Extracellular Regulated Kinase Pathway Research Articles

Isolation and Characterization of Intrinsically Active (MEK-Independent) Mutants of Mpk1/Erk.

The extracellular-regulated kinase (Erk) pathway is a major determinant in the control of diverse cellular processes, such as proliferation, differentiation, survival, and motility. The pathway executes its effects through kinases of the Erk family. Erks are not only critical for a variety of physiological processes, but are also associated with neurodegenerative diseases, cardiovascular diseases, diabetes and a large number of human cancers. However, the exact role of each Erk molecule in these biological and pathological processes is not fully determined. An efficient strategy for revealing these roles is to activate each Erk isoform individually, in a signal independent manner, and to monitor the molecular, physiological, and pathological effects. This could be achieved by developing intrinsically active variants for each Erk isoform and splicing variant and expressing these molecules individually in biological systems. A screening method that selects for relevant and useful active mutants of Erks is described in this chapter. The main principle of the method is to screen for mutants of Erk that function in the total absence of their relevant MEKs. Another principle is that the screen should be unbiased toward particular domains or mechanisms of action. We describe how these principles are combined into a screen that takes advantage of the yeast Mpk1/Erk pathway. Following the description of how intrinsically active Mpk1 molecules are isolated, we provide comprehensive and detailed descriptions of the methods used to characterize their catalytic activity, autophosphorylation capabilities, and phosphorylation status, as well as the methods used to determine the precise phosphorylated sites. The principles of the screen and the methods described here could be easily adapted for any Erk molecule in any organism.

Selumetinib, an Oral Anti-Neoplastic Drug, May Attenuate Cardiac Hypertrophy via Targeting the ERK Pathway.

AimsAlthough extracellular-regulated kinases (ERK) are a well-known central mediator in cardiac hypertrophy, no clinically available ERK antagonist has been tested for preventing cardiac hypertrophy. Selumetinib is a novel oral MEK inhibitor that is currently under Phase II and Phase III clinical investigation for advanced solid tumors. In this study, we investigated whether Selumetinib could inhibit the aberrant ERK activation of the heart in response to stress as well as prevent cardiac hypertrophy.Methods and ResultsIn an in vitro model of PE-induced cardiac hypertrophy, Selumetinib significantly inhibited the ERK activation and prevented enlargement of cardiomyocytes or reactivation of certain fetal genes. In the pathologic cardiac hypertrophy model of ascending aortic constriction, Selumetinib provided significant ERK inhibition in the stressed heart but not in the other organs. This selective ERK inhibition prevented left ventricular (LV) wall thickening, LV mass increase, fetal gene reactivation and cardiac fibrosis. In another distinct physiologic cardiac hypertrophy model of a swimming rat, Selumetinib provided a similar anti-hypertrophy effect, except that no significant fetal gene reactivation or cardiac fibrosis was observed.ConclusionsSelumetinib, a novel oral anti-cancer drug with good safety records in a number of Phase II clinical trials, can inhibit ERK activity in the heart and prevent cardiac hypertrophy. These promising results indicate that Selumetinib could potentially be used to treat cardiac hypertrophy. However, this hypothesis needs to be validated in human clinical trials.

Formyl Peptide Receptor 2 Plays a Deleterious Role During Influenza A Virus Infections.

The pathogenesis of influenza A virus (IAV) infections is a multifactorial process that includes the replication capacity of the virus and a harmful inflammatory response to infection. Formyl peptide receptor 2 (FPR2) emerges as a central receptor in inflammatory processes controlling resolution of acute inflammation. Its role in virus pathogenesis has not been investigated yet. We used pharmacologic approaches to investigate the role of FPR2 during IAV infection in vitro and in vivo. In vitro, FPR2 expressed on A549 cells was activated by IAV, which harbors its ligand, annexin A1, in its envelope. FPR2 activation by IAV promoted viral replication through an extracellular-regulated kinase (ERK)-dependent pathway. In vivo, activating FPR2 by administering the agonist WKYMVm-NH2 decreased survival and increased viral replication and inflammation after IAV infection. This effect was abolished by treating the mice with U0126, a specific ERK pathway inhibitor, showing that, in vivo, the deleterious role of FPR2 also occurs through an ERK-dependent pathway. In contrast, administration of the FPR2 antagonist WRW4 protected mice from lethal IAV infections. These data show that viral replication and IAV pathogenesis depend on FPR2 signaling and suggest that FPR2 may be a promising novel strategy to treat influenza.

