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Sample records for active erbb2 receptors

  1. Inhibition of ErbB-2 Mitogenic and Transforming Activity by RALT, a Mitogen-Induced Signal Transducer Which Binds to the ErbB-2 Kinase Domain†

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    Fiorentino, Loredana; Pertica, Chiara; Fiorini, Monia; Talora, Claudio; Crescenzi, Marco; Castellani, Loriana; Alemà, Stefano; Benedetti, Piero; Segatto, Oreste

    2000-01-01

    The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2–gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer). PMID:11003669

  2. Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain.

    Science.gov (United States)

    Fiorentino, L; Pertica, C; Fiorini, M; Talora, C; Crescenzi, M; Castellani, L; Alemà, S; Benedetti, P; Segatto, O

    2000-10-01

    The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2-gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer).

  3. Photo-activated psoralen binds the ErbB2 catalytic kinase domain, blocking ErbB2 signaling and triggering tumor cell apoptosis.

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    Wenle Xia

    Full Text Available Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85(ErbB2 that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85(ErbB2. Here we show that PUVA reduced p85(ErbB2 phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.

  4. LINGO-1 regulates oligodendrocyte differentiation by inhibiting ErbB2 translocation and activation in lipid rafts.

    Science.gov (United States)

    Lee, Xinhua; Shao, Zhaohui; Sheng, Guoqing; Pepinsky, Blake; Mi, Sha

    2014-05-01

    Oligodendrocyte differentiation is negatively regulated by LINGO-1 and positively regulated by the ErbB2 receptor tyrosine kinase. In wild-type oligodendrocytes, inhibition of ErbB2 blocks differentiation, whereas activation of ErbB2 promotes differentiation. In LINGO-1(-/-) oligodendrocytes, inhibition of ErbB2 blocks oligodendrocyte differentiation; whereas activation of ErbB2 does not enhance differentiation. Biological and biochemical evidence showing that LINGO-1 can directly bind to ErbB2, block ErbB2 translocation into lipid rafts, and inhibit its phosphorylation for activation. The study demonstrates a novel regulatory mechanism of ErbB2 function whereby LINGO-1 suppresses oligodendrocyte differentiation by inhibiting ErbB2 translocation and activation in lipid rafts.

  5. Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models

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    Toscani, Andrés Martín; Sampayo, Rocío G.; Barabas, Federico Martín; Fuentes, Federico; Simian, Marina

    2017-01-01

    ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells. PMID:28306722

  6. Combined inhibition of ErbB1/2 and Notch receptors effectively targets breast ductal carcinoma in situ (DCIS stem/progenitor cell activity regardless of ErbB2 status.

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    Gillian Farnie

    Full Text Available Pathways involved in DCIS stem and progenitor signalling are poorly understood yet are critical to understand DCIS biology and to develop new therapies. Notch and ErbB1/2 receptor signalling cross talk has been demonstrated in invasive breast cancer, but their role in DCIS stem and progenitor cells has not been investigated. We have utilised 2 DCIS cell lines, MCF10DCIS.com (ErbB2-normal and SUM225 (ErbB2-overexpressing and 7 human primary DCIS samples were cultured in 3D matrigel and as mammospheres in the presence, absence or combination of the Notch inhibitor, DAPT, and ErbB1/2 inhibitors, lapatinib or gefitinib. Western blotting was applied to assess downstream signalling. In this study we demonstrate that DAPT reduced acini size and mammosphere formation in MCF10DCIS.com whereas there was no effect in SUM225. Lapatinb reduced acini size and mammosphere formation in SUM225, whereas mammosphere formation and Notch1 activity were increased in MCF10DCIS.com. Combined DAPT/lapatinib treatment was more effective at reducing acini size in both DCIS cell lines. Mammosphere formation in cell lines and human primary DCIS was reduced further by DAPT/lapatinib or DAPT/gefitinib regardless of ErbB2 receptor status. Our pre-clinical human models of DCIS demonstrate that Notch and ErbB1/2 both play a role in DCIS acini growth and stem cell activity. We report for the first time that cross talk between the two pathways in DCIS occurs regardless of ErbB2 receptor status and inhibition of Notch and ErbB1/2 was more efficacious than either alone. These data provide further understanding of DCIS biology and suggest treatment strategies combining Notch and ErbB1/2 inhibitors should be investigated regardless of ErbB2 receptor status.

  7. Combined inhibition of ErbB1/2 and Notch receptors effectively targets breast ductal carcinoma in situ (DCIS) stem/progenitor cell activity regardless of ErbB2 status.

    Science.gov (United States)

    Farnie, Gillian; Willan, Pamela M; Clarke, Robert B; Bundred, Nigel J

    2013-01-01

    Pathways involved in DCIS stem and progenitor signalling are poorly understood yet are critical to understand DCIS biology and to develop new therapies. Notch and ErbB1/2 receptor signalling cross talk has been demonstrated in invasive breast cancer, but their role in DCIS stem and progenitor cells has not been investigated. We have utilised 2 DCIS cell lines, MCF10DCIS.com (ErbB2-normal) and SUM225 (ErbB2-overexpressing) and 7 human primary DCIS samples were cultured in 3D matrigel and as mammospheres in the presence, absence or combination of the Notch inhibitor, DAPT, and ErbB1/2 inhibitors, lapatinib or gefitinib. Western blotting was applied to assess downstream signalling. In this study we demonstrate that DAPT reduced acini size and mammosphere formation in MCF10DCIS.com whereas there was no effect in SUM225. Lapatinb reduced acini size and mammosphere formation in SUM225, whereas mammosphere formation and Notch1 activity were increased in MCF10DCIS.com. Combined DAPT/lapatinib treatment was more effective at reducing acini size in both DCIS cell lines. Mammosphere formation in cell lines and human primary DCIS was reduced further by DAPT/lapatinib or DAPT/gefitinib regardless of ErbB2 receptor status. Our pre-clinical human models of DCIS demonstrate that Notch and ErbB1/2 both play a role in DCIS acini growth and stem cell activity. We report for the first time that cross talk between the two pathways in DCIS occurs regardless of ErbB2 receptor status and inhibition of Notch and ErbB1/2 was more efficacious than either alone. These data provide further understanding of DCIS biology and suggest treatment strategies combining Notch and ErbB1/2 inhibitors should be investigated regardless of ErbB2 receptor status.

  8. Activating Mutations in ERBB2 and Their Impact on Diagnostics and Treatment

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    Herter-Sprie, Grit S.; Greulich, Heidi; Wong, Kwok-Kin

    2013-01-01

    Despite the ongoing “war on cancer,” cancer remains one of the major causes of human morbidity and mortality. A new paradigm of targeted therapies holds the most promise for the future, making identification of tumor-specific therapeutic targets of prime importance. ERBB2/HER2, best known for its role in breast cancer tumorigenesis, can be targeted by two types of pharmacological manipulation: antibody therapy against the extracellular receptor domain and small molecule compounds against the intracellular tyrosine kinase domain. Aberrant activation of ERBB2 by gene amplification has been shown to participate in the pathophysiology of breast, ovarian, gastric, colorectal, lung, brain, and head and neck tumors. However, the advent of next-generation sequencing technologies has enabled efficient identification of activating molecular alterations of ERBB2. In this review, we will focus on the functional role of these somatic mutations that cause ERBB2 receptor activation. We will additionally discuss the current preclinical and clinical therapeutic strategies for targeting mutationally activated ERBB2. PMID:23630663

  9. Activating mutations in ERBB2 and their impact on diagnostics and treatment

    Directory of Open Access Journals (Sweden)

    Grit Sophie Herter-Sprie

    2013-04-01

    Full Text Available Despite the ongoing ‘war on cancer’, cancer remains one of the major causes of human morbidity and mortality. A new paradigm of targeted therapies holds the most promise for the future, making identification of tumor-specific therapeutic targets of prime importance. ERBB2, best known for its role in breast cancer tumorigenesis, can be targeted by two types of pharmacological manipulation: antibody therapy against the extracellular receptor domain and small molecule compounds against the intracellular tyrosine kinase domain. Aberrant activation of ERBB2 by gene amplification has been shown to participate in the pathophysiology of breast, ovarian, gastric, colorectal, lung, brain and head and neck tumors. However, the advent of next-generation sequencing technologies has enabled efficient identification of activating molecular alterations of ERBB2. In this review, we will focus on the functional role of these somatic mutations that cause ERBB2 receptor activation. We will additionally discuss the current preclinical and clinical therapeutic strategies for targeting mutationally activated ERBB2.

  10. ERBB2 influences the subcellular localization of the estrogen receptor in tamoxifen-resistant MCF-7 cells leading to the activation of AKT and RPS6KA2.

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    Pancholi, Sunil; Lykkesfeldt, Anne E; Hilmi, Caroline; Banerjee, Susana; Leary, Alexandra; Drury, Suzanne; Johnston, Stephen; Dowsett, Mitch; Martin, Lesley-Ann

    2008-12-01

    Acquired resistance to endocrine therapies remains a major clinical obstacle in hormone-sensitive breast tumors. We used an MCF-7 breast tumor cell line (Tam(R)-1) resistant to tamoxifen to investigate this mechanism. We demonstrate that Tam(R)-1 express elevated levels of phosphorylated AKT and MAPK3/1-activated RPS6KA2 compared with the parental MCF-7 cell line (MCF-7). There was no change in the level of total ESR between the two cell lines; however, the Tam(R)-1 cells had increased phosphorylation of ESR1 ser(167). SiRNA blockade of AKT or MAPK3/1 had little effect on ESR1 ser(167) phosphorylation, but a combination of the two siRNAs abrogated this. Co-localization studies revealed an association between ERBB2 and ESR1 in the Tam(R)-1 but not MCF-7 cells. ESR1 was redistributed to extranuclear sites in Tam(R)-1 and was less transcriptionally competent compared with MCF-7 suggesting that nuclear ESR1 activity was suppressed in Tam(R)-1. Tamoxifen resistance in the Tam(R)-1 cells could be partially overcome by the ERBB2 inhibitor AG825 in combination with tamoxifen, and this was associated with re-localization of ESR1 to the nucleus. These data demonstrate that tamoxifen-resistant cells have the ability to switch between ERBB2 or ESR1 pathways promoting cell growth and that pharmacological inhibition of ERBB2 may be a therapeutic strategy for overcoming tamoxifen resistance.

  11. erbB2 is required for G protein-coupled receptor signaling in the heart

    OpenAIRE

    Negro, Alejandra; Brar, Bhawanjit K.; Gu, Yusu; Peterson, Kirk L.; Vale, Wylie; Lee, Kuo-Fen

    2006-01-01

    erbB2/Her2, a ligandless receptor kinase, has pleiotropic effects on mammalian development and human disease. The absence of erbB2 signaling in cardiac myocytes results in dilated cardiomyopathy in mice, resembling the cardiotoxic effects observed in a subset of breast cancer patients treated with the anti-Her2 antibody herceptin. Emerging evidence suggests that erbB2 is pivotal for integrating signaling networks involving multiple classes of extracellular signals. However, its role in G prot...

  12. Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells.

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    Munisvaradass, Rusheni; Kumar, Suresh; Govindasamy, Chandramohan; Alnumair, Khalid S; Mok, Pooi Ling

    2017-09-08

    Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non-transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours.

  13. Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3.

    OpenAIRE

    King, C. R.; Borrello, I; Bellot, F; Comoglio, P; Schlessinger, J

    1988-01-01

    The epidermal growth factor receptor (EGF-R) and the erbB-2 proto-oncogene product protein are closely related by their structural homology and their shared enzymatic activity as autophosphorylating tyrosine kinases. We show that in mammary tumor cells (SK-BR-3) EGF causes a rapid increase in tyrosine phosphorylation of the erbB-2 protein. Phosphorylation of erbB-2 does not occur in cells lacking the EGF-R (MDA-MB-453). Phosphorylation of erbB-2 in SK-BR-3 cells is blocked if EGF is prevented...

  14. ErbB2 resembles an autoinhibited invertebrate epidermal growth factor receptor

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    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.; (UPENN-MED)

    2009-09-25

    The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular 'tether' in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, here we show that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor.

  15. Transactivation of EGF receptor and ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis.

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    Yamaoka, Toshimitsu; Yan, Fang; Cao, Hanwei; Hobbs, Stuart S; Dise, Rebecca S; Tong, Wei; Polk, D Brent

    2008-08-19

    TNF is a pleiotropic cytokine that activates both anti- and proapoptotic signaling pathways, with cell fate determined by the balance between these two pathways. Activation of ErbB family members, including EGF receptor (EGFR/ErbB1), promotes cell survival and regulates several signals that overlap with those stimulated by TNF. This study was undertaken to determine the effects of TNF on EGFR and ErbB2 activation and intestinal epithelial cell survival. Mice, young adult mouse colon epithelial cells, and EGFR knockout mouse colon epithelial cells were treated with TNF. Activation of EGFR, ErbB2, Akt, Src, and apoptosis were determined in vivo and in vitro. TNF stimulated EGFR phosphorylation in young adult mouse colon epithelial cells, and loss of EGFR expression or inhibition of kinase activity increased TNF-induced apoptosis, which was prevented in WT but not by kinase-inactive EGFR expression. Similarly, TNF injection stimulated apoptosis in EGFR-kinase-defective mice (EGFR(wa2)) compared with WT mice. TNF also activated ErbB2, and loss of ErbB2 expression increased TNF-induced apoptosis. Furthermore, Src-kinase activity and the expression of both EGFR and ErbB2 were required for TNF-induced cell survival. Akt was shown to be a downstream target of TNF-activated EGFR and ErbB2. These findings demonstrate that EGFR and ErbB2 are critical mediators of TNF-regulated antiapoptotic signals in intestinal epithelial cells. Given evidence for TNF signaling in the development of colitis-associated carcinoma, this observation has significant implications for understanding the role of EGFR in maintaining intestinal epithelial cell homeostasis during cytokine-mediated inflammatory responses.

  16. The rat ErbB2 tyrosine kinase receptor produced in plants is immunogenic in mice and confers protective immunity against ErbB2(+) mammary cancer.

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    Matić, Slavica; Quaglino, Elena; Arata, Lucia; Riccardo, Federica; Pegoraro, Mattia; Vallino, Marta; Cavallo, Federica; Noris, Emanuela

    2016-01-01

    The rat ErbB2 (rErbB2) protein is a 185-kDa glycoprotein belonging to the epidermal growth factor-related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2-pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ-HT expression vector as 6X His tag fusions. All rErbB2 variants (72-74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2(+) mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.

  17. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

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    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( BSC cultures treated with 10 n TBA exhibit increased ( BSC cultures.

  18. HER2/ErbB2 receptor signaling in rat and human prolactinoma cells: strategy for targeted prolactinoma therapy.

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    Fukuoka, Hidenori; Cooper, Odelia; Mizutani, Jun; Tong, Yunguang; Ren, Song-Guang; Bannykh, Serguei; Melmed, Shlomo

    2011-01-01

    Dopamine agonist resistance or intolerance is encountered in approximately 20% of prolactinoma patients. Because human epidermal growth factor receptor 2 (HER2)/ErbB2 is overexpressed in prolactinomas and ErbB receptor ligands regulate prolactin (PRL) gene expression, we tested the role of HER2/ErbB2 in prolactinoma hormone regulation and adenoma cell proliferation to assess the rationale for targeting this receptor for prolactinoma therapy. As we showed prolactinoma HER2 overexpression, we generated constitutively active HER2-stable GH3 cell transfectants (HER2CA). PRL mRNA levels were induced approximately 250-fold and PRL secretion was enhanced 100-fold in HER2CA cells, which also exhibited increased proliferation. Lapatinib, a dual tyrosine kinase inhibitor (TKI) of both epidermal growth factor receptor (EGFR)/ErbB1 and HER2, blocked receptor signaling, and suppressed PRL expression more than gefitinib, a TKI of EGFR/ErbB1. Lapatinib also suppressed colony formation in soft agar more than gefitinib. Oral lapatinib treatment caused tumor shrinkage and serum PRL suppression both in HER2CA transfectant-inoculated Wistar-Furth rats and in estrogen-induced Fischer344 rat prolactinomas. In cultured human cells derived from resected prolactinoma tissue, lapatinib suppressed both PRL mRNA expression and secretion. These results demonstrate that prolactinoma HER2 potently induces PRL and regulates experimental prolactinoma cell proliferation. Because pituitary HER2 signaling is abrogated by TKIs, this receptor could be an effective target for prolactinoma therapy.

  19. A single amino acid substitution is sufficient to modify the mitogenic properties of the epidermal growth factor receptor to resemble that of gp185erbB-2

    DEFF Research Database (Denmark)

    Di Fiore, P P; Helin, K; Kraus, M H

    1992-01-01

    The epidermal growth factor (EGF) receptor (EGFR) and the erbB-2 gene product, gp185erbB-2, exhibit distinct abilities to stimulate mitogenesis in different target cells. By using chimeric molecules between these two receptors, we have previously shown that their intracellular juxtamembrane regio...

  20. Continuous requirement of ErbB2 kinase activity for loss of cell polarity and lumen formation in a novel ErbB2/Neu-driven murine cell line model of metastatic breast cancer

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    Cesar F Ortega-Cava

    2011-01-01

    Full Text Available Background: Well over a quarter of human breast cancers are ErbB2-driven and constitute a distinct subtype with substantially poorer prognosis. Yet, there are substantial gaps in our understanding of how ErbB2 tyrosine kinase activity unleashes a coordinated program of cellular and extracellular alterations that culminate in aggressive breast cancers. Cellular models that exhibit ErbB2 kinase dependency and can induce metastatic breast cancer in immune competent hosts are likely to help bridge this gap. Materials and Methods: Here, we derived and characterized a cell line model obtained from a transgenic ErbB2/Neu-driven mouse mammary adenocarcinoma. Results: The MPPS1 cell line produces metastatic breast cancers when implanted in the mammary fat pads of immune-compromised as well as syngeneic immune-competent hosts. MPPS1 cells maintain high ErbB2 overexpression when propagated in DFCI-1 or related media, and their growth is ErbB2-dependent, as demonstrated by concentration-dependent inhibition of proliferation with the ErbB kinase inhibitor Lapatinib. When grown in 3-dimensional (3-D culture on Matrigel, MPPS1 cells predominantly form large irregular cystic and solid structures. Remarkably, low concentrations of Lapatinib led to a switch to regular acinar growth on Matrigel. Immunofluorescence staining of control vs. Lapatinib-treated acini for markers of epithelial polarity revealed that inhibition of ErbB2 signaling led to rapid resumption of normal mammary epithelium-like cell polarity. Conclusions: The strict dependence of the MPPS1 cell system on ErbB2 signals for proliferation and alterations in cell polarity should allow its use to dissect ErbB2 kinase-dependent signaling pathways that promote loss of cell polarity, a key component of the epithelial mesenchymal transition and aggressiveness of ErbB2-driven breast cancers.

  1. Inhibition of ERBB2-overexpressing Tumors by Recombinant Human Prolidase and Its Enzymatically Inactive Mutant

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    Lu Yang

    2015-05-01

    Full Text Available ERBB2 is an oncogenic receptor tyrosine kinase overexpressed in a subset of human breast cancer and other cancers. We recently found that human prolidase (PEPD, a dipeptidase, is a high affinity ERBB2 ligand and cross-links two ERBB2 monomers. Here, we show that recombinant human PEPD (rhPEPD strongly inhibits ERBB2-overexpressing tumors in mice, whereas it does not impact tumors without ERBB2 overexpression. rhPEPD causes ERBB2 depletion, disrupts oncogenic signaling orchestrated by ERBB2 homodimers and heterodimers, and induces apoptosis. The impact of enzymatically-inactive mutant rhPEPDG278D on ERBB2 is indistinguishable from that of rhPEPD, but rhPEPDG278D is superior to rhPEPD for tumor inhibition. The enzymatic function of rhPEPD stimulates HIF-1α and other pro-survival factors in tumors, which likely attenuates its antitumor activity. rhPEPDG278D is also attractive in that it may not interfere with the physiologic function of endogenous PEPD in normal cells. Collectively, we have identified a human protein as an inhibitory ERBB2 ligand that inhibits ERBB2-overexpressing tumors in vivo. Several anti-ERBB2 agents are on the market but are hampered by drug resistance and high drug cost. rhPEPDG278D may synergize with these agents and may also be highly cost-effective, since it targets ERBB2 with a different mechanism and can be produced in bacteria.

  2. Dynamic transcription factor activity and networks during ErbB2 breast oncogenesis and targeted therapy.

    Science.gov (United States)

    Weiss, M S; Peñalver Bernabé, B; Shin, S; Asztalos, S; Dubbury, S J; Mui, M D; Bellis, A D; Bluver, D; Tonetti, D A; Saez-Rodriguez, J; Broadbelt, L J; Jeruss, J S; Shea, L D

    2014-12-01

    Tissue development and disease progression are multi-stage processes controlled by an evolving set of key regulatory factors, and identifying these factors necessitates a dynamic analysis spanning relevant time scales. Current omics approaches depend on incomplete biological databases to identify critical cellular processes. Herein, we present TRACER (TRanscriptional Activity CEll aRrays), which was employed to quantify the dynamic activity of numerous transcription factor (TFs) simultaneously in 3D and networks for TRACER (NTRACER), a computational algorithm that allows for cellular rewiring to establish dynamic regulatory networks based on activity of TF reporter constructs. We identified major hubs at various stages of culture associated with normal and abnormal tissue growth (i.e., ELK-1 and E2F1, respectively) and the mechanism of action for a targeted therapeutic, lapatinib, through GATA-1, which were confirmed in human ErbB2 positive breast cancer patients and human ErbB2 positive breast cancer cell lines that were either sensitive or resistant to lapatinib.

  3. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

    DEFF Research Database (Denmark)

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne

    2013-01-01

    receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133...... and ERBB2, and 83 % for PGR. Overall agreements, when comparing mRNA expression to immunohistochemistry, were 100 % (ERBB2), 89 % (ESR1) and 83 % (PGR), which was confirmed in the validation sets. Percentage of tumor in the sections did not influence the results. In conclusion, reliable quantification...

  4. Label-Free Quantitation and Mapping of the ErbB2 Tumor Receptor by Multiple Protease Digestion with Data-Dependent (MS1 and Data-Independent (MS2 Acquisitions

    Directory of Open Access Journals (Sweden)

    Jason M. Held

    2013-01-01

    Full Text Available The receptor tyrosine kinase ErbB2 is a breast cancer biomarker whose posttranslational modifications (PTMs are a key indicator of its activation. Quantifying the expression and PTMs of biomarkers such as ErbB2 by selected reaction monitoring (SRM mass spectrometry has several limitations, including minimal coverage and extensive assay development time. Therefore, we assessed the utility of two high resolution, full scan mass spectrometry approaches, MS1 Filtering and SWATH MS2, for targeted ErbB2 proteomics. Endogenous ErbB2 immunoprecipitated from SK-BR-3 cells was in-gel digested with trypsin, chymotrypsin, Asp-N, or trypsin plus Asp-N in triplicate. Data-dependent acquisition with an AB SCIEX TripleTOF 5600 and MS1 Filtering data processing was used to assess peptide and PTM coverage as well as the reproducibility of enzyme digestion. Data-independent acquisition (SWATH was also performed for MS2 quantitation. MS1 Filtering and SWATH MS2 allow quantitation of all detected analytes after acquisition, enabling the use of multiple proteases for quantitative assessment of target proteins. Combining high resolution proteomics with multiprotease digestion enabled quantitative mapping of ErbB2 with excellent reproducibility, improved amino acid sequence and PTM coverage, and decreased assay development time compared to typical SRM assays. These results demonstrate that high resolution quantitative proteomic approaches are an effective tool for targeted biomarker quantitation.

  5. The Mysterious Ways of ErbB2/HER2 Trafficking

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    Vibeke Bertelsen

    2014-08-01

    Full Text Available The EGFR- or ErbB-family of receptor tyrosine kinases consists of EGFR/ErbB1, ErbB2/HER2, ErbB3/HER3 and ErbB4/HER4. Receptor activation and downstream signaling are generally initiated upon ligand-induced receptor homo- or heterodimerization at the plasma membrane, and endocytosis and intracellular membrane transport are crucial for regulation of the signaling outcome. Among the receptors, ErbB2 is special in several ways. Unlike the others, ErbB2 has no known ligand, but is still the favored dimerization partner. Furthermore, while the other receptors are down-regulated either constitutively or upon ligand-binding, ErbB2 is resistant to down-regulation, and also inhibits down-regulation of its partner upon heterodimerization. The reason(s why ErbB2 is resistant to down-regulation are the subject of debate. Contrary to other ErbB-proteins, mature ErbB2 needs Hsp90 as chaperone. Several data suggest that Hsp90 is an important regulator of factors like ErbB2 stability, dimerization and/or signaling. Hsp90 inhibitors induce degradation of ErbB2, but whether Hsp90 directly makes ErbB2 endocytosis resistant is unclear. Exposure to anti-ErbB2 antibodies can also induce down-regulation of ErbB2. Down-regulation induced by Hsp90 inhibitors or antibodies does at least partly involve internalization and endosomal sorting to lysosomes for degradation, but also retrograde trafficking to the nucleus has been reported. In this review, we will discuss different molecular mechanisms suggested to be important for making ErbB2 resistant to down-regulation, and review how membrane trafficking is involved when down-regulation and/or relocalization of ErbB2 is induced.

  6. A Functionally Significant Cross-talk between Androgen Receptor and ErbB2 Pathways in Estrogen Receptor Negative Breast Cancer

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    Ali Naderi

    2008-06-01

    Full Text Available Recent studies have identified novel subgroups in ER-negative breast cancer based on the expression pattern of androgen receptor (AR. One subtype (molecular apocrine has an over-expression of steroid-response genes and ErbB2. Using breast cancer cell lines with molecular apocrine features, we demonstrate a functional cross-talk between AR and ErbB2 pathways. We show that stimulation of AR and ErbB2 pathways leads to the cross-regulation of gene expression for AR, ErbB2, FOXA1, XBP1, TFF3, and KLK3. As opposed to the physiologic transient phosphorylation of extracellular signal–regulated kinase (ERK1/2 observed with the testosterone treatment, we demonstrate that the addition of ErbB2 inhibition leads to a persistent phosphorylation of ERK1/2, which negatively regulates the downstream signaling and cell growth. This suggests a mechanism for the cross-talk involving the ERK pathway. Moreover, testosterone stimulates the proliferation of molecular apocrine breast cell lines, and this effect can be reversed using antiandrogen flutamide and anti-ErbB2 AG825. Conversely, the growth stimulatory effect of heregulin can also be inhibited with flutamide, suggesting a cross-talk between the AR and ErbB2 pathways affecting cell proliferation. Importantly, there is a synergy with the combined use of flutamide and AG825 on cell proliferation and apoptosis, which indicates a therapeutic advantage in the combined blockage of AR and ErbB2 pathways.

  7. A functionally significant cross-talk between androgen receptor and ErbB2 pathways in estrogen receptor negative breast cancer.

    Science.gov (United States)

    Naderi, Ali; Hughes-Davies, Luke

    2008-06-01

    Recent studies have identified novel subgroups in ER-negative breast cancer based on the expression pattern of androgen receptor (AR). One subtype (molecular apocrine) has an over-expression of steroid-response genes and ErbB2. Using breast cancer cell lines with molecular apocrine features, we demonstrate a functional cross-talk between AR and ErbB2 pathways. We show that stimulation of AR and ErbB2 pathways leads to the cross-regulation of gene expression for AR, ErbB2, FOXA1, XBP1, TFF3, and KLK3. As opposed to the physiologic transient phosphorylation of extracellular signal-regulated kinase (ERK1/2) observed with the testosterone treatment, we demonstrate that the addition of ErbB2 inhibition leads to a persistent phosphorylation of ERK1/2, which negatively regulates the downstream signaling and cell growth. This suggests a mechanism for the cross-talk involving the ERK pathway. Moreover, testosterone stimulates the proliferation of molecular apocrine breast cell lines, and this effect can be reversed using antiandrogen flutamide and anti-ErbB2 AG825. Conversely, the growth stimulatory effect of heregulin can also be inhibited with flutamide, suggesting a cross-talk between the AR and ErbB2 pathways affecting cell proliferation. Importantly, there is a synergy with the combined use of flutamide and AG825 on cell proliferation and apoptosis, which indicates a therapeutic advantage in the combined blockage of AR and ErbB2 pathways.

  8. Expression of the EGF receptor family members ErbB2, ErbB3, and ErbB4 in germinal zones of the developing brain and in neurosphere cultures containing CNS stem cells.

    Science.gov (United States)

    Kornblum, H I; Yanni, D S; Easterday, M C; Seroogy, K B

    2000-01-01

    The epidermal growth factor receptor family consists of four related tyrosine kinases: the epidermal growth factor receptor (EGF-R or ErbB), ErbB2, ErbB3, and ErbB4. These receptors are capable of extensive cross-activation upon the binding of their ligands - the EGF family of peptides for EGF-R and the neuregulins for ErbB3 and ErbB4. Since EGF-R is expressed by proliferating cells in the central nervous system (CNS), including multipotent CNS stem cells, we examined the expression of ErbB2, ErbB3 and ErbB4 in the germinal epithelia of the developing rat brain using in situ hybridization. ErbB2 and ErbB4 mRNAs were widely distributed within the germinal zones as early as E12. However, as development proceeded, ErbB2 mRNA was mainly present within the layers of cells immediately adjacent to the ventricular surface - the ventricular zone, while ErbB4 mRNA was predominantly expressed by subventricular zone cells, in the regions where these specialized germinal epithelia were present. ErbB3 mRNA distribution within germinal epithelia was more restricted, primarily confined to the diencephalon and rostral midbrain. Cultured neurospheres, which contain CNS stem cells, expressed ErbB2, ErbB4 and, to a lesser extent, ErbB3 protein as demonstrated by Western blot analysis. This expression declined during following differentiation. Heregulin-beta1, a neuregulin, had no effect on the proliferative capacity of neurospheres. Overall, our results indicate that ErbB2, ErbB3 and ErbB4 may play important and distinct roles in the genesis of the CNS. However, our in vitro data do not support a role for neuregulins in proliferation, per se, of CNS stem cells.

  9. Role of Erbin in ErbB2-dependent breast tumor growth.

    Science.gov (United States)

    Tao, Yanmei; Shen, Chengyong; Luo, Shiwen; Traoré, Wilfried; Marchetto, Sylvie; Santoni, Marie-Josée; Xu, Linlin; Wu, Biao; Shi, Chao; Mei, Jinghong; Bates, Ryan; Liu, Xihui; Zhao, Kai; Xiong, Wen-Cheng; Borg, Jean-Paul; Mei, Lin

    2014-10-21

    ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2), a receptor tyrosine kinase of the ErbB family, is overexpressed in around 25% of breast cancers. In addition to forming a heterodimer with other ErbB receptors in response to ligand stimulation, ErbB2 can be activated in a ligand-independent manner. We report here that Erbin, an ErbB2-interacting protein that was thought to act as an antitumor factor, is specifically expressed in mammary luminal epithelial cells and facilitates ErbB2-dependent proliferation of breast cancer cells and tumorigenesis in MMTV-neu transgenic mice. Disruption of their interaction decreases ErbB2-dependent proliferation, and deletion of the PDZ domain in Erbin hinders ErbB2-dependent tumor development in MMTV-neu mice. Mechanistically, Erbin forms a complex with ErbB2, promotes its interaction with the chaperon protein HSP90, and thus prevents its degradation. Finally, ErbB2 and Erbin expression correlates in human breast tumor tissues. Together, these observations establish Erbin as an ErbB2 regulator for breast tumor formation and progression.

  10. Cardiac-specific over-expression of epidermal growth factor receptor 2 (ErbB2 induces pro-survival pathways and hypertrophic cardiomyopathy in mice.

    Directory of Open Access Journals (Sweden)

    Polina Sysa-Shah

    Full Text Available BACKGROUND: Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored. METHODOLOGY/PRINCIPAL FINDINGS: We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2-3 times larger than those of control littermates, express increased atrial natriuretic peptide and β-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib. CONCLUSIONS/SIGNIFICANCE: These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling

  11. Identification of HTF (HER2 transcription factor) as an AP-2 (activator protein-2) transcription factor and contribution of the HTF binding site to ERBB2 gene overexpression.

    Science.gov (United States)

    Vernimmen, Douglas; Begon, Dominique; Salvador, Christophe; Gofflot, Stéphanie; Grooteclaes, Madeleine; Winkler, Rosita

    2003-02-15

    The ERBB2 gene is overexpressed in 30% of human breast cancers and this is correlated with poor prognosis. Overexpression of the ERBB2 gene is due to increased transcription and gene amplification. Our previous studies have identified a new cis element in the ERBB2 promoter which is involved in the gene's overexpression. This cis element, located 501 bp upstream from the main ERBB2 transcription initiation site, binds a transcription factor called HTF (HER2 transcription factor). We report here the identification of HTF as an AP-2 (activator protein-2) transcription factor. The new cis element is bound by AP-2 with high affinity, compared with a previously described AP-2 binding site located 284 bp downstream. Co-transfection of an AP-2alpha expression vector with a reporter vector containing the newly identified AP-2 binding site in front of a minimal ERBB2 promoter induced a dose-dependent increase in transcriptional activity. We examined the contribution of the new AP-2 binding site to ERBB2 overexpression. For this purpose we abolished the new and/or the previously described AP-2 binding sequence by site-directed mutagenesis. The results show that the two functional AP-2 sites in the first 700 bp of the ERBB2 promoter co-operate to achieve maximal transcriptional activity.

  12. Analysis of protein-protein interactions involved in the activation of the Shc/Grb-2 pathway by the ErbB-2 kinase.

    Science.gov (United States)

    Ricci, A; Lanfrancone, L; Chiari, R; Belardo, G; Pertica, C; Natali, P G; Pelicci, P G; Segatto, O

    1995-10-19

    In murine fibroblasts activation of the Shc/Grb-2 pathway by the ErbB-2 kinase involves tyrosine phosphorylation of Shc products and the formation of Shc/ErbB-2, Shc/Grb-2 and Grb-2/ErbB-2 complexes. Tyr 1139 of ErbB-2 bound to the Grb-2 SH2 domain in vitro as well as in intact cells. Tyr 1221 and 1248 are binding sites of gp185ErbB-2 for Shc SH2 domain in vitro whereas Tyr 1196 and 1248 are major binding sites of ErbB-2 for Shc PTB domain. Inhibition of Shc/ErbB-2 complex formation in intact cells was obtained by simultaneous mutational inactivation of Shc SH2 and Shc PTB binding sites of gp185ErbB-2. Shc/ErbB-2 complexes are formed upon activation of the ErbB-2 kinase and tyrosine phosphorylation of Shc proteins; they are located in both cytosol and cellular membranes. ErbB-2 activation induces also translocation of Grb-2 from cytosol to membranes. This network of protein-protein interactions may reflect the ability of the Shc/Grb-2 pathway to act as a molecular switch controlling different cellular functions regulated by RTK activation. In fact the Ras GDP exchanger mSOS was recruited in Grb-2/ErbB-2 complexes; furthermore besides mSOS, other polypeptides present in either cytosolic or membrane preparations were able to complex in vitro with Grb-2 SH3 domains.

  13. Ligand-associated ERBB2/3 activation confers acquired resistance to FGFR inhibition in FGFR3-dependent cancer cells.

    Science.gov (United States)

    Wang, J; Mikse, O; Liao, R G; Li, Y; Tan, L; Janne, P A; Gray, N S; Wong, K-k; Hammerman, P S

    2015-04-23

    Somatic alterations of fibroblast growth factor receptors (FGFRs) have been described in a wide range of malignancies. A number of anti-FGFR therapies are currently under investigation in clinical trials for subjects with FGFR gene amplifications, mutations and translocations. Here, we develop cell line models of acquired resistance to FGFR inhibition by exposure of cell lines harboring FGFR3 gene amplification and translocation to the selective FGFR inhibitor BGJ398 and multitargeted FGFR inhibitor ponatinib. We show that the acquisition of resistance is rapid, reversible and characterized by an epithelial to mesenchymal transition and a switch from dependency on FGFR3 to ERBB family members. Acquired resistance was associated with demonstrable changes in gene expression including increased production of ERBB2/3 ligands, which were sufficient to drive resistance in the setting of FGFR3 dependency but not dependency on other FGFR family members. These data support the concept that activation of ERBB family members is sufficient to bypass dependency on FGFR3 and suggest that concurrent inhibition of these two pathways may be desirable when targeting FGFR3-dependent cancers.

  14. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kyotani, Yoji, E-mail: cd147@naramed-u.ac.jp [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522 (Japan); Ota, Hiroyo [Second Department of Internal Medicine, Nara Medical University School of Medicine, Kashihara 634-8522 (Japan); Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Itaya-Hironaka, Asako; Yamauchi, Akiyo; Sakuramoto-Tsuchida, Sumiyo [Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Zhao, Jing; Ozawa, Kentaro; Nagayama, Kosuke; Ito, Satoyasu [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Takasawa, Shin [Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Kimura, Hiroshi [Second Department of Internal Medicine, Nara Medical University School of Medicine, Kashihara 634-8522 (Japan); Uno, Masayuki [Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522 (Japan); Yoshizumi, Masanori [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan)

    2013-11-15

    Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation. - Highlights: ●In vitro system for intermittent hypoxia (IH) and sustained hypoxia (SH). ●IH, but not SH, induces the proliferation of rat vascular smooth muscle cell. ●Epiregulin m

  15. ErbB2-Driven Breast Cancer Cell Invasion Depends on a Complex Signaling Network Activating Myeloid Zinc Finger-1-Dependent Cathepsin B Expression

    DEFF Research Database (Denmark)

    Rafn, Bo; Nielsen, Christian Thomas Friberg; Andersen, Sofie Hagel;

    2012-01-01

    signaling network activates the transcription of cathepsin B gene (CTSB) via myeloid zinc finger-1 transcription factor that binds to an ErbB2-responsive enhancer element in the first intron of CTSB. This work provides a model system for ErbB2-induced breast cancer cell invasiveness, reveals a signaling...... network that is crucial for invasion in vitro, and defines a specific role and targets for the identified serine-threonine kinases....

  16. The ErbB2 inhibitor Herceptin (Trastuzumab) promotes axonal outgrowth four weeks after acute nerve transection and repair.

    Science.gov (United States)

    Placheta, Eva; Hendry, J Michael; Wood, Matthew D; Lafontaine, Christine W; Liu, Edward H; Cecilia Alvarez Veronesi, M; Frey, Manfred; Gordon, Tessa; Borschel, Gregory H

    2014-10-17

    Accumulating evidence suggests that neuregulin, a potent Schwann cell mitogen, and its receptor, ErbB2, have an important role in regulating peripheral nerve regeneration. We hypothesized that Herceptin (Trastuzumab), a monoclonal antibody that binds ErbB2, would disrupt ErbB2 signaling, allowing us to evaluate ErbB2's importance in peripheral nerve regeneration. In this study, the extent of peripheral motor and sensory nerve regeneration and distal axonal outgrowth was analyzed two and four weeks after common peroneal (CP) nerve injury in rats. Outcomes analyzed included neuron counts after retrograde labeling, histomorphometry, and protein analysis. The data analysis revealed that there was no impact of Herceptin administration on either the numbers of motor or sensory neurons that regenerated their axons but histomorphometry revealed that Herceptin significantly increased the number of regenerated axons in the distal repaired nerve after 4 weeks. Protein analysis with Western blotting revealed no difference in either expression levels of ErbB2 or the amount of activated, phosphorylated ErbB2 in injured nerves. In conclusion, administration of the ErbB2 receptor inhibitor after nerve transection and surgical repair did not alter the number of regenerating neurons but markedly increased the number of regenerated axons per neuron in the distal nerve stump. Enhanced axon outgrowth in the presence of this ErbB2 inhibitor indicates that ErbB2 signaling may limit the numbers of axons that are emitted from each regenerating neuron.

  17. Genome profiling of ERBB2-amplified breast cancers

    Directory of Open Access Journals (Sweden)

    Ayed Farhat

    2010-10-01

    Full Text Available Abstract Background Around 20% of breast cancers (BC show ERBB2 gene amplification and overexpression of the ERBB2 tyrosine kinase receptor. They are associated with a poor prognosis but can benefit from targeted therapy. A better knowledge of these BCs, genomically and biologically heterogeneous, may help understand their behavior and design new therapeutic strategies. Methods We defined the high resolution genome and gene expression profiles of 54 ERBB2-amplified BCs using 244K oligonucleotide array-comparative genomic hybridization and whole-genome DNA microarrays. Expression of ERBB2, phosphorylated ERBB2, EGFR, IGF1R and FOXA1 proteins was assessed by immunohistochemistry to evaluate the functional ERBB2 status and identify co-expressions. Results First, we identified the ERBB2-C17orf37-GRB7 genomic segment as the minimal common 17q12-q21 amplicon, and CRKRS and IKZF3 as the most frequent centromeric and telomeric amplicon borders, respectively. Second, GISTIC analysis identified 17 other genome regions affected by copy number aberration (CNA (amplifications, gains, losses. The expression of 37 genes of these regions was deregulated. Third, two types of heterogeneity were observed in ERBB2-amplified BCs. The genomic profiles of estrogen receptor-postive (ER+ and negative (ER- ERBB2-amplified BCs were different. The WNT/β-catenin signaling pathway was involved in ER- ERBB2-amplified BCs, and PVT1 and TRPS1 were candidate oncogenes associated with ER+ ERBB2-amplified BCs. The size of the ERBB2 amplicon was different in inflammatory (IBC and non-inflammatory BCs. ERBB2-amplified IBCs were characterized by the downregulated and upregulated mRNA expression of ten and two genes in proportion to CNA, respectively. IHC results showed (i a linear relationship between ERBB2 gene amplification and its gene and protein expressions with a good correlation between ERBB2 expression and phosphorylation status; (ii a potential signaling cross-talk between

  18. Electrocardiographic Characterization of Cardiac Hypertrophy in Mice that Overexpress the ErbB2 Receptor Tyrosine Kinase

    DEFF Research Database (Denmark)

    Sysa-Shah, Polina; Sørensen, Lars L; Abraham, M Roselle

    2015-01-01

    , the ECG recordings of ErbB2(tg) mice were characterized by higher P- and R-wave amplitudes, broader QRS complexes, inverted T waves, and ST interval depression. Pearson's correlation matrix analysis of combined WT and ErbB2(tg) data revealed significant correlation between heart weight and the ECG...... parameters of QT interval (corrected for heart rate), QRS interval, ST height, R amplitude, P amplitude, and PR interval. In addition, the left ventricular posterior wall thickness as determined by echocardiography correlated with ECG-determined ST height, R amplitude, QRS interval; echocardiographic left...... ventricular mass correlated with ECG-determined ST height and PR interval. In summary, we have determined phenotypic ECG criteria to differentiate ErbB2(tg) from WT genotypes in 98.8% of mice. This inexpensive and time-efficient ECG-based phenotypic method might be applied to differentiate between genotypes...

  19. Identification of novel nuclear localization signal within the ErbB-2 protein

    Institute of Scientific and Technical Information of China (English)

    Qiao Qiao CHEN; Xiao Ying CHEN; Yun Yun JIANG; Jing LIU

    2005-01-01

    ErbB2,a member of the receptor tyrosine kinase family,is frequently over-expressed in breast cancer.Proteolysis of the extracellular domain of ErbB2 results in constitutive activation of ErbB2 kinase.Recent study reported that ErbB2 is found in the nucleus.Here,we showed that ErbB2 is imported into the nucleus through a nuclear localization signal (NLS)-mediated mechanism.The NLS sequence KRRQQKIRKYTMRR (aa655-668) contains three clusters of basic amino acids and it is sufficient to target GFP into the nucleus.However,mutation in any basic amino acid cluster of this NLS sequence significantly affects its nuclear localization.Furthermore,it was found that this NLS is essential for the nuclear localization of ErbB2 since the intracellular domain of Erb2 lacking NLS completely abrogates its nuclear translocation.Taken together,our study identified a novel nuclear localization signal and reveals a novel mechanism underlying ErbB2 nuclear trafficking and localization.

  20. Simulation of homology models for the extracellular domains (ECD) of ErbB3, ErbB4 and the ErbB2-ErbB3 complex in their active conformations.

    Science.gov (United States)

    Franco-Gonzalez, Juan Felipe; Ramos, Javier; Cruz, Victor L; Martínez-Salazar, Javier

    2013-02-01

    Epidermal growth factor receptors (EGFR) are associated with a number of biological processes and are becoming increasingly recognized as important therapeutic targets against cancer. In this work, we provide models based on homology for the extracellular domains (ECD) of ErbB3 and ErbB4 in their active conformations, including a Heregulin ligand, followed by further refinement of the models by molecular dynamics simulations at atomistic scale. We compare the results with a model built for ErbB2 based on crystallographic information and analyze the common features observed among members of the family, namely, the periscope movement of the dimerization arm and the hinge displacement of domain IV. Finally, we refine a model for the interaction of the ECDs corresponding to a ErbB2-ErbB3 heterodimer, which is widely recognized to have a high impact in cancer development.

  1. Truncated ErbB2 receptor enhances ErbB1 signaling and induces reversible, ERK-independent loss of epithelial morphology

    DEFF Research Database (Denmark)

    Egeblad, M; Mortensen, Ole Hartvig; Jäättelä, M

    2001-01-01

    breast cancer cells expressing NH(2)-terminally truncated ErbB2 (DeltaNErbB2) were compared with cells overexpressing wild-type ErbB2. Expression of DeltaNErbB2 in MCF-7 cells resulted in sustained activation of extracellular signal-regulated kinases (ERK) 1/2, extensive loss of the epithelial morphology...... and depended on continuous expression of DeltaNErbB2 but not on the activation of the ERK1/2 pathway. Interestingly, the expression of DeltaNErbB2 resulted also in the increased expression and phosphorylation of ErbB1 as well as in the prolonged ligand-induced activation of the ErbB1 signaling pathway...

  2. Differential sensitivity of ERBB2 kinase domain mutations towards lapatinib.

    Directory of Open Access Journals (Sweden)

    Rama Krishna Kancha

    Full Text Available BACKGROUND: Overexpression of the ERBB2 kinase is observed in about one-third of breast cancer patients and the dual ERBB1/ERBB2 kinase inhibitor lapatinib was recently approved for the treatment of advanced ERBB2-positive breast cancer. Mutations in the ERBB2 receptor have recently been reported in breast cancer at diagnosis and also in gastric, colorectal and lung cancer. These mutations may have an impact on the clinical responses achieved with lapatinib in breast cancer and may also have a potential impact on the use of lapatinib in other solid cancers. However, the sensitivity of lapatinib towards clinically observed ERBB2 mutations is not known. METHODOLOGY/PRINCIPAL FINDINGS: We cloned a panel of 8 clinically observed ERBB2 mutations, established stable cell lines and characterized their sensitivity towards lapatinib and alternative ERBB2 inhibitors. Both lapatinib-sensitive and lapatinib-resistant ERBB2 mutations were observed. Interestingly, we were able to generate lapatinib resistance mutations in wt-ERBB2 cells incubated with lapatinib for prolonged periods of time. This indicates that these resistance mutations may also cause secondary resistance in lapatinib-treated patients. Lapatinib-resistant ERBB2 mutations were found to be highly resistant towards AEE788 treatment but remained sensitive towards the dual irreversible inhibitors CL-387785 and WZ-4002. CONCLUSIONS/SIGNIFICANCE: Patients harbouring certain ERBB2 kinase domain mutations at diagnosis may not benefit from lapatinib treatment. Moreover, secondary lapatinib resistance may develop due to kinase domain mutations. Irreversible ERBB2 inhibitors may offer alternative treatment options for breast cancer and other solid tumor patients harbouring lapatinib resistance mutations. In addition, these inhibitors may be of interest in the scenario of secondary lapatinib resistance.

  3. Activation of integrin-ERBB2 signaling in undifferentiated thyroid cancer

    OpenAIRE

    2014-01-01

    Undifferentiated thyroid carcinoma is one of the most aggressive human cancers. Although genetic changes underlying this aggressive cancer remain to be elucidated, RAS mutations have been frequently identified in it. Mice harboring a mutant thyroid hormone receptor ThrbPV (ThrbPV/PV) spontaneously develop differentiated follicular thyroid carcinoma similar to human thyroid cancer. We recently demonstrated that targeting a RAS mutation (KrasG12D) to the thyroid of ThrbPV/PV mice (ThrbPV/PV ...

  4. Activation of EGFR/ERBB2 via pathways involving ERK1/2, P38 MAPK, AKT and FOXO enhances recovery of diabetic hearts from ischemia-reperfusion injury.

    Science.gov (United States)

    Akhtar, Saghir; Yousif, Mariam H M; Chandrasekhar, Bindu; Benter, Ibrahim F

    2012-01-01

    This study characterized the effects of diabetes and/or ischemia on epidermal growth factor receptor, EGFR, and/or erbB2 signaling pathways on cardiac function. Isolated heart perfusion model of global ischemia was used to study the effect of chronic inhibition or acute activation of EGFR/erbB2 signaling on cardiac function in a rat model of type-1 diabetes. Induction of diabetes with streptozotocin impaired recovery of cardiac function (cardiac contractility and hemodynamics) following 40 minutes of global ischemia in isolated hearts. Chronic treatment with AG825 or AG1478, selective inhibitors of erbB2 and EGFR respectively, did not affect hyperglycemia but led to an exacerbation whereas acute administration of the EGFR ligand, epidermal growth factor (EGF), led to an improvement in cardiac recovery in diabetic hearts. Diabetes led to attenuated dimerization and phosphorylation of cardiac erbB2 and EGFR receptors that was associated with reduced signaling via extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 mitogen activated protein (MAP) kinase and AKT (protein kinase B). Ischemia was also associated with reduced cardiac signaling via these molecules whereas EGF-treatment opposed diabetes and/or ischemia induced changes in ERK1/2, p38 MAP kinase, and AKT-FOXO signaling. Losartan treatment improved cardiac function in diabetes but also impaired EGFR phosphorylation in diabetic heart. Co-administration of EGF rescued Losartan-mediated reduction in EGFR phosphorylation and significantly improved cardiac recovery more than with either agent alone. EGFR/erbB2 signaling is an important cardiac survival pathway whose activation, particularly in diabetes, ischemia or following treatment with drugs that inhibit this cascade, significantly improves cardiac function. These findings may have clinical relevance particularly in the treatment of diabetes-induced cardiac dysfunction.

  5. ErbB2-dependent chemotaxis requires microtubule capture and stabilization coordinated by distinct signaling pathways.

    Directory of Open Access Journals (Sweden)

    Khedidja Benseddik

    Full Text Available Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLCγ also contributed to cell guidance. We further showed that PLCγ signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.

  6. Transmembrane Domain Targeting Peptide Antagonizing ErbB2/Neu Inhibits Breast Tumor Growth and Metastasis

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    Alexia Arpel

    2014-09-01

    Full Text Available Breast cancer is still a deadly disease despite major achievements in targeted therapies designed to block ligands or ligand-binding subunits of major tyrosine kinase receptors. Relapse is significant and metastases deleterious, which demands novel strategies for fighting this disease. Here, we report a proof-of-concept experiment demonstrating that small peptides interfering with the transmembrane domain of the tyrosine kinase epidermal growth factor receptor ErbB2 exhibit anticancer properties when used at micromolar dosages in a genetically engineered mouse model of breast cancer. Different assays demonstrate the specificity of the ErbB2-targeting peptide, which induces long-term reduction of ErbB2 phosphorylation and Akt signaling consistent with reduced tumor cell proliferation and increased survival. Microcomputed tomography analysis established the antimetastatic activity of the peptide and its impact on primary tumor growth. This reveals the interior of the cell membrane as an unexplored dimension for drug design.

  7. MGC9753 gene, located within PPP1R1B-STARD3-ERBB2-GRB7 amplicon on human chromosome 17q12, encodes the seven-transmembrane receptor with extracellular six-cystein domain.

    Science.gov (United States)

    Katoh, Masuko; Katoh, Masaru

    2003-06-01

    MYC, ERBB2, MET, FGFR2, CCNE1, MYCN, WNT2, CD44, MDM2, NCOA3, IQGAP1 and STK6 loci are amplified in human gastric cancer. It has been reported that the gene corresponding to EST H16094 is co-amplified with ERBB2 gene in human gastric cancer. Here, we identified and characterized the gene corresponding to EST H16094 by using bioinformatics. BLAST programs revealed that EST H16094 was derived from the uncharacterized MGC9753 gene. Two ORFs were predicted within human MGC9753 mRNA, and ORF1 (nucleotide position 18-980 of NM_033419.1) was predicted as the coding region of human MGC9753 mRNA based on comparative genomics. Nucleotide sequence of mouse Mgc9753 mRNA was next determined in silico by modification of AK052486 cDNA (deleting C at the nucleotide position 37). Human MGC9753 and mouse Mgc9753 proteins were 320-amino-acid seven-transmembrane receptors with the N-terminal six-cysteine domain and an N-glycosylation site (85.0% total-amino-acid identity). Human MGC9753 protein showed 90.6% total-amino-acid identity with human CAB2 aberrant protein, which lacked the third-transmembrane domain of MGC9753 due to frame shifts within ORF. Human MGC9753 gene, consisting of eight exons, were clustered with PPP1R1B, STARD3, TCAP, PNMT, ERBB2, MGC14832 and GRB7 genes within the 120-kb region. PPP1R1B, STARD3, MGC9753, ERBB2 and GRB7 genes are co-amplified in several cases of gastric cancer. This is the first report on comprehensive characterization of the amplicon around the PPP1R1B-STARD3-TCAP-PNMT-MGC9753-ERBB2-MGC14832-GRB7 locus on human chromosome 17q12.

  8. ErbB2 signaling at the crossing between heart failure and cancer

    OpenAIRE

    Vermeulen, Zarha; Segers, Vincent F.M.; De Keulenaer, Gilles W.

    2016-01-01

    The dual role of ErbB2 (or HER-2) in tumor growth and in physiological adaptive reactions of the heart positions ErbB2 at the intersection between cancer and chronic heart failure. Accordingly, ErbB2-targeted inhibitory therapy of cancer may lead to ventricular dysfunction, and activation of ErbB2 for heart failure therapy may induce malignancy. The molecular processes leading to the activation of ErbB2 in tumors and cardiac cells are, however, fundamentally different from each other. Thus, i...

  9. Bispecific antibody to ErbB2 overcomes trastuzumab resistance through comprehensive blockade of ErbB2 heterodimerization.

    Science.gov (United States)

    Li, Bohua; Meng, Yanchun; Zheng, Lei; Zhang, Xunmin; Tong, Qing; Tan, Wenlong; Hu, Shi; Li, Hui; Chen, Yang; Song, Jinjing; Zhang, Ge; Zhao, Lei; Zhang, Dapeng; Hou, Sheng; Qian, Weizhu; Guo, Yajun

    2013-11-01

    The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer. However, resistance to trastuzumab is common. Heterodimerization between ErbB2 and other ErbBs may redundantly trigger cell proliferation signals and confer trastuzumab resistance. Here, we developed a bispecific anti-ErbB2 antibody using trastuzumab and pertuzumab, another ErbB2-specific humanized antibody that binds to a distinct epitope from trastuzumab. This bispecific antibody, denoted as TPL, retained the full binding activities of both parental antibodies and exhibited pharmacokinetic properties similar to those of a conventional immunoglobulin G molecule. Unexpectedly, TPL showed superior ErbB2 heterodimerization-blocking activity over the combination of both parental monoclonal antibodies, possibly through steric hindrance and/or inducing ErbB2 conformational change. Further data indicated that TPL potently abrogated ErbB2 signaling in trastuzumab-resistant breast cancer cell lines. In addition, we showed that TPL was far more effective than trastuzumab plus pertuzumab in inhibiting the growth of trastuzumab-resistant breast cancer cell lines, both in vitro and in vivo. Importantly, TPL treatment eradicated established trastuzumab-resistant tumors in tumor-bearing nude mice. Our results suggest that trastuzumab-resistant breast tumors remain dependent on ErbB2 signaling and that comprehensive blockade of ErbB2 heterodimerization may be an effective therapeutic avenue. The unique potential of TPL to overcome trastuzumab resistance warrants its consideration as a promising treatment in the clinic.

  10. The deaf and the dumb: the biology of ErbB-2 and ErbB-3.

    Science.gov (United States)

    Citri, Ami; Skaria, Kochupurakkal Bose; Yarden, Yosef

    2003-03-10

    ErbB-2 (also called HER2/neu) and ErbB-3 are closely related to the epidermal growth factor receptor (EGFR/ErbB-1), but unlike EGFR, ErbB-2 is a ligandless receptor, whereas ErbB-3 lacks tyrosine kinase activity. Hence, both ErbB-2 and ErbB-3 are active only in the context of ErbB heterodimers, and ErbB-2. ErbB-3 heterodimers, which are driven by neuregulin ligands, are the most prevalent and potent complexes. These stringently controlled heterodimers are repeatedly employed throughout embryonic development and dictate the establishment of several cell lineages through mesenchyme-epithelial inductive processes and the interactions of neurons with muscle, glia, and Schwann cells. Likewise, the potent combination of signaling pathways engaged by the heterodimers drives an aggressive phenotype of tumors of secretory epithelia, including breast and lung cancers. This review highlights recent structural insights into the mechanism of ligand-induced heterodimer formation, and concentrates on signaling pathways employed by ErbB-2 and ErbB-3 in normal and in malignant cells.

