitrilo-triacetic-alkaline phosphatase (Ni-NTA-AP) in blots. The cytosolic fraction of cells incubated with nickel, at concentrations over 100 microM, showed nickel binding components which were absent or present in significantly lower amounts in control cells. These proteins had isoelectric points (pIs) 6.9, 7.7 and 8.5. After 2-DE silver- and protein staining significantly increased abundance of four proteins was observed. Their pI values corresponded to those of the nickel binding ones seen after IEF. A protein with pI 6.9 had a molecular weight estimated to 38 kDa, two proteins with pI around 7.7 showed molecular weights of 57 and 22 kDa, respectively and another protein with pI of 8.5 had a molecular weight of 33 kDa. The increased abundance of these components, both in IEF experiments and in 2-DE, correlated with the nickel concentration in the culture media. N-terminal amino acid sequencing and database search allowed identification of one a protein as phosphoglycerate kinase and another one as annexin II. The involvement of these proteins in cellular functions and their possible implications in the mechanism of nickel toxicity in keratinocytes are discussed. Some of these proteins may be biomarker candidates for effects of nickel exposure in human keratinocytes.     

Identification of novel extracellular signal-regulated kinase docking domain inhibitors

The extracellular signal regulated kinase (ERK1 and ERK2) signal transduction pathways play a critical role in cell proliferation. Hyperactivation of the ERK proteins either through increased expression of membrane-bound growth factor receptors or genetic mutations of upstream proteins is thought to be involved in the pathogenesis of many human cancers. Thus, targeted inhibition of ERK signaling is viewed as a potential approach to prevent cancer cell proliferation. Currently, no specific inhibitors of the ERK proteins exist. Moreover, most kinase inhibitors lack specificity because they target the ATP binding region, which is well conserved among the protein kinase families. Taking advantage of recently identified ERK docking domains, which are reported to facilitate substrate protein interactions, we have used computer-aided drug design (CADD) to identify novel small molecular weight ERK inhibitors. Following a CADD screen of over 800 000 molecules, 80 potential compounds were selected and tested for activity in biological assays. Several compounds inhibited ERK-specific phosphorylation of ribosomal S6 kinase-1 (Rsk-1) or the ternary complex factor Elk-1 (TCF/Elk-1), both of which are involved in promoting cell proliferation. Active compounds showed a dose-dependent reduction in the proliferation of several cancer cell lines as measured by colony survival assays. Direct binding between the active compounds and ERK2 was indicated by fluorescence quenching. These active compounds may serve as lead candidates for development of novel specific inhibitors of ERK-substrate interactions involved in cell proliferation.     

Protein kinase C activation by Helicobacter pylori in human gastric epithelial cells limits interleukin-8 production through suppression of extracellular signal-regulated kinase

Helicobacter pylori (H. pylori) infection of gastric epithelial cells has been shown to induce interleukin (IL)-8 production, but the signal transduction mechanism leading to IL-8 production has not been clearly defined. Here, we investigate the role of protein kinase C (PKC) in the mechanism of induction of IL-8 release by H. pylori in human gastric epithelial cells. In MKN45 cells, H. pylori-induced IL-8 release was enhanced by treatment with PKC inhibitors (GF109203X and calphostin C) and PKC depletion, which completely inhibited PKC activity. Moreover, PKC inhibitors and PKC depletion increased extracellular signal-regulated kinase (ERK) activity and phosphorylation, but not calcium/calmodulin-dependent protein kinase II (CaMK II) activity, in response to H. pylori infection. PKC activated by H. pylori inhibited activation of ERK induced by H. pylori without affecting the CaMK II activity and negatively regulated IL-8 production in human gastric epithelial cells.     

Role of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinase kinase in adenosine A2B receptor-mediated interleukin-8 production in human mast cells

The endogenous nucleoside adenosine is thought to play a role in the pathophysiology of asthma by stimulating mast cells. We previously showed that the human mast cell line HMC-1 expresses A2A and A2B receptors, and that both receptors activate adenylate cyclase via Gs-protein but that only A2B receptors are also coupled to phospholipase C via Gq proteins. Stimulation of A2B but not A2A receptors induced production of interleukin-8 (IL-8) from HMC-1 cells. The mechanism by which adenosine promotes IL-8 synthesis has not been defined. In this study, we tested the hypothesis that mitogen-activated protein kinase (MAPK) signaling pathways are involved in this process. Stimulation of HMC-1 with the stable adenosine analog NECA (5'-N-ethylcarboxamidoadenosine) activated p21(ras) and both p42 and p44 isoforms of extracellular signal-regulated kinase (ERK). NECA (10 microM) induced a 1.9 +/- 0. 06-fold increase in ERK activity, whereas 10 microM of the selective A2A agonist CGS 21680 (4-((N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl)-phenylpropionic acid) had no effect. NECA, in parallel with the activation of ERK, also stimulated the p46 isoform of c-Jun N-terminal kinase (MEK) and p38 MAPK. Furthermore, the selective MAPK/ERK kinase 1 inhibitor PD 98059 (2'-amino-3'-methoxyflavone), and p38 MAPK inhibitors SB 202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole) and SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H- imidaz ole) blocked A2B receptor-mediated production of IL-8. These results indicate that extracellular adenosine can regulate ERK, c-Jun N-terminal kinase, and p38 MAPK signaling cascades and that activation of ERK and p38 MAPK pathways are essential steps in adenosine A2B receptor-dependent stimulation of IL-8 production in HMC-1.     