Resveratrol ameliorates myocardial fibrosis by inhibiting ROS/ERK/TGF-β/periostin pathway in STZ-induced diabetic mice.

BackgroundMyocardial fibrosis is an essential hallmark of diabetic cardiomyopathy (DCM) contributing to cardiac dysfunctions. Resveratrol, an antioxidant, exerts its anti-fibrotic effect via inhibition of oxidative stress, while the underlying molecular mechanism remains largely elusive. Periostin, a fibrogenesis matricellular protein, has been shown to be associated with oxidative stress. In the present study, we investigated the role of periostin in anti-fibrotic effect of resveratrol in streptozocin (STZ)-induced diabetic heart and the underlying mechanisms.MethodsDiabetic mice were induced by STZ injection. After treatment with resveratrol (5 or 25 mg/kg/day i.g) or Saline containing 0.5 % carboxymethyl cellulose (CMC) for 2 months, the hearts were detected for oxidative stress and cardiac fibrosis using western blot, Masson’s trichrome staining and Dihydroethidium (DHE) staining. In in vitro experiments, proliferation and differentiation of fibroblasts under different conditions were investigated through western blot, 3-(4,5)-dimethylthiahiazo (−z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and immunofluorescence staining.ResultsAdministration of resveratrol significantly mitigated oxidative level, interstitial fibrosis and expressions of related proteins in STZ-induced diabetic hearts. In in vitro experiments, resveratrol exhibited anti-proliferative effect on primary mouse cardiac fibroblasts via inhibiting reactive oxygen species (ROS)/extracellular regulated kinase (ERK) pathway and ameliorated myofibroblast differentiation via suppressing ROS/ERK/ transforming growth factor β (TGF-β)/periostin pathway.ConclusionIncreased ROS production, activation of ERK/TGF-β/periostin pathway and myocardial fibrosis are important events in DCM. Alleviated ROS genesis by resveratrol prevents myocardial fibrosis by regulating periostin related signaling pathway. Thus, inhibition of ROS/periostin may represent a novel approach for resveratrol to reverse fibrosis in DCM.

Does mouse embryo primordial germ cell activation start before implantation as suggested by single-cell transcriptomics dynamics?