  11. The Na+/H+ exchanger NHE1, but not the Na+, HCO3- cotransporter NBCn1, regulates motility of MCF7 breast cancer cells expressing constitutively active ErbB2

    DEFF Research Database (Denmark)

    Lauritzen, Gitte; Stock, Christian-Martin; Lemaire, Justine;

    2012-01-01

    We and others have shown central roles of the Na(+)/H(+) exchanger NHE1 in cell motility. The aim of this study was to determine the roles of NHE1 and of the Na(+), HCO(3)(-) cotransporter NBCn1 in motility of serum-starved MCF-7 breast cancer cells expressing constitutively active ErbB2 (¿NErbB2...

  12. ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini

    Energy Technology Data Exchange (ETDEWEB)

    Muthuswamy, Senthil K; Li, Dongmei; Lelievre, Sophie; Bissell, Mina J; Brugge, Joan S

    2001-08-08

    Both ErbB1 and ErbB2 are overexpressed or amplified in breast tumors. To examine the effects of activating ErbB receptors in a context that mimics polarized epithelial cells in vivo, we activated ErbB1 and ErbB2 homodimers in preformed, growth-arrested mammary acini cultured in three-dimensional basement membrane gels. Activation of ErbB2, but not that of ErbB1, led to a reinitiation of cell proliferation and altered the properties of mammary acinar structures. These altered structures share several properties with early-stage tumors, including a loss of proliferative suppression, an absence of lumen, retention of the basement membrane and a lack of invasive properties. ErbB2 activation also disrupted tight junctions and the cell polarity of polarized epithelia, whereas ErbB1 activation did not have any effect. Our results indicate that ErbB receptors differ in their ability to induce early stages of mammary carcinogenesis in vitro and this three-dimensional model system can reveal biological activities of oncogenes that cannot be examined in vitro in standard transformation assays.

  13. Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Vuong, Tram Thu [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway); Bertelsen, Vibeke; Rødland, Marianne Skeie [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Stang, Espen [Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene, E-mail: i.h.madshus@medisin.uio.no [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway)

    2013-02-01

    The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub{sub 4}) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub{sub 4} chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub{sub 4} was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub{sub 4} was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub{sub 4} was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis. -- Highlights: ► A chimera containing ErbB2 and a tetra-Ubiquitin chain internalizes constitutively. ► Receptor fragmentation is not required for endocytosis of ErbB2. ► Ubiquitination is sufficient to induce endocytosis and degradation of ErbB2. ► ErbB2-Ub4 is internalized clathrin-dependently.

  14. Src mutation induces acquired lapatinib resistance in ERBB2-amplified human gastroesophageal adenocarcinoma models.

    Directory of Open Access Journals (Sweden)

    Yong Sang Hong

    Full Text Available ERBB2-directed therapy is now a routine component of therapy for ERBB2-amplified metastatic gastroesophageal adenocarcinomas. However, there is little knowledge of the mechanisms by which these tumors develop acquired resistance to ERBB2 inhibition. To investigate this question we sought to characterize cell line models of ERBB2-amplified gastroesophageal adenocarcinoma with acquired resistance to ERBB2 inhibition. We generated lapatinib-resistant (LR subclones from an initially lapatinib-sensitive ERBB2-amplified esophageal adenocarcinoma cell line, OE19. We subsequently performed genomic characterization and functional analyses of resistant subclones with acquired lapatinib resistance. We identified a novel, acquired SrcE527K mutation in a subset of LR OE19 subclones. Cells with this mutant allele harbour increased Src phosphorylation. Genetic and pharmacologic inhibition of Src resensitized these subclones to lapatinib. Biochemically, Src mutations could activate both the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways in the lapatinib-treated LR OE19 cells. Ectopic expression of SrcE527K mutation also was sufficient to induce lapatinib resistance in drug-naïve cells. These results indicate that pathologic activation of Src is a potential mechanism of acquired resistance to ERBB2 inhibition in ERBB2-amplified gastroesophageal cancer. Although Src mutation has not been described in primary tumor samples, we propose that the Src hyperactivation should be investigated in the settings of acquired resistance to ERBB2 inhibition in esophageal and gastric adenocarcinoma.

  15. Localisation Microscopy of Breast Epithelial ErbB-2 Receptors and Gap Junctions: Trafficking after γ-Irradiation, Neuregulin-1β, and Trastuzumab Application

    Science.gov (United States)

    Pilarczyk, Götz; Nesnidal, Ines; Gunkel, Manuel; Bach, Margund; Bestvater, Felix; Hausmann, Michael

    2017-01-01

    In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin-1β application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon γ-irradiation or alternatively neuregulin-1β application mobilized ErbB receptors in a nucleograde fashion—a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation. PMID:28208769

  16. ERBB2 in cat mammary neoplasias disclosed a positive correlation between RNA and protein low expression levels: a model for erbB-2 negative human breast cancer.

    Directory of Open Access Journals (Sweden)

    Sara Santos

    Full Text Available Human ERBB2 is a proto-oncogene that codes for the erbB-2 epithelial growth factor receptor. In human breast cancer (HBC, erbB-2 protein overexpression has been repeatedly correlated with poor prognosis. In more recent works, underexpression of this gene has been described in HBC. Moreover, it is also recognised that oncogenes that are commonly amplified or deleted encompass point mutations, and some of these are associated with HBC. In cat mammary lesions (CMLs, the overexpression of ERBB2 (27%-59.6% has also been described, mostly at the protein level and although cat mammary neoplasias are considered to be a natural model of HBC, molecular information is still scarce. In the present work, a cat ERBB2 fragment, comprising exons 10 to 15 (ERBB2_10-15 was achieved for the first time. Allelic variants and genomic haplotype analyses were also performed, and differences between normal and CML populations were observed. Three amino acid changes, corresponding to 3 non-synonymous genomic sequence variants that were only detected in CMLs, were proposed to damage the 3D structure of the protein. We analysed the cat ERBB2 gene at the DNA (copy number determination, mRNA (expression levels assessment and protein levels (in extra- and intra protein domains in CML samples and correlated the last two evaluations with clinicopathological features. We found a positive correlation between the expression levels of the ERBB2 RNA and erbB-2 protein, corresponding to the intracellular region. Additionally, we detected a positive correlation between higher mRNA expression and better clinical outcome. Our results suggest that the ERBB2 gene is post-transcriptionally regulated and that proteins with truncations and single point mutations are present in cat mammary neoplastic lesions. We would like to emphasise that the recurrent occurrence of low erbB-2 expression levels in cat mammary tumours, suggests the cat mammary neoplasias as a valuable model for erbB-2

  17. TPD52 represents a survival factor in ERBB2-amplified breast cancer cells.

    Science.gov (United States)

    Roslan, Nuruliza; Bièche, Ivan; Bright, Robert K; Lidereau, Rosette; Chen, Yuyan; Byrne, Jennifer A

    2014-10-01

    TPD52 and ERBB2 co-expression has been persistently reported in human breast cancer and animal models of this disease, but the significance of this is unknown. We identified significant positive associations between relative TPD52 and ERBB2 transcript levels in human diagnostic breast cancer samples, and maximal TPD52 expression in the hormone receptor (HR)- and ERBB2-positive sub-group. High-level TPD52 expression was associated with significantly reduced metastasis-free survival, within the overall cohort (log rank test, P = 8.6 × 10(-4), n = 375) where this was an independent predictor of metastasis-free survival (hazard ratio, 2.69, 95% confidence interval 1.59-4.54, P = 2.2 × 10(-4), n = 359), and the HR- and ERBB2-positive sub-group (log rank test, P = 0.035, n = 47). Transient TPD52 knock-down in the ERBB2-amplified breast cancer cell lines SK-BR-3 and BT-474 produced significant apoptosis, both singly and in combination with transient ERBB2 knock-down. Unlike ERBB2 knock-down, transient TPD52 knock-down produced no reduction in pAKT levels in SK-BR-3 or BT-474 cells. We then derived multiple SK-BR-3 cell lines in which TPD52 levels were stably reduced, and measured significant inverse correlations between pERBB2 and TPD52 levels in viable TPD52-depleted and control cell lines, all of which showed similar proliferative capacities. Our results therefore identify TPD52 as a survival factor in ERBB2-amplified breast cancer cells, and suggest complementary cellular functions for TPD52 and ERBB2.

  18. ErbB-2 blockade and prenyltransferase inhibition alter epidermal growth factor and epidermal growth factor receptor trafficking and enhance (111)In-DTPA-hEGF Auger electron radiation therapy.

    Science.gov (United States)

    Cornelissen, Bart; Darbar, Sonali; Hernandez, Rebecca; Kersemans, Veerle; Tullis, Iain; Barber, Paul R; Smart, Sean; Vojnovic, Borivoj; Reilly, Raymond; Vallis, Katherine A

    2011-05-01

    The intracellular distribution of Auger electron-emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of (111)In-labeled human epidermal growth factor ((111)In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of (111)In-DTPA-hEGF. The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. (111)In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2-blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of (111)In-DTPA-hEGF in MDA-MB-468 (1.3 × 10(6) EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 × 10(6) EGFR per cell; 0.4 × 10(5) ErbB-2 per cell) cell lines. The cytotoxicity of (111)In-DTPA-hEGF (0-64 nM) plus trastuzumab (0-50 μg/mL) or L-778,123 (0-22.5 μM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft-bearing mice treated with (111)In-DTPA-hEGF plus trastuzumab or L-788,123. Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand-receptor complex in MDA-MB-468 cells. Nuclear localization of (111)In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to (111)In-DTPA-hEGF. Nuclear localization and cytotoxicity of (111)In-DTPA-hEGF were significantly

  19. Regulation of ErbB2 localization and function in breast cancer cells by ERM proteins

    Science.gov (United States)

    Asp, Nagham; Kvalvaag, Audun; Sandvig, Kirsten; Pust, Sascha

    2016-01-01

    The ERM protein family is implicated in processes such as signal transduction, protein trafficking, cell proliferation and migration. Consequently, dysregulation of ERM proteins has been described to correlate with carcinogenesis of different cancer types. However, the underlying mechanisms are poorly understood. Here, we demonstrate a novel functional interaction between ERM proteins and the ErbB2 receptor tyrosine kinase in breast cancer cells. We show that the ERM proteins ezrin and radixin are associated with ErbB2 receptors at the plasma membrane, and depletion or functional inhibition of ERM proteins destabilizes the interaction of ErbB2 with ErbB3, Hsp90 and Ebp50. Accompanied by the dissociation of this protein complex, binding of ErbB2 to the ubiquitin-ligase c-Cbl is increased, and ErbB2 becomes dephosphorylated, ubiquitinated and internalized. Furthermore, signaling via Akt- and Erk-mediated pathways is impaired upon ERM inhibition. Finally, interference with ERM functionality leads to receptor degradation and reduced cellular levels of ErbB2 and ErbB3 receptors in breast cancer cells. PMID:27029001

  20. Effects of Neuregulins on Invasion and Metastasis of Non-overexpression ErbB2 Breast Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Ya-mei Zhang; Ting-ting Zhao; Hua-yu Deng

    2009-01-01

    Objective: To explore the effects of neuregulins on ErbB2 receptor signal transduction pathway activation, and invasion and metastasis of non-overexpression ErbB2 breast cancer cell MDA-MB-231. Methods: The expressions of neuregulin were detected by immunocytochemistry and Western blot. MDA-MB-231 cells were treated with ErbB2 kinase inhibitor AG825. Proliferations were measured with MTT assay. Invasion and metastasis of MDA-ME-231 cells were evaluated with transwell chamber. The enzyme activities of MMP-2 and MMP-9 were detected by gelatin zymography. The expressions of MMP-2 and HIF-1α were detected by Western blot.Results: MDA-MB-231 cells expressed a relatively higher level of neuregulin. In Western blot, the positive reaction band was found at 44KD which coincides with the molecular weight of NRG. When MDA-MB-231 cells were treated with AG825, the proliferation was inhibited in a time-dose-dependent manner (P<0.01), invasion and metastasis were also depressed (P<0.05). The enzyme activities of MMP-2 and MMP-9 were lower (P<0.05). The expression levels of MMP-2 and HIF-1α were decreased (P<0.05).Conclusion: Our study indicates that neuregulins are synthesized in MDA-MB-231 cells as transmembrane proteins, neuregulins could activate ErbB2 receptor signal transduction pathway by autocrine or paracrine secretion, and induce invasion and metastasis of MDA-MB-231 cells.

  1. Flotillins as regulators of ErbB2 levels in breast cancer

    DEFF Research Database (Denmark)

    Pust, S; Klokk, T I; Musa, N;

    2013-01-01

    Amplification and overexpression of the receptor tyrosine kinase ErbB2 occur in up to 30% of human breast cancers, and high ErbB2 levels are correlated with poor prognosis for breast cancer patients. In contrast to the epithelial growth factor receptor (ErbB1), ErbB2 is not downregulated by ligand...... from 194 patients diagnosed with carcinomas of the breast showed that flotillin-2 emerged as a potential predictor of prognosis in breast cancer. Depletion of flotillin-1 and flotillin-2 leads to internalization and degradation of ErbB2. Furthermore, flotillin-1 and -2 were found to be in a molecular......-induced mechanisms. Here we show that flotillins are involved in the stabilization of ErbB2 at the plasma membrane. In SKBR3 breast cancer cells and breast cancer tissue, a positive correlation between flotillin and ErbB2 expression levels could be demonstrated. Moreover, the tissue microarray analyses of biopsies...

  2. FASN, ErbB2-mediated glycolysis is required for breast cancer cell migration.

    Science.gov (United States)

    Zhou, Lan; Jiang, Sufang; Fu, Qiang; Smith, Kelly; Tu, Kailing; Li, Hua; Zhao, Yuhua

    2016-05-01

    Both fatty acid synthase (FASN) and ErbB2 have been shown to promote breast cancer cell migration. However, the underlying molecular mechanism remains poorly understood and there is no reported evidence that directly links glycolysis to breast cancer cell migration. In this study, we investigated the role of FASN, ErbB2-mediated glycolysis in breast cancer cell migration. First, we compared lactate dehydrogenase A (LDHA) protein levels, glycolysis and cell migration between FASN, ErbB2-overexpressing SK-BR-3 cells and FASN, ErbB2-low-expressing MCF7 cells. Then, SK-BR-3 cells were treated with cerulenin (Cer), an inhibitor of FASN, and ErbB2, LDHA protein levels, glycolysis, and cell migration were detected. Next, we transiently transfected ErbB2 plasmid into MCF7 cells and detected FASN, LDHA protein levels, glycolysis and cell migration. Heregulin-β1 (HRG-β1) is an activator of ErbB2 and 2-deoxyglucose (2-DG) and oxamate (OX) are inhibitors of glycolysis. MCF7 cells were treated with HRG-β1 alone, HRG-β1 plus 2-DG, OX or cerulenin and glycolysis, and cell migration were measured. We found that FASN, ErbB2-high-expressing SK-BR-3 cells displayed higher levels of glycolysis and migration than FASN, ErbB2-low-expressing MCF7 cells. Inhibition of FASN by cerulenin impaired glycolysis and migration in SK-BR-3 cells. Transient overexpression of ErbB2 in MCF7 cells promotes glycolysis and migration. Moreover, 2-deoxyglucose (2-DG), oxamate (OX), or cerulenin partially reverses heregulin-β1 (HRG-β1)-induced glycolysis and migration in MCF7 cells. In conclusion, this study demonstrates that FASN, ErbB2-mediated glycolysis is required for breast cancer cell migration. These novel findings indicate that targeting FASN, ErbB2-mediated glycolysis may be a new approach to reverse breast cancer cell migration.

  3. Chimeric DNA Vaccines against ErbB2{sup +} Carcinomas: From Mice to Humans

    Energy Technology Data Exchange (ETDEWEB)

    Quaglino, Elena; Riccardo, Federica; Macagno, Marco; Bandini, Silvio; Cojoca, Rodica; Ercole, Elisabetta [Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Turin, 10126 Turin (Italy); Amici, Augusto [Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (Italy); Cavallo, Federica, E-mail: federica.cavallo@unito.it [2 Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (Italy)

    2011-08-10

    DNA vaccination exploits a relatively simple and flexible technique to generate an immune response against microbial and tumor-associated antigens (TAAs). Its effectiveness is enhanced by the application of an electrical shock in the area of plasmid injection (electroporation). In our studies we exploited a sophisticated electroporation device approved for clinical use (Cliniporator, IGEA, Carpi, Italy). As the target antigen is an additional factor that dramatically modulates the efficacy of a vaccine, we selected ErbB2 receptor as a target since it is an ideal oncoantigen. It is overexpressed on the cell membrane by several carcinomas for which it plays an essential role in driving their progression. Most oncoantigens are self-tolerated molecules. To circumvent immune tolerance we generated two plasmids (RHuT and HuRT) coding for chimeric rat/human ErbB2 proteins. Their immunogenicity was compared in wild type mice naturally tolerant for mouse ErbB2, and in transgenic mice that are also tolerant for rat or human ErbB2. In several of these mice, RHuT and HuRT elicited a stronger anti-tumor response than plasmids coding for fully human or fully rat ErbB2. The ability of heterologous moiety to blunt immune tolerance could be exploited to elicit a significant immune response in patients. A clinical trial to delay the recurrence of ErbB2{sup +} carcinomas of the oral cavity, oropharynx and hypopharynx is awaiting the approval of the Italian authorities.

  4. Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand.

    Science.gov (United States)

    Vaidyanath, Arun; Hashizume, Toshihiro; Nagaoka, Tadahiro; Takeyasu, Nao; Satoh, Hitomi; Chen, Ling; Wang, Jiyou; Kasai, Tomonari; Kudoh, Takayuki; Satoh, Ayano; Fu, Li; Seno, Masaharu

    2011-11-01

    Targeting and down-regulation of ErbB2, a member of EGF receptor family, is regarded as one of the key aspect for cancer treatment because it is often overexpressed in breast and ovarian cancer cells. Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand. Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells. In this report, we designed divalent and multivalent forms of EC-1 peptide with the Fc portion of the human IgG and bionanocapsule modified with ZZ-tag on its surface to improve the interaction with ErbB2. These forms showed higher affinity to ErbB2 than that of EC-1 monomer. Furthermore, prominent endosomal accumulation of ErbB2 occurred in SK-BR-3 cells when stimulated with EC-Fc ligand multivalently displayed on the surface of the bionanocapsule, whereas SK-BR-3 cells as themselves displayed stringent mechanism against ErbB2 internalization without stimulation. The multivalent form of EC-1 peptide appeared to internalize ErbB2 more efficiently than divalent form did. This internalization was unaffected by the inhibition of clathrin association, but inhibited when the cholesterol was depleted which explained either caveolar or GPI-AP-early endocytic compartment (GEEC) pathway. Because of the lack of caveolin-1 expression, caveolar machinery may be lost in SK-BR-3 cell line. Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.

  5. ERBB2 increases metastatic potentials specifically in androgen-insensitive prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Jessica Tome-Garcia

    Full Text Available Despite all the blood-based biomarkers used to monitor prostate cancer patients, prostate cancer remains as the second common cause of cancer mortality in men in the United States. This is largely due to a lack of understanding of the molecular pathways that are responsible for the aggressive forms of prostate cancers, the castrate-resistant prostate cancer and the metastatic prostate cancer. Cell signaling pathways activated by the ERBB2 oncogene or the RAS oncogene are frequently found to be altered in metastatic prostate cancers. To evaluate and define the role of the ERBB2/RAS pathway in prostate cancer metastasis, we have evaluated the impact of ERBB2- or RAS-overexpression on the metastatic potentials for four prostate cancer cell lines derived from tumors with different androgen sensitivities. To do so, we transfected the human DU145, LnCaP, and PC3 prostate cancer cells and the murine Myc-CaP prostate cancer cells with the activated form of ERBB2 or H-RAS and assessed their metastatic potentials by three complementary assays, a wound healing assay, a transwell motility assay, and a transwell invasion assay. We showed that while overexpression of ERBB2 increased the metastatic potential of the androgen-insensitive prostate cancer cells (i.e. PC3 and DU145, it did not affect metastatic potentials of the androgen-sensitive prostate cancer cells (i.e. LnCaP and Myc-CaP. In contrast, overexpression of H-RAS only increased the cell motility of Myc-CaP cells, which overexpress the human c-MYC oncogene. Our data suggest that ERBB2 collaborates with androgen signaling to promote prostate cancer metastasis, and that although RAS is one of the critical downstream effectors of ERBB2, it does not phenocopy ERBB2 for its impact on the metastatic potentials of prostate cancer cell lines.

  6. Constitutively active ErbB2 regulates cisplatin-induced cell death in breast cancer cells via pro- and antiapoptotic mechanisms

    DEFF Research Database (Denmark)

    Sigurðsson, Haraldur H; Olesen, Christina Wilkens; Dybboe, Rie

    2015-01-01

    UNLABELLED: Despite the frequent expression of N-terminally truncated ErbB2 (ΔNErbB2/p95HER2) in breast cancer and its association with Herceptin resistance and poor prognosis, it remains poorly understood how ΔNErbB2 affects chemotherapy-induced cell death. Previously it was shown that ΔNErbB2...... upregulates acid extrusion from MCF-7 breast cancer cells and that inhibition of the Na(+)/H(+) exchanger (SLC9A1/NHE1) strongly sensitizes ΔNErbB2-expressing MCF-7 cells to cisplatin chemotherapy. The aim of this study was to identify the mechanism through which ΔNErbB2 regulates cisplatin-induced breast...... cancer cell death, and determine how NHE1 regulates this process. Cisplatin treatment elicited apoptosis, ATM phosphorylation, upregulation of p53, Noxa (PMAIP1), and PUMA (BBC3), and cleavage of caspase-9, -7, fodrin, and PARP-1 in MCF-7 cells. Inducible ΔNErbB2 expression strongly reduced cisplatin...

  7. ErbB2 overexpression upregulates antioxidant enzymes, reduces basal levels of reactive oxygen species, and protects against doxorubicin cardiotoxicity.

    Science.gov (United States)

    Belmonte, Frances; Das, Samarjit; Sysa-Shah, Polina; Sivakumaran, Vidhya; Stanley, Brian; Guo, Xin; Paolocci, Nazareno; Aon, Miguel A; Nagane, Masaki; Kuppusamy, Periannan; Steenbergen, Charles; Gabrielson, Kathleen

    2015-10-01

    Levels of the HER2/ErbB2 protein in the heart are upregulated in some women during breast cancer therapy, and these women are at high risk for developing heart dysfunction after sequential treatment with anti-ErbB2/trastuzumab or doxorubicin. Doxorubicin is known to increase oxidative stress in the heart, and thus we considered the possibility that ErbB2 protein influences the status of cardiac antioxidant defenses in cardiomyocytes. In this study, we measured reactive oxygen species (ROS) in cardiac mitochondria and whole hearts from mice with cardiac-specific overexpression of ErbB2 (ErbB2(tg)) and found that, compared with control mice, high levels of ErbB2 in myocardium result in lower levels of ROS in mitochondria (P = 0.0075) and whole hearts (P = 0.0381). Neonatal cardiomyocytes isolated from ErbB2(tg) hearts have lower ROS levels and less cellular death (P antioxidant enzyme levels and activities, we found that ErbB2(tg) hearts have increased levels of glutathione peroxidase 1 (GPx1) protein (P levels of two known GPx activators, c-Abl (P = 0.0284) and Arg (P antioxidant defenses and suggests a novel mechanism by which anticancer therapies involving ErbB2 antagonists can harm myocardial structure and function. Copyright © 2015 the American Physiological Society.

  8. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

    DEFF Research Database (Denmark)

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne

    2013-01-01

    Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal...... tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor...... + 1,083 blocks), were determined by quantitative reverse transcription polymerase chain reaction for all samples, as well as by microarray for 133 validation samples. In the training set, agreement between high vs. low mRNA expression from whole slide and tumor-enriched sections was absolute for ESR1...

  9. Cooperación oncogénica entre la citoquina pro-inflamatoria SP y el receptor tirosina quinasa ErbB-2/HER2

    OpenAIRE

    Garcia Recio, Susana

    2012-01-01

    INTRODUCCIÓN: La inflamación es un sello distintivo del cáncer. La inflamación crónica está fuertemente relacionada con el desarrollo y la progresión tumoral y suministra al tumor múltiples citoquinas proinflamatorias que pueden modular la señalización de la célula tumoral. Este es el caso de la Sustancia P (SP), una citoquina pro-inflamatoria/neuropéptido presente en el microambiente tumoral perteneciente a la familia de las taquiquininas y que señaliza principalmente a través de su receptor...

  10. AST1306, a novel irreversible inhibitor of the epidermal growth factor receptor 1 and 2, exhibits antitumor activity both in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Hua Xie

    Full Text Available Despite the initial response to the reversible, ATP-competitive quinazoline inhibitors that target ErbB-family, such a subset of cancer patients almost invariably develop resistance. Recent studies have provided compelling evidence that irreversible ErbB inhibitors have the potential to override this resistance. Here, we found that AST1306, a novel anilino-quinazoline compound, inhibited the enzymatic activities of wild-type epidermal growth factor receptor (EGFR and ErbB2 as well as EGFR resistant mutant in both cell-free and cell-based systems. Importantly, AST1306 functions as an irreversible inhibitor, most likely through covalent interaction with Cys797 and Cys805 in the catalytic domains of EGFR and ErbB2, respectively. Further studies showed that AST1306 inactivated pathways downstream of these receptors and thereby inhibited the proliferation of a panel of cancer cell lines. Although the activities of EGFR and ErbB2 were similarly sensitive to AST1306, ErbB2-overexpressing cell lines consistently exhibited more sensitivity to AST1306 antiproliferative effects. Consistent with this, knockdown of ErbB2, but not EGFR, decreased the sensitivity of SK-OV-3 cells to AST1306. In vivo, AST1306 potently suppressed tumor growth in ErbB2-overexpressing adenocarcinoma xenograft and FVB-2/N(neu transgenic breast cancer mouse models, but weakly inhibited the growth of EGFR-overexpressing tumor xenografts. Tumor growth inhibition induced by a single dose of AST1306 in the SK-OV-3 xenograft model was accompanied by a rapid (within 2 h and sustained (≥24 h inhibition of both EGFR and ErbB2, consistent with an irreversible inhibition mechanism. Taken together, these results establish AST1306 as a selective, irreversible ErbB2 and EGFR inhibitor whose growth-inhibitory effects are more potent in ErbB2-overexpressing cells.

  11. Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition

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    Flores Juana M

    2010-07-01

    Full Text Available Abstract Background ErbB2-positive breast cancer is characterized by highly aggressive phenotypes and reduced responsiveness to standard therapies. Although specific ErbB2-targeted therapies have been designed, only a small percentage of patients respond to these treatments and most of them eventually relapse. The existence of this population of particularly aggressive and non-responding or relapsing patients urges the search for novel therapies. The purpose of this study was to determine whether cannabinoids might constitute a new therapeutic tool for the treatment of ErbB2-positive breast tumors. We analyzed their antitumor potential in a well established and clinically relevant model of ErbB2-driven metastatic breast cancer: the MMTV-neu mouse. We also analyzed the expression of cannabinoid targets in a series of 87 human breast tumors. Results Our results show that both Δ9-tetrahydrocannabinol, the most abundant and potent cannabinoid in marijuana, and JWH-133, a non-psychotropic CB2 receptor-selective agonist, reduce tumor growth, tumor number, and the amount/severity of lung metastases in MMTV-neu mice. Histological analyses of the tumors revealed that cannabinoids inhibit cancer cell proliferation, induce cancer cell apoptosis, and impair tumor angiogenesis. Cannabinoid antitumoral action relies, at least partially, on the inhibition of the pro-tumorigenic Akt pathway. We also found that 91% of ErbB2-positive tumors express the non-psychotropic cannabinoid receptor CB2. Conclusions Taken together, these results provide a strong preclinical evidence for the use of cannabinoid-based therapies for the management of ErbB2-positive breast cancer.

  12. ErbB2 inhibition by lapatinib promotes degradation of mutant p53 protein in cancer cells.

    Science.gov (United States)

    Li, Dun; Marchenko, Natalia D

    2017-01-24

    Mutations in the p53 tumor suppressor gene are the most prevalent genetic events in human Her2-positive breast cancer and are associated with poor prognosis and survival. Human clinical data and our in vitro and in vivo studies strongly suggest potent oncogenic cooperation between mutant p53 and Her2 (ErbB2). Yet, the translational significance of mutant p53 in Her2 positive breast cancer, especially with respect to Her2-targeted therapies, has not been evaluated. Our previous work identified novel oncogenic activity of mutant p53 whereby mutp53 amplifies ErbB2 signaling via the mutp53-HSF1-ErbB2 feed-forward loop. Here we report that pharmacological interception of this circuit by ErbB2 inhibitor lapatinib downregulates mutant p53 in vitro and in vivo. We found that ErbB2 inhibition by lapatinib inhibits transcription factor HSF1, and its target Hsp90, followed by mutant p53 degradation in MDM2 dependent manner. Thus, our data suggest that mutant p53 sensitizes cancer cells to lapatinib via two complementary mechanisms: mutant p53 mediated amplification of ErbB2 signaling, and simultaneous annihilation of both potent oncogenic drivers, ErbB2 and mutant p53. Hence, our study could provide valuable information for the optimization of therapeutic protocols to achieve superior clinical effects in the treatment of Her2 positive breast cancer.

  13. Pertuzumab Increases 17-AAG-Induced Degradation of ErbB2, and This Effect Is Further Increased by Combining Pertuzumab with Trastuzumab

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    Juliana Bentes Hughes

    2012-06-01

    Full Text Available ErbB2 is an important oncogenic protein involved in carcinogenesis of, among others, breast, gastric, and ovarian carcinoma. Over-expression of ErbB2 is found in almost 20% of breast cancers, and this results in proliferative and anti-apoptotic signalling. ErbB2 is therefore an important treatment target. Antibodies recognizing full-length ErbB2 are clinically established, and drugs targeting the ErbB2 stabilizing heat shock protein 90 (Hsp90 are under clinical evaluation. We have investigated effects of the ErbB2-binding antibodies trastuzumab and pertuzumab alone and in combination, as well as the effect of the antibodies in combination with the Hsp90 inhibitor 17-AAG. Our results confirm the notion that combination of different ErbB2-binding antibodies more efficiently down-regulates ErbB2 than does one antibody in isolation. Additionally, our data demonstrate that ErbB2 is most efficiently down-regulated upon incubation with anti-ErbB2 antibodies in combination with Hsp90 inhibitors. The combination of anti-ErbB2 antibodies, and especially the combination of antibodies with 17-AAG, did also increase the inhibition of Akt activation of either agent, which could suggest an anti-proliferative effect. In such case, combining these agents could be beneficial in treatment of tumors not responding to trastuzumab only.

  14. Broadening of epitope recognition during immune rejection of ErbB-2-positive tumor prevents growth of ErbB-2-negative tumor.

    Science.gov (United States)

    Pilon, Shari A; Kelly, Carmen; Wei, Wei-Zen

    2003-02-01

    Tumor heterogeneity is a limiting factor in Ag-specific vaccination. Ag-negative variants may arise after tumor cells bearing the immunizing Ags are destroyed. In situ priming to tumor-associated epitopes distinct from and not cross-reactive with the immunizing Ags may be crucial to the ultimate success of cancer vaccination. Immunization of BALB/c mice with DNA encoding wild-type human ErbB-2 (Her-2/neu, E2) or cytoplasmic ErbB-2 (cytE2), activated primarily CD4 or CD8 T cells, respectively, and both vaccines protected against ErbB-2-positive D2F2/E2 tumors. In > or =50% of protected mice, a second challenge of ErbB-2-negative D2F2 tumor cells was rejected. Recognition of non-ErbB-2, tumor-associated Ags was demonstrated by immune cell proliferation upon stimulation with irradiated D2F2 cells. This broadening of epitope recognition was abolished if CD4 T cells were depleted before D2F2/E2 tumor challenge, demonstrating their critical role in Ag priming. Similarly, mice that rejected D2F2/cytE2 tumor cells, which express only MHC I epitopes of ErbB-2, were not protected from a second challenge with D2F2 cells. Depletion of CD8 T cells abolished protection against D2F2, indicating the activation of D2F2-specific CTL. Therefore, long term protection may be achieved by immunization with dominant Ag(s), followed by a general enhancement of CD4 T cell activity to promote priming to multiple tumor-associated Ags.

  15. Expression and function of Neuregulin 1 and its signaling system ERBB2/3 in the enteric nervous system

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    Martina eBarrenschee

    2015-09-01

    Full Text Available Neuregulin 1 (NRG1 is suggested to promote the survival and maintenance of the enteric nervous system (ENS. As deficiency in its corresponding receptor signaling complex ERBB2/ERBB3 leads to postnatal colonic hypo/aganglionosis we assessed the distributional and expressional pattern of the NRG1-ERBB2/ERBB3 system in the human colon and explored the neurotrophic capacity of NRG1 on cultured enteric neurons.Site-specific mRNA expression of the NRG1-ERBB2/3 system was determined in microdissected samples harvested from enteric musculature and ganglia. Localization of NRG1, ERBB2 and ERBB3 was determined by dual-label-immunohistochemistry using pan-neuronal and pan-glial markers. Morphometric analysis was performed on NRG1-stimulated rat enteric nerve cultures to evaluate neurotrophic effects. mRNA expression of the NRG1-ERBB2/3 system was determined by qPCR. Co-localization of NRG1 with neuronal or synaptic markers was analyzed in enteric nerve cultures stimulated with glial cell line-derived neurotrophic factor (GDNF. The NRG1 system was expressed in both neurons and glial cells of enteric ganglia and in nerve fibers. NRG1 significantly enhanced growth parameters in enteric nerve cell cultures and ErB3 mRNA expression was down-regulated upon NRG1 stimulation. GDNF negatively regulates ErbB2 and ErbB3 mRNA expressionThe NRG1-ERBB2/3 system is physiologically present in the human ENS and NRG1 acts as a neurotrophic factor for the ENS. The down-regulation of ErbB3/ErbB2 in GDNF stimulated nerve cell cultures points to an interaction of both neurotrophic factors. Thus, the data may provide a basis to assess disturbed signaling components of the NRG1 system in enteric neuropathies.

  16. erbB-2 autophosphorylation is required for mitogenic action and high-affinity substrate coupling

    DEFF Research Database (Denmark)

    Segatto, O; Lonardo, F; Helin, K;

    1992-01-01

    -mutated chimera. When expressed in NR6 cells, the EGFR/erbB-2 5P mutant was unable to deliver a sizeable mitogenic signal when activated by EGF at physiological levels. In intact cells, the 5P mutant was still able to stimulate phosphorylation of the gamma isozyme of phospholipase C (PLC-gamma), a prototype erb......B-2 substrate, although with a delayed time course, indicating that the 5P mutation decreased the affinity of the erbB-2 kinase for this substrate. This conclusion was further supported by the inability of the 5P mutant to associate with PLC-gamma in co-immunoprecipitation experiments. We infer...

  17. Development of Specific Inhibitors for Breast Cancer-Associated Variants of ErbB2

    Science.gov (United States)

    2015-10-01

    34 conformation ( Ishikawa et al, J Med Chem, 2011, 54, 8030- 8050). We completed the coordinates of ErbB2 with corresponding residues from EGFR coordinates and...activation states and have been refined with restrained MD simulations. As an example of a completed model, Figure 2 shows a ribbon diagram for D769Y

  18. Examining ERBB2 as a candidate gene for susceptibility to leprosy (Hansen's disease) in Brazil.

    Science.gov (United States)

    Araújo, Sérgio Ricardo Fernandes; Jamieson, Sarra Elisabeth; Dupnik, Kathryn Margaret; Monteiro, Glória Regina; Nobre, Maurício Lisboa; Dias, Márcia Sousa; Trindade Neto, Pedro Bezerra; Queiroz, Maria do Carmo Palmeira; Gomes, Carlos Eduardo Maia; Blackwell, Jenefer Mary; Jeronimo, Selma Maria Bezerra

    2014-04-01

    Leprosy remains prevalent in Brazil. ErbB2 is a receptor for leprosy bacilli entering Schwann cells, which mediates Mycobacterium leprae-induced demyelination and the ERBB2 gene lies within a leprosy susceptibility locus on chromosome 17q11-q21. To determine whether polymorphisms at the ERBB2 locus contribute to this linkage peak, three haplotype tagging single nucleotide polymorphisms (tag-SNPs) (rs2517956, rs2952156, rs1058808) were genotyped in 72 families (208 cases; 372 individuals) from the state of Pará (PA). All three tag-SNPs were associated with leprosy per se [best SNP rs2517959 odds ratio (OR) = 2.22; 95% confidence interval (CI) 1.37-3.59; p = 0.001]. Lepromatous (LL) (OR = 3.25; 95% CI 1.37-7.70; p = 0.007) and tuberculoid (TT) (OR = 1.79; 95% CI 1.04-3.05; p = 0.034) leprosy both contributed to the association, which is consistent with the previous linkage to chromosome 17q11-q21 in the population from PA and supports the functional role of ErbB2 in disease pathogenesis. To attempt to replicate these findings, six SNPs (rs2517955, rs2517956, rs1810132, rs2952156, rs1801200, rs1058808) were genotyped in a population-based sample of 570 leprosy cases and 370 controls from the state of Rio Grande do Norte (RN) and the results were analysed using logistic regression analysis. However, none of the associations were replicated in the RN sample, whether analysed for leprosy per se, LL leprosy, TT leprosy, erythema nodosum leprosum or reversal reaction conditions. The role of polymorphisms at ERBB2 in controlling susceptibility to leprosy in Brazil therefore remains unclear.

  19. Mechanisms of Trastuzumab resistance in ErbB2-driven breast cancer and newer opportunities to overcome therapy resistance

    Directory of Open Access Journals (Sweden)

    Tameka A Bailey

    2011-01-01

    Full Text Available The Human Epidermal Growth Factor Receptor 2 (Her2, ErbB2 or Neu is overexpressed in about 20 - 25% of breast cancers and is causally linked to oncogenesis, providing opportunities for targeted therapy. Trastuzumab (Herceptin™, Genentech Inc, San Francisco, CA, a humanized monoclonal antibody against ErbB2, is a successful example of this concept and has vastly improved the response to treatment and overall survival in a majority of ErbB2+ breast cancer patients. However, lack of response in some patients as well as relapse during the course of therapy in others, continue to challenge researchers and clinicians alike towards a better understanding of the fundamental mechanisms of Trastuzumab action and resistance to treatment. The exact in vivo mechanism of action of Trastuzumab remains enigmatic, given its direct effects on the ErbB2 signaling pathway as well as indirect contributions from the immune system, by virtue of the ability of Trastuzumab to elicit Antibody-Dependent Cellular Cytotoxicity. Consequently, multiple mechanisms of resistance have been proposed. We present here a comprehensive review of our current understanding of the mechanisms, both of Trastuzumab action and clinical resistance to Trastuzumab-based therapies. We also review newer strategies (based on ErbB2 receptor biology that are being explored to overcome resistance to Trastuzumab therapy.

  20. erbB2 Overexpression in Uterine Serous Cancer: A Molecular Target for Trastuzumab Therapy

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    Karim S. ElSahwi

    2011-01-01

    Full Text Available Endometrial cancer is the most common female genital tract malignancy in the United States. Type I endometrial cancer is usually diagnosed at an early stage, and has a good prognosis. Type II is very aggressive, and is responsible for most uterine cancer relapses and deaths. Uterine serous adenocarcinomas (USC constitute the majority of Type II variants. They have a higher propensity for lymph node and distant metastases. They are frequently aneuploid and associated with p53 mutations. erbB2 overexpression in USC has been described. The incidence, which is higher in African Americans, ranges from 18–80%. erbB2 overexpression was found to be associated with higher stage, chemoresistance, and worse survival. Trastuzumab a humanized mAb was approved by the FDA for treatment of breast cancers that overexpress erbB2 in combination with standard chemotherapy. Evidence of trastuzumab activity in USC has been reported in vitro, as well as in case reports of advanced and recurrent cases. Promising results were obtained in these heavily pretreated patients either with trastuzumab alone or in combination with chemotherapy. This supports the hypothesis that trastuzumab may very well be an attractive and viable treatment option for advanced stage USC tumors that overexpress the erbB2, and is worthy of further study.

  1. The role of ERBB2 gene polymorphisms in leprosy susceptibility

    Directory of Open Access Journals (Sweden)

    Jamile Leão Rêgo

    Full Text Available Mycobacterium leprae infects skin and peripheral nerves causing deformities and disability. The M. leprae bacterium binds to ErbB2 on the Schwann cell surface causing demyelination and favoring spread of the bacilli and causing nerve injury. Polymorphisms at the ERBB2 gene were previously investigated as genetic risk factors for leprosy in two Brazilian populations but with inconsistent results. Herein we extend the analysis of ERBB2 variants to a third geographically distinct population in Brazil. Our results show that there is no association between the genotyped SNPs and the disease (p > 0.05 in this population. A gene set or pathway analysis under the genomic region of ERBB2 will be necessary to clarify its regulation under M. leprae stimulus.

  2. Nf2/Merlin controls spinal cord neural progenitor function in a Rac1/ErbB2-dependent manner.

    Directory of Open Access Journals (Sweden)

    Cynthia Garcia

    Full Text Available OBJECTIVE: Individuals with the neurofibromatosis type 2 (NF2 cancer predisposition syndrome develop spinal cord glial tumors (ependymomas that likely originate from neural progenitor cells. Whereas many spinal ependymomas exhibit indolent behavior, the only treatment option for clinically symptomatic tumors is surgery. In this regard, medical therapies are unfortunately lacking due to an incomplete understanding of the critical growth control pathways that govern the function of spinal cord (SC neural progenitor cells (NPCs. METHODS: To identify potential therapeutic targets for these tumors, we leveraged primary mouse Nf2-deficient spinal cord neural progenitor cells. RESULTS: We demonstrate that the Nf2 protein, merlin, negatively regulates spinal neural progenitor cell survival and glial differentiation in an ErbB2-dependent manner, and that NF2-associated spinal ependymomas exhibit increased ErbB2 activation. Moreover, we show that Nf2-deficient SC NPC ErbB2 activation results from Rac1-mediated ErbB2 retention at the plasma membrane. SIGNIFICANCE: Collectively, these findings establish ErbB2 as a potential rational therapeutic target for NF2-associated spinal ependymoma.

  3. The scaffold protein p140Cap limits ERBB2-mediated breast cancer progression interfering with Rac GTPase-controlled circuitries

    Science.gov (United States)

    Grasso, Silvia; Chapelle, Jennifer; Salemme, Vincenzo; Aramu, Simona; Russo, Isabella; Vitale, Nicoletta; Verdun di Cantogno, Ludovica; Dallaglio, Katiuscia; Castellano, Isabella; Amici, Augusto; Centonze, Giorgia; Sharma, Nanaocha; Lunardi, Serena; Cabodi, Sara; Cavallo, Federica; Lamolinara, Alessia; Stramucci, Lorenzo; Moiso, Enrico; Provero, Paolo; Albini, Adriana; Sapino, Anna; Staaf, Johan; Di Fiore, Pier Paolo; Bertalot, Giovanni; Pece, Salvatore; Tosoni, Daniela; Confalonieri, Stefano; Iezzi, Manuela; Di Stefano, Paola; Turco, Emilia; Defilippi, Paola

    2017-01-01

    The docking protein p140Cap negatively regulates tumour cell features. Its relevance on breast cancer patient survival, as well as its ability to counteract relevant cancer signalling pathways, are not fully understood. Here we report that in patients with ERBB2-amplified breast cancer, a p140Cap-positive status associates with a significantly lower probability of developing a distant event, and a clear difference in survival. p140Cap dampens ERBB2-positive tumour cell progression, impairing tumour onset and growth in the NeuT mouse model, and counteracting epithelial mesenchymal transition, resulting in decreased metastasis formation. One major mechanism is the ability of p140Cap to interfere with ERBB2-dependent activation of Rac GTPase-controlled circuitries. Our findings point to a specific role of p140Cap in curbing the aggressiveness of ERBB2-amplified breast cancers and suggest that, due to its ability to impinge on specific molecular pathways, p140Cap may represent a predictive biomarker of response to targeted anti-ERBB2 therapies. PMID:28300085

  4. Trigeminal nerve injury ErbB3/ErbB2 promotes mechanical hypersensitivity.

    Science.gov (United States)

    Ma, Fei; Zhang, Liping; Westlund, Karin N

    2012-08-01

    Chronic constriction injury of the trigeminal infraorbital nerve results in transient analgesia followed by whisker pad mechanical allodynia in rats. Neuregulin 1 expressed on axonal membranes binds receptor tyrosine kinase ErbB, promoting Schwann cell development and remyelination. This study investigated whether orofacial mechanical allodynia is signaled by ErbB3-ErbB2 heterodimers in injured nerves. Whisker pad mechanical allodynia (von Frey stimuli) was quantified in wild type rats and in transgenic rats with Sleeping Beauty transposon mutation for neuregulin 1 transgene. Pain-related behavior was retested after intraperitoneal injection of the ErbB2 inhibitor Lapatinib, an agent shown by others to reduce breast cancer pain. Infraorbital nerve injury was evaluated histologically with myelin and neuronal biomarkers. ErbB3 changes over time were measured with western blots. Whisker pad mechanical hypersensitivity began in week 2 in wild type rats (3.11 ± 5.93 g vs. 18.72 ± 0.00 g after sham surgery, n = 9, P wild type rats was alleviated by Lapatinib (15 ± 3.89 g vs. 2.45 ± 1.13 g, n = 6, P wild type and transgenic rats (week 10) coincided with time points when mechanical hypersensitivity was present. The Neuregulin 1-ErbB3-ErbB2 complex is a causal mechanism in nerve injury-induced trigeminal neuropathic pain. Understanding peripheral glial mechanisms after nerve injury will improve neuropathic pain treatment.

  5. Gene amplification and immunohistochemical expression of ERBB2 and EGFR in cervical carcinogenesis. Correlation with cell-cycle markers and HPV presence.

    Science.gov (United States)

    Conesa-Zamora, Pablo; Torres-Moreno, Daniel; Isaac, María A; Pérez-Guillermo, Miguel

    2013-10-01

    Although the members of the epidermal growth factor receptor family ERBB2 and EGFR are important therapeutic targets in the treatment of malignant neoplasias, little is known about their role in cervical carcinogenesis. Our objective was to evaluate the dysfunction of ERBB2 and EGFR at the gene copy number and protein expression level in neoplastic lesions of the uterine cervix with the aim of obtaining information about its role in cervical carcinogenesis and their possible use as therapeutic targets in these diseases. We studied gene amplification and protein expression of ERBB2 and EGFR and their relationship with Ki67, p16 and p53 and HPV presence in 22 normal/benign (N/B) cervices, 20 low-grade squamous intraepithelial lesions (LSILs), 70 high-grade SILs (HSILs) and 32 invasive squamous cervical carcinomas (ISCCs). No cases showed selective amplification of ERBB2 or EGFR but corresponding chromosome-specific probes displayed chromosome 17 and 7 polyploidy associated with the grade of the lesion (plesions (N/B plus LSIL 21.4% vs. HSIL plus ISCC 45.5%; p=0.007). No association was observed between EGFR expression and that of cell-cycle markers or HPV presence. Increased copy number of EGFR and ERBB2 is due to polyploidy of 7 and 17 chromosomes, this being a phenomenon associated with lesion severity and with an increase in the expression of cell-cycle markers. EGFR, but not ERBB2, is expressed in precursor lesions of squamous cervical neoplasia and is related to the neoplastic progression but not to proliferation marker expression and therefore ERBB2 and this calls into question the usefulness of ERBB2 as a therapeutic target.

  6. Examining ERBB2 as a candidate gene for susceptibility to leprosy (Hansen’s disease in Brazil

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    Sérgio Ricardo Fernandes Araújo

    2014-04-01

    Full Text Available Leprosy remains prevalent in Brazil. ErbB2 is a receptor for leprosy bacilli entering Schwann cells, which mediates Mycobacterium leprae-induced demyelination and the ERBB2 gene lies within a leprosy susceptibility locus on chromosome 17q11-q21. To determine whether polymorphisms at the ERBB2 locus contribute to this linkage peak, three haplotype tagging single nucleotide polymorphisms (tag-SNPs (rs2517956, rs2952156, rs1058808 were genotyped in 72 families (208 cases; 372 individuals from the state of Pará (PA. All three tag-SNPs were associated with leprosy per se [best SNP rs2517959 odds ratio (OR = 2.22; 95% confidence interval (CI 1.37-3.59; p = 0.001]. Lepromatous (LL (OR = 3.25; 95% CI 1.37-7.70; p = 0.007 and tuberculoid (TT (OR = 1.79; 95% CI 1.04-3.05; p = 0.034 leprosy both contributed to the association, which is consistent with the previous linkage to chromosome 17q11-q21 in the population from PA and supports the functional role of ErbB2 in disease pathogenesis. To attempt to replicate these findings, six SNPs (rs2517955, rs2517956, rs1810132, rs2952156, rs1801200, rs1058808 were genotyped in a population-based sample of 570 leprosy cases and 370 controls from the state of Rio Grande do Norte (RN and the results were analysed using logistic regression analysis. However, none of the associations were replicated in the RN sample, whether analysed for leprosy per se, LL leprosy, TT leprosy, erythema nodosum leprosum or reversal reaction conditions. The role of polymorphisms at ERBB2 in controlling susceptibility to leprosy in Brazil therefore remains unclear.

  7. Clinical relevance of ErbB-2/HER2 nuclear expression in breast cancer

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    Schillaci Roxana

    2012-02-01

    Full Text Available Abstract Background The biological relevance of nuclear ErbB-2/HER2 (NuclErbB-2 presence in breast tumors remains unexplored. In this study we assessed the clinical significance of ErbB-2 nuclear localization in primary invasive breast cancer. The reporting recommendations for tumor marker prognostic studies (REMARK guidelines were used as reference. Methods Tissue microarrays from a cohort of 273 primary invasive breast carcinomas from women living in Chile, a Latin American country, were examined for membrane (MembErbB-2 and NuclErbB-2 expression by an immunofluorescence (IF protocol we developed. ErbB-2 expression was also evaluated by immunohistochemistry (IHC with a series of antibodies. Correlation between NuclErbB-2 and MembErbB-2, and between NuclErbB-2 and clinicopathological characteristics of tumors was studied. The prognostic value of NuclErbB-2 in overall survival (OS was evaluated using Kaplan-Meier method, and Cox model was used to explore NuclErbB-2 as independent prognostic factor for OS. Results The IF protocol we developed showed significantly higher sensitivity for detection of NuclErbB-2 than IHC procedures, while its specificity and sensitivity to detect MembErbB-2 were comparable to those of IHC procedures. We found 33.6% NuclErbB-2 positivity, 14.2% MembErbB-2 overexpression by IF, and 13.0% MembErbB-2 prevalence by IHC in our cohort. We identified NuclErbB-2 positivity as a significant independent predictor of worse OS in patients with MembErbB-2 overexpression. NuclErbB-2 was also a biomarker of lower OS in tumors that overexpress MembErbB-2 and lack steroid hormone receptors. Conclusions We revealed a novel role for NuclErbB-2 as an independent prognostic factor of poor clinical outcome in MembErbB-2-positive breast tumors. Our work indicates that patients presenting NuclErbB-2 may need new therapeutic strategies involving specific blockage of ErbB-2 nuclear migration.

  8. Inhibition of ErbB-2 induces TFF3 downregulation in breast cancer cell lines.

    Science.gov (United States)

    Yue, Lu; Xiang, Jinyu; Shen, Zan; Wang, Zhihao; Yao, Yasai; Zhou, Quan; Ding, Aiping; Qiu, Wensheng

    2014-07-01

    ErbB-2 gene plays an important role in carcinoma formation whose overexpression was observed in many types of tumors, including breast cancer. Dysregulation of Trefoil factor 3 (TFF3), which is thought to function in the development and progression of breast cancer, was found to be upregulated in ErbB2-overexpressing breast cancers and cells. However, a putative interaction between ErbB-2 and TFF3 in breast cancer remains unknown. To determine whether TFF3 has an important role in breast tumor, its levels were measured by immunohistochemistry in 130 cases of breast infiltrating duct carcinoma and 30 cases of normal breast tissue with a specific monoclonal antibody raised against human TFF3. Patients who were positive for ErbB-2 also had high expression levels of TFF3 (p TFF3 by real-time polymerase chain reaction and Western blotting, respectively. Compared with the control groups, ErbB-2 mRNA expression was decreased in the Lenti-ShERBB2 infection group, and Western blotting indicated a concordant ErbB-2 protein reduction. On the other hand, TFF3 expression at both mRNA and protein levels was significantly downregulated by ErbB-2 silencing in SK-BR-3. These findings are a proof of the foundation for a certain relationships of ErbB-2 and TFF3, which may serve as novel therapeutic markers of ErbB2-overexpressing breast cancers in the future.

  9. Genetic Polymorphisms in the EGFR (R521K and Estrogen Receptor (T594T Genes, EGFR and ErbB-2 Protein Expression, and Breast Cancer Risk in Tunisia

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    Imen Kallel

    2009-01-01

    Full Text Available We evaluated the association of epidermal growth factor receptor (EGFR 142285G>A (R521K and estrogen receptor alpha (ESR1 2014G>A (T594T single nucleotide polymorphisms with breast cancer risk and prognosis in Tunisian patients. EGFR 142285G>A and ESR1 2014G>A were genotyped in a sample of 148 Tunisian breast cancer patients and 303 controls using PCR-RFLP method. Immunohistochemitsry was used to evaluate the expression levels of EGFR, HER2, ESR1, progesterone receptor and BCL2 in tumors. We found no evidence for an association between EGFR R521K polymorphism and breast cancer risk. However, we found that the homozygous GG (Arg genotype was more prevalent in patients with lymph node metastasis (=.03 and high grade tumors (=.011. The ESR1 2014G allele showed significant association with breast cancer risk (=.025. The GG genotype was associated with HER2 overexpression and this association withstood univariate and multivariate analyses (=.009; =.021, resp.. These data suggest that the R521K might be a prognostic factor, because it correlates with both tumor grade and nodule status. The higher expression of HER2 in ESR1 T594T GG patients suggests the possibility that ESR1 gene polymorphisms accompanied by HER2 expression might influence the pathogenesis of breast cancers.

  10. The glucose transporter GLUT1 is required for ErbB2-induced mammary tumorigenesis.

    Science.gov (United States)

    Wellberg, Elizabeth A; Johnson, Stevi; Finlay-Schultz, Jessica; Lewis, Andrew S; Terrell, Kristina L; Sartorius, Carol A; Abel, E Dale; Muller, William J; Anderson, Steven M

    2016-12-20

    Altered tumor cell metabolism is an emerging hallmark of cancer; however, the precise role for glucose in tumor initiation is not known. GLUT1 (SLC2A1) is expressed in breast cancer cells and is likely responsible for avid glucose uptake observed in established tumors. We have shown that GLUT1 was necessary for xenograft tumor formation from primary mammary cells transformed with the polyomavirus middle-T antigen but that it was not necessary for growth after tumors had formed in vivo, suggesting a differential requirement for glucose depending on the stage of tumorigenesis. To determine whether GLUT1 is required early during mammary tumorigenesis, we crossed MMTV-NIC mice, which express activated HER2/NEU/ERBB2 and Cre recombinase, to Slc2a1 (Flox/Flox) (GLUT1(Flox/Flox)) mice to generate NIC-GLUT1(+/+), NIC-GLUT1(Flox/+), and NIC-GLUT1(Flox/Flox) mice. In addition, we evaluated effects of glucose restriction or GLUT1 inhibition on transformation in MCF10A-ERBB2 breast epithelial cells in three-dimensional culture. Finally, we utilized global gene expression profiling data of primary human breast tumors to determine the relationship between SLC2A1 and stage of tumorigenesis. All of the NIC-GLUT1(+/+) mice developed tumors in less than 200 days. In contrast, only 1 NIC-GLUT1(Flox/Flox) mouse and 1 NIC-GLUT1(Flox/+) mouse developed mammary tumors, even after 18 months. Mammary gland development was not disrupted in NIC mice lacking GLUT1; however, epithelial content of mature glands was reduced compared to NIC-GLUT1(Flox/+) mice. In MCF10A-ERBB2 cells, glucose restriction or GLUT1 inhibition blocked transformation induced by activated ERBB2 through reduced cell proliferation. In human breast cancers, SLC2A1 was higher in ductal carcinoma in situ compared to the normal breast, but lower in invasive versus in situ lesions, suggesting the requirement for GLUT1 decreases as tumors progress. This study demonstrates a strict requirement for GLUT1 in the early stages of

  11. A Phase I-II Study of Combined Blockade of the ErbB Receptor Network with Trastuzumab and Gefitinib in Patients with HER2 (ErbB2)-Overexpressing Metastatic Breast Cancer

    Science.gov (United States)

    Arteaga, Carlos L.; O’Neill, Anne; Moulder, Stacy L.; Pins, Michael; Sparano, Joseph A.; Sledge, George W.; Davidson, Nancy E.