异氟醚对乙酰胆碱诱导的细胞外信号调节激酶磷酸化的抑制效应

目的研究异氟醚如何影响乙酰胆碱诱导的细胞内信号传递,探讨挥发性麻醉药影响认知功能的分子机理。方法培养的神经元样PC12细胞随机分成对照组和异氟醚组。对照组正常培养,异氟醚组用1.2%异氟醚处理2 h,分别于处理后0 min、1 h、3 h加入碘化乙酰胆碱(ACh)刺激2 min后收集细胞,采用Westem blot技术检测基础水平和乙酰胆碱诱导下细胞外信号调节激酶1/2(ERK1/2)的磷酸化水平和蛋白激酶B(PKB)的活性。结果单纯ACh刺激可导致ERK1/2磷酸化水平显著增加(P<0.01),异氟醚处理后0 min和1 h乙酰胆碱诱导的ERK1/2磷酸化水平明显降低,3 h时恢复到正常水平。与对ERK的影响效应不同,单独异氟醚处理可迅速增加基础状态下PKB的磷酸化水平(P<0.05),但1 h后即恢复正常水平。结论异氟醚可以长时程减弱乙酰胆碱诱导的ERK活化,从而干扰胞内信号传递,这可能与术后认知功能障碍发生密切相关。     

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

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

银屑病诱发药物对HaCaT角质形成细胞的影响

为了探讨锂盐、普萘洛尔和氯喹是否通过影响银屑病的细胞因子网络从而诱发或加重银屑病，以不同浓度的碳酸锂、盐酸普萘洛尔或二磷酸氯喹处理HaCaT角质形成细胞后加以TNF-α刺激，应用人类细胞因子抗体分析膜技术测定细胞培养液中多种细胞因子和生长因子的分泌情况；采用实时定量PCR法检测IL-8和IL-6 mRNA的表达。人类细胞因子抗体分析膜技术结果显示，碳酸锂明显促进IL-6和TNF-α的产生；盐酸普萘洛尔明显促进IL-6等多种细胞因子和生长因子的产生；二磷酸氯喹也明显促进IL-6的产生。实时定量PCR结果表明，TNF-α能刺激HaCaT角质形成细胞呈剂量依赖性增加IL-8和IL-6 mRNA的表达(P<0.01)；并对IL-8的调节作用更强(P<0.01)；1×10^-6 mol·L^-1盐酸普萘洛尔能显著上调IL-6 mRNA的表达(P<0.05)。碳酸锂、盐酸普萘洛和二磷酸氯喹对HaCaT角质形成细胞表达以及产生某些细胞因子和生长因子具有调节功能。     

ERK/MAPK信号转导途径在急性髓系白血病发病机制中的作用

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

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

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

Signaling pathways for monocyte chemoattractant protein 1-mediated extracellular signal-regulated kinase activation

G protein-coupled receptors (GPCRs) initiate diverse down-stream signaling events in response to ligand stimulation, as rapid activation of the extracellular signal-regulated kinase ERK1 and ERK2. The chemokine monocyte chemoattractant protein-1 (MCP-1) is the agonist for several chemokine receptors that belong to the GPCR superfamily, CCR2 being the most important. Stimulation of mitogen-activated protein kinases (MAPKs) by MCP-1 has been implicated in integrin activation and chemotaxis, but the molecular pathways down-stream of the receptors remain unclear. To dissect the cascade of events leading to MAPK activation upon CCR2 receptor stimulation, several specific inhibitors and mutants of signal transduction proteins were used in monocytic cells endogenously expressing CCR2 and/or in human embryonic kidney-293 cells transfected with CCR2B receptors and epitope-tagged ERK1. We show that ERK activation by MCP-1 involves heterotrimeric Gi protein subunits, protein kinase C, phosphoinositide-3-kinase, and Ras. On the other hand, the activity of cytosolic tyrosine kinases, epidermal growth factor receptor transactivation, or variations in intracellular calcium levels are not required for the mitogenic activation elicited by MCP-1. In addition, we find that internalization of CCR2B itself is not necessary for efficient MCP-1-induced activation of ERK, although a dynamin mutant partially inhibits ERK stimulation. These results suggest that different parallel pathways are being activated that lead to the full activation of the mitogen-activated protein kinase cascade and that internalization of other signaling proteins but not of the receptor is required for complete ERK activation.     

Contribution of extracellular signal-regulated kinases to the IL-1-induced growth inhibition of human melanoma cells A375

The role of ERK1/2 in the IL-1-induced growth inhibition was investigated using human melanoma A375-6 cells. A selective inhibitor of ERK1/2 pathway, PD98059 and a selective inhibitor of p38MAPK, SB203580 each alone significantly reversed the IL-1-induced growth inhibition of A375-6 cells. Co-treatment with PD98059 and SB203580 completely reversed the IL-1-induced growth inhibition. ERK1/2 was constitutively activated in A375-6 cells, and IL-1 further augmented ERK activation. Antiproliferative effect of IL-1 was attenuated by the expression of dominant negative form of ERK2. IL-1 induced cell cycle arrest in G(0)/G(1) phase, expression of p21 and p27 proteins, and down-regulation of cyclin D/cyclin-dependent kinase (CDK) 2 and CDK4 activities. These effects of IL-1 were reversed by PD98059. PD98059 also reversed the IL-1-induced hypophosphorylation of RB protein (pRB) and down-regulation of E2F activity. These findings demonstrate that ERK1/2 contribute to the IL-1-induced growth inhibition through induction of CDK inhibitors, down-regulation of CDK activity, pRB phosphorylation and E2F activity.