Does primordial germ cell (PGC) activation start before mouse embryo implantation, and does the possible regulation of the DNA (cytosine-5-)-methyltransferase 3-like (Dnmt3l) by transcription factor AP-2, gamma (TCFAP2C) have a role in this activation and in the primitive endoderm (PE)-epiblast (EPI) lineage specification? A burst of expression of PGC markers, such as Dppa3/Stella, Ifitm2/Fragilis, Fkbp6 and Prdm4, is observed from embryonic day (E) 3.25, and some of them, together with the late germ cell markers Zp3, Mcf2 and Morc1, become restricted to the EPI subpopulation at E4.5, while the dynamics analysis of the PE-EPI transitions in the single-cell data suggests that TCFAP2C transitorily represses Dnmt3l in EPI cells at E3.5 and such repression is withdrawn with reactivation of Dnmt3l expression in PE and EPI cells at E4.5. In the mouse preimplantation embryo, cells with the same phenotype take different fates based on the orchestration between topological clues (cell polarity, positional history and division orientation) and gene regulatory rules (at transcriptomics and epigenomics level), prompting the proposal of positional, stochastic and combined models explaining the specification mechanism. PGC specification starts at E6.0-6.5 post-implantation. In view of the important role of DNA methylation in developmental events, the cross-talk between some transcription factors and DNA methyltransferases is of particular relevance. TCFAP2C has a CpG DNA methylation motif that is not methylated in pluripotent cells and that could potentially bind on DNMT3L, the stimulatory DNA methyltransferase co-factor that assists in the process of de novo DNA methylation. Chromatin-immunoprecipitation analysis has demonstrated that Dnmt3l is indeed a target of TCFAP2C. We aimed to assess the timing of early preimplantation events and to understand better the segregation of the inner cell mass (ICM) into PE and EPI. We designed a single-cell transcriptomics dynamics computational study to identify markers of the PE-EPI bifurcation in ICM cells through searching for statistically significant (using the Student's t-test method) differently expressed genes (DEGs) between PE and EPI cells from E3.5 to E4.5. The DEGs common for E3.5 and E4.5 were used as the markers defining the steady states. We collected microarray and next-generation sequencing transcriptomics data from public databases from bulk populations and single cells from mice at E3.25, E3.5 and E4.5. The results are based on three independent single-cell transcriptomics data sets, with a fold change of 3 and P-value <0.01 for the DEG selection. The dynamics analysis revealed new transitory E3.5 and steady PE and EPI markers. Among the transitory E3.5 PE markers (Dnmt3l, Dusp4, Cpne8, Akap13, Dcaf12l1, Aaed1, B4galt6, BC100530, Rnpc3, Tfpi, Lgalsl, Ckap4 and Fbxl20), several (Dusp4, Akap13, Cpn8, Dcaf12l1 and Tfpi) are related to the extracellular regulated kinase pathway. We also identified new transitory E3.5 EPI markers (Sgk1, Mal, Ubxn2a, Atg16l2, Gm13102, Tcfap2c, Hexb, Slc1a1, Svip, Liph and Mier3), six new stable PE markers (Sdc4, Cpn1, Dkk1, Havcr1, F2r/Par1 and Slc7a6os) as well as three new stable EPI markers (Zp3, Mcf2 and Hexb), which are known to be late stage germ cell markers. We found that mouse PGC marker activation starts at least at E3.25 preimplantation. The transcriptomics dynamics analyses support the regulation of Dnmt3l expression by TCFAP2C. Since the regulation of Dnmt3l by TCFAP2C is based on computational prediction of DNA methylation motifs, Chip-Seq and transcriptomics data, functional studies are required to validate this result. We identified a collection of previously undescribed E3.5-specific PE and EPI markers, and new steady PE and EPI markers. Identification of these genes, many of which encode cell membrane proteins, will facilitate the isolation and characterization of early PE and EPI populations. Since it is so well established in the literature that mouse PGC specification is a post-implantation event, it was surprising for us to see activation of PGC markers as early as E3.25 preimplantation, and identify the newly found steady EPI markers as late germ cell markers. The discovery of such early activation of PGC markers has important implications in the derivation of germ cells from pluripotent cells (embryonic stem cells or induced pluripotent stem cells), since the initial stages of such derivation resemble early development. The early activation of PGC markers points out the difficulty of separating PGC cells from pluripotent populations. Collectively, our results suggest that the combining of the precision of single-cell omics data with dynamic analysis of time-series data can establish the timing of some developmental stages as earlier than previously thought. Not applicable. This work was supported by grants DFG15/14 and DFG15/020 from Diputación Foral de Gipuzkoa (Spain), and grant II14/00016 from I + D + I National Plan 2013-2016 (Spain) and FEDER funds. The authors declare no conflict of interest.

Activation of extracellular regulated kinase and mechanistic target of rapamycin pathway in focal cortical dysplasia

Neuropathology of resected brain tissue has revealed an association of focal cortical dysplasia (FCD) with drug-resistant epilepsy (DRE). Recent studies have shown that the mechanistic target of rapamycin (mTOR) pathway is hyperactivated in FCD as evidenced by increased phosphorylation of the ribosomal protein S6 (S6) at serine 240/244 (S(240/244) ), a downstream target of mTOR. Moreover, extracellular regulated kinase (ERK) has been shown to phosphorylate S6 at serine 235/236 (S(235/236) ) and tuberous sclerosis complex 2 (TSC2) at serine 664 (S(664) ) leading to hyperactive mTOR signaling. We evaluated ERK phosphorylation of S6 and TSC2 in two types of FCD (FCD I and FCD II) as a candidate mechanism contributing to mTOR pathway dysregulation. Tissue samples from patients with tuberous sclerosis (TS) served as a positive control. Immunostaining for phospho-S6 (pS6(240/244) and pS6(235/236) ), phospho-ERK (pERK), and phospho-TSC2 (pTSC2) was performed on resected brain tissue with FCD and TS. We found increased pS6(240/244) and pS6(235/236) staining in FCD I, FCD II and TS compared to normal-appearing tissue, while pERK and pTSC2 staining was increased only in FCD IIb and TS tissue. Our results suggest that both the ERK and mTOR pathways are dysregulated in FCD and TS; however, the signaling alterations are different for FCD I as compared to FCD II and TS.

Signalling Events and Associated Pathways Related to the Mammalian Sperm Capacitation.