    2010-01-01

    Purpose To determine the safety, and efficacy of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib in combination with trastuzumab in patients with metastatic HER2-positive metastatic breast cancer. Experimental Design Patients with HER2-overexpressing breast cancer were treated with trastuzumab 2 mg/kg/week and gefitinib 250 to 500 mg/day. The primary end point of the study was to increase the proportion progression-free from 50% to 65% at 6 months in chemotherapy-naive patients and from 50% to 70% at 3 months in patients previously treated with chemotherapy in the metastatic setting. Results In the phase I study, all patients treated with gefitinib 500 mg/day developed grade 3 diarrhea. The phase II study was conducted using trastuzumab and gefitinib 250 mg/day. One patient achieved a complete response, 2 had a partial response, and 6 had stable disease for an overall response rate of 9% and a clinical benefit rate of 28% (9 of 32). Median time to progression (TTP) was 3 months (95% confidence interval, 2.3-4.1) in patients with no prior systemic therapy in the metastatic setting (n = 23). In patients treated with prior systemic therapy (n = 9), the median TTP of 5.3 months (95% confidence interval, 2.8-8.1). Overall median survival was 27 months. TTP was similar in EGFR-positive compared with EGFR-negative patients. Conclusions Gefitinib 250 mg/day was the maximal dose that can be safely administered with weekly trastuzumab. Interim analysis of the efficacy suggested that the combination was unlikely to result in clinical benefit compared with trastuzumab alone. These results do not support the use of this combination in patients with HER2-positive breast cancer. PMID:18829509

  12. Aberrations of ERBB2 and TOP2A Genes in Breast Cancer

    DEFF Research Database (Denmark)

    Nielsen, Kirsten Vang; Müller, Sven; Møller, Susanne;

    2009-01-01

    Copy number changes in TOP2A have frequently been linked to ERBB2 (HER2) amplified breast cancers. To study this relationship, copy number changes of ERBB2 and TOP2A were investigated by fluorescence in situ hybridization (FISH) in two cell lines; one characterized by having amplification of both...... genes and the other by having amplification of ERBB2 and deletion of TOP2A. The characteristics are compared to findings on paired ERBB2 and TOP2A data from 649 patients with invasive breast cancer from a previously published biomarker study. The physical localization of FISH signals in metaphase...... compared to TOP2A. In the majority of breast cancer patients, simultaneous aberration of ERBB2 and TOP2A is not explained by simple co-amplification....

  13. Screening to Identify Commonly Used Chinese Herbs That Affect ERBB2 and ESR1 Gene Expression Using the Human Breast Cancer MCF-7 Cell Line

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    Jen-Hwey Chiu

    2014-01-01

    Full Text Available Aim. Our aim the was to screen the commonly used Chinese herbs in order to detect changes in ERBB2 and ESR1 gene expression using MCF-7 cells. Methods. Using the MCF-7 human breast cancer cell line, cell cytotoxicity and proliferation were evaluated by MTT and trypan blue exclusion assays, respectively. A luciferase reporter assay was established by transient transfecting MCF-7 cells with plasmids containing either the ERBB2 or the ESR1 promoter region linked to the luciferase gene. Chinese herbal extracts were used to treat the cells at 24 h after transfection, followed by measurement of their luciferase activity. The screening results were verified by Western blotting to measure HER2 and ERα protein expression. Results. At concentrations that induced little cytotoxicity, thirteen single herbal extracts and five compound recipes were found to increase either ERBB2 or ESR1 luciferase activity. By Western blotting, Si-Wu-Tang, Kuan-Shin-Yin, and Suan-Tsao-Ren-Tang were found to increase either HER2 or ERα protein expression. In addition, Ligusticum chuanxiong was shown to have a great effect on ERBB2 gene expression and synergistically with estrogen to stimulate MCF-7 cell growth. Conclusion. Our results provide important information that should affect clinical treatment strategies among breast cancer patients who are receiving hormonal or targeted therapies.

  14. Effect of c- erbB2 Antisense Oligodeoxynucleotides on Radiosensitivity of Human Ovarian Cancer Cell Line

    Institute of Scientific and Technical Information of China (English)

    RENQing-Lan

    2003-01-01

    Object To explore tile effect of lipofectin - c - erbB2 antisense oligodeoxynucleotides on radiosensitivity of human ovarian cancer cell llne. Methods The expression of c - erbB2 was detected by means of RT - PCR, cellular response to irradiation was evaluated by tile colony forming assay. Results Lipofectin- c - erbB2 antisense oligodeoxynucleotides(AS- ODN) could suppress the expression of c - erbB2 , and significantly decreased the colony forming rate of human ovarian cancer cells after ionizing irradiation (P 0.05 ). Condusion c - erbB2 antisense oligodeoxynueleotides sensitized the SKOV3 to ionizing irradiation through decreasing the expression of e - erbB2 , which might be the result of the fact that c - erbB2 antisense oligodeoxynueleotides inhibit the eelluar signal transductionpathway relating to the radiation- resistant phenotype.

  15. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

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    Benevolo Maria

    2006-09-01

    Full Text Available Abstract Background Aberrant signaling by ErbB-2 (HER 2, Neu, a member of the human Epidermal Growth Factor (EGF receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs to ErbB-2 that involves their functional expression in (a bacteria, (b transient as well as stable transgenic tobacco plants, and (c a newly developed cell-free transcription-translation system. Results An ScFv (ScFv800E6 was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T, displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 × 108 M-1 only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. Conclusion ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology

  16. Sequence Variants and Haplotype Analysis of Cat ERBB2 Gene: A Survey on Spontaneous Cat Mammary Neoplastic and Non-Neoplastic Lesions

    Science.gov (United States)

    Santos, Sara; Bastos, Estela; Baptista, Cláudia S.; Sá, Daniela; Caloustian, Christophe; Guedes-Pinto, Henrique; Gärtner, Fátima; Gut, Ivo G.; Chaves, Raquel

    2012-01-01

    The human ERBB2 proto-oncogene is widely considered a key gene involved in human breast cancer onset and progression. Among spontaneous tumors, mammary tumors are the most frequent cause of cancer death in cats and second most frequent in humans. In fact, naturally occurring tumors in domestic animals, more particularly cat mammary tumors, have been proposed as a good model for human breast cancer, but critical genetic and molecular information is still scarce. The aims of this study include the analysis of the cat ERBB2 gene partial sequences (between exon 17 and 20) in order to characterize a normal and a mammary lesion heterogeneous populations. Cat genomic DNA was extracted from normal frozen samples (n = 16) and from frozen and formalin-fixed paraffin-embedded mammary lesion samples (n = 41). We amplified and sequenced two cat ERBB2 DNA fragments comprising exons 17 to 20. It was possible to identify five sequence variants and six haplotypes in the total population. Two sequence variants and two haplotypes show to be specific for cat mammary tumor samples. Bioinformatics analysis predicts that four of the sequence variants can produce alternative transcripts or activate cryptic splicing sites. Also, a possible association was identified between clinicopathological traits and the variant haplotypes. As far as we know, this is the first attempt to examine ERBB2 genetic variations in cat mammary genome and its possible association with the onset and progression of cat mammary tumors. The demonstration of a possible association between primary tumor size (one of the two most important prognostic factors) and the number of masses with the cat ERBB2 variant haplotypes reveal the importance of the analysis of this gene in veterinary medicine. PMID:22489125

  17. The Role of the Rab Coupling Protein in ErbB2-Driven Mammary Tumorigenesis and Metastasis

    Science.gov (United States)

    2015-10-01

    of the Rab Coupling Protein (RCP) in ErbB2- mediated lung metastasis formation. To accomplish this, we have derived both proficient and deficient...Rab Coupling Protein (RCP), which acts as an effector for Rab11 and Rab25, mediates recycling of the EGFR/α5β1-integrin complex to plasma membrane, a...implicating RCP in cancer progression. For example, RCP- mediated recycling of the integrin 51 is activated by expression of mutant forms of p53 resulting

  18. PNA-mediated modulation and redirection of Her-2 pre-mRNA splicing: specific skipping of erbB-2 exon 19 coding for the ATP catalytic domain

    DEFF Research Database (Denmark)

    Pankratova, Stanislava; Nielsen, Birgit N; Shiraishi, Takehiko;

    2010-01-01

    The Her-2 receptor coded for by the proto-oncogenic erbB-2 gene is a clinically validated target for treatment of a significant genetic subclass of breast cancers, and Her-2 is also overexpressed or mutated in a range of other cancers. In an approach to exploit antisense mediated splicing...... oligomers that specifically induce skipping of exon 19 as this exon is coding for the ATP catalytic domain of Her-2, and if expressed such truncated version of the Her-2 protein should be functionally inactive in a dominant negative fashion. Therefore, antisense compounds having efficient erbB-2 exon 19...

  19. Naked Polyamidoamine Polymers Intrinsically Inhibit Angiotensin II-Mediated EGFR and ErbB2 Transactivation in a Dendrimer Generation- and Surface Chemistry-Dependent Manner.

    Science.gov (United States)

    Akhtar, Saghir; El-Hashim, Ahmed Z; Chandrasekhar, Bindu; Attur, Sreeja; Benter, Ibrahim F

    2016-05-01

    The effects of naked polyamidoamine (PAMAM) dendrimers on renin-angiotensin system (RAS) signaling via Angiotensin (Ang) II-mediated transactivation of the epidermal growth factor receptor (EGFR) and the closely related family member ErbB2 (HER2) were investigated. In primary aortic vascular smooth muscle cells, a cationic fifth-generation (G5) PAMAM dendrimer dose- and time-dependently inhibited Ang II/AT1 receptor-mediated transactivation of EGFR and ErbB2 as well as their downstream signaling via extracellular-regulated kinase 1/2 (ERK1/2). Inhibition even occurred at noncytotoxic concentrations at short (1 h) exposure times and was dependent on dendrimer generation (G7 > G6 > G5 > G4) and surface group chemistry (amino > carboxyl > hydroxyl). Mechanistically, the cationic G5 PAMAM dendrimer inhibited Ang II-mediated transactivation of EGFR and ErbB2 via inhibition of the nonreceptor tyrosine kinase Src. This novel, early onset, intrinsic biological action of PAMAM dendrimers as inhibitors of the Ang II/AT1/Src/EGFR-ErbB2/ERK1/2 signaling pathway could have important toxicological and pharmacological implications.

  20. Macrophage inhibitory cytokine-1 transactivates ErbB family receptors via the activation of Src in SK-BR-3 human breast cancer cells.

    Science.gov (United States)

    Park, Yun Jung; Lee, Hansoo; Lee, Jeong-Hyung

    2010-02-01

    The function of macrophage inhibitory cytokine-1 (MIC-1) in cancer remains controversial, and its signaling pathways remain poorly understood. In this study, we demonstrate that MIC-1 induces the transactivation of EGFR, ErbB2, and ErbB3 through the activation of c-Src in SK-BR-3 breast cells. MIC-1 induced significant phosphorylation of EGFR at Tyr845, ErbB2 at Tyr877, and ErbB3 at Tyr1289 as well as Akt and p38, Erk1/2, and JNK mitogen-activated protein kinases (MAPKs). Treatment of SK-BR-3 cells with MIC-1 increased the phosphorylation level of Src at Tyr416, and induced invasiveness of those cells. Inhibition of c-Src activity resulted in the complete abolition of MIC-1-induced phosphorylation of the EGFR, ErbB2, and ErbB3, as well as invasiveness and matrix metalloproteinase (MMP)-9 expression in SK-BR-3 cells. Collectively, these results show that MIC-1 may participate in the malignant progression of certain cancer cells through the activation of c-Src, which in turn may transactivate ErbB-family receptors.

  1. ErbB2 and bone sialoprotein as markers for metastatic osteosarcoma cells

    Science.gov (United States)

    Valabrega, G; Fagioli, F; Corso, S; Madon, E; Brach del Prever, A; Biasin, E; Linari, A; Aglietta, M; Giordano, S

    2003-01-01

    Osteosarcoma is the most common malignant bone neoplasia occurring in young patients in the first two decades of life, and represents 20% of all primitive malignant bone tumours. At present, treatment of metastatic osteosarcoma is unsatisfactory. High-dose chemotherapy followed by CD34+ leukapheresis rescue may improve these poor results. Neoplastic cells contaminating the apheresis may, however, contribute to relapse. To identify markers suitable for detecting osteosarcoma cells in aphereses we analysed the expression of bone-specific genes (Bone Sialoprotein (BSP) and Osteocalcin) and oncogenes (Met and ErbB2) in 22 patients with metastatic osteosarcoma and six healthy stem cell donors. The expression of these genes in aphereses of patients affected by metastatic osteosarcoma was assessed by RT–PCR and Southern blot analysis. Met and Osteocalcin proved to be not useful markers since they are positive in aphereses of both patients with metastatic osteosarcoma and healthy stem cell donors. On the contrary, BSP was expressed at significant levels in 85% of patients. Moreover, 18% of patients showed a strong and significantly positive (seven to 16 times higher than healthy stem cell donors) ErbB2 expression. In all positive cases, neoplastic tissue also expressed ErbB2. Our data show that ErbB2 can be a useful marker for tumour contamination in aphereses of patients affected by ErbB2-expressing osteosarcomas and that analysis of Bone Sialoprotein expression can be an alternative useful marker. PMID:12569382

  2. The dual targeting of EGFR and ErbB2 with the inhibitor Lapatinib corrects high glucose-induced apoptosis and vascular dysfunction by opposing multiple diabetes-induced signaling changes.

    Science.gov (United States)

    Benter, Ibrahim F; Sarkhou, Fatima; Al-Khaldi, Abeer T; Chandrasekhar, Bindu; Attur, Sreeja; Dhaunsi, Gursev S; Yousif, Mariam H M; Akhtar, Saghir

    2015-01-01

    The epidermal growth factor receptors, EGFR and EGFR2 (ErbB2), appear important mediators of diabetes-induced vascular dysfunction. We investigated whether targeted dual inhibition of EGFR and ErbB2 with Lapatinib would be effective in treating diabetes-induced vascular dysfunction in a rat model of type 1 diabetes. In streptozotocin-induced diabetes, chronic 4-week oral or acute, ex vivo, administration of Lapatinib prevented the development of vascular dysfunction as indicated by the attenuation of the hyper-reactivity of the diabetic mesenteric vascular bed (MVB) to norephinephrine without correcting hyperglycemia. Chronic in vivo or acute ex vivo Lapatinib treatment also significantly attenuated diabetes-induced increases in phosphorylation of EGFR, ErbB2, ERK1/2, AKT, ROCK2 and IkB-alpha as well as normalized the reduced levels of phosphorylated FOXO3A, and eNOS (Ser1177) in the diabetic MVB. Similar results were observed in vascular smooth muscle cells (VSMCs) cultured in high glucose (25 mM) treated with Lapatinib or small interfering RNA (siRNA) targeting the ErbB2 receptor. Lapatinib also prevented high glucose-induced apoptosis in VSMC. Thus, Lapatinib corrects hyperglycemia-induced apoptosis and vascular dysfunction with concomitant reversal of diabetes or high glucose-induced signaling changes in EGFR/ErbB2 and downstream signaling pathways implying that targeted dual inhibition of EGFR/ErbB2 might be an effective vasculoprotective treatment strategy in diabetic patients.

  3. RhNRG-1β Protects the Myocardium against Irradiation-Induced Damage via the ErbB2-ERK-SIRT1 Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Anxin Gu

    Full Text Available Radiation-induced heart disease (RIHD, which is a serious side effect of the radiotherapy applied for various tumors due to the inevitable irradiation of the heart, cannot be treated effectively using current clinical therapies. Here, we demonstrated that rhNRG-1β, an epidermal growth factor (EGF-like protein, protects myocardium tissue against irradiation-induced damage and preserves cardiac function. rhNRG-1β effectively ameliorated irradiation-induced myocardial nuclear damage in both cultured adult rat-derived cardiomyocytes and rat myocardium tissue via NRG/ErbB2 signaling. By activating ErbB2, rhNRG-1β maintained mitochondrial integrity, ATP production, respiratory chain function and the Krebs cycle status in irradiated cardiomyocytes. Moreover, the protection of irradiated cardiomyocytes and myocardium tissue by rhNRG-1β was at least partly mediated by the activation of the ErbB2-ERK-SIRT1 signaling pathway. Long-term observations further showed that rhNRG-1β administered in the peri-irradiation period exerts continuous protective effects on cardiac pump function, the myocardial energy metabolism, cardiomyocyte volume and interstitial fibrosis in the rats receiving radiation via NRG/ErbB2 signaling. Our findings indicate that rhNRG-1β can protect the myocardium against irradiation-induced damage and preserve cardiac function via the ErbB2-ERK-SIRT1 signaling pathway.

  4. Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus

    DEFF Research Database (Denmark)

    Lerdrup, Mads; Bruun, Silas; Grandal, Michael Vibo

    2007-01-01

    inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent...

  5. Development of Specific Inhibitors for Breast Cancer-Associated Variants of ErbB2

    Science.gov (United States)

    2015-10-01

    baculoviruses and used them to infect Sf9 cells in one-liter spinner flasks. We purified the proteins using metal affinity, ion exchange, and gel filtration...antibodies. Similar results were obtained in three separate experiments. Purification of wild-type ErbB2. Sf9 cells (600 ml) were infected with

  6. Early nuclear alterations and immunohistochemical expression of Ki-67, Erb-B2, vascular endothelial growth factor (VEGF), transforming growth factor (TGF-beta1) and integrine-linked kinase (ILK) two days after tamoxifen in breast carcinoma.

    Science.gov (United States)

    Morena, A M L; Oshima, C T F; Gebrim, L H; Egami, M I; Silva, M R R; Segreto, R A; Giannotti Filho, O; Teixeira, V P C; Segreto, H R C

    2004-01-01

    The purpose of the present study was to evaluate breast carcinoma samples before and two days after treatment with tamoxifen in order to analyse early histopathological alterations--particularlynuclear alterations-- as well as immunohistochemical expression of Ki-67, Erb-B2, VEGF, TGF-beta1 and ILK proteins. Twenty one cases of invasive ductal and lobular breast carcinoma were studied. Patients were submitted to biopsy of the lesion and, after confirmation of the diagnosis, they received 20 mg of tamoxifen a day, beginning two days before surgery. The samples obtained during biopsy and after surgery were stained with HE for histopathological diagnosis. Estrogen receptor was positive in 18 cases and negative in 3. The immunohistochemical method was applied for the detection of Ki-67, Erb-B2, protein, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta1) and integrin linked kinase (ILK). Two days after tamoxifen treatment, the following results were observed: 1) decrease in the cell volume, chomatine condensation, nucleoli less evident and clearly defined nuclear limits; 2) significant reduction in the expression of Erb-B2 protein and significant increase in the expression of TGF-beta1 protein; 3) expression of others proteins (Ki-67, VEGF and ILK) was not altered during the indicated time frame. Our results suggest that analyzing nuclear alterations and expression of Erb-B2 and TGF-beta1 proteins would be useful to assess the initial response to tamoxifen.

  7. Involvement of Human Estrogen Related Receptor Alpha 1 (hERR 1) in Breast Cancer and Hormonally Insensitive Disease

    Science.gov (United States)

    2002-08-01

    Jak2 (Janus kinase), which then interacts with and phosphorylates the cytoplasmic tail of ErbB2, thereby activating it (39). Hence, multiple signaling...directly compete Among the first orphan receptors identified were the estro- with estrogen receptor a (ERa) for binding. ERRal ac- gen -related receptors...identified as the major isoform present in HeLa gen responsiveness and as an estrogen-independent ac- cells (Fig. LA) (11, 12). The DBD of human ERRal

  8. Novel Drugs that Target ErbB2

    Science.gov (United States)

    2012-05-01

    of pharmacological properties including antiviral, antibacterial , anti-inflammatory, antimalarial and anticancer activities (1, 2). BA also inhibits...47). Gel mobility shift assays showed that BA also decreased formation of a retarded band containing Sp proteins bound to a GC-rich...analogues exhibit a broad spectrum of pharmacologic properties including antiviral, antibacterial , anti-inflam- matory, antimalarial, and anticancer

  9. Precise ERBB2 copy number assessment in breast cancer by means of molecular inversion probe array analysis.

    Science.gov (United States)

    Christgen, Matthias; van Luttikhuizen, Jana L; Raap, Mieke; Braubach, Peter; Schmidt, Lars; Jonigk, Danny; Feuerhake, Friedrich; Lehmann, Ulrich; Schlegelberger, Brigitte; Kreipe, Hans H; Steinemann, Doris

    2016-12-13

    HER2/ERBB2 amplification/overexpression determines the eligibility of breast cancer patients to HER2-targeted therapy. This study evaluates the agreement between ERBB2 copy number assessment by fluorescence in situ hybridization, a standard method recommended by the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP), and newly available DNA extraction-based methods. A series of n=29 formalin-fixed paraffin-embedded breast cancers were subjected to ERBB2 copy number assessment by fluorescence in situ hybridization (FISH, Vysis, Abbott). Following macrodissection of invasive breast cancer tissue and DNA extraction, ERBB2 copy number was also determined by molecular inversion probe array analysis (MIP, OncoScan, Affymetrix) and next generation sequencing combined with normalized amplicon coverage analysis (NGS/NAC, AmpliSeq, Ion Torrent). ERBB2 copy number values obtained by MIP or NGS/NAC were tightly correlated with ERBB2 copy number values obtained by conventional FISH (rs = 0.940 and rs = 0.894, P copy number detection and should be considered as an ancillary method for clinical HER2 testing.

  10. Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family.

    Directory of Open Access Journals (Sweden)

    Sabine Stindt

    Full Text Available Recently, the epidermal growth factor (EGF receptor (EGFR, a member of the ErbB receptor family, and its down-stream signalling have been identified as co-factors for HCV entry and replication. Since EGFR also functions as a heterodimer with other ErbB receptor family members, the subject of the present study was to investigate a possible viral interference with these cellular components. By using genotype 1b replicon cells as well as an infection-based system we found that while transcript and protein levels of EGFR and ErbB2 were up-regulated or unaffected, respectively, HCV induced a substantial reduction of ErbB3 and ErbB4 expression. Down-regulation of ErbB3 expression by HCV involves specificity protein (Sp1-mediated induction of Neuregulin (NRG1 expression as well as activation of Akt. Consistently, at transcript level disruption of ErbB3 expression by HCV can be prevented by knockdown of NRG1 or Sp1 expression, whereas reconstitution of ErbB3 protein levels requires inhibition of HCV-induced NRG1 expression and of Akt activity. Interestingly, the NRG1-mediated suppression of ErbB3 expression by HCV results in an enhanced expression of EGFR and ErbB2 on the cell surface, which can be mimicked by siRNA-mediated knockdown of ErbB3 expression. These data delineate a novel mechanism enabling HCV to sway the composition of the ErbB family members on the surface of its host cell by an NRG1-driven circuit and unravels a yet unknown cross-regulation between ErbB3 and the two other family members ErbB2 and EGFR. The shift of the receptor surface expression of the ErbB family towards enhanced expression of ErbB2 and EGFR triggered by HCV was found to promote viral RNA replication and infectivity. This suggests that HCV rearranges expression of ErbB family members to adapt the cellular environment to its requirements.

  11. Integrated assessment of potential value of erbB-2 amplification or expression status as novel therapy target in Chinese children and adolescents with osteosarcoma

    Institute of Scientific and Technical Information of China (English)

    SHANG Jian; BI Zheng-gang; JI Hong-fei; WANG Wen-bo

    2009-01-01

    Background Targeted tumor therapies have been making rapid progress in recent years, and the erbB-2 oncogene is a suitable target. There was much discussion about the level of erbB-2 in osteosarcoma. The aim of this study was to investigate the erbB-2 amplification or expression status in osteosarcoma. Methods Fluorescence in situ hybridization (FISH) and DNA probes for erbB-2 and centromere 17 were used to examine the erbB-2 gene amplification status in 32 osteosarcoma samples, and expression of erbB-2 was analyzed by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR).Results None of the 32 osteosarcomas was observed by FISH to have the erbB-2 gene amplified, and no distinguishable membrane staining was seen in any case yet, nevertheless, erbB-2 overexpression was present in 6 tumor samples by RT-PCR.Conclusions The status of erbB-2 gene amplification and membrane overexpression is rare in osteosarcomas, and might suggest that the erbB-2 target agent should not be applied to osteosarcomas as single treatment .

  12. High expression of ErbB2 contributes to cholangiocarcinoma cell invasion and proliferation through AKT/p70S6K

    Institute of Scientific and Technical Information of China (English)

    Warapen; Treekitkarnmongkol; Tuangporn; Suthiphongchai

    2010-01-01

    AIM:To compare the impact of ErbB2 on cell invasion and proliferation in cholangiocarcinoma(CCA) cell lines.METHODS:Level of endogenous ErbB2 expression in three CCA cell lines,namely HuCCA-1,KKU-100 and KKU-M213,was determined by real-time reversetranscriptase polymerase chain reaction.Two ErbB2 inhibitory methods,a small molecule ErbB2 kinase inhibitor(AG825) and siRNA,were used to disrupt ErbB2 function in the cell lines.CCA cell invasion,motility and proliferation under ErbB2-disrupted conditions were d...

  13. p53 and erbB-2 Are Not Associated in Matched Cases of Primary and Metastatic Ovarian Carcinomas

    Directory of Open Access Journals (Sweden)

    Cristina Rodríguez-Burford

    2003-01-01

    Full Text Available Ovarian cancer has a high mortality rate largely due to the limited number of ovarian carcinomas detected at an early stage. Understanding the molecular changes occurring during the progression of ovarian carcinoma would aid in the development of therapies that may inhibit or target metastasis. Primary and metastatic lesions from 54 and 40 patients with advanced ovarian carcinoma, respectively (including matched primary and metastatic lesions from 30 patients were evaluated for nuclear accumulation of p53 (clone BP53-12-1 and cytoplasmic and membranous immunostaining of p185 erbB-2 (clone 3B5 by immunohistochemistry. No differences in the immunostaining of p53 and p185erbB-2 (cytoplasm or membrane were observed between primary and metastatic lesions of the matched cases. Similarly, no differences in the proportion of positive cases of p53 between primary and metastatic lesions of the matched cases was observed. Thus, novel therapies that target p53 or p185erbB-2 can utilize specimens from either primary or metastatic lesions to characterize these targets prior to therapy. Spearman correlations between p53 and p185erbB-2 (cytoplasm or membrane immunohistochemistry scores were insignificant for the matched cases, all primary lesions, and all metastatic lesions. Also, no significant associations occurred between nuclear accumulation of p53 (positive versus negative and phenotypic expression of p185erbB-2 (cytoplasm or membrane immunostaining scores for the matched cases, all primary lesions, and all metastatic lesions. Thus, the nuclear accumulation of p53 and immunostaining of p185erbB-2 in the cytoplasm or on the cellular membranes are independent.

  14. K-RAS point mutation, and amplification of C-MYC and C-ERBB2 in colon adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Tadeusz Pawełczyk

    2004-10-01

    Full Text Available The routine multidisciplinary management of colon cancer is based mainly on tumor staging, histology, grading and vascular invasion. In this approach, important individual information derived from molecular characteristics of the tumor may be missed, especially since significant heterogeneity of molecular aberrations in cancer cells has been observed, and recognition of every of relationships between them may be of value. K-RAS, C-MYC and C-ERBB2 are protooncogenes taking part in carcinogenesis and tumor progression in the colon. They influence cell proliferation, differentiation and survival. K-RAS point mutation, as well as amplification of C-MYC and C-ERBB2 were searched in 84 primary colon adenocarcinomas resected with curative intent. Multiplex polymerase-chain reaction and restriction fragment length polymorphism were performed to assess codon 12 K-RAS point mutation. Amplification of C-MYC and C-ERBB2 genes was evaluated by densitometry after agarose gel separation of the respective multiplex PCR products. No relation was found among mutated and/or amplified genes, and between searched molecular aberrations and pathoclinical features. In multivariate analysis, nodal status appeared to be the only independent prognostic indicator. In colon adenocarcinoma, codon 12 K-RAS point mutation and amplification of C-MYC and C-ERBB2 seem to occur independently in the process of tumor progression. Amplification of C-ERBB2 tends to associate with more advanced stage of disease. Concomitant occurrence of codon 12 K-RAS mutation, C-MYC and C-ERBB2 amplification was of no prognostic value in respect to survival.

  15. A novel mouse monoclonal antibody targeting ErbB2 suppresses breast cancer growth

    Energy Technology Data Exchange (ETDEWEB)

    Kawa, Seiji [Division of Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108-8639 (Japan); Matsushita, Hirohisa; Ohbayashi, Hirokazu [Department of Research and Development, Nichirei Biosciences Inc., Tokyo 104-8402 (Japan); Semba, Kentaro [Department of Life Science and Medical Bio-Science, School of Science and Engineering, Waseda University, Tokyo 169-8555 (Japan); Yamamoto, Tadashi, E-mail: tyamamot@ims.u-tokyo.ac.jp [Division of Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108-8639 (Japan)

    2009-07-03

    Overexpression of ErbB2 in breast cancer is associated with increased recurrence and worse prognosis. Accumulating evidences suggest that molecular targeted therapy is a promising anticancer strategy. In this study, we produced a novel anti-ErbB2 monoclonal antibody, 6G10, that recognized an epitope distinct from the trastuzumab binding site. 6G10 induced aggregation of BT474 breast cancer cells and inhibited proliferation of various breast cancer cell lines including BT474. A growth inhibition assay showed that 6G10 had EC{sub 50} values comparable to trastuzumab, indicating that the drugs have a similar level of potency. Furthermore, intraperitoneal administration of 6G10 completely inhibited the growth of xenografted tumors derived from BT474 and SK-BR-3 cells. These data suggested that 6G10 has great therapeutic potential and could be administered to patients alternatively, or synergistically, with trastuzumab.

  16. Adjuvant letrozole versus tamoxifen according to centrally-assessed ERBB2 status for postmenopausal women with endocrine-responsive early breast cancer: supplementary results from the BIG 1-98 randomised trial

    DEFF Research Database (Denmark)

    Regan, M.M.; Lykkesfeldt, A.E.; Dell'Orto, P.

    2008-01-01

    as ERBB2-positive. In 3533 patients with tumours confirmed to express ER, DFS was poorer in patients with ERBB2-positive tumours (n=239) than in those with ERBB2-negative tumours (n=3294; HR 2-09 [95% CI 1.59-2 -76]; pevidence of heterogeneity in the treatment effect...

  17. Effects of ERBB2 amplicon size and genomic alterations of chromosomes 1, 3, and 10 on patient response to trastuzumab in metastatic breast cancer.

    Science.gov (United States)

    Morrison, Larry E; Jewell, Susan S; Usha, Lydia; Blondin, Beth A; Rao, Ruta D; Tabesh, Bita; Kemper, Matthew; Batus, Marta; Coon, John S

    2007-04-01

    Trastuzumab is widely used for advanced breast cancer patients with ERBB2-amplified tumors. Nevertheless, over half of these patients do not have an objective response. One reason may be altered expression of genes that might compensate for ERBB2 inhibition. We previously mapped the gene-rich region of chromosome 17 telomeric to ERBB2, and reported considerable variability in the telomeric extent of the ERBB2 amplicon. Here we examined whether the variable amplicon size may be associated with patient response to trastuzumab. In addition, we looked at associations between response and several signaling pathway-related genes unrelated to the ERBB2 amplicon, including AKT3, PTEN, PIK3CA, and PTGS2. In 35 patients with ERBB2-amplified metastatic breast cancer, with 40% overall response to trastuzumab, fluorescence in situ hybridization identified the telomeric extent of the ERBB2 amplicon and the status of the several pathway-related genes. Objective response strongly correlated with the telomeric amplicon size, with 62% of patients with shorter amplicons responding, compared with only 7% of patients with longer amplicons (P = 0.0015). Abnormal copy number of PTGS2 was marginally associated with objective response (P = 0.066), while abnormal copy numbers of two reference loci, 1q25 and the chromosome 10 centromere, were significantly associated with response. Pairwise combinations of copy number status of these loci and ERBB2 amplicon size provided stronger associations and identified a group of patients without responders. These results suggest that patient selection for trastuzumab may be improved by considering ERBB2 amplicon size and genomic status of the 1q25, PTGS2, and centromere 10 loci.

  18. Two novel human anti-ErbB2 immunoagents are active on trastuzumab-resistant tumours

    Science.gov (United States)

    Gelardi, T; Damiano, V; Rosa, R; Bianco, R; Cozzolino, R; Tortora, G; Laccetti, P; D'Alessio, G; De Lorenzo, C

    2010-01-01

    Background: Overexpression of ErbB2 receptor in breast cancer is associated with disease progression and poor prognosis. Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer, has proven to be effective; however, a relevant problem for clinical practice is that a high fraction of breast cancer patients shows primary or acquired resistance to trastuzumab treatment. Methods: We tested on trastuzumab-resistant cells two novel human anti-tumour immunoconjugates engineered in our laboratory by fusion of a human anti-ErbB2 scFv, termed Erbicin, with either a human RNase or the Fc region of a human IgG1. Both Erbicin-derived immunoagents (EDIAs) are selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo, target an ErbB2 epitope different from that recognised by trastuzumab and do not show cardiotoxic effects. Results: We report that EDIAs are active also on trastuzumab-resistant tumour cells both in vitro and in vivo, most likely because of the different epitope recognised, as EDIAs, unlike trastuzumab, were found to be able to inhibit the signalling pathway downstream of ErbB2. Conclusion: These results suggest that EDIAs are immunoagents that could not only fulfil the therapeutic need of patients ineligible to trastuzumab treatment due to cardiac dysfunction but also prove to be useful for breast cancer patients unresponsive to trastuzumab treatment. PMID:20051960

  19. Transcriptome analysis of basal and luminal tumor-initiating cells in ErbB2-driven breast cancer

    Directory of Open Access Journals (Sweden)

    Nicholas Borcherding

    2015-06-01

    Full Text Available Breast cancer is the leading cause of cancer-related mortality for females worldwide [1]. Improving early screening strategies and understanding the events that lead to tumor initiation have led to demonstrable improvements in clinical outcome. Our previous work revealed a variance in the tumorigenic capacity between different mammary epithelial cell populations in an MMTV-ErbB2 mouse model. In order to greater understand how different mammary epithelial cells influence the tumorigenic capacity in ErbB2-induced breast cancer, we transplanted different cell populations from pre-neoplastic MMTV-ErbB2 female mice into recipient mice for tumorigenic study. We found that different mammary epithelial cells bear different tumorigenic potentials even when induced by the same ErbB2 proto-oncogene. To understand the difference in tumors formed from different epithelial cells, we performed gene expression profiling using these tumors (GSE64487. Several genes were further validated using real-time reverse transcription polymerase chain reaction (RT-PCR. Here we provide further details on the experimental methods and microarray analysis. This data provides a resource to further understanding how different mammary cell populations can initiate ErbB2-driven tumors and the role of these cell populations as putative tumor-initiating cells (TICs.

  20. C-ERBB2促小鼠卵母细胞成熟及在表皮生长因子调控小鼠卵母细胞体外成熟中的作用%Potential roles of the C-ERBB2 protooncogene in mouse oocyte maturation and in conducting EGF promoting mouse oocyte maturation in vitro

    Institute of Scientific and Technical Information of China (English)

    郑莉萍; 汪小浪; 吴磊; 郑月慧; 高凤兰; 肖秋香; 李芳

    2006-01-01

    目的 探讨C-ERBB2对小鼠卵母细胞成熟的影响及对表皮生长因子在卵母细胞成熟调控中的作用.方法 采用卵母细胞体外培养法,研究反义C-ERBB2寡脱氧核苷酸(C-ERBB2 ASODN)对卵母细胞体外自发成熟及对表皮生长因子(EGF)促卵母细胞成熟的影响;利用RT-PCR检测卵母细胞中C-ERBB2 mRNA的表达及在不同处理因素作用下卵母细胞成熟过程中C-ERBB2 mRNA表达的变化.结果 C-ERBB2 ASODN能呈剂量依赖方式抑制卵母细胞的生发泡破裂(GVBD)率和第一极体(PBⅠ)排出率,并且能抑制EGF对卵母细胞的促成熟作用.卵母细胞中存在C-ERBB2 mRNA,EGF可以增强其表达,C-ERBB2 ASODN可减弱EGF促表达的作用.结论 C-ERBB2与卵母细胞的成熟密切相关.EGF可直接促进裸卵的成熟分裂,其机制可能与上调卵母细胞C-ERBB2的表达有关.

  1. ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients.

    Science.gov (United States)

    Braun, S; Schlimok, G; Heumos, I; Schaller, G; Riethdorf, L; Riethmüller, G; Pantel, K

    2001-03-01

    Occult hematogenous micrometastases are the major cause for metastatic relapse and cancer-related death in patients with operable primary breast cancer. Although sensitive immunocytochemical and molecular methods allow detection of individual breast cancer cells in bone marrow (BM), a major site of metastatic relapse, current detection techniques cannot discriminate between nonviable shed tumor cells and seminal metastatic cells. To address this problem, we analyzed the relevance of erbB2 overexpression on disseminated cytokeratin-18-positive breast cancer cells in the BM of 52 patients with locoregionally restricted primary breast cancer using immunocytochemical double labeling with monoclonal antibody 9G6 to the p185erbB2 oncoprotein. Expression of p185erbB2 on BM micrometastases was detected in 31 of 52 (60%) patients independent of established risk factors such as lymph node involvement, primary tumor size, differentiation grade, or expression of p185erbB2 on primary tumor cells. After a median follow-up of 64 months, patients with p185erbB2-positive BM micrometastases had developed fatal metastatic relapses more frequently than patients with p185erbB2-negative micrometastases (21 versus 7 events; P = 0.032). In multivariate analysis, the presence of p185erbB2-positive micrometastases was an independent prognostic factor with a hazard ratio of 2.78 (95% confidence interval, 1.11-6.96) for overall survival (P = 0.029). We therefore conclude that erbB2 overexpression characterizes a clinically relevant subset of breast cancer micrometastases.

  2. The peroxisome proliferator-activated receptor gamma is an inhibitor of ErbBs activity in human breast cancer cells.

    Science.gov (United States)

    Pignatelli, M; Cortés-Canteli, M; Lai, C; Santos, A; Perez-Castillo, A

    2001-11-01

    One of the most interesting recent developments in the nuclear receptor field has been the identification of natural and synthetic agonists of the peroxisome proliferator-activated receptor (PPAR) family, coupled with a growing recognition that the gamma isoform (PPARgamma) affects pathways important in a variety of human diseases. Here we show that the activation of PPARgamma through the 15-deoxy-Delta-12,14-prostaglandin J(2) (PG-J(2)) ligand causes a dramatic inhibition of ErbB-2 and ErbB-3 tyrosine phosphorylation caused by neuregulin 1 (NRG1) and neuregulin 2 (NRG2) in MCF-7 cells. This effect is accompanied by a very efficient blocking of ErbBs effects upon proliferation, differentiation and cell death in these cells. Preincubation of MCF-7 cells with PG-J(2) before addition of NRG1 and NRG2 had a dramatic growth-suppressive effect accompanied by accumulation of cells in the G0/G1 compartment of the cell cycle, and a marked increase in apoptosis. NRG1 and NRG2 induce G1 progression, which was associated with stimulation of the phosphatidylinositol-3 kinase (PI 3-K) pathway, whereas survival was dependent on ERK1/ERK2 activation. Both pathways were inhibited by PG-J(2). Furthermore, PG-J(2) can abolish the NRG1 and NRG2-induced increase in anchorage-independent growth of these cells. PG-J(2) also blocks phosphorylation of other receptor tyrosine kinases, such as IGF-IR, in MCF-7 cells, and suppress proliferation of other breast cancer cell lines. In summary, our data show a specific inhibitory action of PG-J(2) on the activity of the ErbB receptors in breast cancer cells.

  3. Detection of soluble ERBB2 in breast cancer cell lysates using a combined label-free/fluorescence platform based on Bloch surface waves.

    Science.gov (United States)

    Sinibaldi, Alberto; Sampaoli, Camilla; Danz, Norbert; Munzert, Peter; Sibilio, Leonardo; Sonntag, Frank; Occhicone, Agostino; Falvo, Elisabetta; Tremante, Elisa; Giacomini, Patrizio; Michelotti, Francesco

    2017-06-15

    We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of ERBB2/neu/Her2 in cell lysates. ERBB2 is a pivotal breast cancer biomarker and targetable oncogenic driver associated with aggressive breast cancer subtypes. To quantitate soluble ERBB2, we developed an optical platform that combines label-free and fluorescence detection modes. Such platform makes use of a sandwich assay in which the one-dimensional photonic crystals sustaining Bloch surface waves are tailored with a monoclonal antibody for highly specific biological recognition (BSW biochip). In a second step, a second antibody to ERBB2 quantitatively detects the bound analyte. The strategy of the present approach takes advantage of the combination of label-free and fluorescence techniques, making bio-recognition more robust and sensitive. In the fluorescence operation mode, the platform can attain the limit of detection 0.3ng/mL (1.5pM) for ERBB2 in cell lysates. Such resolution meets the international guidelines and recommendations (15ng/mL) for diagnostic ERBB2 assays that in the future may help to more precisely assign therapies counteracting cancer cell proliferation and metastatic spread.

  4. Differential Gene Expression of BRCA1,ERBB2 and TP53 biomarkers between Human Breast Tissue and Peripheral Blood Samples of Breast Cancer.

    Science.gov (United States)

    Zghair, Abdulrazzaq Neamah; Sinha, Deepak Kumar; Kassim, Arkan; Alfaham, Mohmmad; Sharma, Anil K

    2016-01-01

    Breast cancer is a most common malignancy especially in Iraqi women accounting for high morbidity and mortality. Mutations in BRCA1 gene is one of the important genetic predisposing factors inbreast cancer. Similarly ERBB2 and TP53 are also key prognostic markers in breast cancer treatment.We were interested to explore the gene expression profiles of BRCA1, ERBB2 and TP53 in breast cancer women patients from Iraq so as to assess the potential of such markers in breast cancer treatment. The mRNA levels were significantly over-expressed in tumor tissues in comparison to normal ones with p values (pTP53 and benign tissue samples as well. However in blood samples, no considerable expression of these markers was observed. Out of three selected genes, ERBB2 expression was significantly expressed in comparison to BRCA1 and TP53 in cancer tissue. Mutation analysis of BRCA1, ERBB2 and TP53 has been made to find out the region most susceptible to mutations in these genes The BRCA1 exon 11, ERBB2 16 and TP53 exon 5 displayed increased chances of having mutations. We can conclude from the study that differential gene expression of BRCA1, ERBB2 and TP53 at mRNA levels may act as a diagnostic marker of circulating tumor cells having important prognostic value in breast cancer patients.

  5. ERbB2 Trafficking and Signaling in Human Vestibular Schwannomas

    Science.gov (United States)

    2011-10-01

    patients with macular degeneration and respiratory syncytial virus infections (37). A miR-21Yspecific RNA interference knockdown could be achieved...International Conference on Vestibular Schwannomas and Other CPA Lesions, Barcelona, Spain , June 2007 8 Brown, KD, Clark, J, Hansen MR. Differential...Wallerian degeneration . J Neurosci 1997;17: 1642Y59. 11. Frohnert PW, Stonecypher MS, Carroll SL. Constitutive activation of the neuregulin-1/ErbB receptor

  6. Transactivation of ErbB receptors by leptin in the cardiovascular system: mechanisms, consequences and target for therapy.

    Science.gov (United States)

    Bełtowski, Jerzy; Jazmroz-Wiśniewska, Anna

    2014-01-01

    Many experimental and clinical studies have demonstrated that elevated leptin concentration in patients with obesity/metabolic syndrome contributes to the pathogenesis of cardiovascular disorders including arterial hypertension, atherosclerosis, restenosis after coronary angioplasty and myocardial hypertrophy. Receptor tyrosine kinases belonging to the ErbB family, especially ErbB1 (epidermal growth factor receptor) and ErbB2 are abundantly expressed in the blood vessels and the heart. EGFR is activated not only by its multiple peptide ligands but also by many other factors including angiotensin II, endothelin-1, norepinephrine, thrombin and prorenin; the phenomenon referred to as "transactivation". Augmented EGFR signaling contributes to abnormalities of vascular tone and renal sodium handling as well as vascular remodeling and myocardial hypertrophy through various intracellular mechanisms, in particular extracellular signal-regulated kinases (ERK) and phosphoinositide 3-kinase (PI3K). Recent experimental studies indicate that chronically elevated leptin transactivates the EGFR through the mechanisms requiring reactive oxygen species and cytosolic tyrosine kinase, c-Src. In addition, hyperleptinemia increases ErbB2 activity in the arterial wall. Stimulation of EGFR and ErbB2 downstream signaling pathways such as ERK and PI3K in the vascular wall and the kidney may contribute to the increase in vascular tone, enhanced tubular sodium reabsorption as well as vascular and renal lesions in hyperleptinemic obese subjects.

  7. Effect of spatial inhomogeneities on the membrane surface on receptor dimerization and signal initiation

    Directory of Open Access Journals (Sweden)

    Romica Kerketta

    2016-08-01

    Full Text Available Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as clusters by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones (domains for the two receptor species. The in silico model is parameterized and validated using data from single particle tracking experiments. We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems.

  8. PCNA、neuregulin-1、erbB-2在听神经瘤中的表达与肿瘤生物学行为相关性分析%Relevance between the expression of PCNA, neuregulin-1 and erbB-2 and biological behavior of acoustic neuroma

    Institute of Scientific and Technical Information of China (English)

    冯娟娟; 夏寅

    2011-01-01

    , neuregulin-1 and erbB-2 in acoustic neuroma was determined by immunohistochemistry. Results In all the 27 cases, the positive rate of PCNA was 92.7% (25/27) and the positive rate of neuregulin-1 was 81.4% ( 22/27 ) . ErbB-2 expressed in none of the cases. There was no significant relationship between the expression of PCNA with gender, age, the side and size of the tumors, but there was significant difference between the expression of PCNA with the course(F = 5.011, P=0.015) and clinical growth rate(I= -3.360, P=0.003) of the disease. There was no significant relationship between the expression of neuregulin-1 with gender, age, the side, size and course of the tumors. Although the expression of neuregulin-1 in the tumors of course less than 1 year was higher than that of the course more than 1 year, but the difference was not statistically significant. The expression of neuregulin-1 in the tumors of clinical growth rate less than 10 mm/year was lower than that in the tumors with the clinical growth rate more than 10 mm/year, the difference was not statistically significant. The expression of neuregulin-1 and PCNA had positive correlation ( r = 0.425 , P = 0. 027 ). Conclusion PCNA is related with the growth of acoustic neuroma, and reflects proliferation activities of tumor cells in acoustic neuroma. Neuregulin-1 might participate in the biological behavior of acoustic neuroma. As the functional receptor of neuregulin-1 , the expression of erbB-2 and its role in acoustic neuroma need further study in more cases. The clinical behavior of acoustic neuroma relates to tumor growth . Some characteristics of acoustic neuroma can reflect the trend of tumorgrowth to some extent.

  9. ErbB2 and ErbB3 regulate amputation-induced proliferation and migration during vertebrate regeneration.

    Science.gov (United States)

    Rojas-Muñoz, Agustin; Rajadhyksha, Shibani; Gilmour, Darren; van Bebber, Frauke; Antos, Christopher; Rodríguez Esteban, Concepción; Nüsslein-Volhard, Christiane; Izpisúa Belmonte, Juan Carlos

    2009-03-01

    Epimorphic regeneration is a unique and complex instance of postembryonic growth observed in certain metazoans that is usually triggered by severe injury [Akimenko et al., 2003; Alvarado and Tsonis, 2006; Brockes, 1997; Endo et al., 2004]. Cell division and migration are two fundamental biological processes required for supplying replacement cells during regeneration [Endo et al., 2004; Slack, 2007]. However, the connection between the early stimuli generated after injury and the signals regulating proliferation and migration during regeneration remain largely unknown. Here we show that the oncogenes ErbB2 and ErbB3, two members of the EGFR family, are essential for mounting a successful regeneration response in vertebrates. Importantly, amputation-induced progenitor proliferation and migration are significantly reduced upon genetic and/or chemical modulation of ErbB function. Moreover, we also found that NRG1 and PI3K functionally interact with ErbB2 and ErbB3 during regeneration and interfering with their function also abrogates the capacity of progenitor cells to regenerate lost structures upon amputation. Our findings suggest that ErbB, PI3K and NRG1 are components of a permissive switch for migration and proliferation continuously acting across the amputated fin from early stages of vertebrate regeneration onwards that regulate the expression of the transcription factors lef1 and msxB.

  10. Prolyl isomerase Pin1 is highly expressed in Her2-positive breast cancer and regulates erbB2 protein stability

    Directory of Open Access Journals (Sweden)

    Lu Kun

    2008-12-01

    Full Text Available Abstract Overexpression of HER-2/Neu occurs in about 25–30% of breast cancer patients and is indicative of poor prognosis. While Her2/Neu overexpression is primarily a result of erbB2 amplification, it has recently been recognized that erbB2 levels are also regulated on the protein level. However, factors that regulate Her2/Neu protein stability are less well understood. The prolyl isomerase Pin1 catalyzes the isomerization of specific pSer/Thr-Pro motifs that have been phosphorylated in response to mitogenic signaling. We have previously reported that Pin1-catalyzed post-phosphorylational modification of signal transduction modulates the oncogenic pathways downstream from c-neu. The goal of this study was to examine the expression of prolyl isomerase Pin1 in human Her2+ breast cancer, and to study if Pin1 affects the expression of Her2/Neu itself. Methods Immunohistochemistry for Her2 and Pin1 were performed on two hundred twenty-three human breast cancers, with 59% of the specimen from primary cancers and 41% from metastatic sites. Pin1 inhibition was achieved using siRNA in Her2+ breast cancer cell lines, and its effects were studied using cell viability assays, immunoblotting and immunofluorescence. Results Sixty-four samples (28.7% stained positive for Her2 (IHC 3+, and 54% (122/223 of all breast cancers stained positive for Pin1. Of the Her2-positive cancers 40 (62.5% were also Pin1-positive, based on strong nuclear or nuclear and cytoplasmic staining. Inhibition of Pin1 via RNAi resulted in significant suppression of Her2-positive tumor cell growth in BT474, SKBR3 and AU565 cells. Pin1 inhibition greatly increased the sensitivity of Her2-positive breast cancer cells to the mTOR inhibitor Rapamycin, while it did not increase their sensitivity to Trastuzumab, suggesting that Pin1 might act on Her2 signaling. We found that Pin1 interacted with the protein complex that contains ubiquitinated erbB2 and that Pin1 inhibition accelerated erbB2 degradation, which could be prevented by treatments with the proteasome inhibitor ALLnL. Conclusion Pin1 is a novel regulator of erbB2 that modulates the ubiquitin-mediated degradation of erbB2. The overexpression of Pin1 in a majority of Her2-overexpressing breast cancer may contribute to maintain erbB2 levels. Pin1 inhibition alone and in conjunction with mTOR inhibition suppresses the growth of Her2+ breast cancer cells.

  11. ESR1, ERBB2, and Ki67 mRNA expression predicts stage and grade of non-muscle-invasive bladder carcinoma (NMIBC).

    Science.gov (United States)

    Breyer, Johannes; Wirtz, Ralph M; Laible, Mark; Schlombs, Kornelia; Erben, Philipp; Kriegmair, Maximilian Christian; Stoehr, Robert; Eidt, Sebastian; Denzinger, Stefan; Burger, Maximilian; Hartmann, Arndt; Otto, Wolfgang

    2016-11-01

    Pathological staging and grading are crucial for risk assessment in non-muscle-invasive bladder cancer (NMIBC). Molecular grading might support pathological evaluation and minimize interobserver variability. In this study, the well-established breast cancer markers ESR1, PGR, ERBB2, and MKI67 were evaluated as potential molecular markers to support grading and staging in NMIBC. We retrospectively analyzed clinical data and formalin-fixed paraffin-embedded tissues (FFPE) of patients with NMIBC. Messenger RNA (mRNA) expression of the aforementioned markers was measured by single-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) using RNA-specific TaqMan assays. Relative gene expression was determined by normalization to two reference genes (CALM2 and B2M) using the 40(-ΔΔCT) method and correlated to histopathological stage and grade. Pathological assessment was performed by an experienced uropathologist. Statistical analysis was performed using the SAS software JMP 9.0.0 version and GraphPad Prism 5.04. Of 381 cases of NMIBC, samples of 100 pTa and 255 pT1 cases were included in the final study. Spearman rank correlation revealed significant correlations between grade and expression of MKI67 (r = 0.52, p < 0.0001), ESR1 (r = 0.25, p < 0.0001), and ERBB2 (r = 0.18, p = 0.0008). In Mann-Whitney tests, MKI67 was significantly different between all grades (p < 0.0001), while ESR1 (p = 0.0006) and ERBB2 (p = 0.027) were significantly different between G2 and G3. Higher expression of MKI67 (r = 0.49; p < 0.0001), ERBB2 (r = 0.22; p < 0.0001), and ESR1 (r = 0.18; p = 0.0009) mRNA was positively correlated with higher stage. MKI67 (p < 0.0001), ERBB2 (p = 0.0058), and PGR (p = 0.0007) were significantly different between pTa and pT1. In NMIBC expression of ESR1, ERBB2 and MKI67 are significantly different between stage and grade. This potentially provides objective parameters for pathological

  12. Role of Stat3 and ErbB2 in Breast Cancer

    Science.gov (United States)

    2013-12-01

    precommitted mES transplantation in a mouse model of myocardial infarction. Circ Res 2007, 101:910–918. 42. Anagnostopoulou A, Cao J, Vultur A, Firth KL...activity, in newly established mouse embryo 3T3 fibroblasts. As a control for protein loading, the same extracts were probed for α-tubulin (see Materials... embryos . In addition, to examine Arulanandam et al: TAg Activates cSrc 49 Figure 1. TAg triggers cSrc activation. A, TAg activates cSrc in mouse 3T3

  13. Topoisomerase II alpha gene copy loss has adverse prognostic significance in ERBB2-amplified breast cancer: a retrospective study of paraffin-embedded tumor specimens and medical charts

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    Zhu April

    2008-08-01

    Full Text Available Abstract Background Amplification of the ERBB2 (Her-2/neu oncogene, which occurs in approximately 25% of breast carcinomas, is a known negative prognostic factor. Available data indicate that a variable number of nearby genes on chromosome 17q may be co-amplified or deleted, forming a continuous amplicon of variable size. In approximately 25% of these patients, the amplicon extends to the gene for topoisomerase II alpha (TOP2A, a target for anthracyclines. We sought to understand the significance of these associated genomic changes for breast cancer prognosis and predicting response to therapy. Methods and patients Archival tissue samples from 63 breast cancer patients with ERBB2 amplification, stages 0–IV, were previously analyzed with FISH probes for genes located near ERBB2. In the present study, the clinical outcome data were determined for all patients presenting at stages I–III for whom adequate clinical follow up was available. Results Four amplicon patterns (Classes were identified. These were significantly associated with the clinical outcome, specifically, recurrence of breast cancer. The Amplicon class IV with deleted TOP2A had 67% (6/9 cases with recurrence, whereas the other three classes combined had only 12% (3/25 cases (p-value = 0.004 at the time of last follow-up. TOP2A deletion was also significantly associated with time to recurrence (p-value = 0.0002. After adjusting for age in Cox regression analysis, the association between TOP2A deletion and time to recurrence remains strongly significant (p-value = 0.002 whereas the association with survival is marginally significant (p-value = 0.06. Conclusion TOP2A deletion is associated with poor prognosis in ERBB2-amplified breast carcinomas. Clarification of the mechanism of this association will require additional study.

  14. Chapter 8. Activation mechanisms of chemokine receptors

    DEFF Research Database (Denmark)

    Jensen, Pia C; Rosenkilde, Mette M

    2009-01-01

    Chemokine receptors belong to the large family of 7-transmembrane (7TM) G-protein-coupled receptors. These receptors are targeted and activated by a variety of different ligands, indicating that activation is a result of similar molecular mechanisms but not necessarily similar modes of ligand bin...

  15. Mechanism for the activation of glutamate receptors

    Science.gov (United States)

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  16. Role of ErbB receptors in cancer cell migration and invasion

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    Aline eAppert-Collin

    2015-11-01

    Full Text Available Growth factors mediate their diverse biologic responses (regulation of cellular proliferation, differentiation, migration and survival by binding to and activating cell-surface receptors with intrinsic protein kinase activity named Receptor Tyrosine Kinases (RTKs. About 60 RTKs have been identified and can be classified into more than 16 different receptor families. Their activity is normally tightly controlled and regulated. Overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes or abnormal stimulation by autocrine growth factor loops contribute to constitutive RTK signaling, resulting in alterations in the physiological activities of cells. The ErbB receptor family of RTKs comprises four distinct receptors: the EGFR (also known as ErbB1/HER1, ErbB2 (neu, HER2, ErbB3 (HER3 and ErbB4 (HER4. ErbB family members are often overexpressed, amplified, or mutated in many forms of cancer, making them important therapeutic targets. EGFR has been found to be amplified in gliomas and non-small-cell lung carcinoma while ErbB2 amplifications are seen in breast, ovarian, bladder, non-small-cell lung carcinoma, as well as several other tumor types. Several data have shown that ErbB receptor family and its downstream pathway regulate epithelial-mesenchymal transition, migration, and tumor invasion by modulating extracellular matrix components. Recent findings indicate that extracellular matrix components such as matrikines bind specifically to EGF receptor and promote cell invasion. In this review, we will present an in-depth overview of the structure, mechanisms, cell signaling, and functions of ErbB family receptors in cell adhesion and migration. Furthermore, we will describe in a last part the new strategies developed in anti-cancer therapy to inhibit ErbB family receptor activation.

  17. Assessment of staging, prognosis and mortality of colorectal cancer by tumor markers: receptor erbB-2 and cadherins

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    Jesus Eliane C.

    2005-01-01

    Full Text Available PURPOSE: To evaluate the prognostic significance and correlation with staging and degree of cell differentiation of the tumoral expression of the proteins c-erbB-2 and E-cadherin, in patients with colorectal adenocarcinoma. METHODS: The study included 117 patients with an average age of 63.1 years and an average follow-up duration of 28.1 months. The disease-free interval, survival, incidence of recurrence and specific mortality were evaluated. c-erbB-2 anti-oncoprotein antibodies (Dako were utilized via the streptavidin-biotin technique. Samples were considered to be positive for c-erbB-2 if 10% or more of the tumor cell membranes were stained.The anti-E-cadherin antibodies (Dako, evaluated this protein and is considered positive, if 50% or more of the cell membranes were stained. Statistical analysis was performed using Pearson's chi-squared test, Fisher's exact test, Kaplan-Meier's estimator, the log-rank test and Wilcoxon's test (Breslow version, setting the level of statistical significance at 5% (p<0.05. RESULTS: 52 of 108 patients studied for c-erbB-2 were positive (48,1%, 47 of 93 patients studied for E-cadherin were negative (50,5%. These data do not express any correlation with TNM (tumor, node and metastasis staging and the degree of cell differentiation or with the tumor recurrence rate. The disease-free interval among patients who were positive for c-erbB-2 and negative for E-cadherin was 68.0 months and did not differ from those with c-erbB-2 negative and E-cadherin positive ( 55.0 months - p = 0.5510. The average survival among patients positive for c-erbB-2 and negative for E-cadherin was 75 months without statistical significance difference with the other group ( 61 months - p = 0.5256. Specific mortality occurred in 20.0% of the cases and did not correlate with the expression of c-erbB-2 (p=0,446, E-cadherin (p=0,883. CONCLUSION: The tumoral expression of c-erbB-2 and E-cadherin did not demonstrate a correlation with the staging and degree of cell differentiation, and it did not present prognostic value regarding disease recurrence, disease-free interval, survival and specific mortality among patients with colorectal adenocarcinoma.

  18. ErbB2 and NFκB overexpression as predictors of chemoradiation resistance and putative targets to overcome resistance in muscle-invasive bladder cancer.

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    Fumitaka Koga

    Full Text Available Radical cystectomy for muscle-invasive bladder cancer (MIBC patients frequently impairs their quality of life (QOL due to urinary diversion. To improve their QOL, a bladder-sparing alternative strategy using chemoradiation has been developed. In bladder-sparing protocols, complete response (CR to induction chemoradiation is a prerequisite for bladder preservation and favorable survival. Thus predicting chemoradiation resistance and overcoming it would increase individual MIBC patients' chances of bladder preservation. The aim of this study is to investigate putative molecular targets for treatment aimed at improving chemoradiation response. Expression levels of erbB2, NFκB, p53, and survivin were evaluated immunohistochemically in pretreatment biopsy samples from 35 MIBC patients in whom chemoradiation sensitivity had been pathologically evaluated in cystectomy specimens, and associations of these expression levels with chemoradiation sensitivity and cancer-specific survival (CSS were investigated. Of the 35 patients, 11 (31% achieved pathological CR, while tumors in the remaining 24 patients (69% were chemoradiation-resistant. Multivariate analysis identified erbB2 and NFκB overexpression and hydronephrosis as significant and independent risk factors for chemoradiation resistance with respective relative risks of 11.8 (P = 0.014, 15.4 (P = 0.024 and 14.3 (P = 0.038. The chemoradiation resistance rate was 88.5% for tumors overexpressing erbB2 and/or NFκB, but only 11.1% for those negative for both (P <0.0001. The 5-year CSS rate was 74% overall. Through multivariate analysis, overexpression of erbB2 and/or NFκB was identified as an independent risk factor for bladder cancer death with marginal significance (hazard ratio 21.5, P = 0.056 along with chemoradiation resistance (P = 0.003 and hydronephrosis (P = 0.018. The 5-year CSS rate for the 11 patients achieving pathological CR was 100%, while that for the 24 with chemoradiation-resistant disease was 61% (P = 0.018. Thus, erbB2 and NFκB overexpression are relevant to chemoradiation resistance and are putative targets aimed at overcoming chemoradiation resistance in MIBC.

  19. tabAnti-HER2 (erbB-2 oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO

    Directory of Open Access Journals (Sweden)

    Carrasco-Pancorbo Alegria

    2008-12-01

    Full Text Available Abstract Background The effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2 oncogene-positive and HER2-negative breast carcinomas are considered together. We here investigated the anti-HER2 effects of phenolic fractions directly extracted from Extra Virgin Olive Oil (EVOO in cultured human breast cancer cell lines. Methods Solid phase extraction followed by semi-preparative high-performance liquid chromatography (HPLC was used to isolate phenolic fractions from commercial EVOO. Analytical capillary electrophoresis coupled to mass spectrometry was performed to check for the composition and to confirm the identity of the isolated fractions. EVOO polyphenolic fractions were tested on their tumoricidal ability against HER2-negative and HER2-positive breast cancer in vitro models using MTT, crystal violet staining, and Cell Death ELISA assays. The effects of EVOO polyphenolic fractions on the expression and activation status of HER2 oncoprotein were evaluated using HER2-specific ELISAs and immunoblotting procedures, respectively. Results Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+-pinoresinol and 1-(+-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation

  20. Suppression of human lung cancer cell proliferation and metastasis in vitro by the transducer of ErbB-2.1(TOB1)

    Institute of Scientific and Technical Information of China (English)

    Yang JIAO; Ke-kang SUN; Lin ZHAO; Jia-ying XU; Li-li Wang; Sai-jun FAN

    2012-01-01

    Aim:To investigate the effects of the transducer of ErbB-2.1 (TOB1) on the proliferation,migration and invasion of human lung cancer cells in vitro.Methods:Human lung cancer cell lines (95-D,A549,NCI-H1299,NCI-H1975,NCI-H661,NCI-H446,NCI-H1395,and Calu-3)and the normal human bronchial epithelial (HBE) cell line were tested.The expression levels of TOB1 in the cells were determined with Western blot and RT-PCR analyses.TOB1-overexpressing cell line 95-D/TOB1 was constructed using lipofectamine-induced TOB1 recombinant plasmid transfection and selective G418 cell culture.The A549 cells were transcend-transfected with TOB1-siRNA.MTT assay,flow cytometry and Western blot analysis were used to examine the effects of TOB1 on cancer cell proliferation and wound healing.Transwell invasive assay was performed to evaluate the effects of TOB1 on cancer cell migration and invasion.The activity of MMP2 and MMP9 was measured using gelatin zymography assay.Results:The expression levels of TOB1 in the 8 human lung cancer cell lines were significantly lower than that in HBE cells.TOB1 overexpression inhibited the proliferation of 95-D cells,whereas TOB1 knockdown with TOB1-siRNA promoted the growth of A549 cells.Decreased cell migration and invasion were detected in 95-D/TOB1 cells,and the suppression of TOB1 enhanced the metastasis in A549 cells.TOB1 overexpression not only increased the expression of the phosphatase and tensin homolog (PTEN),an important tumor suppressor,but also regulated the downstream effectors in the PI3K/PTEN signaling pathway,including Akt,ERK1/2,etc.In contrast,decreased expression of TOB1 oppositely regulated the expression of these factors.TOB1 also regulates the gelatinase activity of MMP2 and MMP9 in lung cancer cells.Conclusion:The results demonstrate that the PI3K/PTEN pathway,which is essential for carcinogenesis,angiogenesis,and metastasis,may be one of the possible signaling pathways for regulation of proliferation and metastasis of human lung

  1. CERAPP: Collaborative Estrogen Receptor Activity Prediction Project

    Data.gov (United States)

    U.S. Environmental Protection Agency — Data from a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) demonstrating using predictive computational...

  2. Significance of ERBB2 Overexpression in Therapeutic Resistance and Cancer-Specific Survival in Muscle-Invasive Bladder Cancer Patients Treated With Chemoradiation-Based Selective Bladder-Sparing Approach

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Masaharu [Department of Urology, Tokyo Medical and Dental University, Graduate School, Tokyo (Japan); Koga, Fumitaka, E-mail: f-koga@cick.jp [Department of Urology, Tokyo Medical and Dental University, Graduate School, Tokyo (Japan); Yoshida, Soichiro [Department of Urology, Tokyo Medical and Dental University, Graduate School, Tokyo (Japan); Tamura, Tomoki [Department of Pathology, Tokyo Medical and Dental University, Graduate School, Tokyo (Japan); Fujii, Yasuhisa [Department of Urology, Tokyo Medical and Dental University, Graduate School, Tokyo (Japan); Ito, Eisaku [Department of Pathology, Tokyo Medical and Dental University, Graduate School, Tokyo (Japan); Kihara, Kazunori [Department of Urology, Tokyo Medical and Dental University, Graduate School, Tokyo (Japan)

    2014-10-01

    Purpose: To investigate the associations of ERBB 2 overexpression with chemoradiation therapy (CRT) resistance and cancer-specific survival (CSS) in muscle-invasive bladder cancer (MIBC) patients treated with the CRT-based bladder-sparing protocol. Methods and Materials: From 1997 to 2012, 201 patients with cT2-4aN0M0 bladder cancer were treated with CRT (40 Gy with concurrent cisplatin) following transurethral resection of bladder tumor (TURBT). Basically, patients with tumors that showed good CRT response and were amenable to segmental resection underwent partial cystectomy (PC) with pelvic lymph node dissection for bladder preservation; otherwise, radical cystectomy (RC) was recommended. Included in this study were 119 patients in whom TURBT specimens were available for immunohistochemical analysis of ERBB 2 expression. Following CRT, 30 and 65 patients underwent PC or RC, respectively; the remaining 24 patients did not undergo cystectomy. Tumors were defined as CRT-resistant when patients did not achieve complete response after CRT. Associations of ERBB 2 overexpression with CRT resistance and CSS were evaluated. Results: CRT resistance was observed clinically in 56% (67 of 119 patients) and pathologically (in cystectomy specimens) in 55% (52 of 95 patients). ERBB 2 overexpression was observed in 45 patients (38%). On multivariate analysis, ERBB 2 overexpression was an independent predictor for CRT resistance clinically (odds ratio, 3.6; P=.002) and pathologically (odds ratio, 2.9; P=.031). ERBB 2 overexpression was associated with shorter CSS (5-year CSS rates, 56% vs 87% for the ERBB 2 overexpression group vs the others; P=.001). ERBB 2 overexpression was also an independent risk factor for bladder cancer death at all time points of our bladder-sparing protocol (pre-CRT, post-CRT, and post-cystectomy). Conclusions: ERBB 2 overexpression appears relevant to CRT resistance and unfavorable CSS in MIBC patients treated with the CRT-based bladder

  3. Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance

    Directory of Open Access Journals (Sweden)

    Thieffry Denis

    2009-01-01

    Full Text Available Abstract Background In breast cancer, overexpression of the transmembrane tyrosine kinase ERBB2 is an adverse prognostic marker, and occurs in almost 30% of the patients. For therapeutic intervention, ERBB2 is targeted by monoclonal antibody trastuzumab in adjuvant settings; however, de novo resistance to this antibody is still a serious issue, requiring the identification of additional targets to overcome resistance. In this study, we have combined computational simulations, experimental testing of simulation results, and finally reverse engineering of a protein interaction network to define potential therapeutic strategies for de novo trastuzumab resistant breast cancer. Results First, we employed Boolean logic to model regulatory interactions and simulated single and multiple protein loss-of-functions. Then, our simulation results were tested experimentally by producing single and double knockdowns of the network components and measuring their effects on G1/S transition during cell cycle progression. Combinatorial targeting of ERBB2 and EGFR did not affect the response to trastuzumab in de novo resistant cells, which might be due to decoupling of receptor activation and cell cycle progression. Furthermore, examination of c-MYC in resistant as well as in sensitive cell lines, using a specific chemical inhibitor of c-MYC (alone or in combination with trastuzumab, demonstrated that both trastuzumab sensitive and resistant cells responded to c-MYC perturbation. Conclusion In this study, we connected ERBB signaling with G1/S transition of the cell cycle via two major cell signaling pathways and two key transcription factors, to model an interaction network that allows for the identification of novel targets in the treatment of trastuzumab resistant breast cancer. Applying this new strategy, we found that, in contrast to trastuzumab sensitive breast cancer cells, combinatorial targeting of ERBB receptors or of key signaling intermediates does not

  4. Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting

    Science.gov (United States)

    Singer, Josef; Weichselbaumer, Marlene; Stockner, Thomas; Mechtcheriakova, Diana; Sobanov, Yury; Bajna, Erika; Wrba, Friedrich; Horvat, Reinhard; Thalhammer, Johann G.; Willmann, Michael; Jensen-Jarolim, Erika

    2012-01-01

    To facilitate comparative oncology trials we compared the biological and molecular homologies of canine (dog; Canis lupus familiaris) and human tumor-associated antigens ErbB-1 and -2. Further, we investigated whether they could serve as targets for anti-ErbB-1 (cetuximab) and anti-ErbB-2 antibodies (trastuzumab), which are highly relevant in human clinical oncology. Immunohistochemistry of canine mammary cancer showed ErbB-1 overexpression in 3/10 patients and ErbB-2 in 4/10. We report 91% amino acid homology for ErbB-1 and 92% for ErbB-2 between canine and human molecules. Modeling of canine on human ErbB-1 revealed that the cetuximab epitope only differs by 4 amino acids: Lys443 is replaced by Arg, Ser468 by Asn, Gly471 by Asp, and Asn473 by Lys in canines. The trastuzumab binding site is identical in human and canine ErbB-2 apart from a single amino acid change (Pro557 to Ser). Binding of cetuximab and trastuzumab to canine mammary carcinoma cells CF33, CF41, Sh1b and P114 was confirmed by flow cytometry. Both antibodies significantly inhibited canine tumor cell proliferation partly due to growth arrest in G0/G1 phase. We explain the lower efficiency on the tested canine than on human SKBR3 and A431 cells, by a 2-log lower expression level of the canine ErbB-1 and -2 molecules. Our results indicate significant homology of human and canine Erb-1 and -2 tumor associated antigens. The fact that the canine homologues express the cetuximab and trastuzumab epitopes may facilitate antibody-based immunotherapy in dogs. Importantly, the striking similarities of ErbB-1 and -2 molecules open up avenues towards comparative strategies for targeted drug development. PMID:22424313

  5. 抗p185erbB2人鼠嵌合抗体慢病毒表达载体的构建%Construction of the Lentiviral Expression Vector for Anti-p185erbB2 Mouse/Human Chimeric Antibody

    Institute of Scientific and Technical Information of China (English)

    刘芳; 李力; 张玮; 王琪

    2013-01-01

    本研究构建抗p185erbB2人鼠嵌合抗体慢病毒表达载体,并将其转染293T细胞,明确转染后嵌合抗体基因的表达情况.采用PCR法扩增抗p185erbB2鼠单抗轻、重链可变区基因(vL和vH)和人IgG1的轻、重链恒定区基因(κ和γ1),再利用三引物PCR法将vL和κ,vH和γ1进行拼接,得到嵌合轻链基因(L)和嵌合重链基因(H),分别插入质粒pVAX1/IRES的IRES元件的下、上游.用内切酶将H-IRES-L从pVAX1/H-IRES-L上切下,插入慢病毒载体pWPI中,经相应酶切和测序鉴定,正确构建了慢病毒表达载体pWPI/H-IRES-L.将其转染293T细胞,48 h后通过荧光显微镜检测绿色荧光蛋白(GFP)的表达,RT-PCR和直接ELISA法检测嵌合抗体的表达.结果显示,转染pWPI/H-IRES-L的293T细胞中有嵌合轻链和嵌合重链基因的共同表达,而且表达的嵌合抗体能够与p185erbB2分子特异性结合,为今后抗p185erbB2工程抗体的研究奠定了基础.%This research was to construct the lentiviral expression vector for anti- pl85erbB2 mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-pl85erbB2 mAb and the constant regions of human IgG1 (Κ and γ1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to pl85erbB2 specifically. This research may lay a sound foundation for further study of anti-pl85erbB2engineered antibody.

  6. 人脑胶质瘤中MGMT、erbB2的表达及其意义%Expression and significance of MGMT and erbB2 in human glioma

    Institute of Scientific and Technical Information of China (English)

    孙春明; 王之敏; 周幽心; 左剑玲; 许期年; 王秀英; 周岱

    2009-01-01

    Objective To study the expression and significance of MGMT and erbB2 in human glioma. Methods RT-PCR method was used to determine the mRNA levels of MGMT and erbB2 gene in 42 clinical glioma specimens. Results The expression rates of MGMT and erbB2 in human glioma were 45. 2% and 61. 9%, respectively. The expression of MGMT was closely correlated to that of erbB2 (P<0. 01). Conclusion MGMT and erbB2 are all overexpressed in human gliomas, which is helpful to the drug selection for chemotherapy.%目的 探讨脑胶质瘤组织中耐药相关基因MGMT、erbB2的表达及临床意义.方法 采用RT-PCR法对42例脑胶质瘤标本中MGMT、erbB2 mRNA进行测定.结果 MGMT和erbB2在脑胶质瘤标本中的阳性表达率分别为45.2%和61.9%,两者有极显著相关性(P<0.01).结论 MGMT和erbB2在脑胶质瘤中均有较高表达,对合理选择胶质瘤化疗药物有一定临床意义.

  7. EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

    DEFF Research Database (Denmark)

    Grøvdal, Lene Melsæther; Kim, Jiyoung; Holst, Mikkel Roland

    2012-01-01

    to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition......The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported...... that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance...

  8. Testing ERBB2 p.L755S kinase domain mutation as a druggable target in a patient with advanced colorectal cancer

    Science.gov (United States)

    Aung, Kyaw L.; Stockley, Tracy L.; Serra, Stefano; Kamel-Reid, Suzanne; Bedard, Philippe L.; Siu, Lillian L.

    2016-01-01

    Recent advances in molecular profiling technologies allow genetic driver events in individual tumors to be identified. The hypothesis behind this ongoing molecular profiling effort is that improvement in patients’ clinical outcomes will be achieved by inhibiting these discovered genetic driver events with matched targeted drugs. This hypothesis is currently being tested in oncology clinics with variable early results. Herein, we present our experience with a case of advanced colorectal cancer (CRC) with an ERBB2 p.L755S kinase domain mutation, a BRAF p.N581S mutation, and an APC p.Q1429fs mutation, together with a brief review of the literature describing the biological and clinical significance of ERRB2 kinase domain mutations in CRC. The patient was treated with trastuzumab combined with infusional 5-fluorouracil and leucovorin based on the presence of ERBB2 p.L755S kinase mutation in the tumor and based on the available evidence at the time when standard treatment options had been exhausted. However, there was no therapeutic response illustrating the challenges we face in managing patients with potentially targetable mutations where results from functional in vitro and in vivo studies lag behind those of genomic sequencing studies. Also lagging behind are clinical utility data from oncology clinics, hampering rapid therapeutic advances. Our case also highlights the logistical barriers associated with getting the most optimal therapeutic agents to the right patient in this era of personalized therapeutics based on cancer genomics. PMID:27626067

  9. Downregulation of Epidermal Growth Factor Receptor Expression Contributes to α-TEA's Proapoptotic Effects in Human Ovarian Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Ming-Chieh Shun

    2010-01-01

    Full Text Available RRR-α-tocopherol derivative α-TEA (RRR-α-tocopherol ether-linked acetic acid analog has been shown to be a potent antitumor agent both in vivo and in vitro. In this study, we investigated the effects of α-TEA on the expression of epidermal growth factor receptor (EGFR family members, ErbB1, 2 and 3, and the role of ErbB 2 and 3 in α-TEA-induced apoptosis and suppression of Akt, FLIP and survivin in the cisplatin-sensitive (A2780S and -resistant (A2780/CP70R human ovarian cancer cell lines. Data show that α-TEA's ability to induced apoptosis was associated with reduced expression of ErbB1 (cisplatin-resistant cells, 2 and 3 (both cell types and reduced levels of the phosphorylated (active form of Akt; as well as, reduced levels of FLIP and survivin proteins in both cell types. Ectopic overexpression and siRNA knockdown studies showed that ErbB2, ErbB3, Akt, FLIP and survivin are involved in α-TEA-induce apoptosis and that α-TEA downregulates FLIP and survivin via suppression of pAkt, which is mediated by ErbB2 and ErB3. Thus, α-TEA is a potent pro-apoptotic agent for both cisplatin-sensitive and -resistant ovarian cancer cell lines in cell culture and it produces cell death, at least in part, by downregulation of members of the EGFR family.

  10. New GABA amides activating GABAA-receptors.

    Science.gov (United States)

    Raster, Peter; Späth, Andreas; Bultakova, Svetlana; Gorostiza, Pau; König, Burkhard; Bregestovski, Piotr

    2013-01-01

    We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in whole-cell patch-clamp recordings. New GABA-amides, however, gave moderate activation responses with a clear structure-activity relationship suggesting some of these compounds as promising molecular tools for the functional analysis of GABAA-receptors.

  11. New GABA amides activating GABAA-receptors

    Directory of Open Access Journals (Sweden)

    Peter Raster

    2013-02-01

    Full Text Available We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in whole-cell patch-clamp recordings. New GABA-amides, however, gave moderate activation responses with a clear structure–activity relationship suggesting some of these compounds as promising molecular tools for the functional analysis of GABAA-receptors.

  12. Gene Expression Meta-Analysis identifies Cytokine Pathways and 5q Aberrations involved in Metastasis of ERBB2 Amplified and Basal Breast Cancer

    DEFF Research Database (Denmark)

    Thomassen, Mads; Tan, Qihua; Burton, Mark

    2013-01-01

    Background: Breast tumors have been described by molecular subtypes characterized by pervasively different gene expression profiles. The subtypes are associated with different clinical parameters and origin of precursor cells. However, the biological pathways and chromosomal aberrations that differ...... the subgroups impact metastasis. Results: We have scrutinized publicly available gene expression datasets and identified molecular subtypes in 1,394 breast tumors with outcome data. By analysis of chromosomal regions and pathways using “Gene set enrichment analysis” followed by a meta-analysis, we identified...... show that high expression of 5q14 genes and low levels of TNFR2 pathway genes were associated with poor survival in basal-like cancers. Furthermore, low expression of 5q33 genes and interleukin-12 pathway genes were associated with poor outcome exclusively in ERBB2-like tumors. Conclusion...

  13. Nuclear expression of KLF6 tumor suppressor factor is highly associated with overexpression of ERBB2 oncoprotein in ductal breast carcinomas.

    Directory of Open Access Journals (Sweden)

    Ricardo C Gehrau

    Full Text Available BACKGROUND: Krüppel-like factor 6 (KLF6 is an evolutionarily conserved and ubiquitously expressed protein that belongs to the mammalian Sp1/KLF family of transcriptional regulators. Though KLF6 is a transcription factor and harbors a nuclear localization signal it is not systematically located in the nucleus but it was detected in the cytoplasm of several tissues and cell lines. Hence, it is still not fully settled whether the tumor suppressor function of KLF6 is directly associated with its ability to regulate target genes. METHODOLOGY/PRINCIPAL FINDINGS: In this study we analyzed KLF6 expression and sub-cellular distribution by immunohistochemistry in several normal and tumor tissues in a microarray format representing fifteen human organs. Results indicate that while both nuclear and cytoplasmic distribution of KLF6 is detected in normal breast tissues, breast carcinomas express KLF6 mainly detected in the cytoplasm. Expression of KLF6 was further analyzed in breast cancer tissues overexpressing ERBB2 oncoprotein, which is associated with poor disease prognosis and patient's survival. The analysis of 48 ductal carcinomas revealed a significant population expressing KLF6 predominantly in the nuclear compartment (X(2p = 0.005; Fisher p = 0.003. Moreover, this expression pattern correlates directly with early stage and small ductal breast tumors and linked to metastatic events in lymph nodes. CONCLUSIONS/SIGNIFICANCE: Data are consistent with a preferential localization of KLF6 in the nuclear compartment of early stage and small HER2-ERBB2 overexpressing ductal breast tumor cells, also presenting lymph node metastatic events. Thus, KLF6 tumor suppressor could represent a new molecular marker candidate for tumor prognosis and/or a potential target for therapy strategies.

  14. Mechanism of FGF receptor dimerization and activation

    Science.gov (United States)

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

  15. NMDA receptor activity in neuropsychiatric disorders

    Directory of Open Access Journals (Sweden)

    Shaheen E Lakhan

    2013-06-01

    Full Text Available N-Methyl-D-aspartate (NMDA receptors play a variety of physiologic roles and their proper signaling is essential for cellular homeostasis. Any disruption in this pathway, leading to either enhanced or decreased activity, may result in the manifestation of neuropsychiatric pathologies such as schizophrenia, mood disorders, substance induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus. Here, we explore the notion that the overlap in activity of at least one biochemical pathway, the NMDA receptor pathway, may be the link to understanding the overlap in psychotic symptoms between diseases. This review intends to present a broad overview of those neuropsychiatric disorders for which alternations in NMDA receptor activity is prominent thus suggesting that continued direction of pharmaceutical intervention to this pathway may present a viable option for managing symptoms.

  16. Activation of Neuropeptide FF Receptors by Kisspeptin Receptor Ligands.

    Science.gov (United States)

    Oishi, Shinya; Misu, Ryosuke; Tomita, Kenji; Setsuda, Shohei; Masuda, Ryo; Ohno, Hiroaki; Naniwa, Yousuke; Ieda, Nahoko; Inoue, Naoko; Ohkura, Satoshi; Uenoyama, Yoshihisa; Tsukamura, Hiroko; Maeda, Kei-Ichiro; Hirasawa, Akira; Tsujimoto, Gozoh; Fujii, Nobutaka

    2011-01-13

    Kisspeptin is a member of the RFamide neuropeptide family that is implicated in gonadotropin secretion. Because kisspeptin-GPR54 signaling is implicated in the neuroendocrine regulation of reproduction, GPR54 ligands represent promising therapeutic agents against endocrine secretion disorders. In the present study, the selectivity profiles of GPR54 agonist peptides were investigated for several GPCRs, including RFamide receptors. Kisspeptin-10 exhibited potent binding and activation of neuropeptide FF receptors (NPFFR1 and NPFFR2). In contrast, short peptide agonists bound with much lower affinity to NPFFRs while showing relatively high selectivity toward GPR54. The possible localization of secondary kisspeptin targets was also demonstrated by variation in the levels of GnRH release from the median eminence and the type of GPR54 agonists used. Negligible affinity of the reported NPFFR ligands to GPR54 was observed and indicates the unidirectional cross-reactivity between both ligands.

  17. Role of ErbB family receptor tyrosine kinases in intrahepatic cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Aberrant expression and signaling of epidermal growth factor receptor (ErbB) family receptor tyrosine kinases, most notably that of ErbB2 and ErbB1, have been implicated in the molecular pathogenesis of intrahepatic cholangiocarcinoma. Constitutive overexpression of ErbB2 and/or ErbB1 in malignant cholangiocytes has raised interest in the possibility that agents which selectively target these receptors could potentially be effective in cholangiocarcinoma therapy. However, current experience with such ErbB-directed therapies have at best produced only modest responses in patients with biliary tract cancers. This review provides a comprehensive and critical analysis of both preclinical and clinical studies aimed at assessing the role of altered ErbB2 and/or ErbB1 expression, genetic modifications, and dysregulated signaling on cholangiocarcinoma development and progression. Specific limitations in experimental approaches that have been used to assess human cholangiocarcinoma specimens for ErbB2 and/or ErbB1 overexpression and gene amplification are discussed. In addition, current rodent models of intrahepatic cholangiocarcinogenesis associated with constitutive ErbB2 overexpression are reviewed. Select interactive relationships between ErbB2 or ErbB1 with other relevant molecular signaling pathways associated with intrahepatic cholangiocarcinoma development and progression are also detailed, including those linking ErbB receptors to bile acid, cyclooxygenase-2, interleukin-6/gp130, transmembrane mucins, hepatocyte growth factor/Met, and vascular endothelial growth factor signaling. Lastly, various factors that can limit therapeutic efficacy of ErbB-targeted agents against cholangiocarcinoma are considered.

  18. CERAPP: Collaborative estrogen receptor activity prediction project

    DEFF Research Database (Denmark)

    Mansouri, Kamel; Abdelaziz, Ahmed; Rybacka, Aleksandra

    2016-01-01

    Background: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER......). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. oBjectives: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project...

  19. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

    Science.gov (United States)

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2016-05-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally.

  20. Clinical grade manufacturing of genetically modified, CAR-expressing NK-92 cells for the treatment of ErbB2-positive malignancies.

    Science.gov (United States)

    Nowakowska, Paulina; Romanski, Annette; Miller, Nicole; Odendahl, Marcus; Bonig, Halvard; Zhang, Congcong; Seifried, Erhard; Wels, Winfried S; Tonn, Torsten

    2017-09-06

    The NK-92/5.28.z cell line (also referred to as HER2.taNK) represents a stable, lentiviral-transduced clone of ErbB2 (HER2)-specific, second-generation CAR-expressing derivative of clinically applicable NK-92 cells. This study addresses manufacturing-related issues and aimed to develop a GMP-compliant protocol for the generation of NK-92/5.28.z therapeutic doses starting from a well-characterized GMP-compliant master cell bank. Commercially available GMP-grade culture media and supplements (fresh frozen plasma, platelet lysate) were evaluated for their ability to support expansion of NK-92/5.28.z. Irradiation sensitivity and cytokine release were also investigated. NK-92/5.28.z cells can be grown to clinically applicable cell doses of 5 × 10(8) cells/L in a 5-day batch culture without loss of viability and potency. X-Vivo 10 containing recombinant transferrin supplemented with 5% FFP and 500 IU/mL IL-2 in VueLife 750-C1 bags showed the best results. Platelet lysate was less suited to support NK-92/5.28.z proliferation. Irradiation with 10 Gy completely abrogated NK-92/5.28.z proliferation and preserved viability and potency for at least 24 h. NK-92/5.28.z showed higher baseline cytokine release compared to NK-92, which was significantly increased upon encountering ErbB2(+) targets [GZMB (twofold), IFN-γ (fourfold), IL-8 (24-fold) and IL-10 (fivefold)]. IL-6 was not released by NK cells, but was observed in some stimulated targets. Irradiation resulted in upregulation of IL-8 and downregulation of sFasL, while other cytokines were not impacted. Our concept suggests NK-92/5.28.z maintenance culture from which therapeutic doses up to 5 × 10(9) cells can be expanded in 10 L within 5 days. This established process is feasible to analyze NK-92/5.28.z in phase I/II trials.

  1. 含F/2A序列的抗 P185erbB2人鼠嵌合抗体慢病毒表达载体的构建%Construction of the Lentiviral Expression Vector Containing F/2AFragment for Anti-P185erbB2 Mouse/Human Chimeric Antibody

    Institute of Scientific and Technical Information of China (English)

    刘芳; 李力; 张玮; 王琪

    2012-01-01

    目的 构建含 F/2A 序列的抗 P185erbB2 人鼠嵌合抗体慢病毒表达载体,观察其在 293T 细胞中的表达.方法 用具有自我剪切能力的弗林蛋白酶(Furin)/口蹄疫病毒2A多肽(F/2A)连接人鼠嵌合抗体的重链和轻链,形成一个开放阅读框 (ORF),插入慢病毒表达载体pWPI,构建重组抗P185erbB2全长人鼠嵌合抗体表达载体pWPI/H-F2A-L.以已构建的慢病毒表达载体pWPI/H-IRES-L为对照质粒.应用磷酸钙沉淀法将慢病毒载体 3 质粒系统共转染入 293T 细胞进行包装,测定病毒滴度.再感染 293T 细胞,荧光显微镜下观察 GFP 的表达和转染效率,RT-PCR、ELISA 方法分别检测嵌合抗体 mRNA和蛋白的表达.结果 经测序鉴定,pWPI/H-F2A-L与预期设计一致;pWPI/H-F2A-L组的病毒滴度为4.3×105 TU/ml,而pW-PI/H-IRES-L 组的病毒滴度为3.5×105 TU/ml;两组重组慢病毒的转染效率分别为 87.68%和 79.08%:两组重组慢病毒感染 293T 细胞后,都有嵌合重链和嵌合轻链的表达,由F/2A介导的嵌合抗体的表达水平要高于由 IRES 介导的嵌合抗体.结论 成功构建了含F/2A序列的抗P185erbB2人鼠嵌合抗体慢病毒表达载体,为今后抗P185erbB2工程抗体的研究奠定了基础.%Objective To construct the lentiviral expression vector containing F/2A fragment for anti-P185erbB2 mouse/human chimeric antibody , and detect its expression in 293T cells. Methods The heavy and light chains of chimeric antibody were joined by Furin and 2A ( F/ 2A) self-cleavage peptide and cloned into a lentiviral vector of pWPI , to generate the lentiviral ex-pression vector, pWPI/H-F2A-L Another lentiviral expression vector , pWPI/H4RES-L that had been generated already , was used as control plasmid. 293 T cells were co-transfected with the 3 helper plasmid system by using calcium phosphate precipitation , and then the virus titer was exam-ined. The 293 T cells were infected by the obtained lentiviral particles. The expression of GFP and the transfection efficiency were examined under fluorescent microscope after transfection . The expression of chimeric antibody was examined by RT -PCR and ELISA. Results Sequence analysis showed that the lentiviral expression vector pWPI/H-F2A-L was constructed correctly. The viral titer was exam-ined as 4. 3×105 TU/mL for pWPI/H2A4, group, and 3. 5×105 TU/mL for pWPI/H4RES4, group. The transfection efficiency in these two groups reached to 87. 68% and 79. 08% , respective-ly. After 293T cells were transfected with lentiviral particles , both heavy and light chains of the chi-meric antibody genes could express in the two groups. The expression level of the chimeric anti body linked by F2A was higher than that of the chimeric anti body linked by IRES . Conclusion The lentiviral expression vector containing F/2A fragment for anti-P185erbB2 mouse/human chimeric antibody was successfully constructed for further study of anti -P185erbB2 engineered antibody.

  2. Small Molecule Tyrosine Kinase Inhibitors of ErbB2/HER2/Neu in the Treatment of Aggressive Breast Cancer

    Directory of Open Access Journals (Sweden)

    Richard L. Schroeder

    2014-09-01

    Full Text Available The human epidermal growth factor receptor 2 (HER2 is a member of the erbB class of tyrosine kinase receptors. These proteins are normally expressed at the surface of healthy cells and play critical roles in the signal transduction cascade in a myriad of biochemical pathways responsible for cell growth and differentiation. However, it is widely known that amplification and subsequent overexpression of the HER2 encoding oncogene results in unregulated cell proliferation in an aggressive form of breast cancer known as HER2-positive breast cancer. Existing therapies such as trastuzumab (Herceptin® and lapatinib (Tyverb/Tykerb®, a monoclonal antibody inhibitor and a dual EGFR/HER2 kinase inhibitor, respectively, are currently used in the treatment of HER2-positive cancers, although issues with high recurrence and acquired resistance still remain. Small molecule tyrosine kinase inhibitors provide attractive therapeutic targets, as they are able to block cell signaling associated with many of the proposed mechanisms for HER2 resistance. In this regard we aim to present a review on the available HER2 tyrosine kinase inhibitors, as well as those currently in development. The use of tyrosine kinase inhibitors as sequential or combinatorial therapeutic strategies with other HER family inhibitors is also discussed.

  3. Peroxisome proliferator-activated receptors for hypertension

    Institute of Scientific and Technical Information of China (English)

    Daisuke; Usuda; Tsugiyasu; Kanda

    2014-01-01

    Peroxisome proliferator-activated receptors(PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily, which is composed of four members encoded by distinct genes(α, β, γ, and δ). The genes undergo transactivation or transrepression under specific mechanisms that lead to the induction or repression of target gene expression. As is the case with other nuclear receptors, all four PPAR isoforms contain five or six structural regions in four functional domains; namely, A/B, C, D, and E/F. PPARs have many functions, particularly functions involving control of vascular tone, inflammation, and energy homeostasis, and are, therefore, important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. Hence, PPARs also represent drug targets, and PPARα and PPARγ agonists are used clinically in the treatment of dyslipidemia and type 2 diabetes mellitus, respectively. Because of their pleiotropic effects, they have been identified as active in a number of diseases and are targets for the development of a broad range of therapies for a variety of diseases. It is likely that the range of PPARγ agonist therapeutic actions will result in novel approaches to lifestyle and other diseases. The combination of PPARs with reagents or with other cardiovascular drugs, such as diuretics and angiotensin Ⅱ receptor blockers, should be studied.This article provides a review of PPAR isoform characteristics, a discussion of progress in our understanding of the biological actions of PPARs, and a summary of PPAR agonist development for patient management. We also include a summary of the experimental and clinical evidence obtained from animal studies and clinical trials conducted to evaluate the usefulness and effectiveness of PPAR agonists in the treatment of lifestyle-related diseases.

  4. Peroxisome proliferator-activated receptors for hypertension

    Science.gov (United States)

    Usuda, Daisuke; Kanda, Tsugiyasu

    2014-01-01

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily, which is composed of four members encoded by distinct genes (α, β, γ, and δ). The genes undergo transactivation or transrepression under specific mechanisms that lead to the induction or repression of target gene expression. As is the case with other nuclear receptors, all four PPAR isoforms contain five or six structural regions in four functional domains; namely, A/B, C, D, and E/F. PPARs have many functions, particularly functions involving control of vascular tone, inflammation, and energy homeostasis, and are, therefore, important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. Hence, PPARs also represent drug targets, and PPARα and PPARγ agonists are used clinically in the treatment of dyslipidemia and type 2 diabetes mellitus, respectively. Because of their pleiotropic effects, they have been identified as active in a number of diseases and are targets for the development of a broad range of therapies for a variety of diseases. It is likely that the range of PPARγ agonist therapeutic actions will result in novel approaches to lifestyle and other diseases. The combination of PPARs with reagents or with other cardiovascular drugs, such as diuretics and angiotensin II receptor blockers, should be studied. This article provides a review of PPAR isoform characteristics, a discussion of progress in our understanding of the biological actions of PPARs, and a summary of PPAR agonist development for patient management. We also include a summary of the experimental and clinical evidence obtained from animal studies and clinical trials conducted to evaluate the usefulness and effectiveness of PPAR agonists in the treatment of lifestyle-related diseases. PMID:25228953

  5. Cellular receptors for plasminogen activators recent advances.

    Science.gov (United States)

    Ellis, V

    1997-10-01

    The generation of the broad-specificity protease plasmin by the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) is implicated in a variety of pathophysiological processes, including vascular fibrin dissolution, extracellular matrix degradation and remodeling, and cell migration. A mechanism for the regulation of plasmin generation is through binding of the plasminogen activators to specific cellular receptors: uPA to the glycolipid-anchored membrane protein urokinase-type plasminogen activator receptor (uPAR) and tPA to a number of putative binding sites. The uPA-uPAR complex can interact with a variety of ligands, including plasminogen, vitronectin, and integrins, indicating a multifunctional role for uPAR, regulating not only efficient and spatially restricted plasmin generation but also having the potential to modulate cell adhesion and signal transduction. The cellular binding of tPA, although less well characterized, also has the capacity to regulate plasmin generation and to play a significant role in vessel-wall biology. (Trends Cardiovasc Med 1997;7:227-234). © 1997, Elsevier Science Inc.

  6. Breast cancer cells can switch between estrogen receptor alpha and ErbB signaling and combined treatment against both signaling pathways postpones development of resistance

    DEFF Research Database (Denmark)

    Sonne-Hansen, Katrine; Norrie, Ida C; Emdal, Kristina Bennet

    2010-01-01

    The majority of breast cancers are estrogen responsive, but upon progression of disease other growth promoting pathways are activated, e.g., the ErbB receptor system. The present study focuses on resistance to the pure estrogen antagonist fulvestrant and strategies to treat resistant cells or even...... could be abrogated by combined therapy targeting both receptor systems. Thus, the present study indicates that upon development of antiestrogen resistance, antiestrogen treatment should be continued in combination with signal transduction inhibitors. Further, upfront combination of endocrine therapy...... circumvent development of resistance. Limited effects were observed when targeting EGFR and ErbB2 with the monoclonal antibodies cetuximab, trastuzumab, and pertuzumab, whereas the pan-ErbB inhibitor CI-1033 selectively inhibited growth of fulvestrant resistant cell lines. CI-1033 inhibited Erk but not Akt...

  7. Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

    Science.gov (United States)

    Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

    2014-01-01

    Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to G(i), some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

  8. Peroxisome Proliferator Activated Receptors and Lipoprotein Metabolism

    Directory of Open Access Journals (Sweden)

    Sander Kersten

    2008-01-01

    Full Text Available Plasma lipoproteins are responsible for carrying triglycerides and cholesterol in the blood and ensuring their delivery to target organs. Regulation of lipoprotein metabolism takes place at numerous levels including via changes in gene transcription. An important group of transcription factors that mediates the effect of dietary fatty acids and certain drugs on plasma lipoproteins are the peroxisome proliferator activated receptors (PPARs. Three PPAR isotypes can be distinguished, all of which have a major role in regulating lipoprotein metabolism. PPARα is the molecular target for the fibrate class of drugs. Activation of PPARα in mice and humans markedly reduces hepatic triglyceride production and promotes plasma triglyceride clearance, leading to a clinically significant reduction in plasma triglyceride levels. In addition, plasma high-density lipoprotein (HDL-cholesterol levels are increased upon PPARα activation in humans. PPARγ is the molecular target for the thiazolidinedione class of drugs. Activation of PPARγ in mice and human is generally associated with a modest increase in plasma HDL-cholesterol and a decrease in plasma triglycerides. The latter effect is caused by an increase in lipoprotein lipase-dependent plasma triglyceride clearance. Analogous to PPARα, activation of PPARβ/δ leads to increased plasma HDL-cholesterol and decreased plasma triglyceride levels. In this paper, a fresh perspective on the relation between PPARs and lipoprotein metabolism is presented. The emphasis is on the physiological role of PPARs and the mechanisms underlying the effect of synthetic PPAR agonists on plasma lipoprotein levels.

  9. Identification of Gene Markers for Activation of the Nuclear Receptor Pregnane X Receptor

    Science.gov (United States)

    Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR). Activation of PXR in the mouse liver can lead to increases in liver weight in part through increased hepatocyte replication similar to chemicals that activate other nuclear ...

  10. Identification of Gene Markers for Activation of the Nuclear Receptor Pregnane X Receptor

    Science.gov (United States)

    Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR). Activation of PXR in the mouse liver can lead to increases in liver weight in part through increased hepatocyte replication similar to chemicals that activate other nuclear ...

  11. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  12. Signal transduction of Helicobacter pylori during interaction with host cell protein receptors of epithelial and immune cells

    Science.gov (United States)

    Pachathundikandi, Suneesh Kumar; Tegtmeyer, Nicole; Backert, Steffen

    2013-01-01

    Helicobacter pylori infections can induce pathologies ranging from chronic gastritis, peptic ulceration to gastric cancer. Bacterial isolates harbor numerous well-known adhesins, vacuolating cytotoxin VacA, protease HtrA, urease, peptidoglycan, and type IV secretion systems (T4SS). It appears that H. pylori targets more than 40 known host protein receptors on epithelial or immune cells. A series of T4SS components such as CagL, CagI, CagY, and CagA can bind to the integrin α5β1 receptor. Other targeted membrane-based receptors include the integrins αvβ3, αvβ5, and β2 (CD18), RPTP-α/β, GP130, E-cadherin, fibronectin, laminin, CD46, CD74, ICAM1/LFA1, T-cell receptor, Toll-like receptors, and receptor tyrosine kinases EGFR, ErbB2, ErbB3, and c-Met. In addition, H. pylori is able to activate the intracellular receptors NOD1, NOD2, and NLRP3 with important roles in innate immunity. Here we review the interplay of various bacterial factors with host protein receptors. The contribution of these interactions to signal transduction and pathogenesis is discussed. PMID:24280762

  13. Recruitment of activation receptors at inhibitory NK cell immune synapses.

    Directory of Open Access Journals (Sweden)

    Nicolas Schleinitz

    Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

  14. Co-expression of epidermal growth factor-receptor and c-erb B-2 proto-oncogene product in human salivary-gland adenocarcinoma cell line HSG and the implications for HSG cell autocrine growth.

    Science.gov (United States)

    Kyakumoto, S; Kurokawa, R; Hoshino, M; Ota, M

    1994-07-01

    The autonomous proliferation of HSG cells is mediated by an autocrine growth factor, a 46K epidermal growth factor (EGF)-like molecule. The receptor for this molecule was investigated. Immunoprecipitation and immunoblotting revealed the expression of two possible receptor molecules, EGF-R and p185erbB-2, in HSG cells. Northern blotting also revealed the co-expression of 5.6-kb EGF-R mRNA and 4.6-kb c-erb B-2 mRNA. When the purified EGF-like molecule was added to the cultures, EGF-R but not p185erbB-2 was autophosphorylated. These results suggest that, although both EGF-R and p185erbB-2 are co-expressed in HSG cells, the EGF-R is the genuine receptor for the EGF-like molecule. However, there is a possibility that p185erB-2 is involved in the signal transduction system. This possibility was examined by using specific antibodies to human EGF-R (hEGF-R), p185erbB-2, and EGF to inhibit the functions of these molecules. Addition of these three antibodies to the cultures inhibited the growth of HSG cells. The antibodies to EGF-R and p185erbB-2 also caused morphological changes such as disturbances of the plasma membrane, and some cell death. Surprisingly, the effect of the anti-p185erbB-2 antibody on growth inhibition and morphology was stronger than that of the anti-hEGF-R antibody. Thus, p185erB-2 expressed in HSG cells has an important function in the signal transduction of HSG cell growth.

  15. Thyroid hormone receptors regulate adipogenesis and carcinogenesis via crosstalk signaling with peroxisome proliferator-activated receptors

    Science.gov (United States)

    Lu, Changxue; Cheng, Sheue-Yann

    2012-01-01

    Peroxisome proliferator-activated receptors (PPARs) and thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily. They are ligand-dependent transcription factors that interact with their cognate hormone response elements in the promoters to regulate respective target gene expression to modulate cellular functions. While the transcription activity of each is regulated by their respective ligands, recent studies indicate that via multiple mechanisms PPARs and TRs crosstalk to affect diverse biological functions. Here, we review recent advances in the understanding of the molecular mechanisms and biological impact of crosstalk between these two important nuclear receptors, focusing on their roles in adipogenesis and carcinogenesis. PMID:19741045

  16. Thyroid hormone receptors regulate adipogenesis and carcinogenesis via crosstalk signaling with peroxisome proliferator-activated receptors.

    Science.gov (United States)

    Lu, Changxue; Cheng, Sheue-Yann

    2010-03-01

    Peroxisome proliferator-activated receptors (PPARs) and thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily. They are ligand-dependent transcription factors that interact with their cognate hormone response elements in the promoters to regulate respective target gene expression to modulate cellular functions. While the transcription activity of each is regulated by their respective ligands, recent studies indicate that via multiple mechanisms PPARs and TRs crosstalk to affect diverse biological functions. Here, we review recent advances in the understanding of the molecular mechanisms and biological impact of crosstalk between these two important nuclear receptors, focusing on their roles in adipogenesis and carcinogenesis.

  17. Cell death sensitization of leukemia cells by opioid receptor activation

    Science.gov (United States)

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  18. Activation of glucocorticoid receptors increases 5-HT2A receptor levels

    DEFF Research Database (Denmark)

    Trajkovska, Viktorija; Kirkegaard, Lisbeth; Krey, Gesa;

    2009-01-01

    Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker...... of depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish...... an effect of GR activation on 5-HT2A levels, mature organotypic hippocampal cultures were exposed to corticosterone with or without GR antagonist mifepristone and mineralocorticoid receptor (MR) antagonist spironolactone. In GR under-expressing mice, hippocampal 5-HT2A receptor protein levels were decreased...

  19. Family C 7TM receptor dimerization and activation

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Sheikh, Søren P; Hansen, Jakob Lerche

    2006-01-01

    The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug......-targets today. It is well established that family C 7TM receptors form homo- or hetero-dimers on the cell surface of living cells. The large extra-cellular domains (ECD) have been crystallized as a dimer in the presence and absence of agonist. Upon agonist binding, the dimeric ECD undergoes large conformational...... to be fully defined. This review presents the biochemical support for family C 7TM receptor dimerization and discusses its importance for receptor biosynthesis, surface expression, ligand binding and activation, since lessons learnt here may well be applicable to the whole superfamily of 7TM receptors....

  20. Functional characterization of protease-activated receptor -1 palmitoylation in receptor signaling and trafficking /

    OpenAIRE

    2014-01-01

    G protein-coupled receptors (GPCRs) are the largest family of signaling receptors that respond to diverse stimuli and regulate many physiological responses. GPCRs elicit their cellular responses by coupling to distinct subtypes of heterotrimeric G-proteins composed of G[alpha] and G[beta][gamma] subunits. Activated GPCRs undergo conformational changes that allow the receptor to exchange GDP for GTP on the G[alpha] subunit, which induces dissociation from the [beta][gamma] subunits and subsequ...

  1. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Michigan-Med); (Van Andel)

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  2. The orphan nuclear receptor TR4 is a vitamin A-activated nuclear receptor.

    Science.gov (United States)

    Zhou, X Edward; Suino-Powell, Kelly M; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W; Reynolds, Ross; Engel, James Douglas; Xu, H Eric

    2011-01-28

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  3. Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members.

    Science.gov (United States)

    Schneider, Pascal; Willen, Laure; Smulski, Cristian R

    2014-01-01

    Ligands and receptors of the TNF superfamily are therapeutically relevant targets in a wide range of human diseases. This chapter describes assays based on ELISA, immunoprecipitation, FACS, and reporter cell lines to monitor interactions of tagged receptors and ligands in both soluble and membrane-bound forms using unified detection techniques. A reporter cell assay that is sensitive to ligand oligomerization can identify ligands with high probability of being active on endogenous receptors. Several assays are also suitable to measure the activity of agonist or antagonist antibodies, or to detect interactions with proteoglycans. Finally, self-interaction of membrane-bound receptors can be evidenced using a FRET-based assay. This panel of methods provides a large degree of flexibility to address questions related to the specificity, activation, or inhibition of TNF-TNF receptor interactions in independent assay systems, but does not substitute for further tests in physiologically relevant conditions.

  4. Signal transduction pathway analysis in fibromatosis: receptor and nonreceptor tyrosine kinases.

    Science.gov (United States)

    Cates, Justin M M; Black, Jennifer O; Itani, Doha M; Fasig, John H; Keedy, Vicki L; Hande, Kenneth R; Whited, Brent W; Homlar, Kelly C; Halpern, Jennifer L; Holt, Ginger E; Schwartz, Herbert S; Coffin, Cheryl M

    2012-10-01

    Despite reports of receptor tyrosine kinase activation in desmoid-type fibromatosis, therapeutic benefits of kinase inhibitor therapy are unpredictable. Variability in signal transduction or cellular kinases heretofore unevaluated in desmoid tumors may be responsible for these inconsistent responses. In either case, a better understanding of growth regulatory signaling pathways is necessary to assess the theoretical potential of inhibitor therapy. Immunohistochemical analysis of tyrosine kinases and activated isoforms of downstream signal transduction proteins was performed on a tissue microarray containing 27 cases of desmoid-type fibromatosis and 14 samples of scar; 6 whole sections of normal fibrous tissue were studied for comparison. Platelet-derived growth factor receptor, β type, and focal adhesion kinase 1 were expressed in all desmoid tumors and healing scars but only 80% and 50% of nonproliferative fibrous tissue samples, respectively. Hepatocyte growth factor receptor was detected in 89% of desmoids and all scars tested, but not in any of the fibrous tissue samples. Epidermal growth factor receptor was detected in only 12% of desmoids and not in scar or fibrous tissue. Mast/stem cell growth factor receptor, receptor tyrosine-protein kinase erbB-2, and phosphorylated insulin-like growth factor 1 receptor/insulin receptor were negative in all study cases. Variable levels of phosphorylated downstream signal transduction molecules RAC-α/β/γ serine/threonine-protein kinase, mitogen-activated protein kinase, and signal transducer and activator of transcription-3 were observed in desmoids (58%, 62%, and 67%), scar tissues (100%, 86%, and 86%), and fibrous tissue (33%, 17%, and 17%). These results indicate that tyrosine kinase signaling is active in both fibromatosis and healing scar, but not in most nonproliferating fibrous tissues. Although platelet-derived growth factor receptor, β type, is expressed ubiquitously in desmoids, the kinases driving cell

  5. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    OpenAIRE

    Maryam Rakhshandehroo; Bianca Knoch; Michael Müller; Sander Kersten

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPAR alpha) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPAR alpha serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPAR alpha binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPAR alpha governs biologi...

  6. [Regulation of G protein-coupled receptor kinase activity].

    Science.gov (United States)

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  7. Structural basis for activation of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine

    2002-01-01

    -type and mutant beta2-adrenergic receptors purified from Sf-9 insect cells. Our studies have also raised important questions regarding kinetics of receptors activation. These questions should be addressed in the future by application of techniques that will allow for simultaneous measurement of conformational...

  8. Endomorphins fully activate a cloned human mu opioid receptor.

    Science.gov (United States)

    Gong, J; Strong, J A; Zhang, S; Yue, X; DeHaven, R N; Daubert, J D; Cassel, J A; Yu, G; Mansson, E; Yu, L

    1998-11-13

    Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin-1 and endomorphin-2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin-stimulated increase of cAMP in a dose-dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein-activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose-dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G-protein-activated inwardly rectifying potassium (GIRK) channels.

  9. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

    Science.gov (United States)

    Freund, Jacquelyn; May, Rebecca M; Yang, Enjun; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K; Kambayashi, Taku

    2016-08-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

  10. Signal transduction pathway analysis in fibromatosis: receptor and nonreceptor tyrosine kinases☆

    Science.gov (United States)

    Cates, Justin M. M.; Black, Jennifer O.; Itani, Doha M.; Fasig, John H.; Keedy, Vicki L.; Hande, Kenneth R.; Whited, Brent W.; Homlar, Kelly C.; Halpern, Jennifer L.; Holt, Ginger E.; Schwartz, Herbert S.; Coffin, Cheryl M.

    2014-01-01

    Summary Despite reports of receptor tyrosine kinase activation in desmoid-type fibromatosis, therapeutic benefits of kinase inhibitor therapy are unpredictable. Variability in signal transduction or cellular kinases heretofore unevaluated in desmoid tumors may be responsible for these inconsistent responses. In either case, a better understanding of growth regulatory signaling pathways is necessary to assess the theoretical potential of inhibitor therapy. Immunohistochemical analysis of tyrosine kinases and activated isoforms of downstream signal transduction proteins was performed on a tissue microarray containing 27 cases of desmoid-type fibromatosis and 14 samples of scar; 6 whole sections of normal fibrous tissue were studied for comparison. Platelet-derived growth factor receptor, β type, and focal adhesion kinase 1 were expressed in all desmoid tumors and healing scars but only 80% and 50% of nonproliferative fibrous tissue samples, respectively. Hepatocyte growth factor receptor was detected in 89% of desmoids and all scars tested, but not in any of the fibrous tissue samples. Epidermal growth factor receptor was detected in only 12% of desmoids and not in scar or fibrous tissue. Mast/stem cell growth factor receptor, receptor tyrosine–protein kinase erbB-2, and phosphorylated insulin-like growth factor 1 receptor/insulin receptor were negative in all study cases. Variable levels of phosphorylated downstream signal transduction molecules RAC-α/β/γ serine/threonine-protein kinase, mitogenactivated protein kinase, and signal transducer and activator of transcription-3 were observed in desmoids (58%, 62%, and 67%), scar tissues (100%, 86%, and 86%), and fibrous tissue (33%, 17%, and 17%). These results indicate that tyrosine kinase signaling is active in both fibromatosis and healing scar, but not in most nonproliferating fibrous tissues. Although platelet-derived growth factor receptor, β type, is expressed ubiquitously in desmoids, the kinases driving

  11. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    Science.gov (United States)

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  12. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors. Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly......The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis...... and surveillance. Chemokines are a group of 8-12 kDa large peptides with a secondary structure consisting of a flexible N-terminus and a core-domain usually stabilized by two conserved disulfide bridges. They mainly interact with the extracellular domains of their cognate 7TM receptors. Affinityand activity...