Capacitation is a biological phenomenon occurring prior to fertilization and is a multiple event process. Many physiological and biochemical changes takes place during the process; these changes are related to lipid composition of membrane, intracellular modulation of ion concentration, protein phosphorylation, sperm movement and membrane permeability. These events occur when the sperm is exposed to the new environment of ion concentration in the female reproductive tract. Ions such as bicarbonate and calcium facilitate capacitation by activating adenylyl cyclase, thus initiating protein kinase A (PKA) signalling cascade. Extracellular-regulated kinase pathway is activated by ligand binding to the membrane receptors and intracellular activation by reactive oxygen species (ROS). Activation of these pathways leads to the phosphorylation of different proteins, which is associated with events such as capacitation, hyperactivation and acrosome reaction that are essential for successful fertilization. Extensive studies were carried out on protein phosphorylation in relation to capacitation, but its role still remains ambiguous.

Cavin-2 in oral cancer: A potential predictor for tumor progression.

Cavin-2 (CVN2) affects formation of large caveolae, which are membrane-rich cholesterol domains associated with several functions in signal transduction. Accumulating evidence suggests that CVN2 is present in many cellular types; however, the molecular mechanisms of CVN2 in cancers and its clinical relevance are unknown. We proposed a mechanism by which CVN2 regulates caveolin-1 expression leading to slow cellular proliferation by inactivation of the extracellular regulated kinase (ERK) pathway. Quantitative reverse transcriptase-polymerase chain reaction and immunoblot analyses were used to assess the CVN2 regulation mechanism in oral squamous cell carcinoma (OSCC). Immunohistochemistry (IHC) was performed to analyze the correlation between CVN2 expression and clinical behavior in 115 patients with OSCC. A CVN2 overexpressed model of OSCC cells (oeCVN2 cells) was used for functional experiments. CVN2 expression was down-regulated significantly (P < 0.05) in OSCCs compared with normal counterparts in vitro and in vivo. In addition to the findings that a serum deprivation culture induced up-regulation of CVN2 and slowed cellular proliferation, oeCVN2 cell growth decreased because of cell-cycle arrest at the G1 phase resulting from up-regulated cyclin-dependent kinase inhibitors (p21(Cip1) and p27(Kip1) ) and down-regulated cyclins (cyclin D1, cyclin E) and cyclin-dependent kinases (CDK2, CDK4, and CDK6). Interestingly, CVN2 overexpression facilitated caveolin-1 recruitment and colocalization with each other. We also found decreased ERK phosphorylation levels, an upstream event in cell-cycle arrest. Clinically, IHC data from primary OSCCs showed high tumoral progression in CVN2-negative patients with OSCC. CVN2 may be a possible key regulator of OSCC progression via the CVN2/caveolin-1/ERK pathway and a potential therapeutic target for developing new treatments for OSCCs. © 2015 Wiley Periodicals, Inc.

Co-operation between the AKT and ERK signaling pathways may support growth of deep endometriosis in a fibrotic microenvironment in vitro.