  13. Tonic activation of presynaptic GABAB receptors on rat pallidosubthalamic terminals

    Institute of Scientific and Technical Information of China (English)

    Lei CHEN; Wing-ho YUNG

    2005-01-01

    Aim: The subthalamic nucleus plays a critical role in the regulation of movement,and abnormal activity of its neurons is associated with some basal ganglia motor symptoms. We examined the presence of functional presynaptic GABAB receptors on pallidosubthalamic terminals and tested whether they were tonically active in the in vitro subthalamic slices. Methods: Whole-cell patch-clamp recordings were applied to acutely prepared rat subthalamic nucleus slices. The effects of specific GABAB agonist and antagonist on action potential-independent inhibitory postsynapfic currents (IPSCs), as well as holding current, were examined.Results: Superfusion of baclofen, a GABAB receptor agonist, significantly reduced the frequency of GABAA receptor-mediated miniature IPSCs (mIPSCs), in a Cd2+-sensitive manner, with no effect on the amplitude, indicating presynaptic inhibition on GABA release. In addition, baclofen induced a weak outward current only in a minority of subthalamic neurons. Both the pre- and post-synaptic effects of baclofen were prevented by the specific GABAB receptor antagonist,CGP55845. Furthermore, CGP55845 alone increased the frequency of mIPSCs,but had no effect on the holding current. Conclusion: These findings suggest the functional dominance of presynaptic GABAB receptors on the pallidosubthalamic terminals over the postsynaptic GABAB receptors on subthalamic neurons.Furthermore, the presynaptic, but not the postsynaptic, GABAB receptors are tonically active, suggesting that the presynaptic GABAB receptors in the subthalamic nucleus are potential therapeutic target for the treatment of Parkinson disease.

  14. Structure and dynamics of a constitutively active neurotensin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Krumm, Brian E. [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Lee, Sangbae [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Bhattacharya, Supriyo [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Botos, Istvan [National Inst. of Health (NIH), Bethesda, MD (United States). National Inst. of Diabetes and; White, Courtney F. [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Du, Haijuan [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Vaidehi, Nagarajan [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Grisshammer, Reinhard [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services

    2016-12-07

    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.

  15. Activation of 5-HT7 receptors increases neuronal platelet-derived growth factor β receptor expression.

    Science.gov (United States)

    Vasefi, Maryam S; Kruk, Jeff S; Liu, Hui; Heikkila, John J; Beazely, Michael A

    2012-03-09

    Several antipsychotics have a high affinity for 5-HT7 receptors yet despite intense interest in the 5-HT7 receptor as a potential drug target to treat psychosis, the function and signaling properties of 5-HT7 receptors in neurons remain largely uncharacterized. In primary mouse hippocampal and cortical neurons, as well as in the SH-SY5Y cell line, incubation with 5-HT, 5-carboxamidotryptamine (5-CT), or 5-HT7 receptor-selective agonists increases the expression of platelet-derived growth factor (PDGF)β receptors. The increased PDGFβ receptor expression is cyclic AMP-dependent protein kinase (PKA)-dependent, suggesting that 5-HT7 receptors couple to Gα(s) in primary neurons. Interestingly, up-regulated PDGFβ receptors display an increased basal phosphorylation state at the phospholipase Cγ-activating tyrosine 1021. This novel linkage between the 5-HT7 receptor and the PDGF system may be an important GPCR-neurotrophic factor signaling pathway in neurons.

  16. Multiple switches in G protein-coupled receptor activation.

    Science.gov (United States)

    Ahuja, Shivani; Smith, Steven O

    2009-09-01

    The activation mechanism of G protein-coupled receptors has presented a puzzle that finally may be close to solution. These receptors have a relatively simple architecture consisting of seven transmembrane helices that contain just a handful of highly conserved amino acids, yet they respond to light and a range of chemically diverse ligands. Recent NMR structural studies on the active metarhodopsin II intermediate of the visual receptor rhodopsin, along with the recent crystal structure of the apoprotein opsin, have revealed multiple structural elements or 'switches' that must be simultaneously triggered to achieve full activation. The confluence of several required structural changes is an example of "coincidence counting", which is often used by nature to regulate biological processes. In ligand-activated G protein-coupled receptors, the presence of multiple switches may provide an explanation for the differences between full, partial and inverse agonists.

  17. Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamatergic synapses.

    Science.gov (United States)

    Wang, Xulong; Lippi, Giordano; Carlson, David M; Berg, Darwin K

    2013-12-01

    Astrocytes, an abundant form of glia, are known to promote and modulate synaptic signaling between neurons. They also express α7-containing nicotinic acetylcholine receptors (α7-nAChRs), but the functional relevance of these receptors is unknown. We show here that stimulation of α7-nAChRs on astrocytes releases components that induce hippocampal neurons to acquire more α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors post-synaptically at glutamatergic synapses. The increase is specific in that no change is seen in synaptic NMDA receptor clusters or other markers for glutamatergic synapses, or in markers for GABAergic synapses. Moreover, the increases in AMPA receptors on the neuron surface are accompanied by increases in the frequency of spontaneous miniature synaptic currents mediated by the receptors and increases in the ratio of evoked synaptic currents mediated by AMPA versus NMDA receptors. This suggests that stimulating α7-nAChRs on astrocytes can convert 'silent' glutamatergic synapses to functional status. Astrocyte-derived thrombospondin is necessary but not sufficient for the effect, while tumor necrosis factor-α is sufficient but not necessary. The results identify astrocyte α7-nAChRs as a novel pathway through which nicotinic cholinergic signaling can promote the development of glutamatergic networks, recruiting AMPA receptors to post-synaptic sites and rendering the synapses more functional. We find that activation of nicotinic receptors on astrocytes releases a component that specifically recruits AMPA receptors to glutamatergic synapses. The recruitment appears to occur preferentially at what may be 'silent synapses', that is, synapses that have all the components required for glutamatergic transmission (including NMDA receptors) but lack sufficient AMPA receptors to generate a response. The results are unexpected and open up new possibilities for mechanisms underlying network formation and synaptic plasticity.

  18. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation.

    Science.gov (United States)

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G; Beazely, Michael A

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.

  19. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    Directory of Open Access Journals (Sweden)

    Anshula eSamarajeewa

    2014-11-01

    Full Text Available The serotonin (5-HT type 7 receptor is expressed throughout the CNS including cortical neurons. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA-induced toxicity. The tropomyosin-related kinase B (TrkB receptor is one of the receptors for brain-derived neurotrophic factor (BDNF and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins towards the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.

  20. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    Science.gov (United States)

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  1. Helix 11 Dynamics is Critical for Constitutive Androstane Receptor Activity

    OpenAIRE

    Wright, Edward; Busby, Scott A.; Wisecarver, Sarah; Vincent, Jeremy; Griffin, Patrick R.; Fernandez, Elias J.

    2011-01-01

    The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizi...

  2. Activation of 5-HT6 receptors inhibits corticostriatal glutamatergic transmission.

    Science.gov (United States)

    Tassone, Annalisa; Madeo, Graziella; Schirinzi, Tommaso; Vita, Daniela; Puglisi, Francesca; Ponterio, Giulia; Borsini, Franco; Pisani, Antonio; Bonsi, Paola

    2011-09-01

    We investigated the effect of 5-HT6 receptor subtype activation on glutamatergic transmission by means of whole-cell patch-clamp electrophysiological recordings from medium spiny neurons of the striatum and layer V pyramidal neurons of the prefrontal cortex. To this aim, we took advantage of a novel ligand, ST1936, showing nM affinity and agonist activity at the 5-HT6 receptor subtype. Our data show that 5-HT6 receptor activation by ST1936 reduces the frequency of spontaneous excitatory postsynaptic currents, with an IC50 of 1.3 μM. Moreover, 5-HT6 receptor activation also reduced the amplitude of spontaneous excitatory postsynaptic currents recorded from medium spiny neurons, suggesting a mechanism of action involving postsynaptic 5-HT6 receptors, as further confirmed by the paired-pulse analysis on evoked excitatory postsynaptic currents and by recordings of miniature glutamatergic events. The inhibitory effect of ST1936 on glutamatergic transmission was prevented by the selective 5-HT6 receptor antagonist SB258585 and mimicked by a different agonist, WAY-181187. Conversely, in the cortex ST1936 reduced the frequency, but not the amplitude, of spontaneous excitatory postsynaptic currents suggesting a presynaptic or indirect effect of the 5-HT6 receptor.

  3. Blocking the peroxisome proliferator-activated receptor (PPAR): an overview.

    Science.gov (United States)

    Ammazzalorso, Alessandra; De Filippis, Barbara; Giampietro, Letizia; Amoroso, Rosa

    2013-10-01

    Peroxisome proliferator-activated receptors (PPARs) have been studied extensively over the last few decades and have been assessed as molecular targets for the development of drugs against metabolic disorders. A rapid increase in understanding of the physiology and pharmacology of these receptors has occurred, together with the identification of novel chemical structures that are able to activate the various PPAR subtypes. More recent evidence suggests that moderate activation of these receptors could be favorable in pathological situations due to a decrease in the side effects brought about by PPAR agonists. PPAR partial agonists and antagonists are interesting tools that are currently used to better elucidate the biological processes modulated by this family of nuclear receptors. Herein we present an overview of the various molecular structures that are able to block each of the PPAR subtypes, with a focus on promising therapeutic applications. Copyright © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Nicotinic Receptor Activity Alters Synaptic Plasticity

    Directory of Open Access Journals (Sweden)

    John A. Dani

    2001-01-01

    Full Text Available Studies using specific agonists, antagonists, and lesions have shown that nicotinic cholinergic systems participate in attention, learning, and memory[1,2]. The nicotinic manipulations usually have the greatest influence on difficult tasks or on cognitively impaired subjects[2]. For example, Alzheimer's disease is characterized by a loss of cholinergic projections and nicotinic acetylcholine receptors (nAChRs in the cortex and hippocampus[3]. Nicotine skin patches can improve learning rates and attention in Alzheimer's patients[4].

  5. The peroxisome proliferator-activated receptor: A family of nuclear receptors role in various diseases

    Directory of Open Access Journals (Sweden)

    Sandeep Tyagi

    2011-01-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are ligand-activated transcription factors of nuclear hormone receptor superfamily comprising of the following three subtypes: PPARα, PPARγ, and PPARβ/δ. Activation of PPAR-α reduces triglyceride level and is involved in regulation of energy homeostasis. Activation of PPAR-γ causes insulin sensitization and enhances glucose metabolism, whereas activation of PPAR- β/δ enhances fatty acids metabolism. Thus, PPAR family of nuclear receptors plays a major regulatory role in energy homeostasis and metabolic function. The present review critically analyzes the protective and detrimental effect of PPAR agonists in dyslipidemia, diabetes, adipocyte differentiation, inflammation, cancer, lung diseases, neurodegenerative disorders, fertility or reproduction, pain, and obesity.

  6. Modulation of β-catenin signaling by glucagon receptor activation.

    Directory of Open Access Journals (Sweden)

    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  7. Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Nielsen, Curt A F; Neve, Søren

    2002-01-01

    delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well......The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains...

  8. Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Nielsen, Curt A F; Neve, Søren

    2002-01-01

    delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well......The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains...

  9. Analyzing the activation of the melanocortin-2 receptor of tetrapods.

    Science.gov (United States)

    Dores, Robert M; Liang, Liang

    2014-07-01

    Following the biochemical characterization of the pituitary hormone, adrenocorticotropin (ACTH), in the 1950's, a number of structure/function studies were done which identifies two amino acid motifs in ACTH, the HFRW motif and KKRR motif, as critical for the activation of the "ACTH" receptor on adrenal cortex cells. In the 1990's the "ACTH" receptor was identified as a member of the melanocortin receptor gene family, and given the name melanocortin-2 receptor (MC2R). Since that time a number of studies on both tetrapod and teleost MC2R orthologs have established that these orthologs can only be activated by ACTH, but not by any of the MSH-sized melanocortin ligands, and these orthologs require interaction with the melanocortin-2 receptor accessory protein (MRAP) for functional expression. This review summarizes recent structure/function studies on human ACTH, and points out the importance of the GKPVG motif in ACTH for the activation of the receptor. In this regard, a multiple-step model for the activation of tetrapod and teleost MC2R orthologs is presented, and the evolution of gnathostome MC2R ligand selectivity and the requirement for MRAP interaction is discussed in light of a recent study on a cartilaginous fish MC2R ortholog. This review contains excerpts from the Gorbman/Bern Lecture presented at the Second Meeting of the North American Society for Comparative Endocrinology (NASCE).

  10. Monitoring leptin activity using the chicken leptin receptor.

    Science.gov (United States)

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold.

  11. A human vitamin D receptor mutant activated by cholecalciferol.

    Science.gov (United States)

    Ousley, Amanda M; Castillo, Hilda S; Duraj-Thatte, Anna; Doyle, Donald F; Azizi, Bahareh

    2011-07-01

    The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1α,25-dihydroxyvitamin D(3) (also referred to as 1,25(OH)(2)D(3)) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor was deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC(50) value of 10 μM and 40±14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)(2)D(3). Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1α,25-dihydroxyvitamin D(3) biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC(50) value of 300 nM and 70±11 fold activation in mammalian cell assays. In silico docking analysis of the variant displays a dramatic conformational shift of cholecalciferol in the ligand binding pocket in comparison to the docked analysis of cholecalciferol with wild-type hVDR. This shift is hypothesized to be due to the introduction of two bulkier residues, suggesting that the addition of these bulkier residues introduces molecular interactions between the ligand and receptor, leading to activation with cholecalciferol.

  12. Structure-activity relationships of strychnine analogs at glycine receptors.

    Science.gov (United States)

    Mohsen, Amal M Y; Heller, Eberhard; Holzgrabe, Ulrike; Jensen, Anders A; Zlotos, Darius P

    2014-08-01

    Nine strychnine derivatives including neostrychnine, strychnidine, isostrychnine, 21,22-dihydro-21-hydroxy-22-oxo-strychnine, and several hydrogenated analogs were synthesized, and their antagonistic activities at human α1 and α1β glycine receptors were evaluated. Isostrychnine has shown the best pharmacological profile exhibiting an IC50 value of 1.6 μM at α1 glycine receptors and 3.7-fold preference towards the α1 subtype. SAR Analysis indicates that the lactam moiety and the C(21) = C(22) bond in strychnine are essential structural features for its high antagonistic potency at glycine receptors. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  13. Activation and dynamic network of the M2 muscarinic receptor

    OpenAIRE

    Miao, Yinglong; Nichols, Sara E.; Gasper, Paul M.; Metzger, Vincent T; McCammon, J. Andrew

    2013-01-01

    G-protein-coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. Although significant advances have been made in structural studies of GPCRs, details of their activation mechanism remain unclear. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined recently in an inactive antagonist...

  14. Peroxisome proliferator-activated receptors and cancer: challenges and opportunities.

    Science.gov (United States)

    Youssef, Jihan; Badr, Mostafa

    2011-09-01

    Peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily, function as transcription factors and modulators of gene expression. These actions allow PPARs to regulate a variety of biological processes and to play a significant role in several diseases and conditions. The current literature describes frequently opposing and paradoxical roles for the three PPAR isotypes, PPARα, PPARβ/δ and PPARγ, in cancer. While some studies have implicated PPARs in the promotion and development of cancer, others, in contrast, have presented evidence for a protective role for these receptors against cancer. In some tissues, the expression level of these receptors and/or their activation correlates with a positive outcome against cancer, while, in other tissue types, their expression and activation have the opposite effect. These disparate findings raise the possibility of (i) PPAR receptor-independent effects, including effects on receptors other than PPARs by the utilized ligands; (ii) cancer stage-specific effect; and/or (iii) differences in essential ligand-related pharmacokinetic considerations. In this review, we highlight the latest available studies on the role of the various PPAR isotypes in cancer in several major organs and present challenges as well as promising opportunities in the field. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  15. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    NARCIS (Netherlands)

    Rakhshandehroo, M.; Knoch, B.; Müller, M.R.; Kersten, A.H.

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPAR alpha) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPAR alpha serves as a molecular target for hypolip

  16. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    NARCIS (Netherlands)

    Rakhshandehroo, M.; Knoch, B.; Müller, M.R.; Kersten, A.H.

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPAR alpha) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPAR alpha serves as a molecular target for

  17. Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

    Science.gov (United States)

    Tremblay, Michel G; Herdman, Chelsea; Guillou, François; Mishra, Prakash K; Baril, Joëlle; Bellenfant, Sabrina; Moss, Tom

    2015-06-26

    We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

  18. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  19. Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

    DEFF Research Database (Denmark)

    Klein, H H; Müller, R; Vestergaard, H

    1999-01-01

    % of the receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother......We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ......'s skeletal muscle, suggesting that virtually no truncated receptor was expressed. Receptor kinase activity was, however, reduced by 95 and 91% in the compound heterozygous brothers. This suggests that the mother's mutated allele contributes little to the generation of functional receptor protein...

  20. Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture

    Institute of Scientific and Technical Information of China (English)

    Akira Oda; Hidekazu Tanaka

    2014-01-01

    The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer’s disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which inlfuence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to per-sistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in per-sistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer’s disease.

  1. PSD-95 regulates D1 dopamine receptor resensitization, but not receptor-mediated Gs-protein activation

    Institute of Scientific and Technical Information of China (English)

    Peihua Sun; Jingru Wang; Weihua Gu; Wei Cheng; Guo-zhang Jin; Eitan Friedman; Jie Zheng; Xuechu Zhen

    2009-01-01

    The present study aims to define the role of postsynaptic density (PSD)-95 in the regulation of dopamine (DA) receptor function. We found that PSD-95 physically associates with either D1 or D2 DA receptors in co-transfected HEK-293 cells. Stimulation of DA receptors altered the association between D1 receptor and PSD-95 in a time-depen-dent manner. Functional assays indicated that PSD-95 co-expression did not affect D1 receptor-stimulated cAMP pro-duction, Gs-protein activation or receptor desensitization. However, PSD-95 accelerated the recovery of internalized membrane receptors by promoting receptor recycling, thus resulting in enhanced resensitization of internalized D1 receptors. Our results provide a novel mechanism for regulating DA receptor recycling that may play an important role in postsynaptic DA functional modulation and synaptic neuroplasticity.

  2. Neurotransmitter GABA activates muscle but not α7 nicotinic receptors.

    Science.gov (United States)

    Dionisio, Leonardo; Bergé, Ignacio; Bravo, Matías; Esandi, María Del Carmen; Bouzat, Cecilia

    2015-01-01

    Cys-loop receptors are neurotransmitter-activated ion channels involved in synaptic and extrasynaptic transmission in the brain and are also present in non-neuronal cells. As GABAA and nicotinic receptors (nAChR) belong to this family, we explored by macroscopic and single-channel recordings whether the inhibitory neurotransmitter GABA has the ability to activate excitatory nAChRs. GABA differentially activates nAChR subtypes. It activates muscle nAChRs, with maximal peak currents of about 10% of those elicited by acetylcholine (ACh) and 15-fold higher EC50 with respect to ACh. At the single-channel level, the weak agonism is revealed by the requirement of 20-fold higher concentration of GABA for detectable channel openings, a major population of brief openings, and absence of clusters of openings when compared with ACh. Mutations at key residues of the principal binding-site face of muscle nAChRs (αY190 and αG153) affect GABA activation similarly as ACh activation, whereas a mutation at the complementary face (εG57) shows a selective effect for GABA. Studies with subunit-lacking receptors show that GABA can activate muscle nAChRs through the α/δ interface. Interestingly, single-channel activity elicited by GABA is similar to that elicited by ACh in gain-of-function nAChR mutants associated to congenital myasthenic syndromes, which could be important in the progression of the disorders due to steady exposure to serum GABA. In contrast, GABA cannot elicit single-channel or macroscopic currents of α7 or the chimeric α7-serotonin-type 3 receptor, a feature important for preserving an adequate excitatory/inhibitory balance in the brain as well as for avoiding activation of non-neuronal receptors by serum GABA. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-containing NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Li-Jun Li

    2016-10-01

    Full Text Available NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs, but not GluN2B-containing NMDA receptors (GluN2BRs, to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation.

  4. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

    Science.gov (United States)

    Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

    2016-01-01

    NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation.

  5. Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex.

    Science.gov (United States)

    Damian, Marjorie; Mary, Sophie; Maingot, Mathieu; M'Kadmi, Céline; Gagne, Didier; Leyris, Jean-Philippe; Denoyelle, Séverine; Gaibelet, Gérald; Gavara, Laurent; Garcia de Souza Costa, Mauricio; Perahia, David; Trinquet, Eric; Mouillac, Bernard; Galandrin, Ségolène; Galès, Céline; Fehrentz, Jean-Alain; Floquet, Nicolas; Martinez, Jean; Marie, Jacky; Banères, Jean-Louis

    2015-02-03

    How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.

  6. Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family

    Science.gov (United States)

    2012-01-01

    Accumulating evidence suggests that various diseases, including many types of cancer, result from alteration of subcellular protein localization and compartmentalization. Therefore, it is worthwhile to expand our knowledge in subcellular trafficking of proteins, such as epidermal growth factor receptor (EGFR) and ErbB-2 of the receptor tyrosine kinases, which are highly expressed and activated in human malignancies and frequently correlated with poor prognosis. The well-characterized trafficking of cell surface EGFR is routed, via endocytosis and endosomal sorting, to either the lysosomes for degradation or back to the plasma membrane for recycling. A novel nuclear mode of EGFR signaling pathway has been gradually deciphered in which EGFR is shuttled from the cell surface to the nucleus after endocytosis, and there, it acts as a transcriptional regulator, transmits signals, and is involved in multiple biological functions, including cell proliferation, tumor progression, DNA repair and replication, and chemo- and radio-resistance. Internalized EGFR can also be transported from the cell surface to several intracellular compartments, such as the Golgi apparatus, the endoplasmic reticulum, and the mitochondria, in addition to the nucleus. In this review, we will summarize the functions of nuclear EGFR family and the potential pathways by which EGFR is trafficked from the cell surface to a variety of cellular organelles. A better understanding of the molecular mechanism of EGFR trafficking will shed light on both the receptor biology and potential therapeutic targets of anti-EGFR therapies for clinical application. PMID:22520625

  7. Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family

    Directory of Open Access Journals (Sweden)

    Wang Ying-Nai

    2012-04-01

    Full Text Available Abstract Accumulating evidence suggests that various diseases, including many types of cancer, result from alteration of subcellular protein localization and compartmentalization. Therefore, it is worthwhile to expand our knowledge in subcellular trafficking of proteins, such as epidermal growth factor receptor (EGFR and ErbB-2 of the receptor tyrosine kinases, which are highly expressed and activated in human malignancies and frequently correlated with poor prognosis. The well-characterized trafficking of cell surface EGFR is routed, via endocytosis and endosomal sorting, to either the lysosomes for degradation or back to the plasma membrane for recycling. A novel nuclear mode of EGFR signaling pathway has been gradually deciphered in which EGFR is shuttled from the cell surface to the nucleus after endocytosis, and there, it acts as a transcriptional regulator, transmits signals, and is involved in multiple biological functions, including cell proliferation, tumor progression, DNA repair and replication, and chemo- and radio-resistance. Internalized EGFR can also be transported from the cell surface to several intracellular compartments, such as the Golgi apparatus, the endoplasmic reticulum, and the mitochondria, in addition to the nucleus. In this review, we will summarize the functions of nuclear EGFR family and the potential pathways by which EGFR is trafficked from the cell surface to a variety of cellular organelles. A better understanding of the molecular mechanism of EGFR trafficking will shed light on both the receptor biology and potential therapeutic targets of anti-EGFR therapies for clinical application.

  8. Allosteric activation mechanism of the cys-loop receptors

    Institute of Scientific and Technical Information of China (English)

    Yong-chang CHANG; Wen WU; Jian-liang ZHANG; Yao HUANG

    2009-01-01

    Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of amino-terminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

  9. Immunomodulatory effects of endogenous and synthetic peptides activating opioid receptors.

    Science.gov (United States)

    Pomorska, Dorota K; Gach, Katarzyna; Janecka, Anna

    2014-01-01

    The main role of endogenous opioid peptides is the modulation of pain. Opioid peptides exert their analgesic activity by binding to the opioid receptors distributed widely in the central nervous system (CNS). However, opioid receptors are also found on tissues and organs outside the CNS, including the cells of the immune system, indicating that opioids are capable of exerting additional effects in periphery. Morphine, which is a gold standard in the treatment of chronic pain, is well-known for its immunosuppressive effects. Much less is known about the immunomodulatory effects exerted by endogenous (enkephalins, endorphins, dynorphins and endomorphins) and synthetic peptides activating opioid receptors. In this review we tried to summarize opioid peptide-mediated modulation of immune cell functions which can be stimulatory as well as inhibitory.

  10. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

    Science.gov (United States)

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril

    2014-03-28

    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  11. Mechanism of Activation and Inhibition of the HER4/ErbB4 Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Qiu,C.; Tarrant, M.; Choi, S.; Sathyamurthy, A.; Bose, R.; Banjade, S.; Pal, A.; Bornmann, W.; Lemmon, M.; et al

    2008-01-01

    HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.

  12. Buprenorphine-induced antinociception is mediated by mu-opioid receptors and compromised by concomitant activation of opioid receptor-like receptors.

    Science.gov (United States)

    Lutfy, Kabirullah; Eitan, Shoshana; Bryant, Camron D; Yang, Yu C; Saliminejad, Nazli; Walwyn, Wendy; Kieffer, Brigitte L; Takeshima, Hiroshi; Carroll, F Ivy; Maidment, Nigel T; Evans, Christopher J

    2003-11-12

    Buprenorphine is a mixed opioid receptor agonist-antagonist used clinically for maintenance therapy in opiate addicts and pain management. Dose-response curves for buprenorphine-induced antinociception display ceiling effects or are bell shaped, which have been attributed to the partial agonist activity of buprenorphine at opioid receptors. Recently, buprenorphine has been shown to activate opioid receptor-like (ORL-1) receptors, also known as OP4 receptors. Here we demonstrate that buprenorphine, but not morphine, activates mitogen-activated protein kinase and Akt via ORL-1 receptors. Because the ORL-1 receptor agonist orphanin FQ/nociceptin blocks opioid-induced antinociception, we tested the hypothesis that buprenorphine-induced antinociception might be compromised by concomitant activation of ORL-1 receptors. In support of this hypothesis, the antinociceptive effect of buprenorphine, but not morphine, was markedly enhanced in mice lacking ORL-1 receptors using the tail-flick assay. Additional support for a modulatory role for ORL-1 receptors in buprenorphine-induced antinociception was that coadministration of J-113397, an ORL-1 receptor antagonist, enhanced the antinociceptive efficacy of buprenorphine in wild-type mice but not in mice lacking ORL-1 receptors. The ORL-1 antagonist also eliminated the bell-shaped dose-response curve for buprenorphine-induced antinociception in wild-type mice. Although buprenorphine has been shown to interact with multiple opioid receptors, mice lacking micro-opioid receptors failed to exhibit antinociception after buprenorphine administration. Our results indicate that the antinociceptive effect of buprenorphine in mice is micro-opioid receptor-mediated yet severely compromised by concomitant activation of ORL-1 receptors.

  13. Ah receptor agonist activity in frequently consumed food items

    NARCIS (Netherlands)

    Waard, de W.J.; Aarts, J.M.M.J.G.; Peijnenburg, A.A.C.M.; Kok, de T.M.C.M.; Schooten, van F.J.; Hoogenboom, L.A.P.

    2008-01-01

    The aryl hydrocarbon receptor (AhR) receives much attention for its role in the toxicity of dioxins and dioxin-like polychlorinated biphenyls. However, many other compounds have also been reported to bind and activate AhR, of which natural food components are of special interest from a human health

  14. Ah receptor agonist activity in frequently consumed food items

    NARCIS (Netherlands)

    Waard, de W.J.; Aarts, J.M.M.J.G.; Peijnenburg, A.A.C.M.; Kok, de T.M.C.M.; Schooten, van F.J.; Hoogenboom, L.A.P.

    2008-01-01

    The aryl hydrocarbon receptor (AhR) receives much attention for its role in the toxicity of dioxins and dioxin-like polychlorinated biphenyls. However, many other compounds have also been reported to bind and activate AhR, of which natural food components are of special interest from a human health

  15. The cardiovascular effects of peroxisome proliferator-activated receptor agonists.

    Science.gov (United States)

    Friedland, Sayuri N; Leong, Aaron; Filion, Kristian B; Genest, Jacques; Lega, Iliana C; Mottillo, Salvatore; Poirier, Paul; Reoch, Jennifer; Eisenberg, Mark J

    2012-02-01

    Although peroxisome proliferator-activated receptor agonists are prescribed to improve cardiovascular risk factors, their cardiovascular safety is controversial. We therefore reviewed the literature to identify landmark randomized controlled trials evaluating the effect of peroxisome proliferator-activated receptor gamma agonists (pioglitazone and rosiglitazone), alpha agonists (fenofibrate and gemfibrozil), and pan agonists (bezafibrate, muraglitazar, ragaglitazar, tesaglitazar, and aleglitazar) on cardiovascular outcomes. Pioglitazone may modestly reduce cardiovascular events but also may increase the risk of bladder cancer. Rosiglitazone increases the risk of myocardial infarction and has been withdrawn in European and restricted in the United States. Fibrates improve cardiovascular outcomes only in select subgroups: fenofibrate in diabetic patients with metabolic syndrome, gemfibrozil in patients with dyslipidemia, and bezafibrate in patients with diabetes or metabolic syndrome. The cardiovascular safety of the new pan agonist aleglitazar, currently in phase II trials, remains to be determined. The heterogenous effects of peroxisome proliferator-activated receptor agonists to date highlight the importance of postmarketing surveillance. The critical question of why peroxisome proliferator-activated receptor agonists seem to improve cardiovascular risk factors without significantly improving cardiovascular outcomes requires further investigation.

  16. Bloqueo del receptor del factor de crecimiento semejante a la Insulina Tipo I utilizando oligodeoxinucleótidos antisentido en cáncer de mama experimental Type I insulin-like growth factor receptor antisense strategies in experimental breast cancer

    Directory of Open Access Journals (Sweden)

    Mariana Salatino

    2004-04-01

    Full Text Available Evaluamos el efecto del bloqueo de la expresión del receptor del factor de crecimiento semejante a la insulina tipo I (IGF-IR sobre el crecimiento in vivo de cáncer de mama empleando una estrategia "antisentido". Utilizamos el adenocarcinoma mamario murino progestágeno-dependiente C4HD. La administración intratumoral o sistémica de oligodeoxinucleótidos antisentido fosfotiolados al ARNm del IGF-IR (AS[S]ODN inhibió el crecimiento tumoral. El efecto antitumoral fue específico debido a su dosis-dependencia y a la falta de efecto en ratones tratados con el S[S]ODN "sentido". Los tumores obtenidos de ratones tratados con AS[S]ODN mostraron: disminución en la expresión de IGF-IR y en la fosforilación del sustrato del receptor de insulina-1, inhibición de la activación de PI-3K/Akt, p42/p44MAPK y ErbB-2, mientras que la expresión y activación del receptor de progesterona no se afectó. Es la primera demostración que el crecimiento de cáncer de mama puede ser inhibido por la administración in vivo de AS[S]ODN al IGF-IR.We addressed the effect of targeting type I insulin-like growth factor receptor (IGF-IR, with antisense strategies in in vivo growth of breast cancer cells. We used C4HD tumors from an experimental model of hormonal carcinogenesis in which medroxyprogesterone acetate induced mammary adenocarcinomas in Balb/c mice. Intratumor or systemic administration of phosphorothiolated antisense oligodeoxynucleotides (AS[S]ODN to IGF-IR mRNA resulted in a significant inhibition of C4HD tumor growth. The antitumor effect was specific since inhibition of tumor growth was dose-dependent and no effect was observed in mice treated with sense S[S]ODN. Tumors from AS[S]ODN-treated mice showed a decrease in IGF-IR expression and in insulin receptor substrate-1 tyrosine phosphorylation. Activation of PI-3K/Akt, p42/p44 MAPK and ErbB-2 was abolished in tumors treated with AS[S]ODN. Progesterone receptor expression or activity remained

  17. Regulation of Liver Energy Balance by the Nuclear Receptors Farnesoid X Receptor and Peroxisome Proliferator Activated Receptor α.

    Science.gov (United States)

    Kim, Kang Ho; Moore, David D

    2017-01-01

    The liver undergoes major changes in substrate utilization and metabolic output over the daily feeding and fasting cycle. These changes occur acutely in response to hormones such as insulin and glucagon, with rapid changes in signaling pathways mediated by protein phosphorylation and other post-translational modifications. They are also reflected in chronic alterations in gene expression in response to nutrient-sensitive transcription factors. Among these, the nuclear receptors farnesoid X receptor (FXR) and peroxisome proliferator activated receptor α (PPARα) provide an intriguing, coordinated response to maintain energy balance in the liver. FXR is activated in the fed state by bile acids returning to the liver, while PPARα is activated in the fasted state in response to the free fatty acids produced by adipocyte lipolysis or possibly other signals. Key Messages: Previous studies indicate that FXR and PPARα have opposing effects on each other's primary targets in key metabolic pathways including gluconeogenesis. Our more recent work shows that these 2 nuclear receptors coordinately regulate autophagy: FXR suppresses this pathway of nutrient and energy recovery, while PPARα activates it. Another recent study indicates that FXR activates the complement and coagulation pathway, while earlier studies identify this as a negative target of PPARα. Since secretion is a very energy- and nutrient-intensive process for hepatocytes, it is possible that FXR licenses it in the nutrient-rich fed state, while PPARα represses it to spare resources in the fasted state. Energy balance is a potential connection linking FXR and PPARα regulation of autophagy and secretion, 2 seemingly unrelated aspects of hepatocyte function. FXR and PPARα act coordinately to promote energy balance and homeostasis in the liver by regulating autophagy and potentially protein secretion. It is quite likely that their impact extends to additional pathways relevant to hepatic energy balance, and

  18. A Functional Genetic Screen Identifies the Phosphoinositide 3-kinase Pathway as a Determinant of Resistance to Fibroblast Growth Factor Receptor Inhibitors in FGFR Mutant Urothelial Cell Carcinoma.

    Science.gov (United States)

    Wang, Liqin; Šuštić, Tonći; Leite de Oliveira, Rodrigo; Lieftink, Cor; Halonen, Pasi; van de Ven, Marieke; Beijersbergen, Roderick L; van den Heuvel, Michel M; Bernards, René; van der Heijden, Michiel S

    2017-01-17

    Activating mutations and translocations of the FGFR3 gene are commonly seen in urothelial cell carcinoma (UCC) of the bladder and urinary tract. Several fibroblast growth factor receptor (FGFR) inhibitors are currently in clinical development and response rates appear promising for advanced UCC. A common problem with targeted therapeutics is intrinsic or acquired resistance of the cancer cells. To find potential drug targets that can act synergistically with FGFR inhibition, we performed a synthetic lethality screen for the FGFR inhibitor AZD4547 using a short hairpin RNA library targeting the human kinome in the UCC cell line RT112 (FGFR3-TACC3 translocation). We identified multiple members of the phosphoinositide 3-kinase (PI3K) pathway and found that inhibition of PIK3CA acts synergistically with FGFR inhibitors. The PI3K inhibitor BKM120 acted synergistically with inhibition of FGFR in multiple UCC and lung cancer cell lines having FGFR mutations. Consistently, we observed an elevated PI3K-protein kinase B pathway activity resulting from epidermal growth factor receptor or Erb-B2 receptor tyrosine kinase 3 reactivation caused by FGFR inhibition as the underlying molecular mechanism of the synergy. Our data show that feedback pathways activated by FGFR inhibition converge on the PI3K pathway. These findings provide a strong rationale to test FGFR inhibitors in combination with PI3K inhibitors in cancers harboring genetic activation of FGFR genes.

  19. Identification of prostaglandin E2 receptor subtype 2 as a receptor activated by OxPAPC.

    Science.gov (United States)

    Li, Rongsong; Mouillesseaux, Kevin P; Montoya, Dennis; Cruz, Daniel; Gharavi, Navid; Dun, Martin; Koroniak, Lukasz; Berliner, Judith A

    2006-03-17

    Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), which has been shown to accumulate in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to bind monocytes by activation of endothelial beta1 integrin and subsequent deposition of fibronectin on the apical surface. Our previous studies suggest this function of OxPAPC is mediated via a Gs protein-coupled receptor (GPCR). PEIPC (1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine) is the most active lipid in OxPAPC that activates this pathway. We screened a number of candidate GPCRs for their interaction with OxPAPC and PEIPC, using a reporter gene assay; we identified prostaglandin E2 receptor EP2 and prostaglandin D2 receptor DP as responsive to OxPAPC. We focused on EP2, which is expressed in ECs, monocytes, and macrophages. OxPAPC component PEIPC, but not POVPC, activated EP2 with an EC50 of 108.6 nmol/L. OxPAPC and PEIPC were also able to compete with PGE2 for binding to EP2 in a ligand-binding assay. The EP2 specific agonist butaprost was shown to mimic the effect of OxPAPC on the activation of beta1 integrin and the stimulation of monocyte binding to endothelial cells. Butaprost also mimicked the effect of OxPAPC on the regulation of tumor necrosis factor-alpha and interleukin-10 in monocyte-derived cells. EP2 antagonist AH6809 blocked the activation of EP2 by OxPAPC in HEK293 cells and blocked the interleukin-10 response to PEIPC in monocytic THP-1 cells. These results suggest that EP2 functions as a receptor for OxPAPC and PEIPC, either as the phospholipid ester or the released fatty acid, in both endothelial cells and macrophages.

  20. Facilitation of neocortical presynaptic terminal development by NMDA receptor activation

    OpenAIRE

    2012-01-01

    Abstract Background Neocortical circuits are established through the formation of synapses between cortical neurons, but the molecular mechanisms of synapse formation are only beginning to be understood. The mechanisms that control synaptic vesicle (SV) and active zone (AZ) protein assembly at developing presynaptic terminals have not yet been defined. Similarly, the role of glutamate receptor activation in control of presynaptic development remains unclear. Results Here, we use confocal imag...

  1. Neurohumoral activation in heart failure: the role of adrenergic receptors

    OpenAIRE

    Patricia C. Brum; Rolim, Natale P. L.; BACURAU, Aline V. N.; Alessandra Medeiros

    2006-01-01

    Heart failure (HF) is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality. The development of end-stage HF often involves an initial insult to the myocardium that reduces cardiac output and leads to a compensatory increase in sympathetic nervous system activity. Acutely, the sympathetic hyperactivity through the activation of beta-adrenergic receptors increases heart rate and cardiac contractility, which compensate for decreased cardia...

  2. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Shoko, E-mail: satosho@rs.tus.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Shirakawa, Hitoshi, E-mail: shirakah@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Tomita, Shuhei, E-mail: tomita@med.tottori-u.ac.jp [Division of Molecular Pharmacology, Department of Pathophysiological and Therapeutic Science, Yonago 683-8503 (Japan); Tohkin, Masahiro, E-mail: tohkin@phar.nagoya-cu.ac.jp [Department of Medical Safety Science, Graduate School of Pharmaceutical Science, Nagoya City University, Nagoya 267-8603 (Japan); Gonzalez, Frank J., E-mail: gonzalef@mail.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Komai, Michio, E-mail: mkomai@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan)

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  3. Structural basis for AMPA receptor activation and ligand selectivity

    DEFF Research Database (Denmark)

    Hogner, A; Kastrup, Jette Sandholm Jensen; Jin, R

    2002-01-01

    Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology...... correlation between domain closure and efficacy has been obtained from electrophysiology experiments undertaken on non-desensitising GluR2i(Q)-L483Y receptors expressed in oocytes, providing strong evidence that receptor activation occurs as a result of domain closure. The structural results, combined...

  4. Influence of phasic and tonic dopamine release on receptor activation

    DEFF Research Database (Denmark)

    Dreyer, Jakob Kristoffer Kisbye; Herrik, Kjartan F; Berg, Rune W

    2010-01-01

    Tonic and phasic dopamine release is implicated in learning, motivation, and motor functions. However, the relationship between spike patterns in dopaminergic neurons, the extracellular concentration of dopamine, and activation of dopamine receptors remains unresolved. In the present study, we...... develop a computational model of dopamine signaling that give insight into the relationship between the dynamics of release and occupancy of D(1) and D(2) receptors. The model is derived from first principles using experimental data. It has no free parameters and offers unbiased estimation...

  5. Structure-activity relationships of strychnine analogues at glycine receptors

    DEFF Research Database (Denmark)

    Mohsen, A.M.Y.; Heller, Eberhard; Holzgrabe, Ulrike

    2014-01-01

    Nine strychnine derivatives including neostrychnine, strychnidine, isostrychnine, 21,22-dihydro-21-hydroxy-22-oxo-strychnine, and several hydrogenated analogs were synthesized, and their antagonistic activities at human α1 and α1β glycine receptors were evaluated. Isostrychnine has shown the best...... pharmacological profile exhibiting an IC50 value of 1.6 μM at α1 glycine receptors and 3.7-fold preference towards the α1 subtype. SAR Analysis indicates that the lactam moiety and the C(21)[DOUBLE BOND]C(22) bond in strychnine are essential structural features for its high antagonistic potency at glycine...

  6. The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Netzel-Arnett, S

    2001-01-01

    The urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and ...

  7. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain...

  8. The Role of Novel Substituted Diindolyl Methane Analogues in the Treatment of Triple-Negative and ErbB2-Positive Breast Cancer

    Science.gov (United States)

    2015-05-01

    that NR4A3 expression in the Wistar– Kyoto rat contributed to depressive behavior [183]. Moreover, polymorphisms within the NR4A3 gene are correlated...Nuclear orphan receptor Nor-1 contributes to depressive behavior in the Wistar– Kyoto rat model of depression, Brain Res. 1362 (2010) 32–39. [184] G...healthy cell detection kit was used according to the 155 manufacturer’s protocol and cells were visualized under an EVOS fl, fluorescence 156

  9. Screening of selected pesticides for oestrogen receptor activation in vitro

    DEFF Research Database (Denmark)

    Vinggaard, Anne; Breinholt, Vibeke; Larsen, John Christian

    1999-01-01

    .0, 2.4, and 1.9-fold increase in proliferation of human MCF7 breast cancer cells (E3 clone). The relative proliferation efficiency (RPE) was 43-69%, indicating partial agonism at the oestrogen receptor. Several pesticides did not have any effect oil the proliferation response after 6 days of exposure......Twenty pesticides were tested for their ability to activate the oestrogen receptor in vitro using an,MCF7 cell proliferation assay and a Yeast Oestrogen Screen. The fungicides fenarimol, triadimefon, and triadimenol were identified as weak oestrogen receptor agonists, which at 10 mu M induces a 2......, including. chlorpyrifos, diuron, iprodion, linuron, pentachlorphenol, prochloraz, propioconazol, propyzamine, quintozen, tetrachorvinphos and tetradifon. Some pesticides resulted in a negligible proliferation response, which was nor statistically significant under the present experimental conditions...

  10. Peroxisome proliferator-activated receptor alpha target genes.

    Science.gov (United States)

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  11. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Directory of Open Access Journals (Sweden)

    Maryam Rakhshandehroo

    2010-01-01

    Full Text Available The peroxisome proliferator-activated receptor alpha (PPARα is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  12. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Directory of Open Access Journals (Sweden)

    Ariel Avila

    2013-08-01

    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

  13. Activation of D4 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells.

    Science.gov (United States)

    Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D; Jose, Pedro A; Zeng, Chunyu

    2015-01-01

    The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal angiotensin type 1 (AT1) receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubule cells from Wistar-Kyoto (WKY) rats, but the D4 receptor regulation of AT1 receptor is aberrant in renal proximal tubule cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time- and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na(+)-K(+) ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na(+)-K(+) ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na(+)-K(+) ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na(+)-K(+) ATPase activity in SHR cells. This was confirmed in vivo; pretreatment with PD128077 for 1 week augmented the antihypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension.

  14. Hyaluronic acid induces activation of the κ-opioid receptor.

    Directory of Open Access Journals (Sweden)

    Barbara Zavan

    Full Text Available INTRODUCTION: Nociceptive pain is one of the most common types of pain that originates from an injury involving nociceptors. Approximately 60% of the knee joint innervations are classified as nociceptive. The specific biological mechanism underlying the regulation of nociceptors is relevant for the treatment of symptoms affecting the knee joint. Intra-articular administration of exogenous hyaluronic acid (HA in patients with osteoarthritis (OA appears to be particularly effective in reducing pain and improving patient function. METHODS: We performed an in vitro study conducted in CHO cells that expressed a panel of opioid receptors and in primary rat dorsal root ganglion (DRG neurons to determine if HA induces the activation of opioid peptide receptors (OPr using both aequorin and the fluorescent dye Fura-2/AM. RESULTS: Selective agonists and antagonists for each OPr expressed on CHO cells were used to test the efficacy of our in vitro model followed by stimulation with HA. The results showed that HA induces stimulatory effects on the κ receptor (KOP. These effects of HA were also confirmed in rat DRG neurons, which express endogenously the OPr. CONCLUSIONS: HA activates the KOP receptor in a concentration dependent manner, with a pEC(50 value of 7.57.

  15. Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages.

    Directory of Open Access Journals (Sweden)

    Aline Cristina Abreu Moreira-Souza

    Full Text Available Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.

  16. During hormone depletion or tamoxifen treatment of breast cancer cells the estrogen receptor apoprotein supports cell cycling through the retinoic acid receptor α1 apoprotein.

    Science.gov (United States)

    Salazar, Marcela D; Ratnam, Maya; Patki, Mugdha; Kisovic, Ivana; Trumbly, Robert; Iman, Mohamed; Ratnam, Manohar

    2011-02-07

    Current hormonal adjuvant therapies for breast cancer including tamoxifen treatment and estrogen depletion are overall tumoristatic and are severely limited by the frequent recurrence of the tumors. Regardless of the resistance mechanism, development and progression of the resistant tumors requires the persistence of a basal level of cycling cells during the treatment for which the underlying causes are unclear. In estrogen-sensitive breast cancer cells the effects of hormone depletion and treatment with estrogen, tamoxifen, all-trans retinoic acid (ATRA), fulvestrant, estrogen receptor α (ER) siRNA or retinoic acid receptor α (RARα) siRNA were studied by examining cell growth and cycling, apoptosis, various mRNA and protein expression levels, mRNA profiles and known chromatin associations of RAR. RARα subtype expression was also examined in breast cancer cell lines and tumors by competitive PCR. Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using either siRNA or fulvestrant inhibited basal proliferation by promoting cell cycle arrest, without enrichment for ErbB2/3+ overexpressing cells. The basal expression of RARα1, the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam; RAR-β and -γ were not regulated by apo-ER. Depleting basal RARα1 reproduced the antiproliferative effect of depleting ER whereas its restoration in the ER depleted cells partially rescued the basal cycling. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition; these target genes were generally insensitive to ATRA but were enriched in RAR binding sites in associated chromatin regions. In hormone-sensitive breast

  17. Neurohumoral activation in heart failure: the role of adrenergic receptors

    Directory of Open Access Journals (Sweden)

    Patricia C. Brum

    2006-09-01

    Full Text Available Heart failure (HF is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality. The development of end-stage HF often involves an initial insult to the myocardium that reduces cardiac output and leads to a compensatory increase in sympathetic nervous system activity. Acutely, the sympathetic hyperactivity through the activation of beta-adrenergic receptors increases heart rate and cardiac contractility, which compensate for decreased cardiac output. However, chronic exposure of the heart to elevated levels of catecholamines released from sympathetic nerve terminals and the adrenal gland may lead to further pathologic changes in the heart, resulting in continued elevation of sympathetic tone and a progressive deterioration in cardiac function. On a molecular level, altered beta-adrenergic receptor signaling plays a pivotal role in the genesis and progression of HF. beta-adrenergic receptor number and function are decreased, and downstream mechanisms are altered. In this review we will present an overview of the normal beta-adrenergic receptor pathway in the heart and the consequences of sustained adrenergic activation in HF. The myopathic potential of individual components of the adrenergic signaling will be discussed through the results of research performed in genetic modified animals. Finally, we will discuss the potential clinical impact of beta-adrenergic receptor gene polymorphisms for better understanding the progression of HF.A insuficiência cardíaca (IC é a via final comum da maioria das doenças cardiovasculares e uma das maiores causas de morbi-mortalidade. O desenvolvimento do estágio final da IC freqüentemente envolve um insulto inicial do miocárdio, reduzindo o débito cardíaco e levando ao aumento compensatório da atividade do sistema nervoso simpático (SNS. Existem evidências de que apesar da exposição aguda ser benéfica, exposições crônicas a elevadas concentra

  18. Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development

    Directory of Open Access Journals (Sweden)

    Mahmoud M. Mansour

    2008-01-01

    Full Text Available Exposure to the estrogen receptor alpha (ER ligand diethylstilbesterol (DES between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ER and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPAR. Transcripts for PPARs , , and and 1a splice variant were detected in Sprague-Dawley normal rat penis with PPAR predominating. In addition, PPAR1b and PPAR2 were newly induced by DES. The PPAR transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPAR protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ER and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPAR expression. These results suggest a biological overlap between PPAR and ER and highlight a mechanism for endocrine disruption.

  19. Visualising androgen receptor activity in male and female mice.

    Directory of Open Access Journals (Sweden)

    D Alwyn Dart

    Full Text Available Androgens, required for normal development and fertility of males and females, have vital roles in the reproductive tract, brain, cardiovascular system, smooth muscle and bone. Androgens function via the androgen receptor (AR, a ligand-dependent transcription factor. To assay and localise AR activity in vivo we generated the transgenic "ARE-Luc" mouse, expressing a luciferase reporter gene under the control of activated endogenous AR. In vivo imaging of androgen-mediated luciferase activity revealed several strongly expressing tissues in the male mouse as expected and also in certain female tissues. In males the testes, prostate, seminal vesicles and bone marrow all showed high AR activity. In females, strong activity was seen in the ovaries, uterus, omentum tissue and mammary glands. In both sexes AR expression and activity was also found in salivary glands, the eye (and associated glands, adipose tissue, spleen and, notably, regions of the brain. Luciferase protein expression was found in the same cell layers as androgen receptor expression. Additionally, mouse AR expression and activity correlated well with AR expression in human tissues. The anti-androgen bicalutamide reduced luciferase signal in all tissues. Our model demonstrates that androgens can act in these tissues directly via AR, rather than exclusively via androgen aromatisation to estrogens and activation of the estrogen receptor. Additionally, it visually demonstrates the fundamental importance of AR signalling outside the normal role in the reproductive organs. This model represents an important tool for physiological and developmental analysis of androgen signalling, and for characterization of known and novel androgenic or antiandrogenic compounds.

  20. Monocyte Signal Transduction Receptors in Active and Latent Tuberculosis

    Directory of Open Access Journals (Sweden)

    Magdalena Druszczynska

    2013-01-01

    Full Text Available The mechanisms that promote either resistance or susceptibility to TB disease remain insufficiently understood. Our aim was to compare the expression of cell signaling transduction receptors, CD14, TLR2, CD206, and β2 integrin LFA-1 on monocytes from patients with active TB or nonmycobacterial lung disease and healthy individuals with M.tb latency and uninfected controls to explain the background of the differences between clinical and subclinical forms of M.tb infection. A simultaneous increase in the expression of the membrane bound mCD14 receptor and LFA-1 integrin in patients with active TB may be considered a prodrome of breaking immune control by M.tb bacilli in subjects with the latent TB and absence of clinical symptoms.

  1. Manipulation of P2X Receptor Activities by Light Stimulation

    Directory of Open Access Journals (Sweden)

    Sang Seong Kim

    2016-01-01

    Full Text Available P2X receptors are involved in amplification of inflammatory responses in peripheral nociceptive fibers and in mediating pain-related signals to the CNS. Control of P2X activation has significant importance in managing unwanted hypersensitive neuron responses. To overcome the limitations of chemical ligand treatment, optical stimulation methods of optogenetics and photoswitching achieve efficient control of P2X activation while allowing specificity at the target site and convenient stimulation by light illumination. There are many potential applications for photosensitive elements, such as improved uncaging methods, photoisomerizable ligands, photoswitches, and gold nanoparticles. Each technique has both advantages and downsides, and techniques are selected according to the purpose of the application. Technical advances not only provide novel approaches to manage inflammation or pain mediated by P2X receptors but also suggest a similar approach for controlling other ion channels.

  2. [Peroxisome proliferator-activated receptors (PPAR). Antiproliferative properties].

    Science.gov (United States)

    Hojka, Anna; Rapak, Andrzej

    2011-06-21

    Peroxisome proliferator-activated receptors (PPAR) are transcription factors that belong to the hormone nuclear receptor superfamily. Their main role is control of fatty acid metabolism and to maintain glucose homeostasis. Isotype γ of PPAR can also be implicated in proliferation and cellular differentiation of both normal and cancer cells. Compounds that are PPARγ ligands have a negative influence on cancer cells and can induce apoptosis, inhibit proliferation or induce cellular differentiation of these cells. This review summarizes general information about PPAR and focuses on anticancer activities of PPARγ ligands and their use in combined therapy. Combination treatment using PPARγ ligands and other agents, especially retinoids and specific kinase inhibitors, may be an effective strategy for chemoprevention and treatment of some cancers.

  3. Acute activation, desensitization and smoldering activation of human acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Barbara G Campling

    Full Text Available The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained "smoldering activation" occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β22β2 and (α4β22α4. The (α4β22β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β22α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.

  4. Estrogen receptor- and aryl hydrocarbon receptor- mediated activities of a coal-tar creosote

    Energy Technology Data Exchange (ETDEWEB)

    Fielden, M.R.; Wu, Z.F.; Sinal, C.J.; Jury, H.H.; Bend, J.R.; Hammond, G.L.; Zacharewski, T.R. [Michigan State University, East Lansing, MI (USA). Dept. of Biochemistry

    2000-05-01

    A coal-tar creosote was examined for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind the mouse ER, bind to the human sex hormone-binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfected MCF-7 cells. Based on competitive binding to the mouse ER, creosote contains approximately 165 mg/L of estradiol- equivalents. Creosote effectively transformed the AhR in vitro and induced a Cyp 1a1-regulated luciferase reporter gene in transiently transfected Hepa 1c1c7 cells. Based on dose-response curves, creosote contains approximately 730 mg/L of dioxin-equivalents. Creosote did not exhibit any AhR-mediated antiestrogenic activity in vitro. In vivo, creosote significantly induced liver pentoxyresorufin O- depentylation and ethoxyresorufin-O-deethylation (EROD) in a dose-dependent manner in ovariectomized (OVX) ICR mice, but did not increase uterine weight wet or vaginal cornification, due possibly to AhR-mediated antiestrogenic activity. In OVX DBA/2 mice, a strain less responsive to AhR ligands, creosote induced liver EROD to a lesser extent, but still did not show an increase in uterine wet weight or vaginal cornification. These results demonstrate that coal- tar creosote exhibits AhR- and ER-mediated activity in vitro, but its dioxinlike activity may suppress estrogenic response in vivo.

  5. Estrogen receptor- and aryl hydrocarbon receptor-mediated activities of a coal-tar creosote

    Energy Technology Data Exchange (ETDEWEB)

    Fielden, M.R.; Wu, Z.F.; Sinal, C.J.; Jury, H.H.; Bend, J.R.; Hammond, G.L.; Zacharewski, T.R.

    2000-05-01

    A coal-tar creosote was examined for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind to the mouse ER, bind to the human sex hormone-binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfected MCF-7 cells. Based on competitive binding to the mouse ER, creosote contains approximately 165 mg/L of estradiol-equivalents. Creosote effectively transformed the AhR in vitro and induced a Cyplal-regulated luciferase reporter gene in transiently transfected Hepa 1c1c7 cells. Based on dose-response curves, creosote contains approximately 730 mg/L of dioxin-equivalents. Creosote did not exhibit any AhR-mediated antiestrogenic activity in vitro. In vivo, creosote significantly induced liver pentoxyresorufin O-depentylation and ethoxyresorufin-O-deethylation (EROD) in a dose-dependent manner in ovariectomized (OVX) ICR mice, but did not increase uterine weight wet or vaginal cornification, due possibly to AhR-mediated antiestrogenic activity. In OVX DBA/2 mice, a strain less responsive to AhR ligands, creosote induced liver EROD to a lesser extent, but still did not show an increase in uterine wet weight or vaginal cornification. These results demonstrate that coal-tar creosote exhibits AhR- and ER-mediated activity in vitro, but its dioxinlike activity may suppress estrogenic responses in vivo.

  6. DHEA metabolites activate estrogen receptors alpha and beta

    OpenAIRE

    Michael Miller, Kristy K.; AL-RAYYAN, NUMAN; Ivanova, Margarita M.; Mattingly, Kathleen A.; Ripp, Sharon L.; Klinge, Carolyn M; Prough, Russell A.

    2012-01-01

    Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ) -regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, andro...

  7. A novel hydroxyfuroic acid compound as an insulin receptor activator – structure and activity relationship of a prenylindole moiety to insulin receptor activation

    Directory of Open Access Journals (Sweden)

    Tsai Henry J

    2009-07-01

    Full Text Available Abstract Background Diabetes Mellitus is a chronic disease and many patients of which require frequent subcutaneous insulin injection to maintain proper blood glucose levels. Due to the inconvenience of insulin administration, an orally active insulin replacement has long been a prime target for many pharmaceutical companies. Demethylasterriquinone (DMAQ B1, extracted from tropical fungus, Pseudomassaria sp., has been reported to be an orally effective agent at lowering circulating glucose levels in diabetic (db/db mice; however, the cytotoxicity associated with the quinone moiety has not been addressed thus far. Methods A series of hydroxyfuroic acid compounds were synthesized and tested for their efficacies at activating human insulin receptor. Cytotoxicity to Chinese hamster ovary cells, selectivities over insulin-like growth factor-1 (IGF-1, epidermal growth factor (EGF, and fibroblast growth factor (FGF receptors were examined in this study. Result and Conclusion This study reports a new non-quinone DMAQ B1 derivative, a hydroxyfuroic acid compound (D-410639, which is 128 fold less cytotoxic as DMAQ B1 and as potent as compound 2, a DMAQ B1 synthetic derivative from Merck, at activating human insulin receptor. D-410639 has little activation potential on IGF-1 receptor but is a moderate inhibitor to EGF receptor. Structure and activity relationship of the prenylindole moiety to insulin receptor activation is discussed.

  8. Cannabinoids go nuclear: evidence for activation of peroxisome proliferator-activated receptors

    Science.gov (United States)

    O'Sullivan, S E

    2007-01-01

    Cannabinoids act at two classical cannabinoid receptors (CB1 and CB2), a 7TM orphan receptor and the transmitter-gated channel transient receptor potential vanilloid type-1 receptor. Recent evidence also points to cannabinoids acting at members of the nuclear receptor family, peroxisome proliferator-activated receptors (PPARs, with three subtypes α, β (δ) and γ), which regulate cell differentiation and lipid metabolism. Much evidence now suggests that endocannabinoids are natural activators of PPARα. Oleoylethanolamide regulates feeding and body weight, stimulates fat utilization and has neuroprotective effects mediated through activation of PPARα. Similarly, palmitoylethanolamide regulates feeding and lipid metabolism and has anti-inflammatory properties mediated by PPARα. Other endocannabinoids that activate PPARα include anandamide, virodhamine and noladin. Some (but not all) endocannabinoids also activate PPARγ; anandamide and 2-arachidonoylglycerol have anti-inflammatory properties mediated by PPARγ. Similarly, ajulemic acid, a structural analogue of a metabolite of Δ9-tetrahydrocannabinol (THC), causes anti-inflammatory effects in vivo through PPARγ. THC also activates PPARγ, leading to a time-dependent vasorelaxation in isolated arteries. Other cannabinoids which activate PPARγ include N-arachidonoyl-dopamine, HU210, WIN55212-2 and CP55940. In contrast, little research has been carried out on the effects of cannabinoids at PPARδ. In this newly emerging area, a number of research questions remain unanswered; for example, why do cannabinoids activate some isoforms and not others? How much of the chronic effects of cannabinoids are through activation of nuclear receptors? And importantly, do cannabinoids confer the same neuro- and cardioprotective benefits as other PPARα and PPARγ agonists? This review will summarize the published literature implicating cannabinoid-mediated PPAR effects and discuss the implications thereof. PMID:17704824

  9. Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

    Science.gov (United States)

    Perez, Michel; Lamothe, Marie; Maraval, Catherine; Mirabel, Etienne; Loubat, Chantal; Planty, Bruno; Horn, Clemens; Michaux, Julien; Marrot, Sebastien; Letienne, Robert; Pignier, Christophe; Bocquet, Arnaud; Nadal-Wollbold, Florence; Cussac, Didier; de Vries, Luc; Le Grand, Bruno

    2009-10-08

    Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.

  10. Methylthioadenosine reprograms macrophage activation through adenosine receptor stimulation.

    Directory of Open Access Journals (Sweden)

    Peter A Keyel

    Full Text Available Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA, which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation.

  11. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title ...Receptor tyrosine kinases and the regulation of macrophage activation. Authors Co

  12. ErbB4 Overexpression as an Antagonist of ErbB2/HER2/Neu Induced Human Breast Cancer Cell Proliferation

    Science.gov (United States)

    2006-08-01

    BH3-only proteins, including BID, BIM, BIK, HRK, NOXA , and PUMA, share a single BH3 domain that is essential for cell-killing activity. They are...BH3-only proteins, including BIM, HRK, BBC3, NOXA , and PUMA, are transcriptionally regulated, whereas the apoptotic activities of BID, BAD, and BIK

  13. Bioluminescence imaging of estrogen receptor activity during breast cancer progression.

    Science.gov (United States)

    Vantaggiato, Cristina; Dell'Omo, Giulia; Ramachandran, Balaji; Manni, Isabella; Radaelli, Enrico; Scanziani, Eugenio; Piaggio, Giulia; Maggi, Adriana; Ciana, Paolo

    2016-01-01

    Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation.

  14. Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

    Science.gov (United States)

    Matsumoto, Yasuhiko; Ishii, Masaki; Ishii, Kenichi; Miyaguchi, Wataru; Horie, Ryo; Inagaki, Yoshinori; Hamamoto, Hiroshi; Tatematsu, Ken-ichiro; Uchino, Keiro; Tamura, Toshiki; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2014-12-12

    We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Structure and dynamics of the insulin receptor: implications for receptor activation and drug discovery.

    Science.gov (United States)

    Ye, Libin; Maji, Suvrajit; Sanghera, Narinder; Gopalasingam, Piraveen; Gorbunov, Evgeniy; Tarasov, Sergey; Epstein, Oleg; Klein-Seetharaman, Judith

    2017-07-01

    Recently, major progress has been made in uncovering the mechanisms of how insulin engages its receptor and modulates downstream signal transduction. Here, we present in detail the current structural knowledge surrounding the individual components of the complex, binding sites, and dynamics during the activation process. A novel kinase triggering mechanism, the 'bow-arrow model', is proposed based on current knowledge and computational simulations of this system, in which insulin, after its initial interaction with binding site 1, engages with site 2 between the fibronectin type III (FnIII)-1 and -2 domains, which changes the conformation of FnIII-3 and eventually translates into structural changes across the membrane. This model provides a new perspective on the process of insulin binding to its receptor and, thus, could lead to future novel drug discovery efforts. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. UV ACTIVATION OF RECEPTOR TYROSINE KINASE-ACTIVITY

    NARCIS (Netherlands)

    COFFER, PJ; BURGERING, BMT; PEPPELENBOSCH, MP; BOS, JL; KRUIJER, W

    1995-01-01

    The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumour promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as

  17. Liver x receptors regulate the transcriptional activity of the glucocorticoid receptor: implications for the carbohydrate metabolism.

    Directory of Open Access Journals (Sweden)

    Nancy Nader

    Full Text Available GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase, a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR. The liver X receptors (LXRs, on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR, and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965 activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that

  18. Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism

    Science.gov (United States)

    Nader, Nancy; Ng, Sinnie Sin Man; Wang, Yonghong; Abel, Brent S.; Chrousos, George P.; Kino, Tomoshige

    2012-01-01

    GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of

  19. Peroxisome Proliferator–Activated Receptors and The Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Anna Meiliana

    2009-04-01

    Full Text Available BACKGROUND: Obesity is a growing threat to global health by virtue of its association with insulin resistance, inflammation, hypertension, and dyslipidemia, collectively known as the metabolic syndrome (MetS. The nuclear receptors PPARα and PPARγ are therapeutic targets for hypertriglyceridemia and insulin resistance, respectively, and drugs that modulate these receptors are currently in clinical use. More recent work on the PPARδ has uncovered a dual benefit for both hypertriglyceridemia and insulin resistance, highlighting the broad potential of PPARs in the treatment of metabolic disease. CONTENT: We have learned much about PPARs, the metabolic fat sensors, and the molecular pathways they regulate. Through their distinct tissue distribution and specific target gene activation, the three PPARs together control diverse aspects of fatty acid metabolism, energy balance, insulin sensitivity glucose homeostasis, inflammation, hypertension and atherosclerosis. These studies have advanced our understanding of the etiology for the MetS. Mechanisms revealed by these studies highlight the importance of emerging concepts, such as the endocrine function of adipose tissue, tissue-tissue cross-talk and lipotoxicity, in the pathogenesis of type 2 diabetes mellitus and CVD. SUMMARY: The elucidation of key regulators of energy balance and insulin signaling have revolutionized our understanding of fat and sugar metabolism and their intimate link. The three ‘lipidsensing’ (PPARα, PPARγ and PPARδ exemplify this connection, regulating diverse aspects of lipid and glucose homeostasis, and serving as bonafide therapeutic targets. KEYWORDS: peroxisome proliferator, activated receptor, metabolic syndrome.

  20. Persistently active cannabinoid receptors mute a subpopulation of hippocampal interneurons.

    Science.gov (United States)

    Losonczy, Attila; Biró, Agota A; Nusser, Zoltan

    2004-02-03

    Cortical information processing requires an orchestrated interaction between a large number of pyramidal cells and albeit fewer, but highly diverse GABAergic interneurons (INs). The diversity of INs is thought to reflect functional and structural specializations evolved to control distinct network operations. Consequently, specific cortical functions may be selectively modified by altering the input-output relationship of unique IN populations. Here, we report that persistently active cannabinoid receptors, the site of action of endocannabinoids, and the psychostimulants marijuana and hashish, switch off the output (mute) of a unique class of hippocampal INs. In paired recordings between cholecystokinin-immunopositive, mossy fiber-associated INs, and their target CA3 pyramidal cells, no postsynaptic currents could be evoked with single presynaptic action potentials or with repetitive stimulations at frequencies <25 Hz. Cannabinoid receptor antagonists converted these "mute" synapses into high-fidelity ones. The selective muting of specific GABAergic INs, achieved by persistent presynaptic cannabinoid receptor activation, provides a state-dependent switch in cortical networks.

  1. Receptor conformation and constitutive activity in CCR5 chemokine receptor function and HIV infection.

    Science.gov (United States)

    Flanagan, Colleen A

    2014-01-01

    The CCR5 chemokine receptor mediates the effects of proinflammatory β-chemokines that stimulate chemotaxis, activation, and proliferation of macrophages and T cells. CCR5 is also the major coreceptor that mediates HIV infection in combination with CD4. Chemokine agonists of CCR5 stimulate the activation of cellular calcium and protein kinase signaling pathways that depend on the activation of Gαi and probably also Gαq in some cells. Chemokines also stimulate the recruitment of β-arrestin, which is required for clathrin-dependent receptor internalization and acts as a scaffold protein for the chemotaxis signaling complex that mobilizes the actin cytoskeleton. CCR5 is partially constitutively active for the activation of Gαi, but the physiological significance has not been studied. HIV binding to CCR5 also activates G protein and protein kinase signaling but, in addition, stimulates the production of proinflammatory cytokines, including TNF-α, and mobilizes the actin cytoskeleton to form the fusion pore that allows viral entry and subsequently supports viral replication in the cell. The CCR5 conformation that mediates the fusion of the viral and cell membranes is unknown, but it is probably distinct from the conformation that mediates G protein signaling. Nonpeptide CCR5 blockers are allosteric inverse agonists that increase dissociation of both chemokines and HIV envelope proteins, but this does not correlate with their ability to inhibit HIV infection. Nevertheless, the inverse agonist activity may ameliorate the immune activation that exacerbates AIDS pathogenesis. Inverse agonists of CCR5 have established efficacy for the treatment of AIDS, but may also be useful in preventing HIV infection.

  2. Peroxisome proliferator-activated receptors and renal diseases.

    Science.gov (United States)

    Wu, Jing; Chen, Lihong; Zhang, Dongjuan; Huo, Ming; Zhang, Xiaoyan; Pu, Dan; Guan, Youfei

    2009-01-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-dependent transcription factors. Three isoforms of PPAR, i.e., PPAR-a, -d, and -?, have been identified and are differentially expressed in various tissues, including the kidney. The target genes of PPARs are involved in diverse biological processes, including adipogenesis, lipid metabolism, insulin sensitivity, inflammatory response, reproduction, and cell growth and differentiation. PPARs have been reported to protect against renal injury through indirect systemic effects and/or direct renal effects in diabetic nephropathy, glomerulonephritis, renal cell carcinoma, acute renal failure and chronic renal disease. In this review, we summarize the role of the three identified PPAR isoforms, PPARa, -d, and -?, in renal physiology and discuss the renoprotective effects of PPAR ligands in various kidney diseases.

  3. Structural rearrangement of the intracellular domains during AMPA receptor activation

    DEFF Research Database (Denmark)

    Zachariassen, Linda Grønborg; Katchan, Ljudmila; Jensen, Anna Guldvang

    2016-01-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational...... changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2...... subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch...

  4. Afatinib, an Irreversible EGFR Family Inhibitor, Shows Activity Toward Pancreatic Cancer Cells, Alone and in Combination with Radiotherapy, Independent of KRAS Status.

    Science.gov (United States)

    Huguet, Florence; Fernet, Marie; Giocanti, Nicole; Favaudon, Vincent; Larsen, Annette K

    2016-06-01

    Pancreatic adenocarcinoma is characterized by a high frequency of KRAS mutations and frequent deregulation of the epidermal growth factor receptor (EGFR) and other EGFR family members such as HER2/ErbB2. The EGFR inhibitor erlotinib is approved for treatment of pancreatic cancer, but has shown modest activity in most patients. Here we investigated the activity of afatinib, a second-generation irreversible pan-EGFR family kinase inhibitor, alone or in combination with ionizing radiation, toward pancreatic cancer cells. The influence of afatinib on cell proliferation, cell cycle distribution, clonogenic survival, nuclear fragmentation, ploidy, and centrosome amplification following irradiation was determined. Expression and phosphorylation of HER receptors, Akt, DNA-PKcs, and ERK1/2 was characterized by Western blot analysis. Afatinib was growth-inhibitory for all three cell lines but cytotoxic only toward BxPC3 (KRAS (wt)) and Capan-2 (KRAS (mut)) cells, both of which express high levels of EGFR, HER2, and HER3 receptors. Afatinib increased the radiosensitivity of BxPC3 and Capan-2 cells, prevented the radio-induced phosphorylation of Akt, and induced mitotic catastrophe following irradiation. In comparison, Panc-1 cells (KRAS (mut)) expressing low levels of EGFR family receptors were resistant to afatinib-induced radiosensitization. These results must be confirmed in vivo. Afatinib showed cytotoxic and radiosensitizing effects toward a subset of pancreatic cancer cells which was closely correlated with expression of EGFR, HER2, and HER3 receptors, but not with KRAS status.

  5. Final overall survival analysis of a phase II trial evaluating vinorelbine and lapatinib in women with ErbB2 overexpressing metastatic breast cancer.

    Science.gov (United States)

    Janni, Wolfgang; Sarosiek, Tomasz; Karaszewska, Boguslawa; Pikiel, Joanna; Staroslawska, Elzbieta; Potemski, Piotr; Salat, Christoph; Brain, Etienne; Caglevic, Christian; Briggs, Kathryn; Mahood, Kim; DeSilvio, Michelle; Marini, Luca; Papadimitriou, Christos

    2015-12-01

    Lapatinib plus capecitabine (lap+cap) is approved as treatment for patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC), who have progressed on prior trastuzumab in the metastatic setting. We previously reported progression-free survival (PFS), overall survival (OS) and safety results from this open-label, multicentre, phase II study (VITAL; NCT01013740) conducted in women with HER2 positive MBC, to evaluate the efficacy and safety of lap plus vinorelbine (lap+vin), an important chemotherapy option for MBC, compared with lap+cap. In total, 112 patients were randomised 2:1 to treatment with lap+vin (N = 75) or lap+cap (N = 37). Results showed that the median PFS (primary endpoint) and OS (secondary endpoint) post-randomisation were comparable between treatment arms, with no new safety signals detected. Here, we assessed the final OS in this study at 40 months post-randomisation. At the time of final analyses, 24 (32%) patients were ongoing in the lap+vin arm, compared with 14 (38%) patients in the lap+cap arm (92% in both arms had discontinued treatment). Median OS in the lap+vin arm was 23.3 months (95% confidence intervals [CI]: 18.5, 31.1), compared with 20.3 months (95% CI: 16.4, 31.8) in the lap+cap arm. The median follow-up in the lap+vin arm was 18.86 months (95% CI: 10.68, 26.02), compared with 19.38 (95% CI: 25.56) months in the lap+cap arm. Similar rates of death (56-57%) were observed in both arms. The final OS was consistent with the previously reported data and suggest that lap+vin offers an effective treatment option for women with HER2-positive MBC.

  6. Dual activities of odorants on olfactory and nuclear hormone receptors.

    Science.gov (United States)

    Pick, Horst; Etter, Sylvain; Baud, Olivia; Schmauder, Ralf; Bordoli, Lorenza; Schwede, Torsten; Vogel, Horst

    2009-10-30

    We have screened an odorant compound library and discovered molecules acting as chemical signals that specifically activate both G-protein-coupled olfactory receptors (ORs) on the cell surface of olfactory sensory neurons and the human nuclear estrogen receptor alpha (ER) involved in transcriptional regulation of cellular differentiation and proliferation in a wide variety of tissues. Hence, these apparent dual active odorants induce distinct signal transduction pathways at different subcellular localizations, which affect both neuronal signaling, resulting in odor perception, and the ER-dependent transcriptional control of specific genes. We demonstrate these effects using fluorescence-based in vitro and cellular assays. Among these odorants, we have identified synthetic sandalwood compounds, an important class of molecules used in the fragrance industry. For one estrogenic odorant we have also identified the cognate OR. This prompted us to compare basic molecular recognition principles of odorants on the two structurally and apparent functionally non-related receptors using computational modeling in combination with functional assays. Faced with the increasing evidence that ORs may perform chemosensory functions in a number of tissues outside of the nasal olfactory epithelium, the unraveling of these molecular ligand-receptor interaction principles is of critical importance. In addition the evidence that certain olfactory sensory neurons naturally co-express ORs and ERs may provide a direct functional link between the olfactory and hormonal systems in humans. Our results are therefore useful for defining the structural and functional characteristics of ER-specific odorants and the role of odorant molecules in cellular processes other than olfaction.

  7. Activation profiles of opioid ligands in HEK cells expressing δ opioid receptors

    OpenAIRE

    Clark J; Demirci Hasan; Gharagozlou Parham; Lameh Jelveh

    2002-01-01

    Abstract Background The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only δ opioid receptors. Activation profiles of most of these ligands at δ opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production. Results Naltrexone and nalorphine were classified as antagonists at δ opioid receptor....

  8. Phagocytic receptors activate and immune inhibitory receptor SIRPalpha inhibits phagocytosis through paxillin and cofilin

    Directory of Open Access Journals (Sweden)

    Miri eGitik

    2014-04-01

    Full Text Available The innate-immune function of phagocytosis of apoptotic cells, tissue-debris, pathogens and cancer cells is essential for homeostasis, tissue repair, fighting infection and combating malignancy. Phagocytosis is carried out in the CNS by resident microglia and in both CNS and PNS by recruited macrophages. While phagocytosis proceeds, bystander healthy cells protect themselves by sending a do not eat me message to phagocytes as CD47 on their surface ligates immune inhibitory receptor SIRPα on the surface of phagocytes and SIRPα then produces the signaling which inhibits phagocytosis. This helpful mechanism becomes harmful when tissue-debris and unhealthy cells inhibit their own phagocytosis by employing the same mechanism. However, the inhibitory signaling that SIRPα produces has not been fully revealed. We focus here on how SIRPα inhibits the phagocytosis of the tissue-debris degenerated-myelin which hinders repair in axonal injury and neurodegenerative diseases. We tested whether SIRPα inhibits phagocytosis by regulating cytoskeleton function through paxillin and cofilin since (a the cytoskeleton generates the mechanical forces that drive phagocytosis and (b both paxillin and cofilin control cytoskeleton function. Paxillin and cofilin were transiently activated in microglia as phagocytosis was activated. In contrast, paxillin and cofilin were continuously activated and phagocytosis augmented in microglia in which SIRPα expression was knocked-down by SIRPα-shRNA. Further, levels of phagocytosis, paxillin activation and cofilin activation positively correlated with one another. Taken together, these observations suggest a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by both the activating signaling that phagocytic receptors produce by promoting the activation of paxillin and cofilin and the inhibiting signaling that immune inhibitory SIRPα produces by promoting the inactivation of paxillin and cofilin.

  9. Phagocytic receptors activate and immune inhibitory receptor SIRPα inhibits phagocytosis through paxillin and cofilin.

    Science.gov (United States)

    Gitik, Miri; Kleinhaus, Rachel; Hadas, Smadar; Reichert, Fanny; Rotshenker, Shlomo

    2014-01-01

    The innate immune function of phagocytosis of apoptotic cells, tissue debris, pathogens, and cancer cells is essential for homeostasis, tissue repair, fighting infection, and combating malignancy. Phagocytosis is carried out in the central nervous system (CNS) by resident microglia and in both CNS and peripheral nervous system by recruited macrophages. While phagocytosis proceeds, bystander healthy cells protect themselves by sending a "do not eat me" message to phagocytes as CD47 on their surface ligates immune inhibitory receptor SIRPα on the surface of phagocytes and SIRPα then produces the signaling which inhibits phagocytosis. This helpful mechanism becomes harmful when tissue debris and unhealthy cells inhibit their own phagocytosis by employing the same mechanism. However, the inhibitory signaling that SIRPα produces has not been fully revealed. We focus here on how SIRPα inhibits the phagocytosis of the tissue debris "degenerated myelin" which hinders repair in axonal injury and neurodegenerative diseases. We tested whether SIRPα inhibits phagocytosis by regulating cytoskeleton function through paxillin and cofilin since (a) the cytoskeleton generates the mechanical forces that drive phagocytosis and (b) both paxillin and cofilin control cytoskeleton function. Paxillin and cofilin were transiently activated in microglia as phagocytosis was activated. In contrast, paxillin and cofilin were continuously activated and phagocytosis augmented in microglia in which SIRPα expression was knocked-down by SIRPα-shRNA. Further, levels of phagocytosis, paxillin activation, and cofilin activation positively correlated with one another. Taken together, these observations suggest a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by both the activating signaling that phagocytic receptors produce by promoting the activation of paxillin and cofilin and the inhibiting signaling that immune inhibitory SIRPα produces by promoting the

  10. Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα) in a Mouse Liver Gene Expression Compendium

    Science.gov (United States)

    The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents inc...

  11. Stimulation of cannabinoid receptor 2 (CB2 suppresses microglial activation

    Directory of Open Access Journals (Sweden)

    Fernandez Francisco

    2005-12-01

    Full Text Available Abstract Background Activated microglial cells have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD, multiple sclerosis (MS, and HIV dementia. It is well known that inflammatory mediators such as nitric oxide (NO, cytokines, and chemokines play an important role in microglial cell-associated neuron cell damage. Our previous studies have shown that CD40 signaling is involved in pathological activation of microglial cells. Many data reveal that cannabinoids mediate suppression of inflammation in vitro and in vivo through stimulation of cannabinoid receptor 2 (CB2. Methods In this study, we investigated the effects of a cannabinoid agonist on CD40 expression and function by cultured microglial cells activated by IFN-γ using RT-PCR, Western immunoblotting, flow cytometry, and anti-CB2 small interfering RNA (siRNA analyses. Furthermore, we examined if the stimulation of CB2 could modulate the capacity of microglial cells to phagocytise Aβ1–42 peptide using a phagocytosis assay. Results We found that the selective stimulation of cannabinoid receptor CB2 by JWH-015 suppressed IFN-γ-induced CD40 expression. In addition, this CB2 agonist markedly inhibited IFN-γ-induced phosphorylation of JAK/STAT1. Further, this stimulation was also able to suppress microglial TNF-α and nitric oxide production induced either by IFN-γ or Aβ peptide challenge in the presence of CD40 ligation. Finally, we showed that CB2 activation by JWH-015 markedly attenuated CD40-mediated inhibition of microglial phagocytosis of Aβ1–42 peptide. Taken together, these results provide mechanistic insight into beneficial effects provided by cannabinoid receptor CB2 modulation in neurodegenerative diseases, particularly AD.

  12. Helix 11 Dynamics is Critical for Constitutive Androstane Receptor Activity

    Science.gov (United States)

    Wright, Edward; Busby, Scott A.; Wisecarver, Sarah; Vincent, Jeremy; Griffin, Patrick R.; Fernandez, Elias J.

    2010-01-01

    Summary The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339–345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity. PMID:21220114

  13. DHEA metabolites activate estrogen receptors alpha and beta

    Science.gov (United States)

    Michael Miller, Kristy K.; Al-Rayyan, Numan; Ivanova, Margarita M.; Mattingly, Kathleen A.; Ripp, Sharon L.; Klinge, Carolyn M.; Prough, Russell A.

    2012-01-01

    Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ) -regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, androstenedione, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and 4-hydroxytamoxifen, general P450 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ERβ-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ERβ-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17β-estradiol for ERα and ERβ binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ERα and ERβ in vitro, modulating estrogen target genes in vivo. PMID:23123738

  14. Facilitation of neocortical presynaptic terminal development by NMDA receptor activation

    Directory of Open Access Journals (Sweden)

    Sceniak Michael P

    2012-02-01

    Full Text Available Abstract Background Neocortical circuits are established through the formation of synapses between cortical neurons, but the molecular mechanisms of synapse formation are only beginning to be understood. The mechanisms that control synaptic vesicle (SV and active zone (AZ protein assembly at developing presynaptic terminals have not yet been defined. Similarly, the role of glutamate receptor activation in control of presynaptic development remains unclear. Results Here, we use confocal imaging to demonstrate that NMDA receptor (NMDAR activation regulates accumulation of multiple SV and AZ proteins at nascent presynaptic terminals of visual cortical neurons. NMDAR-dependent regulation of presynaptic assembly occurs even at synapses that lack postsynaptic NMDARs. We also provide evidence that this control of presynaptic terminal development is independent of glia. Conclusions Based on these data, we propose a novel NMDAR-dependent mechanism for control of presynaptic terminal development in excitatory neocortical neurons. Control of presynaptic development by NMDARs could ultimately contribute to activity-dependent development of cortical receptive fields.

  15. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

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    E. Teodorov

    2012-10-01

    Full Text Available The periaqueductal gray (PAG has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc or 0.9% saline (up to 1 mL/kg and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05 because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR. A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05 and lactating female rats (P < 0.01, with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in

  16. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Teodorov, E. [Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Paulo, SP (Brazil); Ferrari, M.F.R. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Fior-Chadi, D.R. [Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Camarini, R. [Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Felício, L.F. [Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  17. Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

    NARCIS (Netherlands)

    Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Elferink, Ronald P. J. Oude; Kuipers, Folkert; Groen, Albert K.

    2009-01-01

    Peroxisome proliferator-activated receptor delta (PPAR delta) is involved in regulation of energy homeostasis. Activation of PPAR delta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobilia

  18. The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma

    DEFF Research Database (Denmark)

    van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars

    2008-01-01

    The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the regulation of lipid and glucose metabolism in adipocytes, by regulating their differentiation, maintenance, and function. The transcriptional activity of PPARgamma is dictated by the set ...

  19. Sulfonylureas and glinides exhibit peroxisome proliferator-activated receptor gamma activity: A combined virtual screening and biological assay approach

    NARCIS (Netherlands)

    Scarsi, M.; Podvinec, M.; Roth, A.; Hug, H.; Kersten, A.H.; Albrecht, H.; Schwede, T.; Meyer, U.A.; Rucker, C.

    2007-01-01

    Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target the peroxisome proliferator-activated receptor (PPAR) improving insulin resistance (thiazolidinediones). Our work shows that sulf

  20. FATTY ACIDS MODULATE TOLL-LIKE RECEPTOR 4 ACTIVATION THROUGH REGULATION OF RECEPTOR DIMERIZATION AND RECRUITMENT INTO LIPID RAFTS

    Science.gov (United States)

    The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies (J Biol Chem 2003, 2004) demonstrated that saturated ...

  1. The Role of Novel Substituted Diindolyl Methane Analogues in the Treatment of Triple-Negative and ErbB2-Positive Breast Cancer

    Science.gov (United States)

    2016-05-01

    DIM10 and DIM 12, 14) and investigate the structure -dependent anticancer activities in TNBC and EPBC using both cell and laboratory animal models...limited research has been done in the area of PDX so far, there is no well-established protocol available for it. Moreover, unlike human cancer cells ... cells were fixed with 0.5% glutaraldehyde and stained with crystal violet for 15 min. The images of scratch area were taken using Olympus microscope

  2. Methamphetamine Increases Locomotion and Dopamine Transporter Activity in Dopamine D5 Receptor-Deficient Mice

    OpenAIRE

    Seiji Hayashizaki; Shinobu Hirai; Yumi Ito; Yoshiko Honda; Yosefu Arime; Ichiro Sora; Haruo Okado; Tohru Kodama; Masahiko Takada

    2013-01-01

    Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behaviora...

  3. Elimination of progressive mammary cancer by repeated administrations of chimeric antigen receptor-modified T cells.

    Science.gov (United States)

    Globerson-Levin, Anat; Waks, Tova; Eshhar, Zelig

    2014-05-01

    Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

  4. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    Science.gov (United States)

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  5. Dopamine receptor activation increases HIV entry into primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Peter J Gaskill

    Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

  6. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    DEFF Research Database (Denmark)

    Barnathan, E S; Kuo, A; Karikó, K

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...

  7. Detection of gene amplification in MYCN, C-MYC, MYCL1, ERBB2, EGFR, AKT2, and human papilloma virus in samples from cervical smear normal cytology, intraepithelial cervical neoplasia (CIN I, II, III, and cervical cancer

    Directory of Open Access Journals (Sweden)

    Dabeiba Adriana García

    2011-06-01

    Full Text Available Introducción: El cáncer cervical es el segundo cáncer más importante en mujeres a nivel mundial y es la segunda causa de muerte por cáncer en mujeres. Se ha demostrado que el proceso de carcinogénesis cervical presenta componentes tanto genéticos como epigenéticos y medio ambientales. En la actualidad, hay gran interés en la búsqueda de marcadores moleculares asociados con la progresión de esta enfermedad, uno de los posibles mecanismos y que además está poco estudiado en cáncer cervical es la amplificación génica de algunos oncogenes como la familia MYC, EGFR y AKT entre otros. Objetivos: Detectar la amplificación génica de MYCN, C-MYC, MYCL1, ERBB2, EGFR y AKT2 además de la presencia del virus de papiloma humano en cepillados cervicales en mujeres con citología normal o con neoplasia intraepitelial cervical (NIC I, II y III o con cáncer cervical. Métodos: Se genotipificó mediante reverse line blot (RLB el virus de papiloma humano (VPH y se determinó el estado de amplificación génica de los genes mencionados mediante PCR en tiempo real utilizando sondas taqman. Resultados: El VPH se encontró presente en 4% de las pacientes con citología normal, en 48% en NIC I, 63.6% en NIC II, 64% en NIC III y 70.8% en cáncer cervical. Los genes MYCN, MYCL1 y ERBB2 mostraron mayor amplificación en lesiones de alto grado y cáncer con diferencias estadísticamente significativas  a las lesiones de bajo grado y citología normal, en 39.1%, 34.7% y 30.4% respectivamente. Además, se encontraron amplificados los genes C-MYC, EGFR y AKT2, en muestras de pacientes con cáncer cervical, en 12%, 18% y 13% respectivamente. Sin embargo, no se observaron diferencias estadísticamente significativas con respecto a las lesiones de alto y bajo grado y citología normal. Conclusión: En las lesiones de alto grado como en cáncer cervical, se encuentra mayor prevalencia del virus al igual que se detectan mayor cantidad de alteraciones gen

  8. Protease activated receptor-2 contributes to heart failure.

    Directory of Open Access Journals (Sweden)

    Silvio Antoniak

    Full Text Available Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs, in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure.

  9. Activation of μ-opioid receptor and Toll-like receptor 4 by plasma from morphine-treated mice.

    Science.gov (United States)

    Xie, Nan; Gomes, Fabio P; Deora, Vandana; Gregory, Kye; Vithanage, Tharindu; Nassar, Zeyad D; Cabot, Peter J; Sturgess, David; Shaw, Paul N; Parat, Marie-Odile

    2017-03-01

    In this study, we quantified the ability of opioids present in biological samples to activate the μ-opioid receptor and TLR4 using cell-based assays. Each assay was standardised, in the presence of plasma, using morphine, its μ receptor-active metabolite morphine-6 glucuronide (M6G) and its μ receptor-inactive, but TLR4-active metabolite morphine-3 glucuronide (M3G). Specificity was verified using antagonists. Morphine- and M6G-spiked plasma samples exhibited μ receptor activation, which M3G-spiked plasma lacked. In contrast, M3G showed moderate but consistent activation of TLR-4. Plasma samples were collected at a number of time points from mice administered morphine (1 or 10mg/kg every 12h for 3days) or saline. Morphine administration led to intermittent μ receptor activation, reversed by μ receptor antagonists, and to TRL4 activation at time points where M3G is measured in plasma. Interestingly, this protocol of morphine administration also led to TLR4-independent NF-κB activation, at time points where M3G was not detected, presumably via elevation of circulating cytokines including, but not limited to, TNFα. Circulating TNFα was increased after three days of morphine administration, and TNFα mRNA elevated in the spleen of morphine-treated mice.

  10. Morphine induces μ opioid receptor endocytosis in guinea pig enteric neurons following prolonged receptor activation

    Science.gov (United States)

    Patierno, Simona; Anselmi, Laura; Jaramillo, Ingrid; Scott, David; Garcia, Rachel; Sternini, Catia

    2010-01-01

    Background & Aims The μ opioid receptor (μOR) undergoes rapid endocytosis following acute stimulation with opioids and most opiates, but not with morphine. We investigated whether prolonged activation of μOR affects morphine’s ability to induce receptor endocytosis in enteric neurons. Methods We compared the effects of morphine, a poor μOR-internalizing opiate, and [D-Ala2, MePhe4,Gly-ol5] enkephalin (DAMGO), a potent μOR-internalizing agonist, on μOR trafficking in enteric neurons and on the expression of dynamin and β-arrestin immunoreactivity in the ileum of guinea pigs rendered tolerant by chronic administration of morphine. Results Morphine (100 µM) strongly induced endocytosis of μOR in tolerant but not naïve neurons (55.7%±9.3% vs. 24.2%±7.3%, P<0.001) whereas DAMGO (10 µM) strongly induced internalization of μOR in neurons from tolerant and naïve animals (63.6%±8.4% and 66.5%±3.6%). Morphine- or DAMGO-induced μOR endocytosis resulted from direct interactions between the ligand and the μOR, because endocytosis was not affected by tetrodotoxin, a blocker of endogenous neurotransmitter release. Ligand-induced μOR internalization was inhibited by pretreatment with the dynamin inhibitor, dynasore. Chronic morphine administration resulted in a significant increase in dynamin and translocation of dynamin immunoreactivity from the intracellular pool to the plasma membrane, but did not affect β arrestin immunoreactivity. Conclusion Chronic activation of μORs increases the ability of morphine to induce μOR endocytosis in enteric neurons, which depends on the level and cellular localization of dynamin, a regulatory protein that has an important role in receptor-mediated signal transduction in cells. PMID:21070774

  11. Evidence that adiponectin receptor 1 activation exacerbates ischemic neuronal death

    Directory of Open Access Journals (Sweden)

    Thundyil John

    2010-08-01

    Full Text Available Abstract Background- Adiponectin is a hormone produced in and released from adipose cells, which has been shown to have anti-diabetic and anti-inflammatory actions in peripheral cells. Two cell surface adiponectin receptors (ADRs mediate the majority of the known biological actions of adiponectin. Thus far, ADR expression in the brain has been demonstrated in the arcuate and the paraventricular nucleus of hypothalamus, where its activation affects food intake. Recent findings suggest that levels of circulating adiponectin increase after an ischemic stroke, but the role of adiponectin receptor activation in stroke pathogenesis and its functional outcome is unclear. Methods- Ischemic stroke was induced in C57BL/6 mice by middle cerebral artery occlusion (MCAO for 1 h, followed by reperfusion. Primary cortical neuronal cultures were established from individual embryonic neocortex. For glucose deprivation (GD, cultured neurons were incubated in glucose-free Locke's medium for 6, 12 or 24 h. For combined oxygen and glucose deprivation (OGD, neurons were incubated in glucose-free Locke's medium in an oxygen-free chamber with 95% N2/5% CO2 atmosphere for either 3, 6, 9, 12 or 24 h. Primary neurons and brain tissues were analysed for Adiponectin and ADRs using reverse transcriptase polymerase chain reaction (RT-PCR, immunoblot and immunochemistry methods. Results- Cortical neurons express ADR1 and ADR2, and that the levels of ADR1 are increased in neurons in response to in vitro or in vivo ischemic conditions. Neurons treated with either globular or trimeric adiponectin exhibited increased vulnerability to oxygen and glucose deprivation which was associated with increased activation of a pro-apoptotic signaling cascade involving p38 mitogen-activated protein kinase (p38MAPK and AMP-activated protein kinase (AMPK. Conclusions- This study reveals a novel pathogenic role for adiponectin and adiponectin receptor activation in ischemic stroke. We show that

  12. Mesenchymal stromal cells expressing ErbB-2/neu elicit protective antibreast tumor immunity in vivo, which is paradoxically suppressed by IFN-gamma and tumor necrosis factor-alpha priming.

    Science.gov (United States)

    Romieu-Mourez, Raphaëlle; François, Moïra; Abate, Amanda; Boivin, Marie-Noëlle; Birman, Elena; Bailey, Dana; Bramson, Jonathan L; Forner, Kathy; Young, Yoon-Kow; Medin, Jeffrey A; Galipeau, Jacques

    2010-10-15

    It is unknown whether mesenchymal stromal cells (MSC) can regulate immune responses targeting tumor autoantigens of low immunogenicity. We tested here whether immunization with MSC could break immune tolerance towards the ErbB-2/HER-2/neu tumor antigen and the effects of priming with IFN-γ and tumor necrosis factor-α (TNF-α) on this process. BALB/c- and C57BL/6-derived MSC were lentivirally transduced to express a kinase-inactive rat neu mutant (MSC/Neu). Immunization of BALB/c mice with nontreated or IFN-γ-primed allogeneic or syngeneic MSC/Neu induced similar levels of anti-neu antibody titers; however, only syngeneic MSC/Neu induced protective neu-specific CD8(+) T cell responses. Compared to immunization with nontreated or IFN-γ-primed syngeneic MSC/Neu, the number of circulating neu-specific CD8(+) T cells and titers of anti-neu antibodies were observed to be decreased after immunizations with IFN-γ- plus TNF-α-primed MSC/Neu. In addition, syngeneic MSC/Neu seemed more efficient than IFN-γ-primed MSC/Neu at inducing a protective therapeutic antitumor immune response resulting in the regression of transplanted neu-expressing mammary tumor cells. In vitro antigen-presenting cell assays performed with paraformaldehyde-fixed or live MSC showed that priming with IFN-γ plus TNF-α, compared to priming with IFN-γ alone, increased antigen presentation as well as the production of immunosuppressive factors. These data suggest that whereas MSC could effectively serve as antigen-presenting cells to induce immune responses aimed at tumor autoantigens, these functions are critically regulated by IFN-γ and TNF-α.

  13. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

    Science.gov (United States)

    Smiley, P L; Stremler, K E; Prescott, S M; Zimmerman, G A; McIntyre, T M

    1991-06-15

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows

  14. Clinical utility of the combination of lapatinib and letrozole in the management of hormone receptor-positive and HER2-positive advanced breast cancer

    Directory of Open Access Journals (Sweden)

    Merriam PA

    2011-10-01

    Full Text Available Priscilla Merriam, William M Sikov Department of Medicine, Division of Hematology-Oncology, Warren Alpert Medical School of Brown University, Providence, RI, USA Abstract: Breast cancers that overexpress human epidermal growth factor receptor-2 (HER2-positive [HER2+] tend to be biologically aggressive and associated with a poor prognosis, even those that coexpress receptors for estrogen and/or progesterone (hormone receptor-positive [HR+]. Optimal therapy for patients with “double-positive” (HR+/HER2+ breast cancers is still being defined. In this subset of patients, the efficacy of targeted endocrine therapies appears to be diminished by cross-activation or “crosstalk” between estrogen receptor-mediated gene transcription and pathways activated by other growth factor receptors, including HER2. Lapatinib is a tyrosine kinase inhibitor which binds reversibly to the intracellular domains of the epidermal growth factor receptor and HER2, interfering with their ability to initiate signal transduction cascades that promote cancer cell proliferation, survival, and metastasis. In a recently published randomized, placebo-controlled Phase III study in postmenopausal HR+ metastatic breast cancer, the addition of lapatinib to the aromatase inhibitor letrozole significantly improved progression-free survival solely in women who were also HER2+. This article reviews the biology of “double-positive” breast cancers and the rationale underlying combining endocrine and HER2-targeted therapies, including the lapatinib/letrozole combination, for these tumors. Results from the Phase III trial are examined, as well as available data on other combinations of HR and HER2-targeted therapies. Ongoing trials and potential future applications of these combinations in both HR+/HER2+ and other subgroups of breast cancer patients are also discussed. Keywords: breast neoplasm, erbB2, estrogen receptor, letrozole, lapatinib

  15. NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs during inflammation induced visceral hypersensitivity

    Directory of Open Access Journals (Sweden)

    Caudle Robert M

    2009-09-01

    Full Text Available Abstract Background Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs, co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG neurons expressing the transient receptor potential vanilloid-1 (TRPV1 receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX prior to inflammation and behavioural testing. Results CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs. Conclusion Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned

  16. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Onuma, Hirohisa; Inukai, Kouichi, E-mail: kinukai@ks.kyorin-u.ac.jp; Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  17. Oleamide activates peroxisome proliferator-activated receptor gamma (PPARγ) in vitro.

    Science.gov (United States)

    Dionisi, Mauro; Alexander, Stephen P H; Bennett, Andrew J

    2012-05-14

    Oleamide (ODA) is a fatty acid primary amide first identified in the cerebrospinal fluid of sleep-deprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. It has recently been reported to exert a hypolipidemic effect in hamsters. Here, we have investigated the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs) as potential targets for ODA action. Activation of PPARα, PPARβ and PPARγ was assessed using recombinant expression in Chinese hamster ovary cells with a luciferase reporter gene assay. Direct binding of ODA to the ligand binding domain of each of the three PPARs was monitored in a cell-free fluorescent ligand competition assay. A well-established assay of PPARγ activity, the differentiation of 3T3-L1 murine fibroblasts into adipocytes, was assessed using an Oil Red O uptake-based assay. ODA, at 10 and 50 μM, was able to transactivate PPARα, PPARβ and PPARγ receptors. ODA bound to the ligand binding domain of all three PPARs, although complete displacement of fluorescent ligand was only evident for PPARγ, at which an IC50 value of 38 μM was estimated. In 3T3-L1 cells, ODA, at 10 and 20 μM, induced adipogenesis. We have, therefore, identified a novel site of action of ODA through PPAR nuclear receptors and shown how ODA should be considered as a weak PPARγ ligand in vitro.

  18. Notch receptors in human choroid plexus tumors.

    Science.gov (United States)

    Beschorner, R; Waidelich, J; Trautmann, K; Psaras, T; Schittenhelm, J

    2013-08-01

    Notch signaling plays a role in development and formation of the normal choroid plexus (nCP), and in formation of various tumors in humans. Activation of Notch3 has been reported to promote tumor growth in invasive gliomas and to initiate formation of choroid plexus tumors (CPT) in mice. We investigated the expression of all currently known Notch receptors (Notch 1-4) in 55 samples of nCP and 88 CPT, including 61 choroid plexus papillomas (CPP), 22 atypical CPP and 5 choroid plexus carcinomas by immunohistochemistry. Notch expression was semiquantitatively evaluated separately for membranous/cytoplasmic and for nuclear staining. In addition, we examined Her2 expression (EGFR2, Her2/neu, ErbB2, CD340) because of its functional link to Notch signaling. All samples were negative for Notch3. Membranous/cytoplasmic expression of Notch1 (pnCP compared to CPT. Nuclear expression of Notch1, -2 and -4 was significantly higher in CPT compared to nCP (pnCP to a predominant nuclear expression in CPT. Her2 was weakly expressed in 42/84 CPT but only in 2/53 nCP (p=0.0001) and positively correlated with nuclear expression of Notch1, -2 and 4 in CPT. In summary, a shift between membranous/cytoplasmic (non-canonical signaling pathway) and nuclear expression (canonical signaling pathway) of Notch1, -2 and -4 and upregulation of Her2 indicate neoplastic transformation in human CP and may reveal new therapeutic approaches.

  19. PDI regulates seizure activity via NMDA receptor redox in rats.

    Science.gov (United States)

    Kim, Ji Yang; Ko, Ah-Rhem; Hyun, Hye-Won; Min, Su-Ji; Kim, Ji-Eun

    2017-02-15

    Redox modulation of cysteine residues is one of the post-translational modifications of N-methyl-D-aspartate receptor (NMDAR). Protein disulfide isomerases (PDI), an endoplasmic reticulum (ER) chaperone, plays a crucial role in catalyzing disulfide bond formation, reduction, and isomerization. In the present study, we found that PDI bound to NMDAR in the normal hippocampus, and that this binding was increased in chronic epileptic rats. In vitro thiol reductase assay revealed that PDI increased the amount of thiols on full-length recombinant NR1 protein. PDI siRNA, 5-5'-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin and PDI antibody reduced seizure susceptibility in response to pilocarpine. In addition, PDI knockdown effectively ameliorated spontaneous seizure activity in chronic epileptic rats. Anticonvulsive effects of PDI siRNA were correlated to the reduction of the amount of free- and nitrosothiols on NMDAR, accompanied by the inhibition of PDI activity. However, PDI knockdown did not lead to alteration in basal neurotransmission or ER stress under physiological condition. These findings provide mechanistic insight into sulfhydration of disulfide bonds on NMDAR by PDI, and suggest that PDI may represent a target of potential therapeutics for epilepsy, which avoids a possible side effect on physiological receptor functionality.

  20. Role of Peroxisome Proliferator-Activated Receptors in Inflammation Control

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    Jihan Youssef

    2004-01-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs were discovered over a decade ago, and were classified as orphan members of the nuclear receptor superfamily. To date, three PPAR subtypes have been discovered and characterized (PPARα, β/δ, γ. Different PPAR subtypes have been shown to play crucial roles in important diseases and conditions such as obesity, diabetes, atherosclerosis, cancer, and fertility. Among the most studied roles of PPARs is their involvement in inflammatory processes. Numerous studies have revealed that agonists of PPARα and PPARγ exert anti-inflammatory effects both in vitro and in vivo. Using the carrageenan-induced paw edema model of inflammation, a recent study in our laboratories showed that these agonists hinder the initiation phase, but not the late phase of the inflammatory process. Furthermore, in the same experimental model, we recently also observed that activation of PPARδ exerted an anti-inflammatory effect. Despite the fact that exclusive dependence of these effects on PPARs has been questioned, the bulk of evidence suggests that all three PPAR subtypes, PPARα,δ,γ, play a significant role in controlling inflammatory responses. Whether these subtypes act via a common mechanism or are independent of each other remains to be elucidated. However, due to the intensity of research efforts in this area, it is anticipated that these efforts will result in the development of PPAR ligands as therapeutic agents for the treatment of inflammatory diseases.

  1. Antitussive activity of Withania somnifera and opioid receptors.

    Science.gov (United States)

    Nosálová, Gabriela; Sivová, Veronika; Ray, Bimalendu; Fraňová, Soňa; Ondrejka, Igor; Flešková, Dana

    2015-01-01

    Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways.

  2. In vitro neuronal network activity in NMDA receptor encephalitis

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    Jantzen Sabine U

    2013-02-01

    Full Text Available Abstract Background Anti-NMDA-encephalitis is caused by antibodies against the N-methyl-D-aspartate receptor (NMDAR and characterized by a severe encephalopathy with psychosis, epileptic seizures and autonomic disturbances. It predominantly occurs in young women and is associated in 59% with an ovarian teratoma. Results We describe effects of cerebrospinal fluid (CSF from an anti-N-methyl-D-aspartate receptor (NMDAR encephalitis patient on in vitro neuronal network activity (ivNNA. In vitro NNA of dissociated primary rat cortical populations was recorded by the microelectrode array (MEA system. The 23-year old patient was severely affected but showed an excellent recovery following multimodal immunomodulatory therapy and removal of an ovarian teratoma. Patient CSF (pCSF taken during the initial weeks after disease onset suppressed global spike- and burst rates of ivNNA in contrast to pCSF sampled after clinical recovery and decrease of NMDAR antibody titers. The synchrony of pCSF-affected ivNNA remained unaltered during the course of the disease. Conclusion Patient CSF directly suppresses global activity of neuronal networks recorded by the MEA system. In contrast, pCSF did not regulate the synchrony of ivNNA suggesting that NMDAR antibodies selectively regulate distinct parameters of ivNNA while sparing their functional connectivity. Thus, assessing ivNNA could represent a new technique to evaluate functional consequences of autoimmune encephalitis-related CSF changes.

  3. Vasopeptidase-activated latent ligands of the histamine receptor-1.

    Science.gov (United States)

    Gera, Lajos; Roy, Caroline; Charest-Morin, Xavier; Marceau, François

    2013-11-01

    Whether peptidases present in vascular cells can activate prodrugs active on vascular cells has been tested with 2 potential latent ligands of the histamine H1 receptor (H1R). First, a peptide consisting of the antihistamine cetirizine (CTZ) condensed at the N-terminus of ε-aminocaproyl-bradykinin (εACA-BK) was evaluated for an antihistamine activity that could be revealed by degradation of the peptide part of the molecule. CTZ-εACA-BK had a submicromolar affinity for the BK B2 receptor (B2R; IC50 of 590 nM, [(3)H]BK binding competition), but a non-negligible affinity for the human H1 receptor (H1R; IC50 of 11 μM for [(3)H]pyrilamine binding). In the human isolated umbilical vein, a system where both endogenous B2R and H1R mediate strong contractions, CTZ-εACA-BK exerted mild antagonist effects on histamine-induced contraction that were not modified by omapatrilat or by a B2R antagonist that prevents endocytosis of the BK conjugate. Cells expressing recombinant ACE or B2R incubated with CTZ-εACA-BK did not release a competitor of [(3)H]pyrilamine binding to H1Rs. Thus, there is no evidence that CTZ-εACA-BK can release free cetirizine in biological environments. The second prodrug was a blocked agonist, L-alanyl-histamine, potentially activated by aminopeptidase N (APN). This compound did not compete for [(3)H]pyrilamine binding to H1Rs. The human umbilical vein contractility assay responded to L-alanyl-histamine (EC50 54.7 μM), but the APN inhibitor amastatin massively (17-fold) reduced its apparent potency. Amastatin did not influence the potency of histamine as a contractile agent. One of the 2 tested latent H1R ligands, L-alanyl-histamine, supported the feasibility of pro-drug activation by vascular ectopeptidases.

  4. Relationship between somatostatin receptors and activation of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 陆良勇; 尤汉宁; 徐芹芳

    2004-01-01

    Background Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs.Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation.SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immunohistochemical staining.Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc.Conclusion The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  5. ERK5 activation by Gq-coupled muscarinic receptors is independent of receptor internalization and β-arrestin recruitment.

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    Guzmán Sánchez-Fernández

    Full Text Available G-protein-coupled receptors (GPCRs are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.

  6. Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction.

    Science.gov (United States)

    Borodinsky, Laura N; Spitzer, Nicholas C

    2007-01-02

    Signaling in the nervous system requires matching of neurotransmitter receptors with cognate neurotransmitters at synapses. The vertebrate neuromuscular junction is the best studied cholinergic synapse, but the mechanisms by which acetylcholine is matched with acetylcholine receptors are not fully understood. Because alterations in neuronal calcium spike activity alter transmitter specification in embryonic spinal neurons, we hypothesized that receptor expression in postsynaptic cells follows changes in transmitter expression to achieve this specific match. We find that embryonic vertebrate striated muscle cells normally express receptors for glutamate, GABA, and glycine as well as for acetylcholine. As maturation progresses, acetylcholine receptor expression prevails. Receptor selection is altered when early neuronal calcium-dependent activity is perturbed, and remaining receptor populations parallel changes in transmitter phenotype. In these cases, glutamatergic, GABAergic, and glycinergic synaptic currents are recorded from muscle cells, demonstrating that activity regulates matching of transmitters and their receptors in the assembly of functional synapses.

  7. Involvement of Activating NK Cell Receptors and Their Modulation in Pathogen Immunity

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    Francesco Marras

    2011-01-01

    Full Text Available Natural Killer (NK cells are endowed with cell-structure-sensing receptors providing inhibitory protection from self-destruction (inhibitory NK receptors, iNKRs, including killer inhibitory receptors and other molecules and rapid triggering potential leading to functional cell activation by Toll-like receptors (TLRs, cytokine receptors, and activating NK cell receptors including natural cytotoxicity receptors (NCRs, i.e., NKp46, NKp46, and NKp44. NCR and NKG2D recognize ligands on infected cells which may be endogenous or may directly bind to some structures derived from invading pathogens. In this paper, we address the known direct or indirect interactions between activating receptors and pathogens and their expression during chronic HIV and HCV infections.

  8. Selective 5-HT7 Receptor Activation May Enhance Synaptic Plasticity Through N-methyl-D-aspartate (NMDA) Receptor Activity in the Visual Cortex.

    Science.gov (United States)

    Xiang, Kangjian; Zhao, Xuefei; Li, Youjun; Zheng, Liang; Wang, Jue; Li, Yan-Hai

    2016-01-01

    Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter that modulates N-methyl-D-aspartate (NMDA) receptor activity by binding to several different 5-HT receptor subtypes. In the present study, we used whole-cell patch-clamp recordings in transverse slice preparations to test the role of 5-HT receptors in modulating the NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in layer II/III pyramidal neurons of the rat visual cortex. We found that the NMDA receptor-mediated component of mEPSCs could be potentiated by exogenously applied 5-HT. Similar results were obtained by exogenously applied 5-CT or 8-OH-DPAT (the 5-HT1A and 5-HT7 receptor agonist). A specific antagonist for the 5-HT7 receptor, SB-269970, completely blocked the increase in NMDA receptor-mediated component of mEPSCs by 5-CT or 8- OH-DPAT. Moreover, the selective 5-HT1A receptor antagonist, WAY-100135, displayed no influence on the enhancement in NMDA receptor-mediated component of mEPSCs by 5-CT or 8-OHDPAT. These results indicated that the increase in NMDA receptor-mediated component of mEPSCs by 5-HT in layer II/III pyramidal neurons of the young rat visual cortex requires activation of 5-HT7 receptors, but not 5-HT1A receptors. These observations might be clinically relevant to schizophrenia and Alzheimer's disease (AD), where enhancing NMDA receptor-mediated neurotransmission is considered to be a promising strategy for treatment of these diseases.

  9. Activation of cardiac ryanodine receptors by cardiac glycosides.

    Science.gov (United States)

    Sagawa, Toshio; Sagawa, Kazuko; Kelly, James E; Tsushima, Robert G; Wasserstrom, J Andrew

    2002-03-01

    This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of approximately 0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 microM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 microM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.

  10. Aryl hydrocarbon receptor ligand activity of commercial health foods.

    Science.gov (United States)

    Amakura, Yoshiaki; Tsutsumi, Tomoaki; Nakamura, Masafumi; Handa, Hiroshi; Yoshimura, Morio; Matsuda, Rieko; Yoshida, Takashi

    2011-06-15

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates toxicological effects by binding to agonists such as dioxins. We previously reported the presence of natural dioxin-like ligands in foods. To further characterise natural ligands with dioxin-like activity, we examined the influence of 50 kinds of commercial supplement and health food on the AhR, using a reporter gene assay. Some samples, prepared using soybean, sesame, or propolis as an ingredient, were revealed to show AhR-binding activity, similar to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at high concentrations. To characterise the AhR-activating substances in eight active samples, the respective extracts were subjected to fractionation with n-hexane, ethyl acetate, and water, followed by estimating their AhR activities. The n-hexane fraction of the propolis extract sample, and the ethyl acetate fractions of the other samples, showed AhR activity similar to that of TCDD, at a high concentration range. HPLC analysis of the active fractions identified isoflavones, such as daidzein and glycitein, and flavones, such as tectochrysin and chrysin, in the samples. Among these compounds, tectochrysin exhibited marked AhR activation. Flavonoids, which are characterised as natural AhR ligands, are known to have representative beneficial effects on human health. The natural AhR ligands identified in this study are known to be useful for human health. Therefore, it is considered that AhR may play a beneficial regulatory role in humans. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. B cell activation triggered by the formation of the small receptor cluster: a computational study.

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    Beata Hat

    2011-10-01

    Full Text Available We proposed a spatially extended model of early events of B cell receptors (BCR activation, which is based on mutual kinase-receptor interactions that are characteristic for the immune receptors and the Src family kinases. These interactions lead to the positive feedback which, together with two nonlinearities resulting from the double phosphorylation of receptors and Michaelis-Menten dephosphorylation kinetics, are responsible for the system bistability. We demonstrated that B cell can be activated by a formation of a tiny cluster of receptors or displacement of the nucleus. The receptors and Src kinases are activated, first locally, in the locus of the receptor cluster or the region where the cytoplasm is the thinnest. Then the traveling wave of activation propagates until activity spreads over the whole cell membrane. In the models in which we assume that the kinases are free to diffuse in the cytoplasm, we found that the fraction of aggregated receptors, capable to initiate B cell activation decreases with the decreasing thickness of cytoplasm and decreasing kinase diffusion. When kinases are restricted to the cell membrane - which is the case for most of the Src family kinases - even a cluster consisting of a tiny fraction of total receptors becomes activatory. Interestingly, the system remains insensitive to the modest changes of total receptor level. The model provides a plausible mechanism of B cells activation due to the formation of small receptors clusters collocalized by binding of polyvalent antigens or arising during the immune synapse formation.