How can deep endometriotic stromal cells proliferate and persist in a fibrotic environment? The serine/threonine kinase AKT and extracellular regulated kinase (ERK) signaling pathways may co-operate to support growth of deep endometriotic lesions by enhancing endometriotic stromal cell proliferation and survival in a fibrotic microenvironment in vitro. Endometriosis, particularly deep infiltrating endometriosis, is characterized histologically by dense fibrous tissue that is primarily composed of type I collagen. This tissue may cause pelvic pain and infertility, which are major clinical issues associated with endometriosis. Proliferation of normal fibroblasts is tightly regulated, and fibrillar, polymerized type I collagen inhibits normal fibroblast proliferation. However, no studies to date have investigated how deep endometriotic stromal cells can proliferate and persist in a fibrotic environment. Endometrial and/or endometriotic tissues from 104 patients (61 with and 43 without endometriosis) of reproductive age with normal menstrual cycles were analyzed. A total of 25 nude mice received a single injection of endometrial fragments from a total of five samples. We evaluated cell proliferation, caspase 3/7 activity, and the AKT and ERK signaling pathways in endometrial and endometriotic stromal cells on three-dimensional (3D) polymerized collagen matrices in vitro. In addition, to determine whether aberrant activation of the AKT and ERK pathways is involved during progression of fibrosis in endometriosis in vivo, we evaluated the expression of phosphorylated AKT and ERK1/2 in endometriotic implants in a nude mouse model of endometriosis. Finally, we evaluated the effects of MK2206 (an AKT inhibitor) and U0126 (a MEK inhibitor) on cell proliferation, caspase 3/7 activity, and phosphorylation of AKT and ERK1/2 of endometriotic stromal cells on 3D polymerized collagen matrices. Proliferation of endometriotic stromal cells was significantly less inhibited than that of endometrial stromal cells (P < 0.05) on 3D polymerized collagen. Levels of phosphorylated AKT, phosphorylated p70S6K and phosphorylated ERK1/2 were significantly higher in endometriotic stromal cells than in endometrial stromal cells at 24 h (P < 0.05) and at 72 h (P < 0.05) on 3D polymerized collagen. Phosphorylated AKT expression was significantly increased on Days 21 and 28 compared with those on Days 3 and 7 (all P < 0.05) in endometriotic implants during progression of fibrosis in a nude mouse model of endometriosis. Inhibition of AKT or ERK1/2 with MK2206 or U0126, respectively, did not significantly increase caspase 3/7 activity in endometriotic stromal cells on either two-dimensional or 3D collagen matrices. Western blot analysis showed that MK2206 alone decreased levels of phosphorylated AKT; however, it increased levels of phosphorylated ERK in endometriotic cells compared with vehicle-treated cells (both P < 0.05). In addition, U0126 treatment decreased levels of phosphorylated ERK; however, it resulted in increased levels of phosphorylated AKT in endometriotic stromal cells compared with vehicle-treated cells (both P < 0.05). Endometriosis involves a number of processes, such as invasion, metastasis, angiogenesis, and apoptosis resistance, and a variety of signaling pathways may be involved in promoting development and progression of the disease. In addition, further animal experiments are required to determine whether the AKT and ERK signaling pathways co-operate to support growth of endometriotic lesions in a fibrotic microenvironment in vivo. Co-targeting the AKT and ERK pathways may be an effective therapeutic strategy for endometriosis treatment. This study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

Propyl gallate inhibits adipogenesis by stimulating extracellular signal-related kinases in human adipose tissue-derived mesenchymal stem cells.

Propyl gallate (PG) used as an additive in various foods has antioxidant and anti-inflammatory effects. Although the functional roles of PG in various cell types are well characterized, it is unknown whether PG has effect on stem cell differentiation. In this study, we demonstrated that PG could inhibit adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells (hAMSCs) by decreasing the accumulation of intracellular lipid droplets. In addition, PG significantly reduced the expression of adipocyte-specific markers including peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein 2 (aP2). PG inhibited adipogenesis in hAMSCs through extracellular regulated kinase (ERK) pathway. Decreased adipogenesis following PG treatment was recovered in response to ERK blocking. Taken together, these results suggest a novel effect of PG on adipocyte differentiation in hAMSCs, supporting a negative role of ERK1/2 pathway in adipogenic differentiation.

Antiangiogenic Activity ofAcer tegmentosumMaxim Water Extract in Vitro and in Vivo

Angiogenesis, the formation of new blood vessels, is critical for tumor growth and metastasis. Notably, tumors themselves can lead to angiogenesis by inducing vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors. Inhibition of angiogenesis is currently perceived as one of the most promising strategies for the blockage of tumor growth. In this study, we investigated the effects of Acer tegmentosum maxim water extract (ATME) on angiogenesis and its underlying signal mechanism. We studied the antiangiogenic activity of ATME by using human umbilical vein endothelial cells (HUVECs). ATME strongly inhibited VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation, as well as vessel sprouting in a rat aortic ring sprouting assay. Moreover, we found that the p44/42 mitogen activated protein (MAP) kinase signaling pathway is involved in the inhibition of angiogenesis by ATME. Moreover, when we performed the in vivo matrigel plug assay, VEGF-induced angiogenesis was potently reduced when compared to that for the control group. Taken together, these results suggest that ATME exhibits potent antiangiogenic activity in vivo and in vitro and that these effects are regulated by the extracellular regulated kinase (ERK) pathway.

Effect of lavage with artificial cerebrospinal fluid on neural cell apoptosis and the ERK pathway after rat traumatic brain injury

To explore the eff ect of lavage with artificial cerebrospinal fluid on neural cell apoptosis and the extracellular regulated kinase (ERK) pathway aft er traumatic brain injury. A total of 192 SD rats were randomly divided into a sham group, a traumatic brain injury model group, a local artificial cerebrospinal fluid group, and a local saline group. Each group was divided into 4 sub-groups by the sacrificed time at 6 h, 12 h, 1 d and 3 d aft er the operation. Th e phosphorylation of extracellular regulated kinase 2 (P-ERK2), TNF-α and cellular apoptosis were examined. Th e levels of P-ERK2 protein and TNF-α protein, as well as the number of apoptotic cells at each time point in the local artificial cerebrospinal fluid group were lower than those in the model group or in the saline group (P<0.05). Lavage with artificial cerebrospinal fluid can reduce apoptosis of neural cells after brain injury through the ERK pathway.

CXCR7 stimulates MAPK signaling to regulate hepatocellular carcinoma progression.

The CXCL12/CXCR4 axis has been posited widely to have significant roles in many primary tumors and metastases. It is known that CXCR7 can also be engaged by CXCL12, but the exact function of CXCR7 is controversial. This prompted us to investigate the expression, specific function and signal transduction of CXCR7 in hepatocellular carcinoma (HCC). In this study, CXCR7 and CXCR4 were differentially expressed in nine cell lines of HCC, and that elevated expression of both CXCR7 and CXCL4 were correlated with highly metastatic ability of HCC cells. Moreover, CXCR7 expression was significantly upregulated in metastatic HCC samples compared with the non-metastatic ones by staining of high-density tissue microarrays constructed from a cohort of 48 human HCC specimens. CXCR7 overexpression enhanced cell growth and invasiveness in vitro, and tumorigenicity and lung metastasis in vivo. By contrast, CXCR7 stable knockdown markedly reduced these malignant behaviors. In addition, it was observed that alterations in CXCR7 expression were positively correlated with the phosphorylation levels of mitogen-activated protein kinase (MAPK) pathway proteins. Targeting extracellular regulated kinase pathway by using U0126 inhibitor or using CCX771, a selective CXCR7 antagonist, drastically reduced CXCR7-mediated cell proliferation. Importantly, by using human biotin-based antibody arrays, several differentially expressed proteins were identified in CXCR7-overexpression and depletion groups. Comparative analysis indicated that upstream regulators including TP53 and IL-6 were involved in CXCR7 signal transduction. CXCR7 expression was further proved to regulate expression of vascular endothelial growth factor A and galectin-3, which may contribute to tumor angiogenesis and invasiveness. Consequently, elevated expression of CXCR7 contributes to HCC growth and invasiveness via activation of MAPK and angiogenesis signaling pathways. Targeting CXCR7 may prevent metastasis and provide a potential therapeutic strategy for HCC.

Efficacy of Patritumab (U3-1287), a Fully Human Anti–Human Epidermal Growth Factor Receptor 3 (Her3) Monoclonal Antibody (Mab), in Head and Neck (H&amp;N) Cancer Models

ABSTRACT Aim: Activation of the receptor tyrosine kinase HER3 and its downstream signaling pathways promote the development of various cancers. HER3 facilitates phosphatidylinositol-3-kinase/AKT signaling via heterodimeric interaction with other HER family members, mediated by binding of the HER3 ligand heregulin (HRG). Patritumab (P) competes with HRG for receptor binding and mediates internalization of HER3. We examined expression of HRG in H&N cancer models and patient samples and evaluated the in vitro and in vivo efficacy of P alone or with cetuximab (C). Methods: Selection of H&N cell lines for reverse transcription polymerase chain reaction was based on microarray data. Tumor cells were treated with 10 µg/mL of P, C, or a combination of both, and cell lysates were analyzed for HER3/pHER3-, AKT/pAKT- and extracellular regulated kinase (ERK)/pERK-levels by Western blotting. The effect of P and C on proliferation and apoptosis was evaluated by bromodeoxyuridine- and caspase-3/7 assays. To address efficacy in vivo, mice bearing tumor xenografts were treated with P and/or C twice weekly, and tumor volumes were assessed. HRG expression was also evaluated in patient samples. Results: HRG expression was frequent in H&N cell lines. HRG mRNA expression was elevated in 67% of patient tumor samples compared with controls. In H&N cell lines, basal HER3 and AKT activation were inhibited by P, with additional blockage of the ERK pathway by co-treatment with C. In vitro combination effects were also observed on the inhibition of proliferation and caspase-3/7 activation. Co-administration of P + C enhanced tumor growth inhibition (TGI) in vivo. Conclusions: High expression frequencies of HRG were observed in H&N tumor cell lines and patient samples, and were associated with single-agent efficacy of P on cell signaling in vitro and on TGI in vivo. The combination of P + C extended the inhibition of signaling pathways, induced apoptosis and inhibited proliferation in vitro, and enforced TGI in vivo. Thus, these data strongly support the combination of P with an anti-epidermal growth factor receptor mAb as a promising strategy for the treatment of H&N cancer. Disclosure: All authors have declared no conflicts of interest.

Insulin promotes proliferation and migration of breast cancer cells through the extracellular regulated kinase pathway.

The present study was undertaken to determine the roles of insulin in the growth of transplanted breast cancer in nude mice, and the proliferation and migration of MCF-7 human breast cancer cells and assess its influence on downstream signaling pathways. In a xenograft mouse model with injection of MCF-7 human breast cancer cells, tumor size was measured every other day. The insulin level and insulin receptor (IR) were increased in the breast cancer patient tissues. Insulin injected subcutaneously around the tumor site in mice caused increase in the size and weight of tumor masses, and promoted proliferation and migration of MCF-7 cells. The effects of insulin on the increase in the proliferation and migration of MCF-7 human breast cancer cells were abolished by pretreatment with the extracellular regulated kinase (ERK) inhibitor PD98059. Insulin increased the phosphorylation of ERK in the MCF-7 cells. These results indicate that insulin promotes the growth of breast cancer in nude mice, and increases the proliferation and migration of MCF-7 human breast cancer cells via the ERK pathway.

Transcription factor Ets1 regulates expression of thioredoxin-interacting protein and inhibits insulin secretion in pancreatic β-cells.

Long-term activation of extracellular-regulated kinase (ERK1/2) pathway has been shown to cause glucotoxicity and inhibit insulin gene expression in β-cells. Transcription factor Ets1 is activated by ERK1/2-mediated phosphorylation at the Thr38 residue. We hypothesize that Ets1 plays an important role in mediating ERK1/2 induced glucotoxicity in β-cells. We determined the role of Ets1 in Min6 cells and isolated mouse islets using overexpression and siRNA mediated knockdown of Ets1. The results show that Ets1 was localized in insulin-staining positive cells but not in glucagon-staining positive cells. Overexpression of Ets1 reduced glucose-stimulated insulin secretion in primary mouse islets. Overexpression of Ets1 in Min6 β-cells and mouse islets increased expression of thioredoxin-interacting protein (TXNIP). Conversely, knockdown of Ets1 by siRNA reduced expression of TXNIP in Min6 cells. Ets1 was associated with the txnip promoter in min6 cells and transfection of 293 cells with Ets1 and p300 synergistically increased txnip promoter reporter activity. Moreover, overexpression of Ets1 inhibited Min6 cell proliferation. Our results suggest that Ets1, by promoting TXNIP expression, negatively regulates β-cell function. Thus, over-activation of Ets1 may contribute to diet-induced β-cell dysfunction.

Role of extracellular regulated kinase pathway in the lipopolysaccharide-induced acute lung injury in rat

bjective To investigate the role of extracellular signal-regulated kinases（ERK） signal pathway in the lipopolysaccharide（LPS）-induced acute lung injury（ALI） in rats. Methods One hundred and twenty male SD rats were randomly divided into four groups（n=30）： Saline group, LPS group, LSP＋U group and Saline＋U group. Each group was divided into five subgroups at 1, 3, 6, 12 h and 24 h time point respectively. Western blot was used to detect the expression of phosphorylation of ERK1/2 and high-mobility group box 1（HMGB-1） in lung tissues, nuclear factor-κB（NF-κB）p65 in the nuclear extracts, which reflected the extent of lung injury. Additionally we examined the concentration of tumor necrosis factor（TNF）-α, interleukin（IL）-10 and inducible nitric oxide synthase（iNOS） in bronchoalveolar lavage fluid（BALF）, the lung water content and the histopathologic changes of lung. In addition, survival rate was investigated between LPS group and LSP＋U group,and each group with other 10 rats. Results With the administration of LPS, the phospho？蛳ERK1/2 substantially increased immediately and subsequently the expression of NF？蛳κB in the nuclear extracts was increased significantly and reached its peak level at 3 h and 12 h. Moreover, HMGB-1, one of the key mediators in the development of sepsis increased significantly after LPS administration. The concentrations of TNF？蛳α,iNOS and IL-10 were also increased. Pathological examination showed that the normal structure of lung was destroyed badly after LPS injection. U0126 effectively inhibited the activation of ERK1/2, blocked LPS-induced NF-κB activation and HMGB-1 expression in lung tissue, reduced the lung water content[（4.59±0.51） vs （5.19±0.10）, P〈0.05], the concentration of TNF-α, iNOS and IL-10[TNF-α, reached a peak at 3 h,（28±3） vs （70±10）（P〈0.05）. iNOS, reached a peak at 12 h, （7 771±957） vs （10 679±1 641）,P〈0.05. IL-10,reached a peak at 6 h,（59±10） vs（91±11）, P〈0.05], and prevented LPS-induced lung injury. Conclusions ERK1/2 plays an important role in the development of LPS-induced ALI, and the activation of ERK1/2 may be one of the mechanisms of LPS-induced ALI. Key words: Extracellular signal-regulated kinases; Nuclear factor-κB; Acute lung injury; Tumor necrosis factor-α; Interleukin-10; Inducible nitric oxide synthase; Lipopolysaccharide

Targeting of Receptor for Advanced Glycation End Products Suppresses Cyst Growth in Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Although a myriad of research groups have attempted to identify a new therapeutic target for ADPKD, no drug has worked well in clinical trials. Our research group has focused on the receptor for advanced glycation end products (RAGE) gene as a novel target for ADPKD. This gene is involved in inflammation and cell proliferation. We have already confirmed that blocking RAGE function attenuates cyst growth in vitro. Based on this previous investigation, our group examined the effect of RAGE on cyst enlargement in vivo. PC2R mice, a severe ADPKD mouse model that we generated, were utilized. An adenovirus containing anti-RAGE shRNA was injected intravenously into this model. We observed that RAGE gene knockdown resulted in loss of kidney weight and volume. Additionally, the cystic area that originated from different nephron segments decreased in size because of down-regulation of the RAGE gene. Blood urea nitrogen and creatinine values tended to be lower after inhibiting RAGE. Based on these results, we confirmed that the RAGE gene could be an effective target for ADPKD treatment.

Molecular mechanisms of protease Nexin-1 on regulating tumorgenesis

Protease uexin-1 (PN-1) is one of the most important members of Serpin superfamily.The abnormal expressions are observed in many human cancers,such as pancreatic cancer,breast cancer,colon cancer,testicular cancer and so on.PN1 participates in tumor cell proliferation,differentiation,apoptosis,angiogenesis and invasion by inhibiting multiple proteases and extracellular signal regulated kinase (ERK) pathways and promotes tumor occurrence,development and metastasis.In prostate cancer particularly,PN-1 inhibits tumor occurrence,development and metastasis by inhibiting urokinase plasminogen activator (uPA),Hh signaling pathway and interacting with matrix metalloproteinase (MMP)-9. Key words: Apoptosis; Neoplasm metastasis; Neoplasm invasiveness; Protease nexin-1

Microbial phenolic metabolites improve glucose-stimulated insulin secretion and protect pancreatic beta cells against tert-butyl hydroperoxide-induced toxicity via ERKs and PKC pathways

Oxidative stress is accepted as one of the causes of beta cell failure in type 2 diabetes. Therefore, identification of natural antioxidant agents that preserve beta cell mass and function is considered an interesting strategy to prevent or treat diabetes. Recent evidences indicated that colonic metabolites derived from flavonoids could possess beneficial effects on various tissues. The aim of this work was to establish the potential anti-diabetic properties of the microbial-derived flavonoid metabolites 3,4-dihydroxyphenylacetic acid (DHPAA), 2,3-dihydroxybenzoic acid (DHBA) and 3-hydroxyphenylpropionic acid (HPPA). To this end, we tested their ability to influence beta cell function and to protect against tert-butyl hydroperoxide-induced beta cell toxicity. DHPAA and HPPA were able to potentiate glucose-stimulated insulin secretion (GSIS) in a beta cell line INS-1E and in rat pancreatic islets. Moreover, pre-treatment of cells with both compounds protected against beta cell dysfunction and death induced by the pro-oxidant. Finally, experiments with pharmacological inhibitors indicate that these effects were mediated by the activation of protein kinase C and the extracellular regulated kinases pathways. Altogether, these findings strongly suggest that the microbial-derived flavonoid metabolites DHPAA and HPPA may have anti-diabetic potential by promoting survival and function of pancreatic beta cells.