  12. Activation of Toll-like receptors by Burkholderia pseudomallei

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    Jansson-Hutson Malinka J

    2008-08-01

    Full Text Available Abstract Background Melioidosis, a lethal tropical infection that is endemic in southeast Asia and northern Australia, is caused by the saprophytic Gram-negative bacterium Burkholderia pseudomallei. Overall mortality approaches 40% yet little is known about mechanisms of host defense. Toll-like receptors (TLRs are host transmembrane receptors that recognize conserved pathogen molecular patterns and induce an inflammatory response. The lipopolysaccharide (LPS of Gram-negative bacteria is a potent inducer of the host innate immune system. TLR4, in association with MD-2, is the archetype receptor for LPS although B. pseudomallei LPS has been previously identified as a TLR2 agonist. We examined TLR signaling induced by B. pseudomallei, B. pseudomallei LPS, and B. pseudomallei lipid A using gain-of-function transfection assays of NF-κB activation and studies of TLR-deficient macrophages. Results In HEK293 cells transfected with murine or human TLRs, CD14, and MD-2, heat-killed B. pseudomallei activated TLR2 (in combination with TLR1 or TLR6 and TLR4. B. pseudomallei LPS and lipid A activated TLR4 and this TLR4-mediated signaling required MD-2. In TLR2-/- macrophages, stimulation with heat-killed B. pseudomallei augmented TNF-α and MIP-2 production whereas in TLR4-/- cells, TNF-α, MIP-2, and IL-10 production was reduced. Cytokine production by macrophages stimulated with B. pseudomallei LPS or lipid A was entirely dependent on TLR4 but was increased in the absence of TLR2. TLR adaptor molecule MyD88 strongly regulated TNF-α production in response to heat-killed B. pseudomallei. Conclusion B. pseudomallei activates TLR2 and TLR4. In the presence of MD-2, B. pseudomallei LPS and lipid A are TLR4 ligands. Although the macrophage cytokine response to B. pseudomallei LPS or lipid A is completely dependent on TLR4, in TLR2-/- macrophages stimulated with B. pseudomallei, B. pseudomallei LPS or lipid A, cytokine production is augmented. Other MyD88

  13. RELATIONSHIP BETWEEN SOMATOSTATIN RECEPTORS AND ACTIVATION OF HEPATIC STELLATE CELL

    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 尤汉宁; 徐芹芳; 陆良勇

    2004-01-01

    Objective To investigate the relationship between expression of somatostatin receptors (SSTRs) and activation of rat hepatic stellate cell (HSC). Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation, and then SSTR1 ~5 mRNA levels in the differentiated first passage HSCs were detected by means of reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1 ~5 in normal as well as fibrotic liver was measured by immunohistochemical staining. Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs and normal rat liver. But SSTR1~3 mRNA appeared as HSCs became wholly activated, and SSTR1 ~3 could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc Conclusion The expression of SSTR1~3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  14. Increased peroxisome proliferator-activated receptor-gamma activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

    Science.gov (United States)

    Wang, Jueqiong; Lu, Liu; Kok, Chung H; Saunders, Verity A; Goyne, Jarrad M; Dang, Phuong; Leclercq, Tamara M; Hughes, Timothy P; White, Deborah L

    2017-02-02

    Imatinib is actively transported by OCT-1 influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Here we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor gamma agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor gamma antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to Bcr-Abl kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor gamma-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor gamma transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; preceptor gamma activation has a negative impact on the intracellular uptake of imatinib and consequent Bcr-Abl kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor gamma activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor gamma agonist pioglitazone was reported to act synergistically with imatinib targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor gamma ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor gamma activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor gamma

  15. New insights into the structural bases of activation of Cys-loop receptors.

    Science.gov (United States)

    Bouzat, Cecilia

    2012-01-01

    Neurotransmitter receptors of the Cys-loop superfamily mediate rapid synaptic transmission throughout the nervous system, and include receptors activated by ACh, GABA, glycine and serotonin. They are involved in physiological processes, including learning and memory, and in neurological disorders, and they are targets for clinically relevant drugs. Cys-loop receptors assemble either from five copies of one type of subunit, giving rise to homomeric receptors, or from several types of subunits, giving rise to heteromeric receptors. Homomeric receptors are invaluable models for probing fundamental relationships between structure and function. Receptors contain a large extracellular domain that carries the binding sites and a transmembrane region that forms the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50Å to the ion channel gate is central to understanding receptor function. Depending on the receptor subtype, occupancy of either two, as in the prototype muscle nicotinic receptor, or three binding sites, as in homomeric receptors, is required for full activation. The conformational changes initiated at the binding sites are propagated to the gate through the interface between the extracellular and transmembrane domains. This region forms a network that relays structural changes from the binding site towards the pore, and also contributes to open channel lifetime and rate of desensitization. Thus, this coupling region controls the beginning and duration of a synaptic response. Here we review recent advances in the molecular mechanism by which Cys-loop receptors are activated with particular emphasis on homomeric receptors.

  16. The role of GH receptor tyrosine phosphorylation in Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, J A; Hansen, L H; Wang, X;

    1997-01-01

    Stimulation of GH receptors leads to rapid activation of Jak2 kinase and subsequent tyrosine phosphorylation of the GH receptor. Three specific tyrosines located in the C-terminal domain of the GH receptor have been identified as being involved in GH-stimulated transcription of the Spi 2.1 promoter....... Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA......-binding activity, whereas the GH receptor mutant lacking all intracellular tyrosines was not. Synthetic tyrosine phosphorylated peptides corresponding to the GH receptor sequence around the three tyrosines inhibited Stat5 DNA-binding activity while their non-phosphorylated counterparts were ineffective. Tyrosine...

  17. SHP-1 phosphatase activity counteracts increased T cell receptor affinity.

    Science.gov (United States)

    Hebeisen, Michael; Baitsch, Lukas; Presotto, Danilo; Baumgaertner, Petra; Romero, Pedro; Michielin, Olivier; Speiser, Daniel E; Rufer, Nathalie

    2013-03-01

    Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K(d) affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity-associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K(d) = 5 to 1 μM), those with supraphysiological affinity (K(d) = 1 μM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8(+) T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity-dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8(+) T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell-mediated immunity.

  18. Prolactinoma ErbB receptor expression and targeted therapy for aggressive tumors.

    Science.gov (United States)

    Cooper, Odelia; Mamelak, Adam; Bannykh, Serguei; Carmichael, John; Bonert, Vivien; Lim, Stephen; Cook-Wiens, Galen; Ben-Shlomo, Anat

    2014-06-01

    As ErbB signaling is a determinant of prolactin synthesis, role of ErbB receptors was tested for prolactinoma outcomes and therapy. The objective of this study was to characterize ErbB receptor expression in prolactinomas and then perform a pilot study treating resistant prolactinomas with a targeted tyrosine kinase inhibitor (TKI). Retrospective analysis of prolactinomas and pilot study for dopamine agonist resistant prolactinomas in tertiary referral center. We performed immunofluorescent staining of a tissue array of 29 resected prolactinoma tissues for EGFR, ErbB2, ErbB3, and ErbB4 correlated with clinical features. Two patients with aggressive resistant prolactinomas enrolled and completed trial. They received lapatinib 1,250 mg daily for 6 months with tumor and hormone assessments. Main outcome measures were positive tumor staining of respective ErbB receptors, therapeutic reduction of prolactin levels and tumor shrinkage. Treated PRL levels and tumor volumes were suppressed in both subjects treated with TKI. EGFR expression was positive in 82 % of adenomas, ErbB2 in 92 %, ErbB3 in 25 %, and ErbB4 in 71 %, with ErbB2 score > EGFR > ErbB4 > ErbB3. Higher ErbB3 expression was associated with optic chiasm compression (p = 0.03), suprasellar extension (p = 0.04), and carotid artery encasement (p = 0.01). Higher DA response rates were observed in tumors with higher ErbB3 expression. Prolactinoma expression of specific ErbB receptors is associated with tumor invasion, symptoms, and response to dopamine agonists. Targeting ErbB receptors may be effective therapy in patients with resistant prolactinomas.

  19. Peroxisome Proliferator-Activated Receptor α Activation Induces Hepatic Steatosis, Suggesting an Adverse Effect

    OpenAIRE

    Fang Yan; Qi Wang; Chao Xu; Mingfeng Cao; Xiaoming Zhou; Tingting Wang; Chunxiao Yu; Fei Jing; Wenbin Chen; Ling Gao; Jiajun Zhao

    2014-01-01

    Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic triglyceride accumulation, ranging from steatosis to steatohepatitis and cirrhosis. NAFLD is a risk factor for cardiovascular diseases and is associated with metabolic syndrome. Antihyperlipidemic drugs are recommended as part of the treatment for NAFLD patients. Although fibrates activate peroxisome proliferator-activated receptor α (PPARα), leading to the reduction of serum triglyceride levels, the effects of these drugs ...

  20. Activation of liver X receptors with T0901317 attenuates cardiac hypertrophy in vivo

    NARCIS (Netherlands)

    Kuipers, Irma; Li, Jiang; Vreeswijk-Baudoin, Inge; Koster, Johan; van der Harst, Pim; Sillje, Herman H. W.; Kuipers, Folkert; van Veldhuisen, Dirk J.; van Gilst, Wiek H.; de Boer, Rudolf A.

    2010-01-01

    Liver X receptor (LXR) is a nuclear receptor regulating cholesterol metabolism. Liver X receptor has also been shown to exert anti-proliferative and anti-inflammatory properties. In this study, we evaluated the effect of LXR activation on cardiac hypertrophy in vitro and in vivo. Treatment with the

  1. Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Benned-Jensen, Tau; Holst, Peter J

    2006-01-01

    Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor-mediate...

  2. Developmental stability of taurine's activation on glycine receptors in cultured neurons of rat auditory cortex.

    Science.gov (United States)

    Tang, Zheng-Quan; Lu, Yun-Gang; Chen, Lin

    2008-01-03

    Taurine is an endogenous amino acid that can activate glycine and/or gamma-aminobutyric acid type A (GABA(A)) receptors in the central nervous system. During natural development, taurine's receptor target undergoes a shift from glycine receptors to GABA(A) receptors in cortical neurons. Here, we demonstrate that taurine's receptor target in cortical neurons remains stable during in vitro development. With whole-cell patch-clamp recordings, we found that taurine always activated glycine receptors, rather than GABA(A) receptors, in neurons of rat auditory cortex cultured for 5-22 days. Our results suggest that the functional sensitivity of glycine and GABA(A) receptors to taurine is critically regulated by their developmental environments.

  3. Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2: Another Target for Thrombin Signaling.

    Science.gov (United States)

    Mihara, Koichiro; Ramachandran, Rithwik; Saifeddine, Mahmoud; Hansen, Kristina K; Renaux, Bernard; Polley, Danny; Gibson, Stacy; Vanderboor, Christina; Hollenberg, Morley D

    2016-05-01

    Thrombin is known to signal to cells by cleaving/activating a G-protein-coupled family of proteinase-activated receptors (PARs). The signaling mechanism involves the proteolytic unmasking of an N-terminal receptor sequence that acts as a tethered receptor-activating ligand. To date, the recognized targets of thrombin cleavage and activation for signaling are PAR1 and PAR4, in which thrombin cleaves at a conserved target arginine to reveal a tethered ligand. PAR2, which like PAR1 is also cleaved at an N-terminal arginine to unmask its tethered ligand, is generally regarded as a target for trypsin but not for thrombin signaling. We now show that thrombin, at concentrations that can be achieved at sites of acute injury or in a tumor microenvironment, can directly activate PAR2 vasorelaxation and signaling, stimulating calcium and mitogen-activated protein kinase responses along with triggeringβ-arrestin recruitment. Thus, PAR2 can be added alongside PAR1 and PAR4 to the targets, whereby thrombin can affect tissue function.

  4. Family C 7TM receptor dimerization and activation

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Sheikh, Søren P; Hansen, Jakob Lerche

    2006-01-01

    The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug-tar...

  5. Upregulation of epidermal growth factor receptor 4 in ora leukoplakia

    Institute of Scientific and Technical Information of China (English)

    Hiroshi Kobayashi; Kenichi Kumagai; Akito Gotoh; Takanori Eguchi; Hiroyuki Yamada; Yoshiki Hamada; Satsuki Suzuki; Ryuji Suzuki

    2013-01-01

    In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ ErbB 1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP), The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP.

  6. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta

    DEFF Research Database (Denmark)

    Yan, Zhen Cheng; Liu, Dao Yan; Zhang, Li Li

    2007-01-01

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow...... or a high-fat diet without and with regular endurance exercise. Exercise in rats on high-fat diet significantly reduced visceral fat mass, blood pressure, and adipocyte size (each p......Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow...

  7. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    Energy Technology Data Exchange (ETDEWEB)

    Yliniemelae, A.; Gynther, J. (Univ. of Kuopio (Finland)); Konschin, H.; Tylli, H. (Univ. of Helsinki (Finland)); Rouvinen, J. (Univ. of Joensuu (Finland))

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  8. Mu-opioid receptor knockout mice show diminished food-anticipatory activity

    NARCIS (Netherlands)

    Kas, Martien J H; van den Bos, Ruud; Baars, Annemarie M; Lubbers, Marianne; Lesscher, Heidi M B; Hillebrand, Jacquelien J G; Schuller, Alwin G; Pintar, John E; Spruijt, Berry M

    2004-01-01

    We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, mu-opioid receptors are activated. To test this hypothesis schedule induced food-anticipatory activity in mu-opioid receptor knockout mice was measured using running wheels. We hypothesized

  9. Repressive effects of resveratrol on androgen receptor transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Wen-feng Shi

    Full Text Available BACKGROUND: The chemopreventive effects of resveratrol (RSV on prostate cancer have been well established; the androgen receptor (AR plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity. METHODOLOGY: The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+ cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(- cells serving as controls. AR(+ cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE. RESULTS: AR in the AR (+ stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment. CONCLUSION: We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

  10. Estrogen receptor alpha binds to peroxisome proliferator-activated receptor response element and negatively interferes with peroxisome proliferator-activated receptor gamma signaling in breast cancer cells.

    Science.gov (United States)

    Bonofiglio, Daniela; Gabriele, Sabrina; Aquila, Saveria; Catalano, Stefania; Gentile, Mariaelena; Middea, Emilia; Giordano, Francesca; Andò, Sebastiano

    2005-09-01

    The molecular mechanisms involved in the repressive effects exerted by estrogen receptors (ER) on peroxisome proliferator-activated receptor (PPAR) gamma-mediated transcriptional activity remain to be elucidated. The aim of the present study was to provide new insight into the crosstalk between ERalpha and PPARgamma pathways in breast cancer cells. Using MCF7 and HeLa cells as model systems, we did transient transfections and electrophoretic mobility shift assay and chromatin immunoprecipitation studies to evaluate the ability of ERalpha to influence PPAR response element-mediated transcription. A possible direct interaction between ERalpha and PPARgamma was ascertained by co-immunoprecipitation assay, whereas their modulatory role in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway was evaluated by determining PI3K activity and AKT phosphorylation. As a biological counterpart, we investigated the growth response to the cognate ligands of both receptors in hormone-dependent MCF7 breast cancer cells. Our data show for the first time that ERalpha binds to PPAR response element and represses its transactivation. Moreover, we have documented the physical and functional interactions of ERalpha and PPARgamma, which also involve the p85 regulatory subunit of PI3K. Interestingly, ERalpha and PPARgamma pathways have an opposite effect on the regulation of the PI3K/AKT transduction cascade, explaining, at least in part, the divergent response exerted by the cognate ligands 17beta-estradiol and BRL49653 on MCF7 cell proliferation. ERalpha physically associates with PPARgamma and functionally interferes with PPARgamma signaling. This crosstalk could be taken into account in setting new pharmacologic strategies for breast cancer disease.

  11. Recovery of network-driven glutamatergic activity in rat hippocampal neurons during chronic glutamate receptor blockade.

    Science.gov (United States)

    Leininger, Eric; Belousov, Andrei B

    2009-01-28

    Previous studies indicated that a long-term decrease in the activity of ionotropic glutamate receptors induces cholinergic activity in rat and mouse hypothalamic neuronal cultures. Here we studied whether a prolonged inactivation of ionotropic glutamate receptors also induces cholinergic activity in hippocampal neurons. Receptor activity was chronically suppressed in rat hippocampal primary neuronal cultures with two proportionally increasing sets of concentrations of NMDA plus non-NMDA receptor antagonists: 100 microM/10 microM AP5/CNQX (1X cultures) and 200 microM/20 microM AP5/CNQX (2X cultures). Using calcium imaging we demonstrate that cholinergic activity does not develop in these cultures. Instead, network-driven glutamate-dependent activity, that normally is detected in hyper-excitable conditions, reappears in each culture group in the presence of these antagonists and can be reversibly suppressed by higher concentrations of AP5/CNQX. This activity is mediated by non-NMDA receptors and is modulated by NMDA receptors. Further, non-NMDA receptors, the general level of glutamate receptor activity and CaMK-dependent signaling are critical for development of this network-driven glutamatergic activity in the presence of receptor antagonists. Using electrophysiology, western blotting and calcium imaging we show that some neuronal parameters are either reduced or not affected by chronic glutamate receptor blockade. However, other parameters (including neuronal excitability, mEPSC frequency, and expression of GluR1, NR1 and betaCaMKII) become up-regulated and, in some cases, proportionally between the non-treated, 1X and 2X cultures. Our data suggest recovery of the network-driven glutamatergic activity after chronic glutamate receptor blockade. This recovery may represent a form of neuronal plasticity that compensates for the prolonged suppression of the activity of glutamate receptors.

  12. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  13. Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors.

    Science.gov (United States)

    Semerdjieva, Sophia; Abdul-Razak, Hayder H; Salim, Sharifah S; Yáñez-Muñoz, Rafael J; Chen, Philip E; Tarabykin, Victor; Alifragis, Pavlos

    2013-04-01

    Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

  14. Immunomodulator CD200 promotes neurotrophic activity by interacting with and activating the fibroblast growth factor receptor

    DEFF Research Database (Denmark)

    Pankratova, Stanislava; Bjornsdottir, Halla; Christensen, Claus;

    2016-01-01

    in the suppression of microglia activation. We for the first time demonstrated that CD200 can interact with and transduce signaling through activation of the fibroblast growth factor receptor (FGFR), thereby inducing neuritogenesis and promoting neuronal survival in primary neurons. CD200-induced FGFR...... phosphorylation was abrogated by CD200R, whereas FGF2-induced FGFR activation was inhibited by CD200. We also identified a sequence motif located in the first Ig-like module of CD200, likely representing the minimal CD200 binding site for FGFR. The FGFR binding motif overlaps with the CD200R binding site......, suggesting that they can compete for CD200 binding in cells that express both receptors. We propose that CD200 in neurons functions as a ligand of FGFR....

  15. Activation of transient receptor potential ankyrin 1 by eugenol.

    Science.gov (United States)

    Chung, G; Im, S T; Kim, Y H; Jung, S J; Rhyu, M-R; Oh, S B

    2014-03-07

    Eugenol is a bioactive plant extract used as an analgesic agent in dentistry. The structural similarity of eugenol to cinnamaldehyde, an active ligand for transient receptor potential ankyrin 1 (TRPA1), suggests that eugenol might produce its effect via TRPA1, in addition to TRPV1 as we reported previously. In this study, we investigated the effect of eugenol on TRPA1, by fura-2-based calcium imaging and patch clamp recording in trigeminal ganglion neurons and in a heterologous expression system. As the result, eugenol induced robust calcium responses in rat trigeminal ganglion neurons that responded to a specific TRPA1 agonist, allyl isothiocyanate (AITC), and not to capsaicin. Capsazepine, a TRPV1 antagonist failed to inhibit eugenol-induced calcium responses in AITC-responding neurons. In addition, eugenol response was observed in trigeminal ganglion neurons from TRPV1 knockout mice and human embryonic kidney 293 cell lines that express human TRPA1, which was inhibited by TRPA1-specific antagonist HC-030031. Eugenol-evoked TRPA1 single channel activity and eugenol-induced TRPA1 currents were dose-dependent with EC50 of 261.5μM. In summary, these results demonstrate that the activation of TRPA1 might account for another molecular mechanism underlying the pharmacological action of eugenol.

  16. Significance of AT1 receptor independent activation of mineralocorticoid receptor in murine diabetic cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Yuji Nagatomo

    Full Text Available BACKGROUND: Diabetes mellitus (DM has deleterious influence on cardiac performance independent of coronary artery disease and hypertension. The objective of the present study was to investigate the role of the renin-angiotensin-aldosterone system, especially angiotensin II type 1a receptor (AT1aR and mineralocorticoid receptor (MR signaling, in left ventricular (LV dysfunction induced by diabetes mellitus (DM. METHODS AND RESULTS: DM was induced by intraperitoneal injection of streptozotocin (200 mg/kg BW in wild-type (WT or AT1aR knockout (KO male mice, and they were bred during 6 or 12 weeks. Some KO mice were administered the MR antagonist eplerenone (100 mg/kg body weight. At 6 weeks, LV diastolic function was impaired in WT-DM, but preserved in KO-DM. At that time point MR mRNA expression was upregulated, NADPH oxidase subunit (p47phox and glutathione peroxidase (GPx1 mRNA expression were upregulated, the staining intensities of LV tissue for 4-hydroxy-2-nonenal was stronger in immunohistochemistry, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL positive cells was increased, Bcl-2 protein expression was significantly downregulated, and the expression of SERCA2a and phosphorylated phospholamban was depressed in WT-DM, while these changes were not seen in KO-DM. At 12 weeks, however, these changes were also noted in KO-DM. Eplerenone arrested those changes. The plasma aldosterone concentration was elevated in WT-DM but not in KO-DM at 6 weeks. It showed 3.7-fold elevation at 12 weeks even in KO-DM, which suggests "aldosterone breakthrough" phenomenon. However, the aldosterone content in LV tissue was unchanged in KO-DM. CONCLUSIONS: DM induced diastolic dysfunction was observed even in KO at 12 weeks, which was ameliorated by minelarocorticoid receptor antagonist, eplerenone. AT1-independent MR activation in the LV might be responsible for the pathogenesis of diabetic cardiomyopathy.

  17. Dihydropyridine receptors actively control gating of ryanodine receptors in resting mouse skeletal muscle fibres

    Science.gov (United States)

    Robin, Gaëlle; Allard, Bruno

    2012-01-01

    Contraction of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) in response to depolarization of the muscle membrane. Depolarization is known to elicit a conformational change of the dihydropyridine receptor (DHPR) in the tubular membrane that controls in a time- and voltage-dependent manner the opening of the ryanodine receptor (RyR), the SR Ca2+ release channel. At rest, it is assumed that RyRs are kept in a closed state imposed by the repressive action of DHPRs; however, a direct control of the RyR gating by the DHPR has up to now never been demonstrated in resting adult muscle. In this study, we monitored slow changes in SR Ca2+ content using the Ca2+ indicator fluo-5N loaded in the SR of voltage-clamped mouse muscle fibres. We first show that external Ca2+ removal induced a reversible SR Ca2+ efflux at −80 mV and prevented SR Ca2+ refilling following depolarization-evoked SR Ca2+ depletion. The dihydropyridine compound nifedipine induced similar effects. The rate of SR Ca2+ efflux was also shown to be controlled in a time- and voltage-dependent manner within a membrane potential range more negative than −50 mV. Finally, intracellular addition of ryanodine produced an irreversible SR Ca2+ efflux and kept the SR in a highly depleted state following depolarization-evoked SR Ca2+ depletion. The fact that resting SR Ca2+ efflux is modulated by conformational changes of DHPRs induced by external Ca2+, nifedipine and voltage demonstrates that DHPRs exert an active control on gating of RyRs in resting skeletal muscle. PMID:23006480

  18. HIV-1 activates macrophages independent of Toll-like receptors.

    Directory of Open Access Journals (Sweden)

    Joseph N Brown

    Full Text Available BACKGROUND: Macrophages provide an interface between innate and adaptive immunity and are important long-lived reservoirs for Human Immunodeficiency Virus Type-1 (HIV-1. Multiple genetic networks involved in regulating signal transduction cascades and immune responses in macrophages are coordinately modulated by HIV-1 infection. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate complex interrelated processes and to assemble an integrated view of activated signaling networks, a systems biology strategy was applied to genomic and proteomic responses by primary human macrophages over the course of HIV-1 infection. Macrophage responses, including cell cycle, calcium, apoptosis, mitogen-activated protein kinases (MAPK, and cytokines/chemokines, to HIV-1 were temporally regulated, in the absence of cell proliferation. In contrast, Toll-like receptor (TLR pathways remained unaltered by HIV-1, although TLRs 3, 4, 7, and 8 were expressed and responded to ligand stimulation in macrophages. HIV-1 failed to activate phosphorylation of IRAK-1 or IRF-3, modulate intracellular protein levels of Mx1, an interferon-stimulated gene, or stimulate secretion of TNF, IL-1beta, or IL-6. Activation of pathways other than TLR was inadequate to stimulate, via cross-talk mechanisms through molecular hubs, the production of proinflammatory cytokines typical of a TLR response. HIV-1 sensitized macrophage responses to TLR ligands, and the magnitude of viral priming was related to virus replication. CONCLUSIONS/SIGNIFICANCE: HIV-1 induced a primed, proinflammatory state, M1(HIV, which increased the responsiveness of macrophages to TLR ligands. HIV-1 might passively evade pattern recognition, actively inhibit or suppress recognition and signaling, or require dynamic interactions between macrophages and other cells, such as lymphocytes or endothelial cells. HIV-1 evasion of TLR recognition and simultaneous priming of macrophages may represent a strategy for viral survival, contribute

  19. Oleamide activates peroxisome proliferator-activated receptor gamma (PPARγ in vitro

    Directory of Open Access Journals (Sweden)

    Dionisi Mauro

    2012-05-01

    Full Text Available Abstract Background Oleamide (ODA is a fatty acid primary amide first identified in the cerebrospinal fluid of sleep-deprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. It has recently been reported to exert a hypolipidemic effect in hamsters. Here, we have investigated the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs as potential targets for ODA action. Results Activation of PPARα, PPARβ and PPARγ was assessed using recombinant expression in Chinese hamster ovary cells with a luciferase reporter gene assay. Direct binding of ODA to the ligand binding domain of each of the three PPARs was monitored in a cell-free fluorescent ligand competition assay. A well-established assay of PPARγ activity, the differentiation of 3T3-L1 murine fibroblasts into adipocytes, was assessed using an Oil Red O uptake-based assay. ODA, at 10 and 50 μM, was able to transactivate PPARα, PPARβ and PPARγ receptors. ODA bound to the ligand binding domain of all three PPARs, although complete displacement of fluorescent ligand was only evident for PPARγ, at which an IC50 value of 38 μM was estimated. In 3T3-L1 cells, ODA, at 10 and 20 μM, induced adipogenesis. Conclusions We have, therefore, identified a novel site of action of ODA through PPAR nuclear receptors and shown how ODA should be considered as a weak PPARγ ligand in vitro.

  20. Molecular Mechanism of Peroxisome Proliferator-Activated Receptor alpha Activation by WY14643: a New Mode of Ligand Recognition and Receptor Stabilization

    NARCIS (Netherlands)

    Bernardes, Amanda; Telles de Souza, Paulo C; Muniz, João R C; Ricci, Clarisse G; Ayers, Stephen D; Parekh, Nili M; Godoy, André S; Trivella, Daniela B B; Reinach, Peter; Webb, Paul; Skaf, Munir S; Polikarpov, Igor

    2013-01-01

    Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPAR alpha ligands effectively treat dyslipidemia and have significant

  1. Adipocyte insulin receptor activity maintains adipose tissue mass and lifespan.

    Science.gov (United States)

    Friesen, Max; Hudak, Carolyn S; Warren, Curtis R; Xia, Fang; Cowan, Chad A

    2016-08-05

    Type 2 diabetes follows a well-defined progressive pathogenesis, beginning with insulin resistance in metabolic tissues such as the adipose. Intracellular signaling downstream of insulin receptor activation regulates critical metabolic functions of adipose tissue, including glucose uptake, lipogenesis, lipolysis and adipokine secretion. Previous studies have used the aP2 promoter to drive Cre recombinase expression in adipose tissue. Insulin receptor (IR) knockout mice created using this aP2-Cre strategy (FIRKO mice) were protected from obesity and glucose intolerance. Later studies demonstrated the promiscuity of the aP2 promoter, casting doubts upon the tissue specificity of aP2-Cre models. It is our goal to use the increased precision of the Adipoq promoter to investigate adipocyte-specific IR function. Towards this end we generated an adipocyte-specific IR knockout (AIRKO) mouse using an Adipoq-driven Cre recombinase. Here we report AIRKO mice are less insulin sensitive throughout life, and less glucose tolerant than wild-type (WT) littermates at the age of 16 weeks. In contrast to WT littermates, the insulin sensitivity of AIRKO mice is unaffected by age or dietary regimen. At any age, AIRKO mice are comparably insulin resistant to old or obese WT mice and have a significantly reduced lifespan. Similar results were obtained when these phenotypes were re-examined in FIRKO mice. We also found that the AIRKO mouse is protected from high-fat diet-induced weight gain, corresponding with a 90% reduction in tissue weight of major adipose depots compared to WT littermates. Adipose tissue mass reduction is accompanied by hepatomegaly and increased hepatic steatosis. These data indicate that adipocyte IR function is crucial to systemic energy metabolism and has profound effects on adiposity, hepatic homeostasis and lifespan. Copyright © 2016. Published by Elsevier Inc.

  2. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    Science.gov (United States)

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms.

  3. Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha.

    Science.gov (United States)

    Yueh, Mei-Fei; Li, Tao; Evans, Ronald M; Hammock, Bruce; Tukey, Robert H

    2012-01-01

    Triclocarban (3,4,4'-trichlorocarbanilide, TCC) is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs). To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR) and estrogen receptor alpha (ERα) activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR) because no induction occurred in hUGT1Car(-/-) mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for potential human

  4. GabaB receptors activation in the NTS blocks the glycemic responses induced by carotid body receptor stimulation.

    Science.gov (United States)

    Lemus, Mónica; Montero, Sergio; Cadenas, José Luis; Lara, José Jesús; Tejeda-Chávez, Héctor Rafael; Alvarez-Buylla, Ramón; de Alvarez-Buylla, Elena Roces

    2008-08-18

    The carotid body receptors participate in glucose regulation sensing glucose levels in blood entering the cephalic circulation. The carotid body receptors information, is initially processed within the nucleus tractus solitarius (NTS) and elicits changes in circulating glucose and brain glucose uptake. Previous work has shown that gamma-aminobutyric acid (GABA) in NTS modulates respiratory reflexes, but the role of GABA within NTS in glucose regulation remains unknown. Here we show that GABA(B) receptor agonist (baclofen) or antagonists (phaclofen and CGP55845A) locally injected into NTS modified arterial glucose levels and brain glucose retention. Control injections outside NTS did not elicit these responses. In contrast, GABA(A) agonist and antagonist (muscimol or bicuculline) produced no significant changes in blood glucose levels. When these GABAergic drugs were applied before carotid body receptors stimulation, again, only GABA(B) agonist or antagonist significantly affected glycemic responses; baclofen microinjection significantly reduced the hyperglycemic response and brain glucose retention observed after carotid body receptors stimulation, while phaclofen produced the opposite effect, increasing significantly hyperglycemia and brain glucose retention. These results indicate that activation of GABA(B), but not GABA(A), receptors in the NTS modulates the glycemic responses after anoxic stimulation of the carotid body receptors, and suggest the presence of a tonic inhibitory mechanism in the NTS to avoid hyperglycemia.

  5. Biased signaling by peptide agonists of protease activated receptor 2.

    Science.gov (United States)

    Jiang, Yuhong; Yau, Mei-Kwan; Kok, W Mei; Lim, Junxian; Wu, Kai-Chen; Liu, Ligong; Hill, Timothy A; Suen, Jacky Y; Fairlie, David P

    2017-02-07

    Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 μM) but not ERK1/2 phosphorylation (EC50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

  6. Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity

    DEFF Research Database (Denmark)

    Holst, P J; Rosenkilde, M M; Manfra, D;

    2001-01-01

    ORF74 (or KSHV-vGPCR) is a highly constitutively active G protein-coupled receptor encoded by HHV8 that is regulated both positively and negatively by endogenous chemokines. When expressed in transgenic mice, this chemokine receptor induces an angioproliferative disease closely resembling Kaposi...... sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does...

  7. Biology and therapeutic applications of peroxisome proliferator- activated receptors.

    Science.gov (United States)

    Menendez-Gutierrez, Maria P; Roszer, Tamas; Ricote, Mercedes

    2012-01-01

    Peroxisome proliferator-activated receptors (PPARs) are ligand dependent transcription factors. The three mammalian PPARs are key regulators of fatty acid and lipoprotein metabolism, glucose homeostasis, cellular proliferation/ differentiation and the immune response. PPARs are therefore important targets in the treatment of metabolic disorders such as insulin resistance and type 2 diabetes mellitus, and are also of interest in relation to chronic inflammatory diseases such as atherosclerosis, arthritis, chronic pulmonary inflammation, pancreatitis, inflammatory bowel disease, and psoriasis. Recent advances have attributed novel functions to PPARs in blood pressure regulation, neuroinflammation, nerve-cell protection, inflammatory pain reduction, and the hypothalamic control of metabolism. The abundant pleiotropic actions of PPARs suggest that PPAR agonists have enormous therapeutic potential. However, current PPAR-based therapies often have undesired side effects due to the concomitant activation of PPARs in non-target cells. There is therefore growing interest in the development of cell-specific PPAR agonists and improvement of the clinical use of PPAR ligands. This review gives an overview of PPAR functions and discusses the current and potential medical implications of PPAR ligands in various pathologies, ranging from metabolic disorders to cardiovascular disease, chronic inflammation, neurodegenerative disorders and cancer.

  8. Peroxisome proliferator-activated receptors, metabolic syndrome and cardiovascular disease

    Science.gov (United States)

    Azhar, Salman

    2011-01-01

    Metabolic syndrome (MetS) is a constellation of risk factors including insulin resistance, central obesity, dyslipidemia and hypertension that markedly increase the risk of Type 2 diabetes (T2DM) and cardiovascular disease (CVD). The peroxisome proliferators-activated receptor (PPAR) isotypes, PPARα, PPARδ/β and PPARγ are ligand-activated nuclear transcription factors, which modulate the expression of an array of genes that play a central role in regulating glucose, lipid and cholesterol metabolism, where imbalance can lead to obesity, T2DM and CVD. They are also drug targets, and currently, PPARα (fibrates) and PPARγ (thiazolodinediones) agonists are in clinical use for treating dyslipidemia and T2DM, respectively. These metabolic characteristics of the PPARs, coupled with their involvement in metabolic diseases, mean extensive efforts are underway worldwide to develop new and efficacious PPAR-based therapies for the treatment of additional maladies associated with the MetS. This article presents an overview of the functional characteristics of three PPAR isotypes, discusses recent advances in our understanding of the diverse biological actions of PPARs, particularly in the vascular system, and summarizes the developmental status of new single, dual, pan (multiple) and partial PPAR agonists for the clinical management of key components of MetS, T2DM and CVD. It also summarizes the clinical outcomes from various clinical trials aimed at evaluating the atheroprotective actions of currently used fibrates and thiazolodinediones. PMID:20932114

  9. Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation

    OpenAIRE

    2016-01-01

    Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire coul...

  10. Activation of Cannabinoid Receptor 2 Enhances Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yong-Xin Sun

    2015-01-01

    Full Text Available Bone marrow derived mesenchymal stem cells (BM-MSCs are considered as the most promising cells source for bone engineering. Cannabinoid (CB receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2 receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA was used to study the role of CB2 receptor in osteogenic differentiation. Results showed activation of CB2 receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2 receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2 receptor. Finally, bone marrow samples demonstrated that expression of CB2 receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2 receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2 receptor may be related to osteoporosis.

  11. NMDA receptor activation regulates sociability by its effect on mTOR signaling activity

    Science.gov (United States)

    Burket, Jessica A.; Benson, Andrew D.; Tang, Amy H.; Deutsch, Stephen I.

    2017-01-01

    Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORCl in neurons (e.g., cerebellar Purkinje cells). mTORCl is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORCl, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORCl overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORCl activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are “drivers” of mTORCl activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders. PMID:25703582

  12. NMDA receptor activation regulates sociability by its effect on mTOR signaling activity.

    Science.gov (United States)

    Burket, Jessica A; Benson, Andrew D; Tang, Amy H; Deutsch, Stephen I

    2015-07-01

    Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORC1 in neurons (e.g., cerebellar Purkinje cells). mTORC1 is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORC1, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORC1 overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORC1 activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are "drivers" of mTORC1 activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders.

  13. [5-HT1A/5-HT7 receptor interplay: Chronic activation of 5-HT7 receptors decreases the functional activity of 5-HT1A receptor and its сontent in the mouse brain].

    Science.gov (United States)

    Kondaurova, E M; Bazovkina, D V; Naumenko, V S

    2017-01-01

    Serotonin receptors 5-HT1A and 5-HT7 are involved in the development of various psychopathologies. Some data indicate that there is an interplay between 5-HT1A 5-HT7 receptors that could be implicated in the regulation of their function. This work analyzed the effects of chronic 5-HT7 activation on the functional activity of 5-HT7 and 5-HT1A receptors, on the corresponding protein levels, and on the expression of genes encoding 5-HT7 and 5-HT1A receptors in the mouse brain. Chronic administration of the 5-HT7 selective agonist LP44 (20.5 nmol, i.c.v., 14 days) produced considerable desensitization of both 5-HT7 and 5-HT1A receptors. In LP44-treated mice, the hypothermic responses mediated by both 5-HT7 and 5-HT1A receptors were attenuated. Moreover, the levels of 5-HT1A receptor protein in the midbrain and the frontal cortex of LP44-treated mice were significantly decreased. However, the brain levels of 5-HT7 receptor protein did not differ between LP44-treated and control mice. Chronic LP44 treatment did not alter the expression of the 5-HT7 and 5-HT1A receptor genes in all investigated brain structure. These data suggest that 5-HT7 receptors participate in the posttranscriptional regulation of the 5-HT1A receptors functioning.

  14. Study of Drug-Protein Covalent Interactions by Mass Spectrometry. A case study: Epidermal Growth Factor Receptor

    OpenAIRE

    Moretti, Elisa

    2011-01-01

    Study of Drug-Protein Covalent Interactions by Mass Spectrometry A case study: Epidermal Growth Factor Receptor Abstract The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein belonging to the human EGFR family, which is in turn composed of four members: EGFR (ErbB1), ErbB2, ErbB3 and ErbB4. It is characterized by the presence of an extracellular ligand-binding domain, a transmembrane region, and a cytoplasmatic domain that is endowed with a tyrosine kinase (TK)...

  15. Common structural basis for constitutive activity of the ghrelin receptor family

    DEFF Research Database (Denmark)

    Holst, Birgitte; Holliday, Nicholas D; Bach, Anders

    2004-01-01

    Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand-independent sig......, the structural basis for which is determined by an aromatic cluster on the inner face of the extracellular ends of TMs VI and VII.......-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G......(q), phospholipase C pathway was approximately 50% of their maximal capacity as determined through inositol phosphate accumulation. These two receptors also showed very high constitutive activity in activation of cAMP response element-driven transcription. GPR39 displayed a clear but lower degree of constitutive...

  16. Stereostructure-activity studies on agonists at the AMPA and kainate subtypes of ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Johansen, Tommy N; Greenwood, Jeremy R; Frydenvang, Karla Andrea

    2003-01-01

    -methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of ionotropic Glu receptors in the presence or absence of an agonist has provided important information about ligand-receptor interaction mechanisms. The availability of these binding domain crystal structures has formed the basis for rational...... design of ligands, especially for the AMPA and kainate subtypes of ionotropic Glu receptors. This mini-review will focus on structure-activity relationships on AMPA and kainate receptor agonists with special emphasis on stereochemical and three-dimensional aspects....

  17. Plasma soluble urokinase plasminogen activator receptor in children with urinary tract infection

    DEFF Research Database (Denmark)

    Wittenhagen, Per; Andersen, Jesper Brandt; Hansen, Anita

    2011-01-01

    In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection.......In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection....

  18. Activation of toll-like receptors and dendritic cells by a broad range of bacterial molecules

    NARCIS (Netherlands)

    Boele, L.C.L.; Bajramovic, J.J.; Vries, A.M.M.B.C. de; Voskamp-Visser, I.A.I.; Kaman, W.E.; Kleij, D. van der

    2009-01-01

    Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, the

  19. Peroxisome proliferators-activated receptor (PPAR) regulation in cardiac metabolism and disease

    NARCIS (Netherlands)

    el Azzouzi, H.|info:eu-repo/dai/nl/304072796

    2009-01-01

    Peroxisome proliferators-activated receptors (PPARs) are members of the nuclear receptor family of ligand activated transcription factors and consist of the three isoforms, PPAR, PPAR/ and PPAR. Considerable evidence has established the importance of PPARs in myocardial lipid homeostasis and

  20. Activation of toll-like receptors and dendritic cells by a broad range of bacterial molecules

    NARCIS (Netherlands)

    Boele, L.C.L.; Bajramovic, J.J.; Vries, A.M.M.B.C. de; Voskamp-Visser, I.A.I.; Kaman, W.E.; Kleij, D. van der

    2009-01-01

    Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, the

  1. Peroxisome proliferators-activated receptor (PPAR) regulation in cardiac metabolism and disease

    NARCIS (Netherlands)

    el Azzouzi, H.

    2009-01-01

    Peroxisome proliferators-activated receptors (PPARs) are members of the nuclear receptor family of ligand activated transcription factors and consist of the three isoforms, PPAR, PPAR/ and PPAR. Considerable evidence has established the importance of PPARs in myocardial lipid homeostasis and car

  2. 5-Fluorouracil combined with apigenin enhances anticancer activity through induction of apoptosis in human breast cancer MDA-MB-453 cells.

    Science.gov (United States)

    Choi, Eun J; Kim, Gun-Hee

    2009-12-01

    We investigated the effects of combined treatment with 5-fluorouracil and apigenin on proliferation and apoptosis, as well as the underlying mechanism, in human breast cancer MDA-MB-453 cells. The MDA-MB-453 cells, which have been shown to overexpress ErbB2, were resistant to 5-fluorouracil; 5-fluorouracil exhibited a small dose-dependent anti-proliferative effect, with an IC50 of 90 microM. Interestingly, combined treatment with apigenin significantly decreased the resistance. Cellular proliferation was significantly inhibited in cells exposed to 5-fluorouracil at its IC50 and apigenin (5, 10, 50 and 100 microM), compared with proliferation in cells exposed to 5-fluorouracil alone. This inhibition in turn led to apoptosis, as evidenced by an increased number of apoptotic cells and the activation of caspase-3. To investigate the mechanism by which the combination of 5-fluorouracil and apigenin induces apoptosis, ErbB2 expression was analyzed. The level of ErbB2 was unchanged by 5-fluorouracil alone but was drastically reduced in cells treated with 5-fluorouracil plus apigenin. Moreover, compared with 5-fluorouracil alone, 5-fluorouracil in combination with apigenin at concentrations >10 microM exerted a pro-apoptotic effect via the inhibition of Akt expression. Taken together, our results suggest that 5-fluorouracil acts synergistically with apigenin inhibiting cell growth and inducing apoptosis via the down-regulation of ErbB2 expression and Akt signaling.

  3. Agonist-biased signaling via proteinase activated receptor-2: differential activation of calcium and mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Ramachandran, Rithwik; Mihara, Koichiro; Mathur, Maneesh; Rochdi, Moulay Driss; Bouvier, Michel; Defea, Kathryn; Hollenberg, Morley D

    2009-10-01

    We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR(2)) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR(2) and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) kinase. The TL receptor mutants, rPAR(2)-Leu(37)Ser(38), rPAR(2)-Ala(37-38), and rPAR(2)-Ala(39-42) were compared with the trypsin-revealed wild-type rPAR(2) TL sequence, S(37)LIGRL(42)-. Upon trypsin activation, all constructs stimulated MAP kinase signaling, but only the wt-rPAR(2) and rPAR(2)-Ala(39-42) triggered calcium signaling. Furthermore, the TL-derived synthetic peptide SLAAAA-NH2 failed to cause PAR(2)-mediated calcium signaling but did activate MAP kinase, whereas SLIGRL-NH2 triggered both calcium and MAP kinase signaling by all receptors. The peptides AAIGRL-NH2 and LSIGRL-NH2 triggered neither calcium nor MAP kinase signals. Neither rPAR(2)-Ala(37-38) nor rPAR(2)-Leu(37)Ser(38) constructs recruited beta-arrestins-1 or -2 in response to trypsin stimulation, whereas both beta-arrestins were recruited to these mutants by SLIGRL-NH2. The lack of trypsin-triggered beta-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsin-stimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of Galpha(i) (pertussis toxin), Galpha(q) [N-cyclohexyl-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide (GP2A)], Src kinase [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], or the epidermal growth factor (EGF) receptor [4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27362). The data indicate that the

  4. Regulatory network of inflammation downstream of proteinase-activated receptors

    Directory of Open Access Journals (Sweden)

    Hurst Robert E

    2007-03-01

    Full Text Available Abstract Background Protease-activated receptors (PAR are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS, substance P (SP, and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. Results Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen. 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA and gene ontology (GO. Selected transcripts were target validated by quantitative PCR (Q-PCR. Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and

  5. Phytoceramide and sphingoid bases derived from brewer's yeast Saccharomyces pastorianus activate peroxisome proliferator-activated receptors

    Directory of Open Access Journals (Sweden)

    Mitsutake Susumu

    2011-08-01

    Full Text Available Abstract Background Peroxisome proliferator-activated receptors (PPARs are ligand-activated transcription factors that regulate lipid and glucose metabolism. PPARα is highly expressed in the liver and controls genes involved in lipid catabolism. We previously reported that synthetic sphingolipid analogs, part of which contains shorter-length fatty acid chains than natural sphingolipids, stimulated the transcriptional activities of PPARs. Sphingosine and dihydrosphingosine (DHS are abundant sphingoid bases, and ceramide and dihydroceramide are major ceramide species in mammals. In contrast, phytosphingosine (PHS and DHS are the main sphingoid bases in fungi. PHS and phytoceramide exist in particular tissues such as the epidermis in mammals, and involvement of ceramide species in PPARβ activation in cultured keratinocytes has been reported. The purpose of the present study is to investigate whether natural sphingolipids with C18 fatty acid and yeast-derived sphingoid bases activate PPARs as PPAR agonists. Method Lipids of brewer's yeast contain PHS- and DHS-based sphingolipids. To obtain the sphingoid bases, lipids were extracted from brewer's yeast and acid-hydrolyzed. The sphingoid base fraction was purified and quantified. To assess the effects of sphingolipids on PPAR activation, luciferase reporter assay was carried out. NIH/3T3 and human hepatoma (HepG2 cells were transfected with expression vectors for PPARs and retinoid × receptors, and PPAR responsive element reporter vector. When indicated, the PPAR/Gal4 chimera system was performed to enhance the credibility of experiments. Sphingolipids were added to the cells and the dual luciferase reporter assay was performed to determine the transcriptional activity of PPARs. Results We observed that phytoceramide increased the transcriptional activities of PPARs significantly, whereas ceramide and dihydroceramide did not change PPAR activities. Phytoceramide also increased transactivation of

  6. Phytoceramide and sphingoid bases derived from brewer's yeast Saccharomyces pastorianus activate peroxisome proliferator-activated receptors.

    Science.gov (United States)

    Murakami, Itsuo; Wakasa, Yukari; Yamashita, Shinji; Kurihara, Toshio; Zama, Kota; Kobayashi, Naoyuki; Mizutani, Yukiko; Mitsutake, Susumu; Shigyo, Tatsuro; Igarashi, Yasuyuki

    2011-08-24

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate lipid and glucose metabolism. PPARα is highly expressed in the liver and controls genes involved in lipid catabolism. We previously reported that synthetic sphingolipid analogs, part of which contains shorter-length fatty acid chains than natural sphingolipids, stimulated the transcriptional activities of PPARs. Sphingosine and dihydrosphingosine (DHS) are abundant sphingoid bases, and ceramide and dihydroceramide are major ceramide species in mammals. In contrast, phytosphingosine (PHS) and DHS are the main sphingoid bases in fungi. PHS and phytoceramide exist in particular tissues such as the epidermis in mammals, and involvement of ceramide species in PPARβ activation in cultured keratinocytes has been reported. The purpose of the present study is to investigate whether natural sphingolipids with C18 fatty acid and yeast-derived sphingoid bases activate PPARs as PPAR agonists. Lipids of brewer's yeast contain PHS- and DHS-based sphingolipids. To obtain the sphingoid bases, lipids were extracted from brewer's yeast and acid-hydrolyzed. The sphingoid base fraction was purified and quantified. To assess the effects of sphingolipids on PPAR activation, luciferase reporter assay was carried out. NIH/3T3 and human hepatoma (HepG2) cells were transfected with expression vectors for PPARs and retinoid × receptors, and PPAR responsive element reporter vector. When indicated, the PPAR/Gal4 chimera system was performed to enhance the credibility of experiments. Sphingolipids were added to the cells and the dual luciferase reporter assay was performed to determine the transcriptional activity of PPARs. We observed that phytoceramide increased the transcriptional activities of PPARs significantly, whereas ceramide and dihydroceramide did not change PPAR activities. Phytoceramide also increased transactivation of PPAR/Gal4 chimera receptors. Yeast-derived sphingoid

  7. Five layers of receptor signalling in γδ T cell differentiation and activation

    Directory of Open Access Journals (Sweden)

    Sérgio T. Ribeiro

    2015-01-01

    Full Text Available The contributions of gamma-delta T cells to immunity to infection or tumours critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate gamma-delta T cell physiology. The understanding of how environmental signals are integrated by gamma-delta T cells is critical for their manipulation in clinical settings. Here we discuss how different classes of surface receptors impact on human and murine gamma-delta T cell differentiation, activation and expansion. In particular, we review the role of five receptor types: the T cell receptor (TCR, costimulatory receptors, cytokine receptors, NK receptors and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of gamma-delta T cells; the cytokine receptors IL-2R, IL-7R and IL-15R, which drive functional differentiation and expansion of gamma-delta T cells; the NK receptor NKG2D and its contribution to gamma-delta T cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control gamma-delta T cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signalling in gamma-delta T cell differentiation and activation, and discuss its implications for the manipulation of gamma-delta T cells in immunotherapy.

  8. Troglitazone inhibits cell proliferation by attenuation of epidermal growth factor receptor signaling independent of peroxisome proliferator-activated receptor γ

    Institute of Scientific and Technical Information of China (English)

    Xiaoqi Li; Xuanming Yang; Youli Xu; Xuejun Jiang; Xin Li; Fajun Nan; Hong Tang

    2009-01-01

    Peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily of ligand-dependent transcription factors. Recent results have shown that agonists of PPARy, such as troglitazone (TGZ), can inhibit cell proliferation and promote cell differentiation independent of PPARγ. In the present study, we provide evidence that TGZ may bind directly to EGFR and trigger its signaling and internalization independent of PPARγ. Detailed studies revealed that prolonged incubation with TGZ effectively attenuated EGFR signaling by target-ing the receptor to the endo-lysosomal degradation machinery. Although the extracellular signal-regulated kinase-signaling pathway was transiently activated by TGZ in EGFR overexpressing cancer cells, inhibition of EGF-induced Akt phosphorylation most likely accounted for the growth arrest of tumor cells caused by TGZ at pharmacologically achievable concentrations. Therefore, we have provided a new line of evidence indicating that TGZ inhibits cell pro-liferation by promoting EGFR degradation and attenuating Akt phosphorylation.

  9. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    Science.gov (United States)

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  10. Epidermal growth factor receptor transactivation by intracellular prostaglandin E2-activated prostaglandin E2 receptors. Role in retinoic acid receptor-β up-regulation.

    Science.gov (United States)

    Fernández-Martínez, Ana B; Lucio Cazaña, Francisco J

    2013-09-01

    The pharmacological modulation of renoprotective factor vascular endothelial growth factor-A (VEGF-A) in the proximal tubule has therapeutic interest. In human proximal tubular HK-2 cells, treatment with all-trans retinoic acid or prostaglandin E2 (PGE2) triggers the production of VEGF-A. The pathway involves an initial increase in intracellular PGE2, followed by activation of EP receptors (PGE2 receptors, most likely an intracellular subset) and increase in retinoic acid receptor-β (RARβ) expression. RARβ then up-regulates transcription factor hypoxia-inducible factor-1α (HIF-1α), which increases the transcription and production of VEGF-A. Here we studied the role in this pathway of epidermal growth factor receptor (EGFR) transactivation by EP receptors. We found that EGFR inhibitor AG1478 prevented the increase in VEGF-A production induced by PGE2- and all-trans retinoic acid. This effect was due to the inhibition of the transcriptional up-regulation of RARβ, which resulted in loss of the RARβ-dependent transcriptional up-regulation of HIF-1α. PGE2 and all-trans retinoic acid also increased EGFR phosphorylation and this effect was sensitive to antagonists of EP receptors. The role of intracellular PGE2 was indicated by two facts; i) PGE2-induced EGFR phosphorylation was substantially prevented by inhibitor of prostaglandin uptake transporter bromocresol green and ii) all-trans retinoic acid treatment, which enhanced intracellular but not extracellular PGE2, had lower effect on EGFR phosphorylation upon pre-treatment with cyclooxygenase inhibitor diclofenac. Thus, EGFR transactivation by intracellular PGE2-activated EP receptors results in the sequential activation of RARβ and HIF-1α leading to increased production of VEGF-A and it may be a target for the therapeutic modulation of HIF-1α/VEGF-A. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen

    2015-01-01

    The presence of AT2 receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT2 receptors including their presence in mitochondria and the role in the induction...... densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high...

  12. Is receptor oligomerization causally linked to activation of the EGF receptor kinase?

    Science.gov (United States)

    Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Transduction of a signal from an extracellular peptide hormone to produce an intracellular response is often mediated by a cell surface receptor, which is usually a glycoprotein. The secondary intracellular signal(s) generated after hormone binding to the receptor have been intensively studied. The nature of the primary signal generated by ligand binding to the receptor is understood less well in most cases. The particular case of the epidermal growth factor (EGF) receptor is analyzed, and evidence for or against two dissimilar models of primary signal transduction is reviewed. Evidence for the most widely accepted current model is found to be unconvincing. Evidence for the other model is substantial but indirect; a direct test of this model remains to be done.

  13. Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    Directory of Open Access Journals (Sweden)

    Pangburn Heather A

    2005-09-01

    Full Text Available Abstract Background Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs is associated with a decreased mortality from colorectal cancer (CRC. NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2 signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF receptor (EGFR. Methods HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068, total EGFR, phosphorylated ERK1/2 (pERK1/2, total ERK1/2, activated caspase-3, and α-tubulin. Results EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. Conclusion These results suggest that

  14. Protease-Activated Receptors and other G-Protein-Coupled Receptors: the Melanoma Connection

    OpenAIRE

    Rosero, Rebecca A.; Villares, Gabriel J.; Bar-Eli, Menashe

    2016-01-01

    The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors ...

  15. Activation of NTS A2a adenosine receptors differentially resets baroreflex control of renal vs. adrenal sympathetic nerve activity.

    Science.gov (United States)

    Ichinose, Tomoko K; O'Leary, Donal S; Scislo, Tadeusz J

    2009-04-01

    The role of nucleus of solitary tract (NTS) A(2a) adenosine receptors in baroreflex mechanisms is controversial. Stimulation of these receptors releases glutamate within the NTS and elicits baroreflex-like decreases in mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), whereas inhibition of these receptors attenuates HR baroreflex responses. In contrast, stimulation of NTS A(2a) adenosine receptors increases preganglionic adrenal sympathetic nerve activity (pre-ASNA), and the depressor and sympathoinhibitory responses are not markedly affected by sinoaortic denervation and blockade of NTS glutamatergic transmission. To elucidate the role of NTS A(2a) adenosine receptors in baroreflex function, we compared full baroreflex stimulus-response curves for HR, RSNA, and pre-ASNA (intravenous nitroprusside/phenylephrine) before and after bilateral NTS microinjections of selective adenosine A(2a) receptor agonist (CGS-21680; 2.0, 20 pmol/50 nl), selective A(2a) receptor antagonist (ZM-241385; 40 pmol/100 nl), and nonselective A(1) + A(2a) receptor antagonist (8-SPT; 1 nmol/100 nl) in urethane/alpha-chloralose anesthetized rats. Activation of A(2a) receptors decreased the range, upper plateau, and gain of baroreflex-response curves for RSNA, whereas these parameters all increased for pre-ASNA, consistent with direct effects of the agonist on regional sympathetic activity. However, no resetting of baroreflex-response curves along the MAP axis occurred despite the marked decreases in baseline MAP. The antagonists had no marked effects on baseline variables or baroreflex-response functions. We conclude that the activation of NTS A(2a) adenosine receptors differentially alters baroreflex control of HR, RSNA, and pre-ASNA mostly via non-baroreflex mechanism(s), and these receptors have virtually no tonic action on baroreflex control of these sympathetic outputs.

  16. Pharmacological activation of 5-HT7 receptors reduces nerve injury-induced mechanical and thermal hypersensitivity.

    Science.gov (United States)

    Brenchat, Alex; Nadal, Xavier; Romero, Luz; Ovalle, Sergio; Muro, Asunción; Sánchez-Arroyos, Ricard; Portillo-Salido, Enrique; Pujol, Marta; Montero, Ana; Codony, Xavier; Burgueño, Javier; Zamanillo, Daniel; Hamon, Michel; Maldonado, Rafael; Vela, José Miguel

    2010-06-01

    The involvement of the 5-HT(7) receptor in nociception and pain, particularly chronic pain (i.e., neuropathic pain), has been poorly investigated. In the present study, we examined whether the 5-HT(7) receptor participates in some modulatory control of nerve injury-evoked mechanical hypersensitivity and thermal (heat) hyperalgesia in mice. Activation of 5-HT(7) receptors by systemic administration of the selective 5-HT(7) receptor agonist AS-19 (1 and 10mg/kg) exerted a clear-cut reduction of mechanical and thermal hypersensitivities that were reversed by co-administering the selective 5-HT(7) receptor antagonist SB-258719. Interestingly, blocking of 5-HT(7) receptors with SB-258719 (2.5 and 10mg/kg) enhanced mechanical (but not thermal) hypersensitivity in nerve-injured mice and induced mechanical hypersensitivity in sham-operated mice. Effectiveness of the treatment with a 5-HT(7) receptor agonist was maintained after repeated systemic administration: no tolerance to the antiallodynic and antihyperalgesic effects was developed following treatment with the selective 5-HT(7) receptor agonist E-57431 (10mg/kg) twice daily for 11 days. The 5-HT(7) receptor co-localized with GABAergic cells in the dorsal horn of the spinal cord, suggesting that the activation of spinal inhibitory GABAergic interneurons could contribute to the analgesic effects of 5-HT(7) receptor agonists. In addition, a significant increase of 5-HT(7) receptors was found by immunohistochemistry in the ipsilateral dorsal horn of the spinal cord after nerve injury, suggesting a "pain"-triggered regulation of receptor expression. These results support the idea that the 5-HT(7) receptor subtype is involved in the control of pain and point to a new potential use of 5-HT(7) receptor agonists for the treatment of neuropathic pain.

  17. The angiotensin II type 1 receptor antagonist telmisartan inhibits cell proliferation and tumor growth of esophageal adenocarcinoma via the AMPKα/mTOR pathway in vitro and in vivo.

    Science.gov (United States)

    Fujihara, Shintaro; Morishita, Asahiro; Ogawa, Kana; Tadokoro, Tomoko; Chiyo, Taiga; Kato, Kiyohito; Kobara, Hideki; Mori, Hirohito; Iwama, Hisakazu; Masaki, Tsutomu

    2017-01-31

    Telmisartan, a widely used antihypertensive drug, is an angiotensin II type 1 (AT1) receptor blocker (ARB). This drug inhibits cancer cell proliferation, but the underlying mechanisms in various cancers, including esophageal cancer, remain unknown. The aim of the present study was to evaluate the effects of telmisartan on human esophageal cancer cell proliferation in vitro and in vivo. We assessed the effects of telmisartan on human esophageal adenocarcinoma (EAC) cells using the cell lines OE19, OE33, and SKGT-4. Telmisartan inhibited the proliferation of these three cell lines via blockade of the G0 to G1 cell cycle transition. This blockade was accompanied by a strong decrease in cyclin D1, cyclin E, and other cell cycle-related proteins. Notably, the AMP-activated protein kinase (AMPK) pathway, a fuel sensor signaling pathway, was enhanced by telmisartan. Compound C, which inhibits the two catalytic subunits of AMPK, enhanced the expression of cyclin E, leading to G0/G1 arrest in human EAC cells. In addition, telmisartan reduced the phosphorylation of epidermal growth factor receptor (p-EGFR) and ERBB2 in vitro. In our in vivo study, intraperitoneal injection of telmisartan led to a 73.2% reduction in tumor growth in mice bearing xenografts derived from OE19 cells. Furthermore, miRNA expression was significantly altered by telmisartan in vitro and in vivo. In conclusion, telmisartan suppressed human EAC cell proliferation and tumor growth by inducing cell cycle arrest via the AMPK/mTOR pathway.

  18. Topical Rosiglitazone Treatment Improves Ulcerative Colitis by Restoring Peroxisome Proliferator-Activated Receptor-gamma Activity

    DEFF Research Database (Denmark)

    Pedersen, G.; Brynskov, Jørn

    2010-01-01

    OBJECTIVES: Impaired epithelial expression of peroxisome proliferator-activated receptor-gamma (PPAR gamma) has been described in animal colitis models and briefly in patients with ulcerative colitis, but the functional significance in humans is not well defined. We examined PPAR gamma expression...... and functional activity in human colonic epithelium and explored the potential of topical treatment with rosiglitazone (a PPAR gamma ligand) in patients with ulcerative colitis. METHODS: Spontaneous and rosiglitazone-mediated PPAR gamma and adipophillin expression (a gene transcriptionally activated by PPAR...... for 14 days. RESULTS: PPAR gamma expression was fourfold reduced in epithelial cells from inflamed compared with uninflamed mucosa and controls. Adipophillin levels were decreased in parallel. Rosiglitazone induced a concentration-dependent increase in adipophillin levels and restored PPAR gamma activity...

  19. Quantitative structure-activity relationships of selective antagonists of glucagon receptor using QuaSAR descriptors.

    Science.gov (United States)

    Manoj Kumar, Palanivelu; Karthikeyan, Chandrabose; Hari Narayana Moorthy, Narayana Subbiah; Trivedi, Piyush

    2006-11-01

    In the present paper, quantitative structure activity relationship (QSAR) approach was applied to understand the affinity and selectivity of a novel series of triaryl imidazole derivatives towards glucagon receptor. Statistically significant and highly predictive QSARs were derived for glucagon receptor inhibition by triaryl imidazoles using QuaSAR descriptors of molecular operating environment (MOE) employing computer-assisted multiple regression procedure. The generated QSAR models revealed that factors related to hydrophobicity, molecular shape and geometry predominantly influences glucagon receptor binding affinity of the triaryl imidazoles indicating the relevance of shape specific steric interactions between the molecule and the receptor. Further, QSAR models formulated for selective inhibition of glucagon receptor over p38 mitogen activated protein (MAP) kinase of the compounds in the series highlights that the same structural features, which influence the glucagon receptor affinity, also contribute to their selective inhibition.

  20. Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor α and β and androgen receptor.

    Science.gov (United States)

    Watanabe, Yoko; Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Ohta, Shigeru; Kitamura, Shigeyuki

    2013-07-01

    The structure-activity relationships of parabens which are widely used as preservatives for transcriptional activities mediated by human estrogen receptor α (hERα), hERβ and androgen receptor (hAR) were investigated. Fourteen of 17 parabens exhibited hERα and/or hERβ agonistic activity at concentrations of ≤ 1 × 10(-5)M, whereas none of the 17 parabens showed AR agonistic or antagonistic activity. Among 12 parabens with linear alkyl chains ranging in length from C₁ to C₁₂, heptylparaben (C₇) and pentylparaben (C₅) showed the most potent ERα and ERβ agonistic activity in the order of 10(-7)M and 10(-8)M, respectively, and the activities decreased in a stepwise manner as the alkyl chain was shortened to C₁ or lengthened to C₁₂. Most parabens showing estrogenic activity exhibited ERβ-agonistic activity at lower concentrations than those inducing ERα-agonistic activity. The estrogenic activity of butylparaben was markedly decreased by incubation with rat liver microsomes, and the decrease of activity was blocked by a carboxylesterase inhibitor. These results indicate that parabens are selective agonists for ERβ over ERα; their interactions with ERα/β are dependent on the size and bulkiness of the alkyl groups; and they are metabolized by carboxylesterases, leading to attenuation of their estrogenic activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.

    Science.gov (United States)

    te Kamp, Verena; Lindner, Ricco; Jahnke, Heinz-Georg; Krinke, Dana; Kostelnik, Katja B; Beck-Sickinger, Annette G; Robitzki, Andrea A

    2015-05-15

    Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer.

  2. Method and tool for prognosticating HIV infection in a subject by measuring soluble urokinase plasminogen activator receptor, degradation products thereof, and urokinase plasminogen activator receptor

    DEFF Research Database (Denmark)

    2000-01-01

    Method of diagnosing and/or prognosticating HIV infection in a subject comprising the steps of: (a) performing in vitro a measurement of the level of a marker in the form of (i) urokinase plasminogen activator receptor (uPAR), (ii) soluble urokinase plasminogen activator receptor (suPAR), (iii......) urokinase-type plasminogen activator (uPA), (iv) one or more degradation products of (i), (ii), or (iii), and/or (v) an mRNA for (i), (ii) or (iii), in a biological fluid sample from a subject, and (b) using the measurement value obtained to evaluate the state of the subject....

  3. Activity of protease-activated receptors in primary cultured human myenteric neurons

    Directory of Open Access Journals (Sweden)

    Eva Maria Kugler

    2012-09-01

    Full Text Available Activity of the four known protease-activated receptors (PARs has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (AP. Application of the PAR1-AP (TFLLR or PAR4-AP (GYPGQV evoked spike discharge in 79% or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs of PAR1/PAR2 in 51%, PAR1/PAR4 in 43% and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system.

  4. Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists: design, synthesis, structure-activity relationships, physicochemical properties and biological activity

    NARCIS (Netherlands)

    Blaazer, A.R.; Lange, J.H.M.; van der Neut, M.A.W.; Mulder, A.; den Boon, F.S.; Werkman, T.R.; Kruse, C.G.; Wadman, W.J.

    2011-01-01

    The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands are reported. Based on the aminoalkylindole class of cannabinoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl

  5. Src Family Kinases and Receptors: Analysis of Three Activation Mechanisms by Dynamic Systems Modeling

    OpenAIRE

    Fuß, Hendrik; Dubitzky, Werner; Downes, C. Stephen; Kurth, Mary Jo

    2007-01-01

    Src family kinases (SFKs) interact with a number of cellular receptors. They participate in diverse signaling pathways and cellular functions. Most of the receptors involved in SFK signaling are characterized by similar modes of regulation. This computational study discusses a general kinetic model of SFK-receptor interaction. The analysis of the model reveals three major ways of SFK activation: release of inhibition by C-terminal Src kinase, weakening of the inhibitory intramolecular phospho...

  6. Mechanisms involved in VPAC receptors activation and regulation: lessons from pharmacological and mutagenesis studies.

    Directory of Open Access Journals (Sweden)

    Ingrid eLanger

    2012-10-01

    Full Text Available VIP plays diverse and important role in human physiology and physiopathology and their receptors constitute potential targets for the treatment of several diseases such as neurodegenerative disorder, asthma, diabetes and inflammatory diseases. This article reviews the current knowledge regarding the two VIP receptors, VPAC1 and VPAC2, with respect to mechanisms involved in receptor activation, G protein coupling, signaling, regulation and oligomerization.

  7. Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration.

    Science.gov (United States)

    Galvao, Joana; Elvas, Filipe; Martins, Tiago; Cordeiro, M Francesca; Ambrósio, António Francisco; Santiago, Ana Raquel

    2015-11-01

    Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 μM) prevented apoptosis (TUNEL(+)-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL(+)-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL(+)-cells/mm(2) in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL(+) cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V(+)-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases.

  8. Enhanced antitumor activity of cabazitaxel targeting CD44+ receptor ...

    African Journals Online (AJOL)

    prolonged circulation and slow release of the drug, as well as internalization of the nanocarrier into cancer cells. ... delivery of the anticancer drug via the CD44 receptor. ..... clinical pharmacology of the taxanes docetaxel and paclitaxel--a ...

  9. Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: treprostinil is a potent DP1 and EP2 agonist.

    Science.gov (United States)

    Whittle, Brendan J; Silverstein, Adam M; Mottola, David M; Clapp, Lucie H

    2012-07-01

    The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP1 and IP receptors (Ki 1.1 and 3.9 nM, respectively), low affinity for FP, EP3 or EP4 receptors, and very low affinity for EP2, DP1 or TP receptors. By contrast, treprostinil had high affinity for the DP1, EP2 and IP receptors (Ki 4.4, 3.6 and 32 nM, respectively), low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells expressing the human IP receptor and stimulating calcium influx in cells expressing EP1 receptors (EC50 0.37 and 0.3 nM, respectively) with the rank order of activity on the other receptors comparable to the binding assays. As with binding studies, treprostinil elevated cyclic AMP with a similar high potency in cells expressing DP1, IP and EP2 receptors (EC50 0.6, 1.9 and 6.2 nM, respectively), but had low activity at the other receptors. Activation of IP, DP1 and EP2 receptors, as with treprostinil, can all result in vasodilatation of human pulmonary arteries. However, activation of EP1 receptors can provoke vasoconstriction, and hence may offset the IP-receptor mediated vasodilator effects of iloprost. Treprostinil may therefore differ from iloprost in its overall beneficial pulmonary vasorelaxant profile and other pharmacological actions, especially in diseases where the IP receptor is down-regulated. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Pharmacology and toxicology of fibrates as hypolipidemic drugs mediated by nuclear receptor peroxisome proliferator—activated receptor

    Institute of Scientific and Technical Information of China (English)

    SugaT

    2002-01-01

    PPAR(peroxisome proliferator-activated receptor) is a family of nuclear receptor.In recent years,it has been focused for the discovery and development of new drugs which are mediated by PPARs.Fibrate hypolipidemic drugs are the specific and potent ligands to PPAR alpha and have been widely used for the treatment of hyperlipidemia.But these drugs induce hepatocarcinogenesis in rodent animals after the long-term administration.However,there are species differences on these phenomena which are not seen in mammals ioncluding human.To clarify the mechanism of carcinogenesis by these drugs in important for the evaluation of safety of these drugs in human.

  11. Xenobiotic-induced hepatocyte proliferation associated with constitutive active/androstane receptor (CAR or peroxisome proliferator-activated receptor α (PPARα is enhanced by pregnane X receptor (PXR activation in mice.

    Directory of Open Access Journals (Sweden)

    Ryota Shizu

    Full Text Available Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR, constitutive active/androstane receptor (CAR and peroxisome proliferator-activated receptor α (PPARα play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPARα activation induces hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In the present study, we have investigated the role of PXR in the xenobiotic-induced hepatocyte proliferation with or without CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy]benzene (TCPOBOP and phenobarbital, or PPARα activation by Wy-14643 in mice. Treatment with TCPOBOP or phenobarbital increased the percentage of Ki-67-positive nuclei as well as mRNA levels of cell proliferation-related genes in livers as expected. On the other hand, treatment with the PXR activator pregnenolone 16α-carbonitrile (PCN alone showed no such effects. Surprisingly, PCN co-treatment significantly augmented the hepatocyte proliferation induced by CAR activation with TCPOBOP or phenobarbital in wild-type mice but not in PXR-deficient mice. Intriguingly, PXR activation also augmented the hepatocyte proliferation induced by Wy-14643 treatment. Moreover, PCN treatment increased the RNA content of hepatocytes, suggesting the induction of G0/G1 transition, and reduced mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation. Our present findings indicate that xenobiotic-induced hepatocyte proliferation mediated by CAR or PPARα is enhanced by PXR co-activation despite that PXR activation alone does not cause the cell proliferation in mouse livers. Thus PXR may play a novel and unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics.

  12. Cellular phosphatases facilitate combinatorial processing of receptor-activated signals

    Directory of Open Access Journals (Sweden)

    Siddiqui Zaved

    2008-09-01

    Full Text Available Abstract Background Although reciprocal regulation of protein phosphorylation represents a key aspect of signal transduction, a larger perspective on how these various interactions integrate to contribute towards signal processing is presently unclear. For example, a key unanswered question is that of how phosphatase-mediated regulation of phosphorylation at the individual nodes of the signaling network translates into modulation of the net signal output and, thereby, the cellular phenotypic response. Results To address the above question we, in the present study, examined the dynamics of signaling from the B cell antigen receptor (BCR under conditions where individual cellular phosphatases were selectively depleted by siRNA. Results from such experiments revealed a highly enmeshed structure for the signaling network where each signaling node was linked to multiple phosphatases on the one hand, and each phosphatase to several nodes on the other. This resulted in a configuration where individual signaling intermediates could be influenced by a spectrum of regulatory phosphatases, but with the composition of the spectrum differing from one intermediate to another. Consequently, each node differentially experienced perturbations in phosphatase activity, yielding a unique fingerprint of nodal signals characteristic to that perturbation. This heterogeneity in nodal experiences, to a given perturbation, led to combinatorial manipulation of the corresponding signaling axes for the downstream transcription factors. Conclusion Our cumulative results reveal that it is the tight integration of phosphatases into the signaling network that provides the plasticity by which perturbation-specific information can be transmitted in the form of a multivariate output to the downstream transcription factor network. This output in turn specifies a context-defined response, when translated into the resulting gene expression profile.

  13. The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Jansen Sandra

    2011-02-01

    Full Text Available Abstract Background Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure mediates activation of the immune response in bacterial infection of the central nervous system (CNS. The chemotactic G-protein-coupled receptor (GPCR formyl-peptide-receptor like-1 (FPRL1 plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD. Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. Methods Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP and Neisseria meningitides (NM. Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2 phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene expression and signal transduction were determined. Results We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between

  14. Tumor-Suppressive Activity of Lunatic Fringe in Prostate through Differential Modulation of Notch Receptor Activation

    Directory of Open Access Journals (Sweden)

    Shubing Zhang

    2014-02-01

    Full Text Available Elevated Notch ligand and receptor expression has been associated with aggressive forms of prostate cancer, suggesting a role for Notch signaling in regulation of prostate tumor initiation and progression. Here, we report a critical role for Lunatic Fringe (Lfng, which encodes an O-fucosylpeptide 3-ß-N-acetylglucosaminyltransferase known to modify epidermal growth factor repeats of Notch receptor proteins, in regulation of prostate epithelial differentiation and proliferation, as well as in prostate tumor suppression. Deletion of Lfng in mice caused altered Notch activation in the prostate, associated with elevated accumulation of Notch1, Notch2, and Notch4 intracellular domains, decreased levels of the putative Notch3 intracellular fragment, as well as increased expression of Hes1, Hes5, and Hey2. Loss of Lfng resulted in expansion of the basal layer, increased proliferation of both luminal and basal cells, and ultimately, prostatic intraepithelial neoplasia. The Lfng-null prostate showed down-regulation of prostatic tumor suppressor gene NKX3.1 and increased androgen receptor expression. Interestingly, expression of LFNG and NKX3.1 were positively correlated in publically available human prostate cancer data sets. Knockdown of LFNG in DU-145 prostate cancer cells led to expansion of CD44+CD24− and CD49f+CD24− stem/progenitor-like cell population associated with enhanced prostatosphere-forming capacity. Taken together, these data revealed a tumor-suppressive role for Lfng in the prostate through differential regulation of Notch signaling.

  15. Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation

    DEFF Research Database (Denmark)

    Pedersen, Mikkel W; Pedersen, Nina; Damstrup, Lars;

    2005-01-01

    moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes...... by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1...

  16. The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection

    OpenAIRE

    Kawabata, Atsufumi; Kinoshita, Mitsuhiro; Nishikawa, Hiroyuki; Kuroda, Ryotaro; Nishida, Minoru; Araki, Hiromasa; Arizono, Naoki; Oda, Yasuo; Kakehi, Kazuaki

    2001-01-01

    Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2–activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsai...

  17. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium

    OpenAIRE

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R.; Aleksunes, Lauren M; Thomas, Russell S.; Applegate, Dawn; Klaassen, Curtis D.; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression comp...

  18. Autophagy regulator BECN1 suppresses mammary tumorigenesis driven by WNT1 activation and following parity.

    Science.gov (United States)

    Cicchini, Michelle; Chakrabarti, Rumela; Kongara, Sameera; Price, Sandy; Nahar, Ritu; Lozy, Fred; Zhong, Hua; Vazquez, Alexei; Kang, Yibin; Karantza, Vassiliki

    2014-01-01

    Earlier studies reported allelic deletion of the essential autophagy regulator BECN1 in breast cancers implicating BECN1 loss, and likely defective autophagy, in tumorigenesis. Recent studies have questioned the tumor suppressive role of autophagy, as autophagy-related gene (Atg) defects generally suppress tumorigenesis in well-characterized mouse tumor models. We now report that, while it delays or does not alter mammary tumorigenesis driven by Palb2 loss or ERBB2 and PyMT overexpression, monoallelic Becn1 loss promotes mammary tumor development in 2 specific contexts, namely following parity and in association with wingless-type MMTV integration site family, member 1 (WNT1) activation. Our studies demonstrate that Becn1 heterozygosity, which results in immature mammary epithelial cell expansion and aberrant TNFRSF11A/TNR11/RANK (tumor necrosis factor receptor superfamily, member 11a, NFKB activator) signaling, promotes mammary tumorigenesis in multiparous FVB/N mice and in cooperation with the progenitor cell-transforming WNT1 oncogene. Similar to our Becn1(+/-);MMTV-Wnt1 mouse model, low BECN1 expression and an activated WNT pathway gene signature correlate with the triple-negative subtype, TNFRSF11A axis activation and poor prognosis in human breast cancers. Our results suggest that BECN1 may have nonautophagy-related roles in mammary development, provide insight in the seemingly paradoxical roles of BECN1 in tumorigenesis, and constitute the basis for further studies on the pathophysiology and treatment of clinically aggressive triple negative breast cancers (TNBCs).

  19. An angiotensin II type 1 receptor activation switch patch revealed through evolutionary trace analysis

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Yao, Rong; Ma, Jian-Nong;

    2010-01-01

    in the cytoplasmic parts of TM2, TM3, and TM6 to form an activation switch that is common to all family A 7TM receptors. We tested this hypothesis in the rat Angiotensin II (Ang II) type 1a (AT1a) receptor. The receptor has important roles in the cardiovascular system, but has also frequently been applied as a model...... for 7TM receptor activation and signaling. Six mutations: F66A, L67R, L70R, L119R, D125A, and I245F were targeted to the putative switch and assayed for changes in activation state by their ligand binding, signaling, and trafficking properties. All but one receptor mutant (that was not expressed well...

  20. Activation of peroxisome proliferator-activated receptor alpha in rat spinal cord after peripheral noxious stimulation.

    Science.gov (United States)

    Benani, A; Heurtaux, T; Netter, P; Minn, A

    2004-10-07

    Following recurrent noxious stimulation, both functional modification and structural reorganization such as activation of the arachidonate cascade or axon sprouting occur in the central nervous system (CNS). It has been recently proposed that these alterations observed during chronic pain state were supported by an intensification of the lipid metabolism. In this regard, it has been shown that mRNA coding for several fatty acid metabolizing enzymes are up-regulated in the rat lumbar spinal cord in response to persistent nociception induced by a peripheral inflammation. As peroxisome proliferators-activated receptor (PPAR) could mediate such effects, we therefore investigated the activation of this transcription factor in the rat spinal cord following subcutaneous injection of complete Freund's adjuvant (CFA) into a hind paw. In this study, we compared the DNA-binding activity of nuclear proteins extracted from healthy and inflamed rats toward a PPAR response element. Using electrophoretic mobility-shift assay (EMSA), we found that only the PPARalpha isoform was activated in the rat spinal cord after CFA injection. This activation occurred rapidly, as early as 30 min post-CFA injection, and was persistent up to 10 h, reaching a maximum at 6h after CFA injection. In view of the consequences of PPARalpha activation in other tissues, these results suggest that fatty acid utilization is enhanced in the CNS during chronic pain state. Although the physiopathological relevance of PPARalpha activation during hyperalgesia needs further investigation, we provided here a new player in the molecular modeling of pain pathways.

  1. A2A adenosine receptor antagonism enhances synaptic and motor effects of cocaine via CB1 cannabinoid receptor activation.

    Directory of Open Access Journals (Sweden)

    Alessandro Tozzi

    Full Text Available BACKGROUND: Cocaine increases the level of endogenous dopamine (DA in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. PRINCIPAL FINDINGS: Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. CONCLUSIONS: The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine.

  2. Activation of histamine H3 receptors in human nasal mucosa inhibits sympathetic vasoconstriction.

    Science.gov (United States)

    Varty, LoriAnn M; Gustafson, Eric; Laverty, Maureen; Hey, John A

    2004-01-19

    The peripheral histamine H3 receptor is a presynaptic heterologous receptor located on postganglionic sympathetic nerve fibers innervating sympathetic effector systems such as blood vessels and the heart. An extensive body of evidence shows that activation of the histamine H3 receptor attenuates sympathetic tone by presynaptic inhibition of noradrenaline release. It is proposed that this sympathoinhibitory action, in vivo, leads to reduced vasoconstriction, thereby eliciting a vasodilatory effect. In humans, the peripheral histamine H3 receptor has also been shown to exert a sympathoinhibitory function on specific peripheral autonomic effector systems. For example, human saphenous vein and heart possess functional presynaptic histamine H3 receptors on the sympathetic nerve terminals that upon activation decrease the sympathetic tone to these respective organs. The present studies were conducted to define the role of histamine H3 receptors on neurogenic sympathetic vasoconstrictor responses in human nasal turbinate mucosa. Contractility studies were conducted to evaluate the effect of histamine H3 receptor activation on sympathetic vasoconstriction in surgically isolated human nasal turbinate mucosa. We found that the histamine H3 receptor agonist, (R)-alpha-methylhistamine (30 and 300 nM), inhibited electrical field stimulation-induced (neurogenic) sympathetic vasoconstriction in a concentration-dependent fashion. Pretreatment with the selective histamine H3 receptor antagonist, clobenpropit (100 nM), blocked the sympathoinhibitory effect of (R)-alpha-methylhistamine on the neurogenic sympathetic vasoconstriction. In addition, analysis of Taqman mRNA expression studies showed a specific, high level of distribution of the histamine H3 receptor localized in the human nasal mucosa. Taken together, these studies indicate that histamine H3 receptors modulate vascular contractile responses in human nasal mucosa most likely by inhibiting noradrenaline release from

  3. Enhancement of CA3 hippocampal network activity by activation of group II metabotropic glutamate receptors.

    Science.gov (United States)

    Ster, Jeanne; Mateos, José María; Grewe, Benjamin Friedrich; Coiret, Guyllaume; Corti, Corrado; Corsi, Mauro; Helmchen, Fritjof; Gerber, Urs

    2011-06-14

    Impaired function or expression of group II metabotropic glutamate receptors (mGluRIIs) is observed in brain disorders such as schizophrenia. This class of receptor is thought to modulate activity of neuronal circuits primarily by inhibiting neurotransmitter release. Here, we characterize a postsynaptic excitatory response mediated by somato-dendritic mGluRIIs in hippocampal CA3 pyramidal cells and in stratum oriens interneurons. The specific mGluRII agonists DCG-IV or LCCG-1 induced an inward current blocked by the mGluRII antagonist LY341495. Experiments with transgenic mice revealed a significant reduction of the inward current in mGluR3(-/-) but not in mGluR2(-/-) mice. The excitatory response was associated with periods of synchronized activity at theta frequency. Furthermore, cholinergically induced network oscillations exhibited decreased frequency when mGluRIIs were blocked. Thus, our data indicate that hippocampal responses are modulated not only by presynaptic mGluRIIs that reduce glutamate release but also by postsynaptic mGluRIIs that depolarize neurons and enhance CA3 network activity.

  4. Affinities and intrinsic activities of dopamine receptor agonists for the hD(21) and hD(4.4) receptors

    NARCIS (Netherlands)

    Lahti, RA; Mutin, A; Cochrane, EV; Tepper, PG; Dijkstra, D; Wikstrom, H; Tamminga, CA

    1996-01-01

    The affinity and intrinsic activity of dopamine receptor agonists were determined at the human dopamine hD(21) and hD(4.4) receptors. (-)-3-Hydroxy-N-n-propylpiperidine ((-)3-PPP) had an intrinsic activity of 46% and 83%, whereas (+)-N-propylnorapomorphine ((+)-NPA) had intrinsic activities of 61%

  5. Different efficacy of adenosine and NECA derivatives at the human A3 adenosine receptor: insight into the receptor activation switch.

    Science.gov (United States)

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Kachler, Sonja; Falgner, Nico; Marucci, Gabriella; Thomas, Ajiroghene; Cristalli, Gloria; Volpini, Rosaria; Klotz, Karl-Norbert

    2014-01-15

    A3 Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A3 adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A3 adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N(6)-position. In contrast, the adenosine-5'-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A3 agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5'-substituents on ligand-target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation.

  6. Kaempferol inhibits cancer cell growth by antagonizing estrogen-related receptor α and γ activities.

    Science.gov (United States)

    Wang, Haibin; Gao, Minghui; Wang, Junjian

    2013-11-01

    Kaempferol is a dietary flavonoid that can function as a selective estrogen receptor modulator (SERM). Estrogen-related receptors alpha and gamma (ERRα and ERRγ) are orphan nuclear receptors that play important roles in mitochondrial biogenesis and cancer development. We have shown that kaempferol can functionally antagonize the activities of ERRs based on both response element reporter systems and target gene analysis. Kaempferol modulation of mitochondrial function and suppression cancer cell growth has been confirmed. These findings suggest that kaempferol may exert their anti-cancer activities through antagonizing ERRs activities.

  7. A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor

    DEFF Research Database (Denmark)

    Grundmann, Manuel; Tikhonova, Irina G; Hudson, Brian D

    2016-01-01

    Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand...

  8. Allosteric regulation of G protein-coupled receptor activity by phospholipids.

    Science.gov (United States)

    Dawaliby, Rosie; Trubbia, Cataldo; Delporte, Cédric; Masureel, Matthieu; Van Antwerpen, Pierre; Kobilka, Brian K; Govaerts, Cédric

    2016-01-01

    Lipids are emerging as key regulators of membrane protein structure and activity. These effects can be attributed either to the modification of bilayer properties (thickness, curvature and surface tension) or to the binding of specific lipids to the protein surface. For G protein-coupled receptors (GPCRs), the effects of phospholipids on receptor structure and activity remain poorly understood. Here we reconstituted purified β2-adrenergic receptor (β2R) in high-density lipoparticles to systematically characterize the effect of biologically relevant phospholipids on receptor activity. We observed that the lipid headgroup type affected ligand binding (agonist and antagonist) and receptor activation. Specifically, phosphatidylgycerol markedly favored agonist binding and facilitated receptor activation, whereas phosphatidylethanolamine favored antagonist binding and stabilized the inactive state of the receptor. We then showed that these effects could be recapitulated with detergent-solubilized lipids, demonstrating that the functional modulation occurred in the absence of a bilayer. Our data suggest that phospholipids act as direct allosteric modulators of GPCR activity.

  9. Discoidin domain receptor 1 is activated independently of beta(1) integrin

    DEFF Research Database (Denmark)

    Vogel, W; Brakebusch, C; Fässler, R

    2000-01-01

    Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor (DDR) tyrosine kinases, DDR1 and DDR2. It is presently unclear whether collagen-induced DDR receptor activation, which occurs with very slow kinetics, involves additional proteins...... with kinase activity or membrane-anchored proteins serving as coreceptors. In particular, the role of the collagen-binding integrins alpha(1)beta(1) or alpha(2)beta(1) in the DDR activation process is undefined. Here, we provide three lines of evidence suggesting that DDR1 signaling is distinct from integrin...... activation. First we demonstrate that the enzymatic activity of DDR1 is essential for receptor tyrosine phosphorylation. Collagen-induced DDR receptor autophosphorylation can be blocked either by a dominant negative mutant or by a preparation of recombinant extracellular domain. Second, we show DDR1 signals...

  10. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    Science.gov (United States)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  11. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR.

    Science.gov (United States)

    Calkin, Anna C; Tontonoz, Peter

    2012-03-14

    Nuclear receptors are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body. Given that modulation of lipid and glucose metabolism has been linked to diseases including type 2 diabetes, obesity and atherosclerosis, a greater understanding of pathways that regulate metabolism in physiology and disease is crucial. The liver X receptors (LXRs) and the farnesoid X receptors (FXRs) are activated by oxysterols and bile acids, respectively. Mounting evidence indicates that these nuclear receptors have essential roles, not only in the regulation of cholesterol and bile acid metabolism but also in the integration of sterol, fatty acid and glucose metabolism.

  12. Role of Triggering Receptor Expressed on Myeloid Cells in the Activation of Innate Immunity

    Directory of Open Access Journals (Sweden)

    V. G. Matveyeva

    2011-01-01

    Full Text Available The innate immune system plays a key role in triggering a systemic inflammatory response (SIR. The triggering receptor expressed on myeloid cells (TREM-1, which is located on neutrophils and monocytes, is involved in SIR, by regulating the effector mechanisms of innate immunity. Hyperproduction of proinflammatory cytokines is a pathogenetic component of the hyperergic phase of acute systemic inflammation. The simultaneous activation of Toll-like receptors and TREM-1 increases the production of cytokines manifold. This is compensatory and adaptive, however, resulting in damage to organs and tissues during excessive production of cytokines. Key words: triggering receptor expressed on myeloid cells, Toll-like receptors, cytokines, inflammation.

  13. DMPD: Proximal effects of Toll-like receptor activation in dendritic cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17142025 Proximal effects of Toll-like receptor activation in dendritic cells. Watt...) (.svg) (.html) (.csml) Show Proximal effects of Toll-like receptor activation in dendritic cells. PubmedID... 17142025 Title Proximal effects of Toll-like receptor activation in dendritic ce

  14. Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

    Science.gov (United States)

    Sirohi, Sunil; Walker, Brendan M

    2015-11-01

    Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function.

  15. Phospholipase A2-modified low-density lipoprotein activates macrophage peroxisome proliferator-activated receptors.

    Science.gov (United States)

    Namgaladze, Dmitry; Morbitzer, Daniel; von Knethen, Andreas; Brüne, Bernhard

    2010-02-01

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors modulating metabolic and inflammatory responses of phagocytes to stimuli such as fatty acids and their metabolites. We studied the role of PPARs in macrophages exposed to low-density lipoprotein (LDL) modified by secretory phospholipase A(2) (PLA). By analyzing PPAR ligand-binding domain luciferase reporter activation, we observed that PLA-LDL transactivates PPARalpha and PPARdelta, but not PPARgamma. We confirmed that PLA-LDL induced PPAR response element reporter activation by endogenous PPARalpha and PPARdelta in human THP-1 macrophages. By using THP-1 cells with a stable knockdown of PPARalpha and PPARdelta, we showed that PLA-LDL-activated PPARdelta altered macrophage gene expression related to lipid metabolism and lipid droplet formation. Although PPARalpha/delta silencing did not affect cholesterol and triglyceride accumulation in PLA-LDL-treated macrophages, PPARdelta activation by PLA-LDL attenuated macrophage inflammatory gene expression induced by interferon gamma and lipopolysaccharide. PPARdelta activation by PLA-LDL does not influence lipid accumulation in PLA-LDL-treated macrophages. However, it attenuates macrophage inflammatory responses, thus contributing to an anti-inflammatory cell phenotype.

  16. Activation, internalization, and recycling of the serotonin 2A receptor by dopamine

    Science.gov (United States)

    Bhattacharyya, Samarjit; Raote, Ishier; Bhattacharya, Aditi; Miledi, Ricardo; Panicker, Mitradas M.

    2006-01-01

    Serotonergic and dopaminergic systems, and their functional interactions, have been implicated in the pathophysiology of various CNS disorders. Here, we use recombinant serotonin (5-HT) 2A (5-HT2A) receptors to further investigate direct interactions between dopamine and 5-HT receptors. Previous studies in Xenopus oocytes showed that dopamine, although not the cognate ligand for the 5-HT2A receptor, acts as a partial-efficacy agonist. At micromolar concentrations, dopamine also acts as a partial-efficacy agonist on 5-HT2A receptors in HEK293 cells. Like 5-HT, dopamine also induces receptor-internalization in these cells, although at significantly higher concentrations than 5-HT. Interestingly, if the receptors are first sensitized or “primed” by subthreshold concentrations of 5-HT, then dopamine-induced internalization occurs at concentrations ≈10-fold lower than when dopamine is used alone. Furthermore, unlike 5-HT-mediated internalization, dopamine-mediated receptor internalization, alone, or after sensitization by 5-HT, does not depend on PKC. Dopamine-internalized receptors recycle to the surface at rates similar to those of 5-HT-internalized receptors. Our results suggest a previously uncharacterized role for dopamine in the direct activation and internalization of 5-HT2A receptors that may have clinical relevance to the function of serotonergic systems in anxiety, depression, and schizophrenia and also to the treatment of these disorders. PMID:17005723

  17. Activated receptors for peroxisomic proliferators. Its role in the atherosclerosis, obesity and high blood pressure.

    Directory of Open Access Journals (Sweden)

    Mikhail Benet Rodríguez

    2004-08-01

    Full Text Available The receptors activated by peroxisome proliferators are a family of factors of transcription that belong to the superfamily of the steroid receptors and include tree subtypes which are PPARá, PPAR©¬ and PPAR ã. These receptors join to direct hexameric repetitions in the form of heterodimers with the retinoid receptor. PPAR receptors regulate the expressions of a great variety of genes that codify the proteins that are implied in the lipid metabolism, the energetic homeostasis, the cellular differentiation and the formation of tumours. This review describes the features, regulation and target genes of the PPAR receptor and the physiopathological and pharmacological implications of the regulation of the lipid and glucose metabolism, the energetic homeostasis ,hypertension and endothelial dysfunction.

  18. GABA-B receptor activation and conflict behavior

    Energy Technology Data Exchange (ETDEWEB)

    Ketelaars, C.E.J.; Bollen, E.L.; Rigter, H.; Bruinvels, J.

    1988-01-01

    Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on (/sup 3/H)-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables.

  19. Research Resource: Androgen Receptor Activity Is Regulated Through the Mobilization of Cell Surface Receptor Networks.

    Science.gov (United States)

    Hsiao, Jordy J; Ng, Brandon H; Smits, Melinda M; Martinez, Harryl D; Jasavala, Rohini J; Hinkson, Izumi V; Fermin, Damian; Eng, Jimmy K; Nesvizhskii, Alexey I; Wright, Michael E

    2015-08-01

    The aberrant expression of androgen receptor (AR)-dependent transcriptional programs is a defining pathology of the development and progression of prostate cancers. Transcriptional cofactors that bind AR are critical determinants of prostate tumorigenesis. To gain a deeper understanding of the proteins linked to AR-dependent gene transcription, we performed a DNA-affinity chromatography-based proteomic screen designed to identify proteins involved in AR-mediated gene transcription in prostate tumor cells. Functional experiments validated the coregulator roles of known AR-binding proteins in AR-mediated transcription in prostate tumor cells. More importantly, novel coregulatory functions were detected in components of well-established cell surface receptor-dependent signal transduction pathways. Further experimentation demonstrated that components of the TNF, TGF-β, IL receptor, and epidermal growth factor signaling pathways modulated AR-dependent gene transcription and androgen-dependent proliferation in prostate tumor cells. Collectively, our proteomic dataset demonstrates that the cell surface receptor- and AR-dependent pathways are highly integrated, and provides a molecular framework for understanding how disparate signal-transduction pathways can influence AR-dependent transcriptional programs linked to the development and progression of human prostate cancers.

  20. The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Olsen, Kristine Boisen; Erikstrup, Niels;

    2011-01-01

    The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately...

  1. In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein

    NARCIS (Netherlands)

    G.G.J.M. Kuiper (George); P.E. de Ruiter (Petra); J. Trapman (Jan); G.W. Jenster (Guido); A.O. Brinkmann (Albert)

    1993-01-01

    textabstractTranslation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with

  2. Expression and function of heregulin-α and its receptors in the mouse mammary gland

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Heregulin-α (HRGα) is a cytokine secreted by the mammary mesenchyme, adjacent to lobuloalveolar structures. To understand the role of HRGα and its receptors in mammary glands, and the underlying mechanisms, we performed this study to determine the expression and localization of HRGα and its receptors ErbB2 and ErbB3. We also determined the role of HRGα in the development of mammary glands, β-casein expression and secretion, Rab3A protein expression and the phosphorylation of HRGα signaling molecules using confocal laser scanning microscopy, tissue culture, capillary electrophoresis, Western blotting and enzyme-linked immunosorbent assays. We found that a peak was on pregnancy day 15. Changes of ErbB2 and ErbB3 expression were positively and linearly correlated with HRGα, indicating that HRGα positively regulates ErbB2 and ErbB3 expression. During pregnancy, HRGα enhanced the phosphorylation of STAT5, p42/p44, p38, PKC and Rab3A protein expression, stimulated the proliferation and differentiation of the ductal epithelial cells of mammary glands, and increased and maintained the expression and secretion of β-casein. During lactation, HRGα enhanced the phosphorylation of STAT5 and p38, inhibited the phosphorylation of PKC and Rab3A protein expression, maintained the morphology of the mammary glands and increased the secretion of lactoprotein to reduce the expression of β-casein in mammary epithelial cells. During involution, HRGα induced the phosphorylation of STAT3 and Rab3A protein expression, and inhibited the phosphorylation of PKC to stimulate the degeneration of mammary epithelial cells. It also inhibited the secretion of β-casein, resulting in increased levels of β-casein in mammary epithelial cells.

  3. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    Science.gov (United States)

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  4. Redirected Primary Human Chimeric Antigen Receptor Natural Killer Cells As an “Off-the-Shelf Immunotherapy” for Improvement in Cancer Treatment

    Science.gov (United States)

    Oberschmidt, Olaf; Kloess, Stephan; Koehl, Ulrike

    2017-01-01

    Primary human natural killer (NK) cells recognize and subsequently eliminate virus infected cells, tumor cells, or other aberrant cells. However, cancer cells are able to develop tumor immune escape mechanisms to undermine this immune control. To overcome this obstacle, NK cells can be genetically modified to express chimeric antigen receptors (CARs) in order to improve specific recognition of cancer surface markers (e.g., CD19, CD20, and ErbB2). After target recognition, intracellular CAR domain signaling (CD3ζ, CD28, 4-1BB, and 2B4) leads to activation of PI3K or DNAX proteins (DAP10, DAP12) and finally to enhanced cytotoxicity, proliferation, and/or interferon γ release. This mini-review summarizes both the first preclinical trials with CAR-engineered primary human NK cells and the translational implications for “off-the-shelf immunotherapy” in cancer treatment. Signal transduction in NK cells as well as optimization of CAR signaling will be described, becoming more and more a focal point of interest in addition to redirected T cells. Finally, strategies to overcome off-target effects will be discussed in order to improve future clinical trials and to avoid attacking healthy tissues. PMID:28649246

  5. Cell death-independent activities of the death receptors CD95, TRAILR1, and TRAILR2.

    Science.gov (United States)

    Siegmund, Daniela; Lang, Isabell; Wajant, Harald

    2017-04-01

    Since their identification more than 20 years ago, the death receptors CD95, TRAILR1, and TRAILR2 have been intensively studied with respect to their cell death-inducing activities. These receptors, however, can also trigger a variety of cell death-independent cellular responses reaching from the activation of proinflammatory gene transcription programs over the stimulation of proliferation and differentiation to induction of cell migration. The cell death-inducing signaling mechanisms of CD95 and the TRAIL death receptors are well understood. In contrast, despite the increasing recognition of the biological and pathophysiological relevance of the cell death-independent activities of CD95, TRAILR1, and TRAILR2, the corresponding signaling mechanisms are less understood and give no fully coherent picture. This review is focused on the cell death-independent activities of CD95 and the TRAIL death receptors and addresses mainly three questions: (a) how are these receptors linked to noncell death pathways at the molecular level, (b) which factors determine the balance of cell death and cell death-independent activities of CD95 and the TRAIL death receptors at the cellular level, and (c) what are the consequences of the cell death-independent functions of these receptors for their role in cancer and inflammatory diseases. © 2016 Federation of European Biochemical Societies.

  6. Effects of histamine H1 receptor signaling on glucocorticoid receptor activity. Role of canonical and non-canonical pathways

    Science.gov (United States)

    Zappia, Carlos Daniel; Granja-Galeano, Gina; Fernández, Natalia; Shayo, Carina; Davio, Carlos; Fitzsimons, Carlos P.; Monczor, Federico

    2015-01-01

    Histamine H1 receptor (H1R) antagonists and glucocorticoid receptor (GR) agonists are used to treat inflammatory conditions such as allergic rhinitis, atopic dermatitis and asthma. Consistent with the high morbidity levels of such inflammatory conditions, these receptors are the targets of a vast number of approved drugs, and in many situations their ligands are co-administered. However, this drug association has no clear rationale and has arisen from clinical practice. We hypothesized that H1R signaling could affect GR-mediated activity, impacting on its transcriptional outcome. Indeed, our results show a dual regulation of GR activity by the H1R: a potentiation mediated by G-protein βγ subunits and a parallel inhibitory effect mediated by Gαq-PLC pathway. Activation of the H1R by its full agonists resulted in a composite potentiating effect. Intriguingly, inactivation of the Gαq-PLC pathway by H1R inverse agonists resulted also in a potentiation of GR activity. Moreover, histamine and clinically relevant antihistamines synergized with the GR agonist dexamethasone to induce gene transactivation and transrepression in a gene-specific manner. Our work provides a delineation of molecular mechanisms underlying the widespread clinical association of antihistamines and GR agonists, which may contribute to future dosage optimization and reduction of well-described side effects associated with glucocorticoid administration. PMID:26635083

  7. Effects of histamine H1 receptor signaling on glucocorticoid receptor activity. Role of canonical and non-canonical pathways.

    Science.gov (United States)

    Zappia, Carlos Daniel; Granja-Galeano, Gina; Fernández, Natalia; Shayo, Carina; Davio, Carlos; Fitzsimons, Carlos P; Monczor, Federico

    2015-12-04

    Histamine H1 receptor (H1R) antagonists and glucocorticoid receptor (GR) agonists are used to treat inflammatory conditions such as allergic rhinitis, atopic dermatitis and asthma. Consistent with the high morbidity levels of such inflammatory conditions, these receptors are the targets of a vast number of approved drugs, and in many situations their ligands are co-administered. However, this drug association has no clear rationale and has arisen from clinical practice. We hypothesized that H1R signaling could affect GR-mediated activity, impacting on its transcriptional outcome. Indeed, our results show a dual regulation of GR activity by the H1R: a potentiation mediated by G-protein βγ subunits and a parallel inhibitory effect mediated by Gαq-PLC pathway. Activation of the H1R by its full agonists resulted in a composite potentiating effect. Intriguingly, inactivation of the Gαq-PLC pathway by H1R inverse agonists resulted also in a potentiation of GR activity. Moreover, histamine and clinically relevant antihistamines synergized with the GR agonist dexamethasone to induce gene transactivation and transrepression in a gene-specific manner. Our work provides a delineation of molecular mechanisms underlying the widespread clinical association of antihistamines and GR agonists, which may contribute to future dosage optimization and reduction of well-described side effects associated with glucocorticoid administration.

  8. Peripheral Sensitization Increases Opioid Receptor Expression and Activation by Crotalphine in Rats

    Science.gov (United States)

    Zambelli, Vanessa Olzon; Fernandes, Ana Carolina de Oliveira; Gutierrez, Vanessa Pacciari; Ferreira, Julio Cesar Batista; Parada, Carlos Amilcar; Mochly-Rosen, Daria; Cury, Yara

    2014-01-01

    Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the µ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective µ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids. PMID:24594607

  9. Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

    Science.gov (United States)

    Butts, Arielle; Martin, Jennifer A; DiDone, Louis; Bradley, Erin K; Mutz, Mitchell; Krysan, Damian J

    2015-01-01

    Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM) tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

  10. Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

    Directory of Open Access Journals (Sweden)

    Arielle Butts

    Full Text Available Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1 the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2 an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3 electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

  11. Activity of L-alpha-amino acids at the promiscuous goldfish odorant receptor 5.24

    DEFF Research Database (Denmark)

    Christiansen, Bolette; Wellendorph, Petrine; Bräuner-Osborne, Hans

    2006-01-01

    The goldfish odorant receptor 5.24 is a member of family C of G protein-coupled receptors and is closely related to the human receptor GPRC6A. Receptor 5.24 has previously been shown to have binding affinity for L-alpha-amino acids, especially the basic amino acids arginine and lysine. Here we...... a preference for basic amino acids....... report the agonist activities of the 20 proteinogenic L-alpha-amino acids, and L-ornithine and L-citrulline, measured in an intracellular calcium release assay in mammalian tsA cells. The results show that receptor 5.24 is broadly activated by 19 of the tested L-alpha-amino acids and displays...

  12. An Angiotensin II type 1 receptor activation switch patch revealed through Evolutionary Trace analysis

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Yao, Rong; Ma, Jian-Nong

    2010-01-01

    in the cytoplasmic parts of TM2, TM3, and TM6 to form an activation switch that is common to all family A 7TM receptors. We tested this hypothesis in the rat Angiotensin II (Ang II) type 1a (AT1a) receptor. The receptor has important roles in the cardiovascular system, but has also frequently been applied as a model......) displayed phenotypes associated with changed activation state, such as increased agonist affinity or basal activity, promiscuous activation, or constitutive internalization highlighting the importance of testing different signaling pathways. We conclude that this evolutionary important patch mediates...... to be completely resolved. Evolutionary Trace (ET) analysis is a computational method, which identifies clusters of functionally important residues by integrating information on evolutionary important residue variations with receptor structure. Combined with known mutational data, ET predicted a patch of residues...

  13. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    LENUS (Irish Health Repository)

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

  14. Effects related to gene-gene interactions of peroxisome proliferator-activated receptor on essential hypertension

    Institute of Scientific and Technical Information of China (English)

    俞浩

    2013-01-01

    Objective To explore the impact of the gene-gene interaction among the single nucleotide polymorphisms(SNPs) of peroxisome proliferator-activated receptorα/δ/γ on essential hypertension(EH).Methods

  15. Identification of (beta-carboxyethyl)-rhodanine derivatives exhibiting peroxisome proliferator-activated receptor gamma activity.

    Science.gov (United States)

    Choi, Jiwon; Ko, Yoonae; Lee, Hui Sun; Park, Yun Sun; Yang, Young; Yoon, Sukjoon

    2010-01-01

    We applied an improved virtual screening scheme combining ligand-centric and receptor-centric methods for the identification of a new series of PPARgamma agonists known as (beta-carboxyethyl)-rhodanine derivatives which include a thiazolidin-based core structure, 2-thioxo-thiazolidine-4-one. An in vitro assay confirmed the nanomolar binding affinity in one of the (beta-carboxyethyl)-rhodanine derivatives, SP1818. It showed a PPARgamma agonistic activity similar to that of a known PPARgamma drug, pioglitazone, in a cell-based transactivation assay. Furthermore, the structure-activity relationships of the rhodanine derivatives were investigated through comparative molecular field analysis. We also characterized the inconsistency between the in vitro binding affinity and cell-based transactivation ability by using a set of property-based molecular descriptors. The binding mode analysis provided new insight concerning their agonistic effect on PPARgamma.

  16. Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Okazaki, Shogo [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nakatani, Fumi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Masuko, Kazue; Tsuchihashi, Kenji [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ueda, Shiho; Masuko, Takashi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nagano, Osamu, E-mail: osmna@sb3.so-net.ne.jp [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

    2016-01-29

    The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

  17. Multiple activities of insect repellents on odorant receptors in mosquitoes

    Science.gov (United States)

    Several lines of evidence suggest that insect repellent molecules reduce mosquito-host contacts by interacting with odorants and odorant receptors (ORs) ultimately affecting olfactory-driven behaviors. We describe the molecular effects of ten insect repellents and a pyrethroid insecticide with known...

  18. The phosphoproteome of toll-like receptor-activated macrophages

    DEFF Research Database (Denmark)

    Weintz, Gabriele; Olsen, Jesper Velgaard; Frühauf, Katja;

    2010-01-01

    Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome...

  19. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    2000-01-01

    Nine structurally different polycyclic aromatic hydrocarbons (PAHs) were tested for their ability to either agonize or antagonize the human androgen receptor (hAR) in a sensitive reporter gene assay based on CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector. Benz...

  20. Altered activity profile of a tertiary silanol analog of multi-targeting nuclear receptor modulator T0901317.

    Science.gov (United States)

    Toyama, Hirozumi; Sato, Shoko; Shirakawa, Hitoshi; Komai, Michio; Hashimoto, Yuichi; Fujii, Shinya

    2016-04-01

    We report the design, synthesis, and physicochemical/biological evaluation of novel silanol derivative 6 (sila-T) as a silanol analog of multi-target nuclear receptor modulator T0901317 (5). Compound 6 showed intermediate hydrophobicity between the corresponding alcohol 13 and perfluoroalcohol 5. While 5 exhibited potent activities toward liver X receptor α and β, farnesoid X receptor, pregnane X receptor (PXR) and retinoic acid receptor-related orphan receptor (ROR)γ, silanol 6 exhibited activity only toward PXR and RORs. Incorporation of silanol instead of perfluoroalcohol is a promising option for developing novel target-selective, biologically active compounds.

  1. Telmisartan attenuates peritoneal fibrosis via peroxisome proliferator-activated receptoractivation in rats.

    Science.gov (United States)

    Su, Xuesong; Yu, Rui; Yang, Xu; Zhou, Guangyu; Wang, Yanqiu; Li, Li; Li, Detian

    2015-06-01

    Peritoneal dialysis (PD) is an effective treatment for patients with end-stage renal diseases, but long-term continuous PD causes peritoneal fibrosis (PF). This study aims to evaluate the anti-fibrotic effect of telmisartan on a rat model of PF and to investigate the underlying mechanisms. Five-sixths kidney nephrectomy and PD were used to establish the PF rat model. Glucose (2.5%) was used to establish an in vitro model in rat peritoneal mesothelial cells (PMC). Haematoxylin-eosin staining was used to examine the structural alterations. Masson's trichrome staining was used to observe the tissue fibrosis in peritoneal membrane of rats. Real-time polymerase chain reaction was used to measure messenger RNA expressions of profibrotic factors. Western blotting was used to determine protein expressions of profibrotic factors, peroxisome proliferator-activated receptor-γ, and mitogen-activated protein kinases (MAPK). Results demonstrated that administration of telmisartan dose-dependently attenuated the thickening of the peritoneal membrane and the fibrosis induced by long-term PD fluid exposure in rats. In addition, telmisartan treatment inhibited the upregulation of profibrotic factors induced by PD in the peritoneum of rats and by high-concentration glucose in PMC. Telmisartan was also effective in inhibiting PD and high-concentration, glucose-induced phosphorylation of MAPK in the peritoneum and PMC. Furthermore, peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 blocked these protective effects of telmisartan in PMC. The results suggest that telmisartan is effective in attenuating PD-induced PF, and this effect may be associated with the inhibition of profibrotic factor expression and MAPK phosphorylation via PPARγ activation. © 2015 Wiley Publishing Asia Pty Ltd.

  2. Antifibrotic effect by activation of peroxisome proliferator-activated receptor-gamma in corneal fibroblasts.

    Science.gov (United States)

    Pan, Hongwei; Chen, Jiansu; Xu, Jintang; Chen, Miaojiao; Ma, Rong

    2009-11-10

    The transformation of quiescent keratocytes to active phenotypes and the ensuing fibrotic response play important roles in corneal scar formation. This study aims to observe the antifibrotic effect of peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist on corneal fibroblasts cultured in vitro, and to explore the potential application of peroxisome proliferator-activated receptor agonist to the prevention of corneal opacity following wound repair. Rabbit corneal keratocytes were cultured in a medium containing 10% serum to induce their transformation to fibroblasts and myofibroblasts, which are similar to those that repair corneas. After incubation with the PPARgamma agonist pioglitazone at different concentrations, the effect of pioglitazone on the migration, contractility, and viability of corneal fibroblasts was examined. The secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 was determined by gelatin zymography, and the synthesis of collagen I and fibronectin was investigated by western blotting. Treatment with pioglitazone at concentrations ranging from 1 to 10 mum significantly decreased corneal fibroblast migration, as determined by scrape-wound assay, inhibited corneal fibroblast-induced collagen lattice contraction, and reduced MMP-2 and MMP-9 secretion into the supernatant of cell cultures in a dose-dependent manner. The expression of fibronectin was significantly decreased, while the expression of collagen I was only decreased when treated with 10 mum pioglitazone. Cell viability was not evidently changed compared to the control. This in vitro study demonstrated the anti-fibrotic effect of pioglitazone, suggesting that activation of PPARgamma may be a new approach for the treatment of corneal opacity and scar formation in the corneal wound healing process.

  3. Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation.

    Science.gov (United States)

    Manni, Sandro; Mineev, Konstantin S; Usmanova, Dinara; Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kirpichnikov, Mikhail P; Winter, Jonas; Matkovic, Milos; Deupi, Xavier; Arseniev, Alexander S; Ballmer-Hofer, Kurt

    2014-08-05

    Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase