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  1. Updates on HIPK2: a resourceful oncosuppressor for clearing cancer

    PubMed Central

    2012-01-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a multitalented protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. HIPK2 phosphorylates oncosuppressor p53 for apoptotic activation. In addition, also p53-independent apoptotic pathways are regulated by HIPK2 and can be exploited for anticancer purpose too. Therefore, HIPK2 activity is considered a central switch in targeting tumor cells toward apoptosis upon genotoxic damage and the preservation and/or restoration of HIPK2 function is crucial for an efficient tumor response to therapies. As a proof of principle, HIPK2 knockdown impairs p53 function, induces chemoresistance, angiogenesis, and tumor growth in vivo, on the contrary, HIPK2 overexpression activates apoptotic pathways, counteracts hypoxia, inhibits angiogenesis, and induces chemosensitivity both in p53-dependent and -independent ways. The role of HIPK2 in restraining tumor development was also confirmed by studies with HIPK2 knockout mice. Recent findings demonstrated that HIPK2 inhibitions do exist in tumors and depend by several mechanisms including HIPK2 cytoplasmic localization, protein degradation, and loss of heterozygosity (LOH), recapitulating the biological outcome obtained by RNA interference studies in tumor cells, such as p53 inactivation, resistance to therapies, apoptosis inhibition, and tumor progression. These findings may lead to new diagnostic and therapeutic approaches for treating cancer patients. This review will focus on the last updates about HIPK2 contribution in tumorigenesis and cancer treatment. PMID:22889244

  2. Counteracting MDM2-induced HIPK2 downregulation restores HIPK2/p53 apoptotic signaling in cancer cells.

    PubMed

    Nardinocchi, Lavinia; Puca, Rosa; Givol, David; D'Orazi, Gabriella

    2010-10-01

    Homeodomain-interacting protein kinase-2 (HIPK2) is a crucial regulator of p53 apoptotic function by phosphorylating serine 46 (Ser46) in response to DNA damage. In tumors with wild-type p53, its tumor suppressor function is often impaired by MDM2 overexpression that targets p53 for proteasomal degradation. Likewise, MDM2 targets HIPK2 for protein degradation impairing p53-apoptotic function. Here we report that zinc antagonised MDM2-induced HIPK2 degradation as well as p53 ubiquitination. The zinc inhibitory effect on MDM2 activity leads to HIPK2-induced p53Ser46 phosphorylation and p53 pro-apoptotic transcriptional activity. These results suggest that zinc derivatives are potential molecules to target the MDM2-induced HIPK2/p53 inhibition. PMID:20849851

  3. HIPK2 sustains apoptotic response by phosphorylating Che-1/AATF and promoting its degradation.

    PubMed

    De Nicola, F; Catena, V; Rinaldo, C; Bruno, T; Iezzi, S; Sorino, C; Desantis, A; Camerini, S; Crescenzi, M; Floridi, A; Passananti, C; Soddu, S; Fanciulli, M

    2014-01-01

    Che-1/AATF is an RNA polymerase II-binding protein that is involved in the regulation of gene transcription, which undergoes stabilization and accumulation in response to DNA damage. We have previously demonstrated that following apoptotic induction, Che-1 protein levels are downregulated through its interaction with the E3 ligase HDM2, which leads to Che-1 degradation by ubiquitylation. This interaction is mediated by Pin1, which determines a phosphorylation-dependent conformational change. Here we demonstrate that HIPK2, a proapoptotic kinase, is involved in Che-1 degradation. HIPK2 interacts with Che-1 and, upon genotoxic stress, phosphorylates it at specific residues. This event strongly increases HDM2/Che-1 interaction and degradation of Che-1 protein via ubiquitin-dependent proteasomal system. In agreement with these findings, we found that HIPK2 depletion strongly decreases Che-1 ubiquitylation and degradation. Notably, Che-1 overexpression strongly counteracts HIPK2-induced apoptosis. Our results establish Che-1 as a new HIPK2 target and confirm its important role in the cellular response to DNA damage. PMID:25210797

  4. Downregulation of HIPK2 Increases Resistance of Bladder Cancer Cell to Cisplatin by Regulating Wip1

    PubMed Central

    Lin, Jun; Zhang, Qiang; Lu, Yi; Xue, Wenrui; Xu, Yue; Zhu, Yichen; Hu, Xiaopeng

    2014-01-01

    Cisplatin-based combination chemotherapy regimen is a reasonable alternative to cystectomy in advanced/metastatic bladder cancer, but acquisition of cisplatin resistance is common in patients with bladder cancer. Previous studies showed that loss of homeodomain-interacting protein kinase-2 (HIPK2) contributes to cell proliferation and tumorigenesis. However, the role of HIPK2 in regulating chemoresistance of cancer cell is not fully understood. In the present study, we found that HIPK2 mRNA and protein levels are significantly decreased in cisplatin-resistant bladder cancer cell in vivo and in vitro. Downregulation of HIPK2 increases the cell viability in a dose- and time-dependent manner during cisplatin treatment, whereas overexpression of HIPK2 reduces the cell viability. HIPK2 overexpression partially overcomes cisplatin resistance in RT4-CisR cell. Furthermore, we showed that Wip1 (wild-type p53-induced phosphatase 1) expression is upregulated in RT4-CisR cell compared with RT4 cell, and HIPK2 negatively regulates Wip1 expression in bladder cancer cell. HIPK2 and Wip1 expression is also negatively correlated after cisplatin-based combination chemotherapy in vivo. Finally, we demonstrated that overexpression of HIPK2 sensitizes chemoresistant bladder cancer cell to cisplatin by regulating Wip1 expression. Conclusions These data suggest that HIPK2/Wip1 signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of bladder cancer. PMID:24846322

  5. The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4.

    PubMed

    Yamada, D; Pérez-Torrado, R; Filion, G; Caly, M; Jammart, B; Devignot, V; Sasai, N; Ravassard, P; Mallet, J; Sastre-Garau, X; Schmitz, M L; Defossez, P-A

    2009-07-01

    HIPK2 is a eukaryotic Serine-Threonine kinase that controls cellular proliferation and survival in response to exogenous signals. Here, we show that the human transcription factor ZBTB4 is a new target of HIPK2. The two proteins interact in vitro, colocalize and associate in vivo, and HIPK2 phosphorylates several conserved residues of ZBTB4. Overexpressing HIPK2 causes the degradation of ZBTB4, whereas overexpressing a kinase-deficient mutant of HIPK2 has no effect. The chemical activation of HIPK2 also decreases the amount of ZBTB4 in cells. Conversely, the inhibition of HIPK2 by drugs or by RNA interference causes a large increase in ZBTB4 levels. This negative regulation of ZBTB4 by HIPK2 occurs under normal conditions of cell growth. In addition, the degradation is increased by DNA damage. These findings have two consequences. First, we have recently shown that ZBTB4 inhibits the transcription of p21. Therefore, the activation of p21 by HIPK2 is two-pronged: stimulation of the activator p53, and simultaneous repression of the inhibitor ZBTB4. Second, ZBTB4 is also known to bind methylated DNA and repress methylated sequences. Consequently, our findings raise the possibility that HIPK2 might influence the epigenetic regulation of gene expression at loci that remain to be identified. PMID:19448668

  6. Heat shock modulates the subcellular localization, stability, and activity of HIPK2.

    PubMed

    Upadhyay, Mamta; Bhadauriya, Pratibha; Ganesh, Subramaniam

    2016-04-15

    The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress - such as hypoxia, oxidative stress, or UV damage - is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 and the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors. PMID:26972256

  7. PML tumor suppressor is regulated by HIPK2-mediated phosphorylation in response to DNA damage.

    PubMed

    Gresko, E; Ritterhoff, S; Sevilla-Perez, J; Roscic, A; Fröbius, K; Kotevic, I; Vichalkovski, A; Hess, D; Hemmings, B A; Schmitz, M L

    2009-02-01

    The promyelocytic leukemia (PML) tumor suppressor protein, a central regulator of cell proliferation and apoptosis, is frequently fused to the retinoic acid receptor-alpha (RARalpha) in acute PML. Here we show the interaction of PML with another tumor suppressor protein, the serine/threonine kinase homeodomain-interacting protein kinase (HIPK2). In response to DNA damage, HIPK2 phosphorylates PML at serines 8 and 38. Although HIPK2-mediated phosphorylation of PML occurs early during the DNA damage response, the oncogenic PML-RARalpha fusion protein is phosphorylated with significantly delayed kinetics. DNA damage or HIPK2 expression leads to the stabilization of PML and PML-RARalpha proteins. The N-terminal phosphorylation sites contribute to the DNA damage-induced PML SUMOylation and are required for the ability of PML to cooperate with HIPK2 for the induction of cell death. PMID:19015637

  8. From top to bottom: the two faces of HIPK2 for regulation of the hypoxic response.

    PubMed

    Calzado, Marco A; De La Vega, Laureano; Munoz, Eduardo; Schmitz, M Lienhard

    2009-06-01

    Oxygen deprivation (hypoxia) triggers a complex network of signaling pathways that result in changed gene expression patterns in order to cope with this challenge. Recent work has identified the serine/threonine kinase HIPK2 as a novel regulatory protein participating in hypoxic gene regulation. HIPK2 can affect apical as well as downstream events during the hypoxic response. Under normoxic conditions, HIPK2-mediated phosphorylation of the ubiquitin E3 ligase Siah2 weakens mutual binding and destabilizes the phosphorylated E3 ligase. Low oxygen levels result in strongly increased HIPK2/Siah2 interactions that lead to efficient polyubiquitylation and proteasomal degradation of the kinase. At the apical level, the Siah2 inhibiting phosphorylations are lost, thus allowing Siah2-dependent proteolysis of dioxygenases which in turn allows for activation of transcription factor HIF. Downstream events of the hypoxic response are affected by the proteasomal elimination of HIPK2 from gene repressing complexes, an event that allows for full induction of gene expression. Thus HIPK2 can regulate a subset of HIF-dependent and -independent genes during the hypoxic response. PMID:19448429

  9. Identification of Hipk2 as an essential regulator of white fat development

    PubMed Central

    Sjölund, Jonas; Pelorosso, Facundo G.; Quigley, David A.; DelRosario, Reyno; Balmain, Allan

    2014-01-01

    Homeodomain-interacting protein kinase 2 (Hipk2) has previously been implicated in the control of several transcription factors involved in embryonic development, apoptosis, cell proliferation, and tumor development, but very little is understood about the exact mechanisms through which Hipk2 influences these processes. Analysis of gene expression in normal tissues from genetically heterogeneous mouse or human populations can reveal network motifs associated with the structural or functional components of the tissue, and may predict roles for genes of unknown function. Here we have applied this network strategy to uncover a role for the Hipk2 gene in the transcriptional system controlling adipogenesis. Both in vitro and in vivo models were used to show that knockdown or loss of Hipk2 specifically inhibits white adipose cell differentiation and tissue development. In addition, loss of Hipk2 leads to induction of pockets of multilocular brown fat-like cells in remaining white adipose depots, which express markers of brown and beige fat such as uncoupling protein 1 and transmembrane protein 26. These changes are accompanied by increased insulin sensitivity in Hipk2 knockout mice and reduced high-fat diet–induced weight gain, highlighting a potential role for this kinase in diseases such as diabetes and obesity. Our study underscores the versatility and power of a readily available tissue, such as skin, for network modeling of systemic transcriptional programs involved in multiple pathways, including lipid metabolism and adipogenesis. PMID:24785298

  10. HIPK2 phosphorylates ΔNp63α and promotes its degradation in response to DNA damage.

    PubMed

    Lazzari, C; Prodosmo, A; Siepi, F; Rinaldo, C; Galli, F; Gentileschi, M; Bartolazzi, A; Costanzo, A; Sacchi, A; Guerrini, L; Soddu, S

    2011-12-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is an emerging player in cell response to genotoxic agents that senses damage intensity and contributes to the cell's choice between cell cycle arrest and apoptosis. Phosphorylation of p53 at S46, an apoptosis-specific p53 posttranslational modification, is the most characterized HIPK2 function in response to lethal doses of ultraviolet (UV), ionizing radiation or different anticancer drugs, such as cisplatin, roscovitine and doxorubicin (DOX). Indeed, like p53, HIPK2 has been shown to contribute to the effectiveness of these treatments. Interestingly, p53-independent mechanisms of HIPK2-induced apoptosis were described for UV and tumor growth factor-β treatments; however, it is unknown whether these mechanisms are relevant for the responses to anticancer drugs. Because of the importance of the so-called 'p53-independent apoptosis and drug response' in human cancer chemotherapy, we asked whether p53-independent factor(s) might be involved in HIPK2-mediated chemosensitivity. Here, we show that HIPK2 depletion by RNA interference induces resistance to different anticancer drugs even in p53-null cells, suggesting the involvement of HIPK2 targets other than p53 in response to chemotherapy. In particular, we found that HIPK2 phosphorylates and promotes proteasomal degradation of ΔNp63α, a prosurvival ΔN isoform of the p53 family member, p63. Indeed, effective cell response to different genotoxic agents was shown to require phosphorylation-induced proteasomal degradation of ΔNp63α. In DOX-treated cells, we show that HIPK2 depletion interferes with ΔNp63α degradation, and expression of a HIPK2-resistant ΔNp63α-Δ390 mutant induces chemoresistance. We identify T397 as the ΔNp63α residue phosphorylated by HIPK2, and show that the non-phosphorylatable ΔNp63α-T397A mutant is not degraded in the face of either HIPK2 overexpression or DOX treatment. These results indicate ΔNp63α as a novel target of HIPK2 in

  11. How cells switch HIPK2 on and off.

    PubMed

    Sombroek, D; Hofmann, T G

    2009-02-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is an emerging regulator of cell growth and apoptosis in various cell types, tissues and organisms. Previous work indicates that HIPK2 is a potential tumour suppressor and DNA damage-responsive kinase, which phosphorylation-dependently activates the apoptotic programme by engaging diverse downstream targets, including tumour suppressor p53 and the anti-apoptotic transcriptional corepressor C-terminal binding protein. The regulation of HIPK2, however, remained largely obscure. Recent studies show that HIPK2 activity is mainly controlled at the post-transcriptional level through targeted proteolysis. Caspase-dependent processing triggers HIPK2 hyperactivation, whereas the ubiquitin-proteasome system (UPS) keeps HIPK2 in check by targeting it for degradation. Both HIPK2 hyperactivation and HIPK2 degradation are under the control of transcription factor p53. Negative regulation of HIPK2 by the UPS is abolished in response to DNA damage, which facilitates HIPK2 stabilization and activation. Here we discuss these findings in the context of DNA damage signalling and tumour suppression. PMID:18974774

  12. MYCN sensitizes human neuroblastoma to apoptosis by HIPK2 activation through a DNA damage response.

    PubMed

    Petroni, Marialaura; Veschi, Veronica; Prodosmo, Andrea; Rinaldo, Cinzia; Massimi, Isabella; Carbonari, Maurizio; Dominici, Carlo; McDowell, Heather P; Rinaldi, Christian; Screpanti, Isabella; Frati, Luigi; Bartolazzi, Armando; Gulino, Alberto; Soddu, Silvia; Giannini, Giuseppe

    2011-01-01

    MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53(S46) kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53(S46) phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53(S46) phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma. PMID:21173028

  13. HIPK2 modification code for cell death and survival

    PubMed Central

    Wook Choi, Dong; Yong Choi, Cheol

    2014-01-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine protein kinase that participates in the regulation of diverse cellular activities as a transcriptional cofactor and signal transducer. HIPK2 senses various signaling cues that in turn phosphorylate downstream substrates to coordinate developmental processes, cell cycle regulation, cell proliferation, differentiation, and the DNA damage response. HIPK2 functions are affected by its catalytic activity, stability, and subcellular localization, which in turn are dynamically regulated by diverse post-translational modifications such as polyubiquitination, SUMOylation, phosphorylation, and acetylation. HIPK2 is not modified with small molecules and/or peptides individually or independently, but in a combinatorial manner that is referred to as the “HIPK2 modification code.” HIPK2 integrates various signaling cues and senses different doses of DNA damage and ROS stimuli, which are reflected by unique patterns of HIPK2 modification. Hence, the HIPK2 modification code differentially contributes to cellular homeostasis and determination of cell fate depending on cellular context. PMID:27308327

  14. Posttranslational modifications regulate HIPK2, a driver of proliferative diseases.

    PubMed

    Saul, Vera V; Schmitz, M Lienhard

    2013-09-01

    The serine/threonine kinase homeodomain-interacting protein kinase (HIPK2) is a tumor suppressor and functions as an evolutionary conserved regulator of signaling and gene expression. This kinase regulates a surprisingly vast array of biological processes that range from the DNA damage response and apoptosis to hypoxia signaling and cell proliferation. Recent studies show the tight control of HIPK2 by hierarchically occurring posttranslational modifications such as phosphorylation, small ubiquitin-like modifier modification, acetylation, and ubiquitination. The physiological function of HIPK2 as a regulator of cell proliferation and survival has a downside: proliferative diseases. Dysregulation of HIPK2 can result in increased proliferation of cell populations as it occurs in cancer or fibrosis. We discuss various models that could explain how inappropriate expression, modification, or localization of HIPK2 can be a driver for these proliferative diseases. PMID:23616089

  15. The WD40-repeat protein Han11 functions as a scaffold protein to control HIPK2 and MEKK1 kinase functions

    PubMed Central

    Ritterhoff, Stefanie; Farah, Carla M; Grabitzki, Julia; Lochnit, Günter; Skurat, Alexander V; Schmitz, Michael Lienhard

    2010-01-01

    Protein kinases are organized in hierarchical networks that are assembled and regulated by scaffold proteins. Here, we identify the evolutionary conserved WD40-repeat protein Han11 as an interactor of the kinase homeodomain-interacting protein kinase 2 (HIPK2). In vitro experiments showed the direct binding of Han11 to HIPK2, but also to the kinases DYRK1a, DYRK1b and mitogen-activated protein kinase kinase kinase 1 (MEKK1). Han11 was required to allow coupling of MEKK1 to DYRK1 and HIPK2. Knockdown experiments in Caenorhabditis elegans showed the relevance of the Han11 orthologs Swan-1 and Swan-2 for the osmotic stress response. Downregulation of Han11 in human cells lowered the threshold and amplitude of HIPK2- and MEKK1-triggered signalling events and changed the kinetics of kinase induction. Han11 knockdown changed the amplitude and time dependence of HIPK2-driven transcription in response to DNA damage and also interfered with MEKK1-triggered gene expression and stress signalling. Impaired signal transmission also occurred upon interference with stoichiometrically assembled signalling complexes by Han11 overexpression. Collectively, these experiments identify Han11 as a novel scaffold protein regulating kinase signalling by HIPK2 and MEKK1. PMID:20940704

  16. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

    PubMed Central

    Glenewinkel, Florian; Cohen, Michael J.; King, Cason R.; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S.; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  17. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2.

    PubMed

    Glenewinkel, Florian; Cohen, Michael J; King, Cason R; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  18. Transcriptional corepressors HIPK1 and HIPK2 control angiogenesis via TGF-β-TAK1-dependent mechanism.

    PubMed

    Shang, Yulei; Doan, Christina N; Arnold, Thomas D; Lee, Sebum; Tang, Amy A; Reichardt, Louis F; Huang, Eric J

    2013-01-01

    Several critical events dictate the successful establishment of nascent vasculature in yolk sac and in the developing embryos. These include aggregation of angioblasts to form the primitive vascular plexus, followed by the proliferation, differentiation, migration, and coalescence of endothelial cells. Although transforming growth factor-β (TGF-β) is known to regulate various aspects of vascular development, the signaling mechanism of TGF-β remains unclear. Here we show that homeodomain interacting protein kinases, HIPK1 and HIPK2, are transcriptional corepressors that regulate TGF-β-dependent angiogenesis during embryonic development. Loss of HIPK1 and HIPK2 leads to marked up-regulations of several potent angiogenic genes, including Mmp10 and Vegf, which result in excessive endothelial proliferation and poor adherens junction formation. This robust phenotype can be recapitulated by siRNA knockdown of Hipk1 and Hipk2 in human umbilical vein endothelial cells, as well as in endothelial cell-specific TGF-β type II receptor (TβRII) conditional mutants. The effects of HIPK proteins are mediated through its interaction with MEF2C, and this interaction can be further enhanced by TGF-β in a TAK1-dependent manner. Remarkably, TGF-β-TAK1 signaling activates HIPK2 by phosphorylating a highly conserved tyrosine residue Y-361 within the kinase domain. Point mutation in this tyrosine completely eliminates the effect of HIPK2 as a transcriptional corepressor in luciferase assays. Our results reveal a previously unrecognized role of HIPK proteins in connecting TGF-β signaling pathway with the transcriptional programs critical for angiogenesis in early embryonic development. PMID:23565059

  19. miR-141 regulates TGF-β1-induced epithelial-mesenchymal transition through repression of HIPK2 expression in renal tubular epithelial cells

    PubMed Central

    HUANG, YUANHANG; TONG, JUNRONG; HE, FENG; YU, XINPEI; FAN, LIMING; HU, JING; TAN, JIANGPING; CHEN, ZHENGLIANG

    2015-01-01

    Epithelial-mesenchymal transition (EMT) plays a critical role in embryonic development, wound healing, tissue regeneration, cancer progression and organ fibrosis. The proximal tubular epithelial cells undergo EMT, resulting in matrix-producing fibroblasts and thereby contribute to the pathogenesis of renal fibrosis. The profibrotic cytokine, TGF-β, is now recognized as the main pathogenic driver that has been shown to induce EMT in tubular epithelial cells. Increasing evidence indicate that HIPK2 dysfunction may play a role in fibroblasts behavior, and therefore, HIPK2 may be considered as a novel potential target for anti-fibrosis therapy. Recently, members of the miR-200 family (miR-200a, b and c and miR-141) have been shown to inhibit EMT. However, the steps of the multifactorial renal fibrosis progression that these miRNAs regulate, particularly miR-141, are unclear. To study the functional importance of miR-141 in EMT, a well-established in vitro EMT assay was used to demonstrate renal tubulointerstitial fibrosis; transforming growth factor-β1-induced EMT in HK-2 cells. Overexpression of miR-141 in HK-2 cells, either with or without TGF-β1 treatment, hindered EMT by enhancing E-cadherin and decreasing vimentin and fibroblast-specific protein 1 expression. miR-141 expression was repressed during EMT in a dose- and time-dependent manner through upregulation of HIPK2 expression. Ectopic expression of HIPK2 promoted EMT by decreasing E-cadherin. Furthermore, co-transfection of miR-141 with the HIPK2 ORF clone partially inhibited EMT by restoring E-cadherin expression. miR-141 downregulated the expression of HIPK2 via direct interaction with the 3′-untranslated region of HIPK2. Taken together, these findings aid in the understanding of the role and mechanism of miR-141 in regulating renal fibrosis via the TGF-β1/miR-141/HIPK2/EMT axis, and miR-141 may represent novel biomarkers and therapeutic targets in the treatment of renal fibrosis. PMID:25421593

  20. FHL2 mediates p53-induced transcriptional activation through a direct association with HIPK2

    SciTech Connect

    Lee, Sang-Wang . E-mail: umsj@sejong.ac.kr

    2006-01-27

    To understand the molecular mechanism underlying HIPK2 regulation of the transcriptional activation by p53, we sought to identify the protein that interacts with HIPK2. From our yeast two-hybrid screen, we found that four and a half LIM domains 2 (FHL2) could bind to the C-terminal half of HIPK2. Further assays in yeast mapped the minimal interaction domain to amino acids 812-907 in HIPK2. The interaction was confirmed using a GST pull-down assay in vitro, and an immunoprecipitation (IP) assay and fluorescence microscopy in vivo. FHL2 alone spread throughout both the cytoplasm and nucleus but was redistributed to dot-like structures in the nucleus when HIPK2 was coexpressed in HEK293 cells. When tethered to the Gal4-responsive promoter through the Gal4 DBD fusion, FHL2 showed autonomous transcriptional activity that was enhanced by wild-type HIPK2, but not by the kinase-defective mutant. In addition, FHL2 increased the p53-dependent transcriptional activation and had an additive effect on the activation when coexpressed with HIPK2, which was again not observed with the kinase-defective mutant of HIPK2. Finally, we found a ternary complex of p53, HIPK2, and FHL2 using IP, and their recruitment to the p53-responsive p21Waf1 promoter in chromatin IP assays. Overall, our findings indicate that FHL2 can also regulate p53 via a direct association with HIPK2.

  1. Cutaneous HPV23 E6 prevents p53 phosphorylation through interaction with HIPK2.

    PubMed

    Muschik, Dorothea; Braspenning-Wesch, Ilona; Stockfleth, Eggert; Rösl, Frank; Hofmann, Thomas G; Nindl, Ingo

    2011-01-01

    Ultraviolet irradiation (UV) is the major risk factor for the development of skin cancer. Moreover, increasing evidence suggests cutaneotropic human papillomaviruses (HPV) from the beta genus to play a causal role as a co-factor in the development of cutaneous squamous cell carcinoma. Homeodomain-interacting protein kinase 2 (HIPK2) operates as a potential suppressor in skin tumorigenesis and is stabilized by UV-damage. HIPK2 is an important regulator of apoptosis, which forms a complex with the tumor suppressor p53, mediating p53 phosphorylation at Ser 46 and thus promoting pro-apoptotic gene expression. In our study, we demonstrate that cutaneous HPV23 E6 protein directly targets HIPK2 function. Accordingly, HPV23 E6 interacts with HIPK2 both in vitro and in vivo. Furthermore, upon massive UVB-damage HPV23 E6 co-localizes with endogenous HIPK2 at nuclear bodies. Functionally, we demonstrate that HPV23 E6 inhibits HIPK2-mediated p53 Ser 46 phosphorylation through enforcing dissociation of the HIPK2/p53 complex. In addition, HPV23 E6 co-accumulates with endogenous HIPK2 upon UV damage suggesting a mechanism by which HPV23 E6 keeps HIPK2 in check after UV damage. Thus, cutaneous HPV23 E6 prevents HIPK2-mediated p53 Ser 46 phosphorylation, which may favour survival of UV-damaged keratinocytes and skin carcinogenesis by apoptosis evasion. PMID:22110707

  2. Zyxin is a critical regulator of the apoptotic HIPK2-p53 signaling axis.

    PubMed

    Crone, Johanna; Glas, Carolina; Schultheiss, Kathrin; Moehlenbrink, Jutta; Krieghoff-Henning, Eva; Hofmann, Thomas G

    2011-03-15

    HIPK2 activates the apoptotic arm of the DNA damage response by phosphorylating tumor suppressor p53 at serine 46. Unstressed cells keep HIPK2 levels low through targeted polyubiquitination and subsequent proteasomal degradation. Here we identify the LIM domain protein Zyxin as a novel regulator of the HIPK2-p53 signaling axis in response to DNA damage. Remarkably, depletion of endogenous Zyxin, which colocalizes with HIPK2 at the cytoskeleton and in the cell nucleus, stimulates proteasome-dependent HIPK2 degradation. In contrast, ectopic expression of Zyxin stabilizes HIPK2, even upon enforced expression of its ubiquitin ligase Siah-1. Consistently, Zyxin physically interacts with Siah-1, and knock-down of Siah-1 rescues HIPK2 expression in Zyxin-depleted cancer cells. Mechanistically, our data suggest that Zyxin regulates Siah-1 activity through interference with Siah-1 dimerization. Furthermore, we show that endogenous Zyxin coaccumulates with HIPK2 in response to DNA damage in cancer cells, and that depletion of endogenous Zyxin results in reduced HIPK2 protein levels and compromises DNA damage-induced p53 Ser46 phosphorylation and caspase activation. These findings suggest an unforeseen role for Zyxin in DNA damage-induced cell fate control through modulating the HIPK2-p53 signaling axis. PMID:21248071

  3. Control of HIPK2 stability by ubiquitin ligase Siah-1 and checkpoint kinases ATM and ATR.

    PubMed

    Winter, Melanie; Sombroek, Dirk; Dauth, Ilka; Moehlenbrink, Jutta; Scheuermann, Karin; Crone, Johanna; Hofmann, Thomas G

    2008-07-01

    The tumour suppressor HIPK2 is an important regulator of cell death induced by DNA damage, but how its activity is regulated remains largely unclear. Here we demonstrate that HIPK2 is an unstable protein that colocalizes and interacts with the E3 ubiquitin ligase Siah-1 in unstressed cells. Siah-1 knockdown increases HIPK2 stability and steady-state levels, whereas Siah-1 expression facilitates HIPK2 polyubiquitination, degradation and thereby inactivation. During recovery from sublethal DNA damage, HIPK2, which is stabilized on DNA damage, is degraded through a Siah-1-dependent, p53-controlled pathway. Downregulation of Siah-1 inhibits HIPK2 degradation and recovery from damage, driving the cells into apoptosis. We have also demonstrated that DNA damage triggers disruption of the HIPK2-Siah-1 complex, resulting in HIPK2 stabilization and activation. Disruption of the HIPK2-Siah-1 complex is mediated by the ATM/ATR pathway and involves ATM/ATR-dependent phosphorylation of Siah-1 at Ser 19. Our results provide a molecular framework for HIPK2 regulation in unstressed and damaged cells. PMID:18536714

  4. PEBP2-beta/CBF-beta-dependent phosphorylation of RUNX1 and p300 by HIPK2: implications for leukemogenesis.

    PubMed

    Wee, Hee-Jun; Voon, Dominic Chih-Cheng; Bae, Suk-Chul; Ito, Yoshiaki

    2008-11-01

    The heterodimeric transcription factor RUNX1/PEBP2-beta (also known as AML1/CBF-beta) is essential for definitive hematopoiesis. Here, we show that interaction with PEBP2-beta leads to the phosphorylation of RUNX1, which in turn induces p300 phosphorylation. This is mediated by homeodomain interacting kinase 2 (HIPK2), targeting Ser(249), Ser(273), and Thr(276) in RUNX1, in a manner that is also dependent on the RUNX1 PY motif. Importantly, we observed the in vitro disruption of this phosphorylation cascade by multiple leukemogenic genetic defects targeting RUNX1/CBFB. In particular, the oncogenic protein PEBP2-beta-SMMHC prevents RUNX1/p300 phosphorylation by sequestering HIPK2 to mislocalized RUNX1/beta-SMMHC complexes. Therefore, phosphorylation of RUNX1 appears a critical step in its association with and phosphorylation of p300, and its disruption may be a common theme in RUNX1-associated leukemogenesis. PMID:18695000

  5. MDM4/HIPK2/p53 cytoplasmic assembly uncovers coordinated repression of molecules with anti-apoptotic activity during early DNA damage response.

    PubMed

    Mancini, F; Pieroni, L; Monteleone, V; Lucà, R; Fici, L; Luca, E; Urbani, A; Xiong, S; Soddu, S; Masetti, R; Lozano, G; Pontecorvi, A; Moretti, F

    2016-01-14

    The p53 inhibitor, MDM4 (MDMX) is a cytoplasmic protein with p53-activating function under DNA damage conditions. Particularly, MDM4 promotes phosphorylation of p53 at Ser46, a modification that precedes different p53 activities. We investigated the mechanism by which MDM4 promotes this p53 modification and its consequences in untransformed mammary epithelial cells and tissues. In response to severe DNA damage, MDM4 stimulates p53Ser46(P) by binding and stabilizing serine-threonine kinase HIPK2. Under these conditions, the p53-inhibitory complex, MDM4/MDM2, dissociates and this allows MDM4 to promote p53/HIPK2 functional interaction. Comparative proteomic analysis of DNA damage-treated cells versus -untreated cells evidenced a diffuse downregulation of proteins with anti-apoptotic activity, some of which were targets of p53Ser46(P)/HIPK2 repressive activity. Importantly, MDM4 depletion abolishes the downregulation of these proteins indicating the requirement of MDM4 to promote p53-mediated transcriptional repression. Consistently, MDM4-mediated HIPK2/p53 activation precedes HIPK2/p53 nuclear translocation and activity. Noteworthy, repression of these proteins was evident also in mammary glands of mice subjected to γ-irradiation and was significantly enhanced in transgenic mice overexpressing MDM4. This study evidences the flexibility of MDM2/MDM4 heterodimer, which allows the development of a positive activity of cytoplasmic MDM4 towards p53-mediated transcriptional function. Noteworthy, this activity uncovers coordinated repression of molecules with shared anti-apoptotic function which precedes active cell apoptosis and that are frequently overexpressed and/or markers of tumour phenotype in human cancer. PMID:25961923

  6. MDM4/HIPK2/p53 cytoplasmic assembly uncovers coordinated repression of molecules with anti-apoptotic activity during early DNA damage response

    PubMed Central

    Mancini, F; Pieroni, L; Monteleone, V; Lucà, R; Fici, L; Luca, E; Urbani, A; Xiong, S; Soddu, S; Masetti, R; Lozano, G; Pontecorvi, A; Moretti, F

    2016-01-01

    The p53 inhibitor, MDM4 (MDMX) is a cytoplasmic protein with p53-activating function under DNA damage conditions. Particularly, MDM4 promotes phosphorylation of p53 at Ser46, a modification that precedes different p53 activities. We investigated the mechanism by which MDM4 promotes this p53 modification and its consequences in untransformed mammary epithelial cells and tissues. In response to severe DNA damage, MDM4 stimulates p53Ser46P by binding and stabilizing serine–threonine kinase HIPK2. Under these conditions, the p53-inhibitory complex, MDM4/MDM2, dissociates and this allows MDM4 to promote p53/HIPK2 functional interaction. Comparative proteomic analysis of DNA damage-treated cells versus -untreated cells evidenced a diffuse downregulation of proteins with anti-apoptotic activity, some of which were targets of p53Ser46P/HIPK2 repressive activity. Importantly, MDM4 depletion abolishes the downregulation of these proteins indicating the requirement of MDM4 to promote p53-mediated transcriptional repression. Consistently, MDM4-mediated HIPK2/p53 activation precedes HIPK2/p53 nuclear translocation and activity. Noteworthy, repression of these proteins was evident also in mammary glands of mice subjected to γ-irradiation and was significantly enhanced in transgenic mice overexpressing MDM4. This study evidences the flexibility of MDM2/MDM4 heterodimer, which allows the development of a positive activity of cytoplasmic MDM4 towards p53-mediated transcriptional function. Noteworthy, this activity uncovers coordinated repression of molecules with shared anti-apoptotic function which precedes active cell apoptosis and that are frequently overexpressed and/or markers of tumour phenotype in human cancer. PMID:25961923

  7. Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization

    PubMed Central

    Polonio-Vallon, Tilman; Kirkpatrick, Joanna; Krijgsveld, Jeroen; Hofmann, Thomas G

    2014-01-01

    Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm. PMID:24196445

  8. Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization.

    PubMed

    Polonio-Vallon, Tilman; Kirkpatrick, Joanna; Krijgsveld, Jeroen; Hofmann, Thomas G

    2014-01-01

    Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm. PMID:24196445

  9. HIPK2: a versatile switchboard regulating the transcription machinery and cell death.

    PubMed

    Calzado, Marco A; Renner, Florian; Roscic, Ana; Schmitz, M Lienhard

    2007-01-15

    Homeodomain interacting protein kinase 2 (HIPK2) is an evolutionary conserved serine/threonine kinase that regulates gene expression by phosphorylation of transcription factors and accessory components of the transcription machinery. HIPK2 is activated in response to DNA-damaging agents or morphogenic signals and accordingly HIPK2-guided gene expression programs trigger differentiation and development or alternatively apoptosis. The kinase contributes to the regulation of remarkably diverse pathways such as p53 activation or Wnt signaling. Here we discuss recent findings from biochemical and functional experiments that allow a deeper understanding of the pleiotropic effects mediated by HIPK2. PMID:17245128

  10. Autophosphorylation and Pin1 binding coordinate DNA damage-induced HIPK2 activation and cell death.

    PubMed

    Bitomsky, Nadja; Conrad, Elisa; Moritz, Christian; Polonio-Vallon, Tilman; Sombroek, Dirk; Schultheiss, Kathrin; Glas, Carolina; Greiner, Vera; Herbel, Christoph; Mantovani, Fiamma; del Sal, Giannino; Peri, Francesca; Hofmann, Thomas G

    2013-11-01

    Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1-HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis. PMID:24145406

  11. HIPK2: A tumour suppressor that controls DNA damage-induced cell fate and cytokinesis.

    PubMed

    Hofmann, Thomas G; Glas, Carolina; Bitomsky, Nadja

    2013-01-01

    In response to DNA-damage, cells have to decide between different cell fate programmes. Activation of the tumour suppressor HIPK2 specifies the DNA damage response (DDR) and tips the cell fate balance towards an apoptotic response. HIPK2 is activated by the checkpoint kinase ATM, and triggers apoptosis through regulatory phosphorylation of a set of cellular key molecules including the tumour suppressor p53 and the anti-apoptotic corepressor CtBP. Recent work has identified HIPK2 as a regulator of the ultimate step in cytokinesis: the abscission of the mother and daughter cells. Since proper cytokinesis is essential for genome stability and maintenance of correct ploidy, this finding sheds new light on the tumour suppressor function of HIPK2. Here we highlight the molecular mechanisms coordinating HIPK2 function and discuss its emerging role as a tumour suppressor. PMID:23169233

  12. HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation.

    PubMed

    Siepi, Francesca; Gatti, Veronica; Camerini, Serena; Crescenzi, Marco; Soddu, Silvia

    2013-06-01

    HIPK2 (homeodomain-interacting protein kinase-2) binds to and phosphorylates, at Ser and Thr residues, a large number of targets involved in cell division and cell fate decision in response to different physiological or stress stimuli. Inactivation of HIPK2 has been observed in human and mouse cancers supporting its role as a tumor suppressor. Despite the biological relevance of this kinase, very little is known on how HIPK2 becomes catalytically active. Based on sequence homologies, HIPK2 has been taxonomically classified as a subfamily member of the dual-specificity tyrosine-regulated kinases (DYRKs) and the activation-loop Y354 of HIPK2 has been found phosphorylated in different cells; however, the relevance of this Y phosphorylation is presently unknown. Here, we show that HIPK2, which is extensively phosphorylated at S/T sites throughout its functional domains, becomes catalytically active by autophosphorylation at the activation-loop Y354. In particular, we found that, in analogy to DYRKs, HIPK2-Y354 phosphorylation is an autocatalytic event and its prevention, through Y354 substitution with non-phosphorylatable amino acids or by using the kinase inhibitor purvalanol A, induces a strong reduction of the HIPK2 S/T-kinase activity on different substrates. Interestingly, at variance from DYRKs, inhibition of HIPK2-Y354 phosphorylation induces a strong out-of-target Y-kinase activity in cis and a strong cytoplasmic relocalization of the kinase. Together, these results demonstrate that the catalytic activity, substrate specificity, and subcellular localization of HIPK2 are regulated by autophosphorylation of its activation-loop Y354. PMID:23485397

  13. HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation

    PubMed Central

    Siepi, Francesca; Gatti, Veronica; Camerini, Serena; Crescenzi, Marco; Soddu, Silvia

    2013-01-01

    HIPK2 (homeodomain-interacting protein kinase-2) binds to and phosphorylates, at Ser and Thr residues, a large number of targets involved in cell division and cell fate decision in response to different physiological or stress stimuli. Inactivation of HIPK2 has been observed in human and mouse cancers supporting its role as a tumor suppressor. Despite the biological relevance of this kinase, very little is known on how HIPK2 becomes catalytically active. Based on sequence homologies, HIPK2 has been taxonomically classified as a subfamily member of the dual-specificity tyrosine-regulated kinases (DYRKs) and the activation-loop Y354 of HIPK2 has been found phosphorylated in different cells; however, the relevance of this Y phosphorylation is presently unknown. Here, we show that HIPK2, which is extensively phosphorylated at S/T sites throughout its functional domains, becomes catalytically active by autophosphorylation at the activation-loop Y354. In particular, we found that, in analogy to DYRKs, HIPK2-Y354 phosphorylation is an autocatalytic event and its prevention, through Y354 substitution with non-phosphorylatable amino acids or by using the kinase inhibitor purvalanol A, induces a strong reduction of the HIPK2 S/T-kinase activity on different substrates. Interestingly, at variance from DYRKs, inhibition of HIPK2-Y354 phosphorylation induces a strong out-of-target Y-kinase activity in cis and a strong cytoplasmic relocalization of the kinase. Together, these results demonstrate that the catalytic activity, substrate specificity, and subcellular localization of HIPK2 are regulated by autophosphorylation of its activation-loop Y354. PMID:23485397

  14. Activation of HIPK2 Promotes ER Stress-Mediated Neurodegeneration in Amyotrophic Lateral Sclerosis.

    PubMed

    Lee, Sebum; Shang, Yulei; Redmond, Stephanie A; Urisman, Anatoly; Tang, Amy A; Li, Kathy H; Burlingame, Alma L; Pak, Ryan A; Jovičić, Ana; Gitler, Aaron D; Wang, Jinhua; Gray, Nathanael S; Seeley, William W; Siddique, Teepu; Bigio, Eileen H; Lee, Virginia M-Y; Trojanowski, John Q; Chan, Jonah R; Huang, Eric J

    2016-07-01

    Persistent accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress, a prominent feature in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here we report the identification of homeodomain interacting protein kinase 2 (HIPK2) as the essential link that promotes ER-stress-induced cell death via the IRE1α-ASK1-JNK pathway. ER stress, induced by tunicamycin or SOD1(G93A), activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain. In SOD1(G93A) mice, loss of HIPK2 delays disease onset, reduces cell death in spinal motor neurons, mitigates glial pathology, and improves survival. Remarkably, HIPK2 activation positively correlates with TDP-43 proteinopathy in NEFH-tTA/tetO-hTDP-43ΔNLS mice, sporadic ALS and C9ORF72 ALS, and blocking HIPK2 kinase activity protects motor neurons from TDP-43 cytotoxicity. These results reveal a previously unrecognized role of HIPK2 activation in ER-stress-mediated neurodegeneration and its potential role as a biomarker and therapeutic target for ALS. VIDEO ABSTRACT. PMID:27321923

  15. Transcriptional regulation of ferritin and antioxidant genes by HIPK2 under genotoxic stress.

    PubMed

    Hailemariam, Kiros; Iwasaki, Kenta; Huang, Bo-Wen; Sakamoto, Kensuke; Tsuji, Yoshiaki

    2010-11-15

    ATF1 (activating transcription factor 1), a stimulus-induced CREB family transcription factor, plays important roles in cell survival and proliferation. Phosphorylation of ATF1 at Ser63 by PKA (cAMP-dependent protein kinase) and related kinases was the only known post-translational regulatory mechanism of ATF1. Here, we found that HIPK2 (homeodomain-interacting protein kinase 2), a DNA-damage-responsive nuclear kinase, is a new ATF1 kinase that phosphorylates Ser198 but not Ser63. ATF1 phosphorylation by HIPK2 activated ATF1 transcription function in the GAL4-reporter system. ATF1 is a transcriptional repressor of ferritin H, the major intracellular iron storage gene, through an ARE (antioxidant-responsive element). HIPK2 overrode the ATF1-mediated ARE repression in a kinase-activity-dependent manner in HepG2 cells. Furthermore, DNA-damage-inducing agents doxorubicin, etoposide and sodium arsenite induced ferritin H mRNA expression in HIPK2(+/+) MEF cells, whereas it was significantly impaired in HIPK2(-/-) MEF cells. Induction of other ARE-regulated detoxification genes such as NQO1 (NADPH quinone oxidoreductase 1), GST (glutathione S-transferase) and HO1 (heme oxygenase 1) by genotoxic stress was also decreased in HIPK2-deficient cells. Taken together, these results suggest that HIPK2 is a new ATF1 kinase involved in the regulation of ferritin H and other antioxidant detoxification genes in genotoxic stress conditions. PMID:20980392

  16. Restoring wtp53 activity in HIPK2 depleted MCF7 cells by modulating metallothionein and zinc.

    PubMed

    Puca, Rosa; Nardinocchi, Lavinia; Bossi, Gianluca; Sacchi, Ada; Rechavi, Gideon; Givol, David; D'Orazi, Gabriella

    2009-01-01

    The maintenance of p53 transactivation activity is important for p53 apoptotic function. We have shown that stable knockdown of HIPK2 induces p53 misfolding with inhibition of p53 target gene transcription. In this study we established a lentiviral-based system for doxycyclin (Dox)-induced conditional interference of HIPK2 expression to evaluate the molecular mechanisms involved in p53 deregulation. We found that HIPK2 knockdown induced metallothionein 2A (MT2A) upregulation as assessed by RT-PCR analysis, increased promoter acetylation, and increased promoter luciferase activity. The MT2A upregulation correlated with resistance to Adriamycin (ADR)-driven apoptosis and with p53 inhibition. Thus, acute knockdown of HIPK2 (HIPK2i) induced misfolded p53 protein in MCF7 breast cancer cells and inhibited p53 DNA-binding and transcription activities in response to ADR treatment. Previous works show that MT may modulate p53 activity through zinc exchange. Here, we found that inhibition of MT2A expression by siRNA in the HIPK2i cells restored p53 transcription activity. Similarly zinc supplementation to HIPK2i cells restored p53 transcription activity and drug-induced apoptosis. These data support the notion that MT2A is involved in p53 deregulation and strengthen the possibility that combination of chemotherapy and zinc might be useful to treat tumors with inactive wtp53. PMID:18996371

  17. Interplay between Mdm2 and HIPK2 in the DNA damage response.

    PubMed

    Zhang, Xiao-Peng; Liu, Feng; Wang, Wei

    2014-07-01

    The tumour suppressor p53 is activated to induce cell-cycle arrest or apoptosis in the DNA damage response (DDR). p53 phosphorylation at Ser46 by HIPK2 (homeodomain-interacting protein kinase 2) is a critical event in apoptosis induction. Interestingly, HIPK2 is degraded by Mdm2 (a negative regulator of p53), whereas Mdm2 is downregulated by HIPK2 through several mechanisms. Here, we develop a four-module network model for the p53 pathway to clarify the role of interplay between Mdm2 and HIPK2 in the DDR evoked by ultraviolet radiation. By numerical simulations, we reveal that Mdm2-dependent HIPK2 degradation promotes cell survival after mild DNA damage and that inhibition of HIPK2 degradation is sufficient to trigger apoptosis. In response to severe damage, p53 phosphorylation at Ser46 is promoted by the accumulation of HIPK2 due to downregulation of nuclear Mdm2 in the later phase of the response. Meanwhile, the concentration of p53 switches from moderate to high levels, contributing to apoptosis induction. We show that the presence of three mechanisms for Mdm2 downregulation, i.e. repression of mdm2 expression, inhibition of its nuclear entry and HIPK2-induced degradation, guarantees the apoptosis of irreparably damaged cells. Our results agree well with multiple experimental observations, and testable predictions are also made. This work advances our understanding of the regulation of p53 activity in the DDR and suggests that HIPK2 should be a significant target for cancer therapy. PMID:24829283

  18. An inducible autoregulatory loop between HIPK2 and Siah2 at the apex of the hypoxic response.

    PubMed

    Calzado, Marco A; de la Vega, Laureano; Möller, Andreas; Bowtell, David D L; Schmitz, M Lienhard

    2009-01-01

    Oxygen deprivation (hypoxia) results in reprogrammed gene expression patterns that induce multifaceted cellular responses. Here we identify a regulated interaction between the serine/threonine kinase HIPK2 and the ubiquitin E3 ligase Siah2 as a mechanism controlling the hypoxic response. Under normoxic conditions, several mechanisms ensure HIPK2 stability: only a fraction of HIPK2 is found in association with Siah2, whereas HIPK2-mediated phosphorylation of this E3 ligase at positions 26, 28 and 68 weakens mutual binding and destabilizes its phosphorylated interaction partner. Hypoxic conditions allow a markedly increased HIPK2/Siah2 interaction and result in efficient polyubiquitylation and proteasomal degradation of the kinase. Accordingly, hypoxia-induced HIPK2 elimination is markedly reduced in Siah2-deficient cells. As HIPK2 has an important role as a negative regulator of gene expression, its elimination from promoter-associated repressor complexes allows the induction of a substantial fraction of hypoxia-induced genes. PMID:19043406

  19. HIPK2 controls cytokinesis and prevents tetraploidization by phosphorylating histone H2B at the midbody.

    PubMed

    Rinaldo, Cinzia; Moncada, Alice; Gradi, Alessandra; Ciuffini, Laura; D'Eliseo, Donatella; Siepi, Francesca; Prodosmo, Andrea; Giorgi, Alessandra; Pierantoni, Giovanna M; Trapasso, Francesco; Guarguaglini, Giulia; Bartolazzi, Armando; Cundari, Enrico; Schininà, M Eugenia; Fusco, Alfredo; Soddu, Silvia

    2012-07-13

    Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization. PMID:22658722

  20. HIPK2 kinase activity depends on cis-autophosphorylation of its activation loop.

    PubMed

    Saul, Vera V; de la Vega, Laureano; Milanovic, Maja; Krüger, Marcus; Braun, Thomas; Fritz-Wolf, Karin; Becker, Katja; Schmitz, M Lienhard

    2013-02-01

    The multitude of mechanisms regulating the activity of protein kinases includes phosphorylation of amino acids contained in the activation loop. Here we show that the serine/threonine kinase HIPK2 (homeodomain-interacting protein kinase 2) is heavily modified by autophosphorylation, which occurs by cis-autophosphorylation at the activation loop and by trans-autophosphorylation at other phosphorylation sites. Cis-autophosphorylation of HIPK2 at Y354 and S357 in the activation loop is essential for its kinase function and the binding to substrates and the interaction partner Pin1. HIPK2 activation loop phosphorylation is also required for its biological activity as a regulator of gene expression and cell proliferation. Phosphorylation of HIPK2 at Y354 alone is not sufficient for full HIPK2 activity, which is in marked contrast to some dual-specificity tyrosine-phosphorylated and regulated kinases where tyrosine phosphorylation is absolutely essential. This study shows that differential phosphorylation of HIPK2 provides a mechanism for controlling and specifying the signal output from this kinase. PMID:23000554

  1. Role of the SUMO-interacting motif in HIPK2 targeting to the PML nuclear bodies and regulation of p53

    SciTech Connect

    Sung, Ki Sa; Lee, Yun-Ah; Kim, Eui Tae; Lee, Seung-Rock; Ahn, Jin-Hyun; Choi, Cheol Yong

    2011-04-15

    Homeodomain-interacting protein kinase 2 (HIPK2) is a key regulator of various transcription factors including p53 and CtBP in the DNA damage signaling pathway. PML-nuclear body (NB) is required for HIPK2-mediated p53 phosphorylation at Ser46 and induction of apoptosis. Although PML-NB targeting of HIPK2 has been shown, much is not clear about the molecular mechanism of HIPK2 recruitment to PML-NBs. Here we show that HIPK2 colocalizes specifically with PML-I and PML-IV. Mutational analysis showed that HIPK2 recruitment to PML-IV-NBs is mediated by the SUMO-interaction motifs (SIMs) of both PML-IV and HIPK2. Wild-type HIPK2 associated with SUMO-conjugated PML-IV at a higher affinity than with un-conjugated PML-IV, while the association of a HIPK2 SIM mutant with SUMO-modified PML-IV was impaired. In colony formation assays, HIPK2 strongly suppressed cell proliferation, but HIPK2 SIM mutants did not. In addition, activation and phosphorylation of p53 at the Ser46 residue were impaired by HIPK2 SIM mutants. These results suggest that SIM-mediated HIPK2 targeting to PML-NBs is crucial for HIPK2-mediated p53 activation and induction of apoptosis.

  2. Role of the SUMO-interacting motif in HIPK2 targeting to the PML nuclear bodies and regulation of p53.

    PubMed

    Sung, Ki Sa; Lee, Yun-Ah; Kim, Eui Tae; Lee, Seung-Rock; Ahn, Jin-Hyun; Choi, Cheol Yong

    2011-04-15

    Homeodomain-interacting protein kinase 2 (HIPK2) is a key regulator of various transcription factors including p53 and CtBP in the DNA damage signaling pathway. PML-nuclear body (NB) is required for HIPK2-mediated p53 phosphorylation at Ser46 and induction of apoptosis. Although PML-NB targeting of HIPK2 has been shown, much is not clear about the molecular mechanism of HIPK2 recruitment to PML-NBs. Here we show that HIPK2 colocalizes specifically with PML-I and PML-IV. Mutational analysis showed that HIPK2 recruitment to PML-IV-NBs is mediated by the SUMO-interaction motifs (SIMs) of both PML-IV and HIPK2. Wild-type HIPK2 associated with SUMO-conjugated PML-IV at a higher affinity than with un-conjugated PML-IV, while the association of a HIPK2 SIM mutant with SUMO-modified PML-IV was impaired. In colony formation assays, HIPK2 strongly suppressed cell proliferation, but HIPK2 SIM mutants did not. In addition, activation and phosphorylation of p53 at the Ser46 residue were impaired by HIPK2 SIM mutants. These results suggest that SIM-mediated HIPK2 targeting to PML-NBs is crucial for HIPK2-mediated p53 activation and induction of apoptosis. PMID:21192925

  3. Apoptosis induced by a HIPK2 full-length-specific siRNA is due to off-target effects rather than prevalence of HIPK2-Δe8 isoform.

    PubMed

    Di Rocco, Giuliana; Verdina, Alessandra; Gatti, Veronica; Virdia, Ilaria; Toietta, Gabriele; Todaro, Matilde; Stassi, Giorgio; Soddu, Silvia

    2016-01-12

    Small interfering RNAs (siRNAs) are widely used to study gene function and extensively exploited for their potential therapeutic applications. HIPK2 is an evolutionary conserved kinase that binds and phosphorylates several proteins directly or indirectly related to apoptosis. Recently, an alternatively spliced isoform skipping 81 nucleotides of exon 8 (Hipk2-Δe8) has been described. Selective depletion of Hipk2 full-length (Hipk2-FL) with a specific siRNA that spares the Hipk2-Δe8 isoform has been shown to strongly induce apoptosis, suggesting an unpredicted dominant-negative effect of Hipk2-FL over the Δe8 isoform. From this observation, we sought to take advantage and assessed the therapeutic potential of generating Hipk2 isoform unbalance in tumor-initiating cells derived from colorectal cancer patients. Strong reduction of cell viability was induced in vitro and in vivo by the originally described exon 8-specific siRNA, supporting a potential therapeutic application. However, validation analyses performed with additional exon8-specific siRNAs with different stabilities showed that all exon8-targeting siRNAs can induce comparable Hipk2 isoform unbalance but only the originally reported e8-siRNA promotes cell death. These data show that loss of viability does not depend on the prevalence of Hipk2-Δe8 isoform but it is rather due to microRNA-like off-target effects. PMID:26625198

  4. PML-RARα and its phosphorylation regulate pml oligomerization and HIPK2 stability.

    PubMed

    Shima, Yutaka; Honma, Yuki; Kitabayashi, Issay

    2013-07-15

    The PML gene is frequently fused to the retinoic acid receptor α (RARα) gene in acute promyelocytic leukemia (APL), generating a characteristic PML-RARα oncogenic chimera. PML-RARα disrupts the discrete nuclear speckles termed nuclear bodies, which are formed in PML, suggesting that nuclear body disruption is involved in leukemogenesis. Nuclear body formation that relies upon PML oligomerization and its stabilization of the hypoxia-inducible protein kinase (HIPK)-2 is disrupted by expression of the PML-RARα chimera. Here, we report that disruption of nuclear bodies is also mediated by PML-RARα inhibition of PML oligomerization. PKA-mediated phosphorylation of PML-RARα blocked its ability to inhibit PML oligomerization and destabilize HIPK2. Our results establish that both PML oligomerization and HIPK2 stabilization at nuclear bodies are important for APL cell differentiation, offering insights into the basis for the most common prodifferentiation therapies of APL used clinically. PMID:23722549

  5. Homeodomain-interacting protein kinase 2 (HIPK2) targets {beta}-catenin for phosphorylation and proteasomal degradation

    SciTech Connect

    Kim, Eun-A; Kim, Ji Eon; Sung, Ki Sa; Choi, Dong Wook; Lee, Byeong Jae; Choi, Cheol Yong

    2010-04-16

    The regulation of intracellular {beta}-catenin levels is central in the Wnt/{beta}-catenin signaling cascade and the activation of the Wnt target genes. Here, we show that homeodomain-interacting protein kinase 2 (HIPK2) acts as a negative regulator of the Wnt/{beta}-catenin pathway. Knock-down of endogenous HIPK2 increases the stability of {beta}-catenin and results in the accumulation of {beta}-catenin in the nucleus, consequently enhancing the expression of Wnt target genes and cell proliferation both in vivo and in cultured cells. HIPK2 inhibits TCF/LEF-mediated target gene activation via degradation of {beta}-catenin. HIPK2 phosphorylates {beta}-catenin at its Ser33 and Ser37 residues without the aid of a priming kinase. Substitutions of Ser33 and Ser37 for alanines abolished the degradation of {beta}-catenin associated with HIPK2. In ex vivo mouse model, HIPK2 knock-down resulted in accumulation of {beta}-catenin, thereby potentiated {beta}-catenin-mediated cell proliferation and tumor formation. Furthermore, the axis duplication induced by the ectopic expression of {beta}-catenin was blocked by co-injection of HIPK2 mRNAs into Xenopus embryos. Taken together, HIPK2 appears to function as a novel negative regulator of {beta}-catenin through its phosphorylation and proteasomal degradation.

  6. Homeodomain-interacting protein kinase 2 (HIPK2) targets beta-catenin for phosphorylation and proteasomal degradation.

    PubMed

    Kim, Eun-A; Kim, Ji Eon; Sung, Ki Sa; Choi, Dong Wook; Lee, Byeong Jae; Choi, Cheol Yong

    2010-04-16

    The regulation of intracellular beta-catenin levels is central in the Wnt/beta-catenin signaling cascade and the activation of the Wnt target genes. Here, we show that homeodomain-interacting protein kinase 2 (HIPK2) acts as a negative regulator of the Wnt/beta-catenin pathway. Knock-down of endogenous HIPK2 increases the stability of beta-catenin and results in the accumulation of beta-catenin in the nucleus, consequently enhancing the expression of Wnt target genes and cell proliferation both in vivo and in cultured cells. HIPK2 inhibits TCF/LEF-mediated target gene activation via degradation of beta-catenin. HIPK2 phosphorylates beta-catenin at its Ser33 and Ser37 residues without the aid of a priming kinase. Substitutions of Ser33 and Ser37 for alanines abolished the degradation of beta-catenin associated with HIPK2. In ex vivo mouse model, HIPK2 knock-down resulted in accumulation of beta-catenin, thereby potentiated beta-catenin-mediated cell proliferation and tumor formation. Furthermore, the axis duplication induced by the ectopic expression of beta-catenin was blocked by co-injection of HIPK2 mRNAs into Xenopus embryos. Taken together, HIPK2 appears to function as a novel negative regulator of beta-catenin through its phosphorylation and proteasomal degradation. PMID:20307497

  7. HIPK2: a multitalented partner for transcription factors in DNA damage response and development.

    PubMed

    Rinaldo, Cinzia; Prodosmo, Andrea; Siepi, Francesca; Soddu, Silvia

    2007-08-01

    Protein phosphorylation is a widely diffuse and versatile post-translational modification that controls many cellular processes, from signal transduction to gene transcription. The homeodomain-interacting protein kinases (HIPKs) belong to a new family of serine-threonine kinases first identified as corepressors for homeodomain transcription factors. Different screenings for the identification of new partners of transcription factors have indicated that HIPK2, the best characterized member of the HIPK family, is a multitalented coregulator of an increasing number of transcription factors and cofactors. The aim of this review is to describe the different mechanisms through which HIPK2 regulates gene transcription. PMID:17713576

  8. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism.

    PubMed

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  9. Hypoxia suppresses chemotherapeutic drug-induced p53 Serine 46 phosphorylation by triggering HIPK2 degradation.

    PubMed

    Moehlenbrink, Jutta; Bitomsky, Nadja; Hofmann, Thomas G

    2010-06-01

    The molecular mechanisms by which hypoxic tumor cells escape radio- and chemotherapy are largely unclear. Homeodomain-interacting protein kinase 2 (HIPK2) drives the apoptotic program in response to DNA-damaging chemotherapeutic drug treatment by phosphorylating the tumor suppressor protein p53 at Ser46. HIPK2 is kept inactive in unstressed cells through ubiquitination and degradation facilitated by the ubiquitin ligases WSB1 and Siah1. Here, we demonstrate that HIPK2 is degraded during hypoxia in a proteasome-dependent and partially Siah1-dependent fashion. Concordantly, hypoxic tumor cells show an impaired p53 Ser46 phosphorylation in response to treatment with the chemotherapeutic Adriamycin. Remarkably, proteasome-inhibition rescues HIPK2 expression in hypoxic hepatoma cells and restores p53 Ser46 phosphorylation and caspase activity after Adriamycin treatment. Our findings suggest a molecular mechanism by which hypoxic cancer cells can escape chemotherapeutic drug treatment and suggest proteasome-inhibition as a promising approach to sensitise hypoxic cancer cells to therapy. PMID:20018442

  10. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

    PubMed Central

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  11. WIP1, a homeostatic regulator of the DNA damage response, is targeted by HIPK2 for phosphorylation and degradation.

    PubMed

    Choi, Dong Wook; Na, Wooju; Kabir, Mohammad Humayun; Yi, Eunbi; Kwon, Seonjeong; Yeom, Jeonghun; Ahn, Jang-Won; Choi, Hee-Hyun; Lee, Youngha; Seo, Kyoung Wan; Shin, Min Kyoo; Park, Se-Ho; Yoo, Hae Yong; Isono, Kyo-Ichi; Koseki, Haruhiko; Kim, Seong-Tae; Lee, Cheolju; Kwon, Yunhee Kim; Choi, Cheol Yong

    2013-08-01

    WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPKα2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATM-mediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IR-induced γ-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/- mice upon γ-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR. PMID:23871434

  12. Phosphorylation-dependent control of Pc2 SUMO E3 ligase activity by its substrate protein HIPK2.

    PubMed

    Roscic, Ana; Möller, Andreas; Calzado, Marco A; Renner, Florian; Wimmer, Verena C; Gresko, Ekaterina; Lüdi, Katharina Schmid; Schmitz, M Lienhard

    2006-10-01

    Sumoylation serves to control key cellular functions, but the regulation of SUMO E3 ligase activity is largely unknown. Here we show that the polycomb group protein Pc2 binds to and colocalizes with homeodomain interacting protein kinase 2 (HIPK2) and serves as a SUMO E3 ligase for this kinase. DNA damage-induced HIPK2 directly phosphorylates Pc2 at multiple sites, which in turn controls Pc2 sumoylation and intranuclear localization. Inducible phosphorylation of Pc2 at threonine 495 is required for its ability to increase HIPK2 sumoylation in response to DNA damage, thereby establishing an autoregulatory feedback loop between a SUMO substrate and its cognate E3 ligase. Sumoylation enhances the ability of HIPK2 to mediate transcriptional repression, thus providing a mechanistic link for DNA damage-induced transcriptional silencing. PMID:17018294

  13. MDM2-regulated degradation of HIPK2 prevents p53Ser46 phosphorylation and DNA damage-induced apoptosis.

    PubMed

    Rinaldo, Cinzia; Prodosmo, Andrea; Mancini, Francesca; Iacovelli, Stefano; Sacchi, Ada; Moretti, Fabiola; Soddu, Silvia

    2007-03-01

    In response to DNA damage, p53 induces either cell-cycle arrest or apoptosis by differential transcription of several target genes and through transcription-independent apoptotic functions. p53 phosphorylation at Ser46 by HIPK2 is one determinant of the outcome because it takes place only upon severe, nonrepairable DNA damage that irreversibly drives cells to apoptosis. Here, we show that p53 represses its proapoptotic activator HIPK2 via MDM2-mediated degradation, whereas a degradation-resistant HIPK2 mutant has increased apoptotic activity. Upon cytostatic, nonsevere DNA damage, inhibition of HIPK2 degradation is sufficient to induce p53Ser46 phosphorylation and apoptosis, converting growth-arresting stimuli to apoptotic ones. These findings establish HIPK2 as an MDM2 target and support a model in which, upon nonsevere DNA damage, p53 represses its own phosphorylation at Ser46 due to HIPK2 degradation, supporting the notion that the cell-cycle-arresting functions of p53 include active inhibition of the apoptotic ones. PMID:17349959

  14. HIF-1α antagonizes p53-mediated apoptosis by triggering HIPK2 degradation

    PubMed Central

    Nardinocchi, Lavinia; Puca, Rosa; D'Orazi, Gabriella

    2011-01-01

    Many human diseases are characterized by the development of tissue hypoxia. Hypoxia-inducible factor (HIF) is a transcription factor that regulates fundamental cellular processes in response to changes in oxygen concentration, such as angiogenesis, survival, and alterations in metabolism. The levels of HIF-1α subunit are increased in most solid tumors not only by low oxygen but also by growth factors and oncogenes and correlate with patient prognosis and treatment failure. The link between HIF-1α and apoptosis, a major determinant of cancer progression and treatment outcome, is poorly understood. Here we show that HIF-1α protects against drug-induced apoptosis by antagonizing the function of the tumor suppressor p53. HIF-1α upregulation induced proteasomal degradation of homeodomain-interacting protein kinase-2 (HIPK2), the p53 apoptotic activator. Inhibition of HIF-1α by siRNA, HIF-1α-dominant negative or by zinc re-established the HIPK2 levels and the p53-mediated chemosensitivity in tumor cells. Our findings identify a novel circuitry between HIF-1α and p53, and provide a paradigm for HIPK2 dictating cell response to antitumor therapies. PMID:21248371

  15. Regulation of p53 activity by HIPK2: molecular mechanisms and therapeutical implications in human cancer cells.

    PubMed

    Puca, R; Nardinocchi, L; Givol, D; D'Orazi, G

    2010-08-01

    The p53 protein is the most studied tumor suppressor and the p53 pathway has been shown to mediate cellular stress responses that are disrupted when cancer develops. After DNA damage, p53 is activated as transcription factor to directly induce the expression of target genes involved in cell-cycle arrest, DNA repair, senescence and, importantly, apoptosis. Post-translational modifications of p53 are essential for the activation of p53 and for selection of target genes. The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) is a crucial regulator of p53 apoptotic function by phosphorylating its N-terminal serine 46 (Ser46) and facilitating Lys382 acetylation at the C-terminus. HIPK2 is activated by numerous genotoxic agents and can be deregulated in tumors by several conditions including hypoxia. Recent findings suggest that HIPK2 active/inactive protein can affect p53 function in multiple and unexpected ways. This makes p53 as well as HIPK2 interesting targets for cancer therapy. Hence, understanding the role of HIPK2 as p53 activator may provide important insights in the process of tumor progression, and may also serve as the crucial point in the diagnostic and therapeutical aspects of cancer. PMID:20514025

  16. Daxx cooperates with the Axin/HIPK2/p53 complex to induce cell death.

    PubMed

    Li, Qinxi; Wang, Xuan; Wu, Xiaoling; Rui, Yanning; Liu, Wei; Wang, Jifeng; Wang, Xinghao; Liou, Yih-Cherng; Ye, Zhiyun; Lin, Sheng-Cai

    2007-01-01

    Daxx, a death domain-associated protein, has been implicated in proapoptosis, antiapoptosis, and transcriptional regulation. Many factors known to play critically important roles in controlling apoptosis and gene transcription have been shown to associate with Daxx, including the Ser/Thr protein kinase HIPK2, promyelocytic leukemia protein, histone deacetylases, and the chromatin remodeling protein ATRX. Although it is clear that Daxx may exert multiple functions, the underlying mechanisms remain far from clear. Here, we show that Axin, originally identified for its scaffolding role to control beta-catenin levels in Wnt signaling, strongly associates with Daxx at endogenous levels. The Daxx/Axin complex formation is enhanced by UV irradiation. Axin tethers Daxx to the tumor suppressor p53, and cooperates with Daxx, but not DaxxDeltaAxin, which is unable to interact with Axin, to stimulate HIPK2-mediated Ser(46) phosphorylation and transcriptional activity of p53. Interestingly, Axin and Daxx seem to selectively activate p53 target genes, with strong activation of PUMA, but not p21 or Bax. Daxx-stimulated p53 transcriptional activity was significantly diminished by small interfering RNA against Axin; Daxx fails to inhibit colony formation in Axin(-/-) cells. Moreover, UV-induced cell death was attenuated by the knockdown of Axin and Daxx. All these results show that Daxx cooperates with Axin to stimulate p53, and implicate a direct role for Axin, HIPK2, and p53 in the proapoptotic function of Daxx. We have hence unraveled a novel aspect of p53 activation and shed new light on the ultimate understanding of the Daxx protein, perhaps most pertinently, in relation to stress-induced cell death. PMID:17210684

  17. HIPK2 is a new drug target for anti-fibrosis therapy in kidney disease.

    PubMed

    Nugent, Melinda M; Lee, Kyung; He, John Cijiang

    2015-01-01

    In vitro and animal studies continue to elucidate the mechanisms of fibrosis and have led to advancements in treatment for idiopathic pulmonary fibrosis and cirrhosis, but the search for treatments for renal fibrosis has been more disappointing. Here, we will discuss homeodomain-interacting-protein kinase 2 (HIPK2), a novel regulator of fibrosis that acts upstream of major fibrosis signaling pathways. Its key role in renal fibrosis has been validated in vitro and in several murine models of chronic kidney diseases (CKD). PMID:25972814

  18. Hipk2 and PP1c cooperate to maintain Dvl protein levels required for Wnt signal transduction.

    PubMed

    Shimizu, Nobuyuki; Ishitani, Shizuka; Sato, Atsushi; Shibuya, Hiroshi; Ishitani, Tohru

    2014-09-11

    The phosphoprotein Dishevelled (Dvl) is a common essential component of Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. Here, we show that homeodomain-interacting protein kinase 2 (Hipk2) facilitates protein phosphatase 1 catalytic subunit (PP1c)-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/β-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. We also show that Wnt-3a, a Wnt/β-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/β-catenin signaling. PMID:25159144

  19. The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage.

    PubMed

    Reuven, Nina; Adler, Julia; Porat, Ziv; Polonio-Vallon, Tilman; Hofmann, Thomas G; Shaul, Yosef

    2015-07-01

    The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate. PMID:25944899

  20. Interaction and cooperation of the CCAAT-box enhancer-binding protein β (C/EBPβ) with the homeodomain-interacting protein kinase 2 (Hipk2).

    PubMed

    Steinmann, Simone; Coulibaly, Anna; Ohnheiser, Johanna; Jakobs, Anke; Klempnauer, Karl-Heinz

    2013-08-01

    CCAAT box/enhancer-binding protein β (C/EBPβ) is a bZip transcription factor that plays crucial roles in important cellular processes such as differentiation and proliferation of specific cell types. Previously, we showed that C/EBPβ cooperates with the coactivator p300 through a novel mechanism that involves the C/EBPβ-induced phosphorylation of multiple sites in the carboxyl-terminal domain of p300 by protein kinase Hipk2. We have now examined the interaction and cooperation of C/EBPβ, p300, and Hipk2 in more detail. We show that Hipk2 and C/EBPβ are direct physical binding partners whose interaction is mediated by sequences located in the amino-terminal and central domains of Hipk2 and the amino-terminal part of C/EBPβ. In addition to phosphorylating p300 recruited to C/EBPβ, Hipk2 also phosphorylates C/EBPβ at sites that have previously been shown to plays key roles in the regulation of C/EBPβ activity. Silencing of Hipk2 expression disrupts adipocyte differentiation of 3T3-L1 cells, a physiological C/EBPβ-dependent differentiation process indicating that the cooperation of C/EBPβ and Hipk2 is functionally relevant. Finally, we demonstrate that C/EBPα, a related C/EBP family member whose amino-terminal sequences differ significantly from that of C/EBPβ, is unable to interact and cooperate with Hipk2. Instead, our data suggest that C/EBPα cooperates with the protein kinase Jnk to induce phosphorylation of p300. Overall, our data identify Hipk2 as a novel regulator of C/EBPβ and implicate different protein kinases in the cooperation of p300 with C/EBPβ and C/EBPα. PMID:23782693

  1. Overexpression of Dlx2 leads to postnatal condyle degradation

    PubMed Central

    Dai, Jiewen; Si, Jiawen; Zhu, Xiaofang; Zhang, Lei; Wu, Dandan; Lu, Jingting; Ouyang, Ningjuan; Wang, Xudong; Shen, Guofang

    2016-01-01

    Distal-less homeobox 2 (Dlx2), a member of the Dlx family of transcription factors, is important for the development of craniofacial tissues. Previous studies based on knock-out mutant mice revealed that Dlx2 primarily disturbed the development of tissues from maxillary arch. The present study used a transgenic mouse model to specifically overexpress Dlx2 in neural crest cells in order to investigate the role of Dlx2 overexpression in post-natal condyle in mice. The model was constructed and the phenotype observed using gross observation, micro-CT scan and histological examination. The model determined that overexpression of Dlx2 may lead to postnatal condyle malformation, subchondral bone degradation and irregular histological structure of the condylar cartilage. In addition, the expression of osteocalcin in the condyle region was markedly downregulated, whereas expression of msh homeobox 2 was upregulated. The results of the present study suggest that Dlx2 overexpression in cranial neural crest cells would disrupt the development of post-natal condyle, which demonstrates that the expression level and the spatiotemporal expression patterns of Dlx2 may be important in regulating the development of post-natal condyle in mice, and also offered a possible temporal-mandibular joint osteoarthritis model animal for future studies. PMID:27315306

  2. Roles of HIPK1 and HIPK2 in AML1- and p300-dependent transcription, hematopoiesis and blood vessel formation.

    PubMed

    Aikawa, Yukiko; Nguyen, Lan Anh; Isono, Kyoichi; Takakura, Nobuyuki; Tagata, Yusuke; Schmitz, M Lienhard; Koseki, Haruhiko; Kitabayashi, Issay

    2006-09-01

    Histone acetyltransferases (HATs) p300 and CREB-binding protein (CBP) function as co-activators for a variety of sequence-specific transcription factors, including AML1. Here, we report that homeodomain-interacting protein kinase-2 (HIPK2) forms a complex with AML1 and p300, and phosphorylates both AML1 and p300 to stimulate transcription activation as well as HAT activities. Phosphorylation of p300 is triggered by phosphorylated AML1 as well as by PU.1, c-MYB, c-JUN and c-FOS, and is inhibited by dominant-negative HIPK2. Phosphorylation of p300 and AML1 is impaired in Hipk1/2 double-deficient mouse embryos. Double-deficient mice exhibit defects in primitive/definitive hematopoiesis, vasculogenesis, angiogenesis and neural tube closure. These phenotypes are in part similar to those observed in p300- and CBP-deficient mice. HIPK2 also phosphorylates another co-activator, MOZ, in an AML1-dependent manner. We discuss a possible mechanism by which transcription factors could regulate local histone acetylation and transcription of their target genes. PMID:16917507

  3. XAF1 directs apoptotic switch of p53 signaling through activation of HIPK2 and ZNF313.

    PubMed

    Lee, Min-Goo; Han, Jikhyon; Jeong, Seong-In; Her, Nam-Gu; Lee, Jin-Hee; Ha, Tae-Kyu; Kang, Min-Ju; Ryu, Byung-Kyu; Chi, Sung-Gil

    2014-10-28

    X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a tumor suppressor that is frequently inactivated in many human cancers. However, the molecular mechanism underlying its growth-inhibitory function remains largely unknown. Here, we report that XAF1 forms a positive feedback loop with p53 and acts as a molecular switch in p53-mediated cell-fate decisions favoring apoptosis over cell-cycle arrest. XAF1 binds directly to the N-terminal proline-rich domain of p53 and thus interferes with E3 ubiquitin ligase MDM2 binding and ubiquitination of p53. XAF1 stimulates homeodomain-interacting protein kinase 2 (HIPK2)-mediated Ser-46 phosphorylation of p53 by blocking E3 ubiquitin ligase Siah2 interaction with and ubiquitination of HIPK2. XAF1 also steps up the termination of p53-mediated cell-cycle arrest by activating zinc finger protein 313 (ZNF313), a p21(WAF1)-targeting ubiquitin E3 ligase. XAF1 interacts with p53, Siah2, and ZNF313 through the zinc finger domains 5, 6, and 7, respectively, and truncated XAF1 isoforms preferentially expressed in cancer cells fail to form a feedback loop with p53. Together, this study uncovers a novel role for XAF1 in p53 stress response, adding a new layer of complexity to the mechanisms by which p53 determines cell-fate decisions. PMID:25313037

  4. Prolyl isomerase Pin1 and protein kinase HIPK2 cooperate to promote cortical neurogenesis by suppressing Groucho/TLE:Hes1-mediated inhibition of neuronal differentiation.

    PubMed

    Ciarapica, R; Methot, L; Tang, Y; Lo, R; Dali, R; Buscarlet, M; Locatelli, F; del Sal, G; Rota, R; Stifani, S

    2014-02-01

    The Groucho/transducin-like Enhancer of split 1 (Gro/TLE1):Hes1 transcriptional repression complex acts in cerebral cortical neural progenitor cells to inhibit neuronal differentiation. The molecular mechanisms that regulate the anti-neurogenic function of the Gro/TLE1:Hes1 complex during cortical neurogenesis remain to be defined. Here we show that prolyl isomerase Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1) and homeodomain-interacting protein kinase 2 (HIPK2) are expressed in cortical neural progenitor cells and form a complex that interacts with the Gro/TLE1:Hes1 complex. This association depends on the enzymatic activities of both HIPK2 and Pin1, as well as on the association of Gro/TLE1 with Hes1, but is independent of the previously described Hes1-activated phosphorylation of Gro/TLE1. Interaction with the Pin1:HIPK2 complex results in Gro/TLE1 hyperphosphorylation and weakens both the transcriptional repression activity and the anti-neurogenic function of the Gro/TLE1:Hes1 complex. These results provide evidence that HIPK2 and Pin1 work together to promote cortical neurogenesis, at least in part, by suppressing Gro/TLE1:Hes1-mediated inhibition of neuronal differentiation. PMID:24270405

  5. Homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and modulates its DNA binding affinity.

    PubMed

    Zhang, Qingchun; Wang, Yinsheng

    2007-12-01

    The chromosomal high-mobility group A (HMGA) proteins, composed of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation, and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated, HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook. PMID:17960875

  6. Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization.

    PubMed

    An, Rong; da Silva Xavier, Gabriela; Semplici, Francesca; Vakhshouri, Saharnaz; Hao, Huai-Xiang; Rutter, Jared; Pagano, Mario A; Meggio, Flavio; Pinna, Lorenzo A; Rutter, Guy A

    2010-08-20

    Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature beta-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in beta-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 beta-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1 in vitro; phosphate incorporation substantially decreases in PDX1 S269A mutant. Silencing of HIPK2 led to a 51+/-0.2% decrease in Ser-269 phosphorylation in MIN6 beta-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function. PMID:20637728

  7. NORE1A is a Ras senescence effector that controls the apoptotic/senescent balance of p53 via HIPK2.

    PubMed

    Donninger, Howard; Calvisi, Diego F; Barnoud, Thibaut; Clark, Jennifer; Schmidt, M Lee; Vos, Michele D; Clark, Geoffrey J

    2015-03-16

    The Ras oncoprotein is a key driver of cancer. However, Ras also provokes senescence, which serves as a major barrier to Ras-driven transformation. Ras senescence pathways remain poorly characterized. NORE1A is a novel Ras effector that serves as a tumor suppressor. It is frequently inactivated in tumors. We show that NORE1A is a powerful Ras senescence effector and that down-regulation of NORE1A suppresses senescence induction by Ras and enhances Ras transformation. We show that Ras induces the formation of a complex between NORE1A and the kinase HIPK2, enhancing HIPK2 association with p53. HIPK2 is a tumor suppressor that can induce either proapoptotic or prosenescent posttranslational modifications of p53. NORE1A acts to suppress its proapoptotic phosphorylation of p53 but enhance its prosenescent acetylation of p53. Thus, we identify a major new Ras signaling pathway that links Ras to the control of specific protein acetylation and show how NORE1A allows Ras to qualitatively modify p53 function to promote senescence. PMID:25778922

  8. NORE1A is a Ras senescence effector that controls the apoptotic/senescent balance of p53 via HIPK2

    PubMed Central

    Donninger, Howard; Calvisi, Diego F.; Barnoud, Thibaut; Clark, Jennifer; Schmidt, M. Lee; Vos, Michele D.

    2015-01-01

    The Ras oncoprotein is a key driver of cancer. However, Ras also provokes senescence, which serves as a major barrier to Ras-driven transformation. Ras senescence pathways remain poorly characterized. NORE1A is a novel Ras effector that serves as a tumor suppressor. It is frequently inactivated in tumors. We show that NORE1A is a powerful Ras senescence effector and that down-regulation of NORE1A suppresses senescence induction by Ras and enhances Ras transformation. We show that Ras induces the formation of a complex between NORE1A and the kinase HIPK2, enhancing HIPK2 association with p53. HIPK2 is a tumor suppressor that can induce either proapoptotic or prosenescent posttranslational modifications of p53. NORE1A acts to suppress its proapoptotic phosphorylation of p53 but enhance its prosenescent acetylation of p53. Thus, we identify a major new Ras signaling pathway that links Ras to the control of specific protein acetylation and show how NORE1A allows Ras to qualitatively modify p53 function to promote senescence. PMID:25778922

  9. Dual retrovirus integration tagging: identification of new signaling molecules Fiz1 and Hipk2 that are involved in the IL-7 signaling pathway in B lymphoblastic lymphomas.

    PubMed

    Tsuruyama, Tatsuaki; Imai, Yukiko; Takeuchi, Haruya; Hiratsuka, Takuya; Maruyama, Yasuhiro; Kanaya, Kazuya; Kaszynski, Richard; Jin, Guang; Okuno, Tomoko; Ozeki, Munetaka; Nakamura, Takuro; Takakuwa, Tetsuya; Manabe, Toshiaki; Tamaki, Keiji; Hiai, Hiroshi

    2010-07-01

    IL-7R, FLT3, and CD43 are surface antigens expressed during the transition from pro-B to pre-B cells in BM. To understand interactions between their signaling pathways, we analyzed spontaneous mouse B-LBLs with dual MLV integration into Stat5a and Fiz1 or Stat5a and Hipk2. MLV integration resulted in up-regulation of these genes in lymphoma cells compared with normal pro-B cells from the BM. In lymphomas with both integrations into Stat5a and Fiz1, increases in phosphorylated STAT5A and expression of c-Myc, a target gene of STAT5A, were observed following stimulation of the FLT3. Clones with the dual integrations grew faster in IL-7 and FLT3L-supplemented medium than clones with Stat5a integration alone. On the other hand, in lymphomas with integrations into Stat5a and Hipk2, increases in phosphorylated STAT5A and expression of c-Myc were observed following cross-linking of CD43. In conclusion, FLT3 and CD43 signaling pathways involve STAT5A via Fiz1 and Hipk2 in B-LBLs. Identification of the dual MLV integration sites in B-LBLs, therefore, will provide an excellent tool for identification of the signaling pathways in B-LBLs. PMID:20360400

  10. Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase

    PubMed Central

    Lee, Kwon Ho; Lee, Sang Ki; Kim, Hyo Shin; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Eun Ji; Lee, Ji Young; Park, Myoung Soo; Chang, Seok Jong; Cho, Chung-Hyun; Park, Jin Bong

    2009-01-01

    The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells. PMID:20054488

  11. Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

    PubMed

    Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin

    2015-03-27

    Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. PMID:25659434

  12. Tumor suppressor protein Pdcd4 interacts with Daxx and modulates the stability of Daxx and the Hipk2-dependent phosphorylation of p53 at serine 46.

    PubMed

    Kumar, N; Wethkamp, N; Waters, L C; Carr, M D; Klempnauer, K-H

    2013-01-01

    The tumor suppressor protein Pdcd4 is a nuclear/cytoplasmic shuttling protein that has been implicated in the development of several types of human cancer. In the nucleus, Pdcd4 affects the transcription of specific genes by modulating the activity of several transcription factors. We have identified the Daxx protein as a novel interaction partner of Pdcd4. Daxx is a scaffold protein with roles in diverse processes, including transcriptional regulation, DNA-damage signaling, apoptosis and chromatin remodeling. We show that the interaction of both proteins is mediated by the N-terminal domain of Pdcd4 and the central part of Daxx, and that binding to Pdcd4 stimulates the degradation of Daxx, presumably by disrupting the interaction of Daxx with the de-ubiquitinylating enzyme Hausp. Daxx has previously been shown to serve as a scaffold for protein kinase Hipk2 and tumor suppressor protein p53 and to stimulate the phosphorylation of p53 at serine 46 (Ser-46) in response to genotoxic stress. We show that Pdcd4 also disrupts the Daxx-Hipk2 interaction and inhibits the phosphorylation of p53. We also show that ultraviolet irradiation decreases the expression of Pdcd4. Taken together, our results support a model in which Pdcd4 serves to suppress the phosphorylation of p53 in the absence of DNA damage, while the suppressive effect of Pdcd4 is abrogated after DNA damage owing to the decrease of Pdcd4. Overall, our data demonstrate that Pdcd4 is a novel modulator of Daxx function and provide evidence for a role of Pdcd4 in restraining p53 activity in unstressed cells. PMID:23536002

  13. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato.

    PubMed

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K; Arias, Cintia L; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E; González, María de la Cruz; Hernández, Jose A; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-02-01

    Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the

  14. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato

    PubMed Central

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K.; Arias, Cintia L.; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E.; González, María de la Cruz; Hernández, Jose A.; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-01-01

    Drought stress conditions modify source–sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of

  15. Overexpression of the Type 1 Adenylyl Cyclase in the Forebrain Leads to Deficits of Behavioral Inhibition

    PubMed Central

    Cao, Hong; Saraf, Amit; Zweifel, Larry S.

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition. PMID:25568126

  16. ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice

    PubMed Central

    Vaisman, Boris L.; Lambert, Gilles; Amar, Marcelo; Joyce, Charles; Ito, Toshimitsu; Shamburek, Robert D.; Cain, William J.; Fruchart-Najib, Jamila; Neufeld, Edward D.; Remaley, Alan T.; Brewer, H. Bryan; Santamarina-Fojo, Silvia

    2001-01-01

    The discovery of the ABCA1 lipid transporter has generated interest in modulating human plasma HDL levels and atherogenic risk by enhancing ABCA1 gene expression. To determine if increased ABCA1 expression modulates HDL metabolism in vivo, we generated transgenic mice that overexpress human ABCA1 (hABCA1-Tg). Hepatic and macrophage expression of hABCA1 enhanced macrophage cholesterol efflux to apoA-I; increased plasma cholesterol, cholesteryl esters (CEs), free cholesterol, phospholipids, HDL cholesterol, and apoA-I and apoB levels; and led to the accumulation of apoE-rich HDL1. ABCA1 transgene expression delayed 125I-apoA-I catabolism in both liver and kidney, leading to increased plasma apoA-I levels, but had no effect on apoB secretion after infusion of Triton WR1339. Although the plasma clearance of HDL-CE was not significantly altered in hABCA1-Tg mice, the net hepatic delivery of exogenous 3H-CEt-HDL, which is dependent on the HDL pool size, was increased 1.5-fold. In addition, the cholesterol and phospholipid concentrations in hABCA1-Tg bile were increased 1.8-fold. These studies show that steady-state overexpression of ABCA1 in vivo (a) raises plasma apoB levels without altering apoB secretion and (b) raises plasma HDL-C and apoA-I levels, facilitating hepatic reverse cholesterol transport and biliary cholesterol excretion. Similar metabolic changes may modify atherogenic risk in humans. PMID:11457883

  17. Overexpressed CacyBP/SIP leads to the suppression of growth in renal cell carcinoma

    SciTech Connect

    Sun, Shiren; Ning, Xiaoxuan; Liu, Jie; Liu, Lili; Chen, Yu; Han, Shuang; Zhang, Yanqi; Liang, Jie; Wu, Kaichun; Fan, Daiming . E-mail: fandaim@fmmu.edu.cn

    2007-05-18

    Calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP), a target protein of S100, has been identified as a component of a novel ubiquitinylation complex leading to {beta}-catenin degradation, which was found to be related to the malignant phenotypes of gastric cancer. However, the roles of CacyBP/SIP in renal cell carcinoma still remain unclear. In the present study, we had analyzed the expression of the CacyBP/SIP protein in human renal cancer cells and clinical tissue samples. The possible roles of CacyBP/SIP in regulating the malignant phenotype of renal cancer cells were also investigated. The results demonstrated that the expression of CacyBP/SIP was markedly down-regulated in renal cell carcinoma tissues and cell lines. Ectopic overexpression of CacyBP/SIP in A498 cells inhibited the proliferation of this cell and delayed cell cycle progression significantly, which might be related to the down-regulation of Cyclin D1 through reducing {beta}-catenin protein. CacyBP/SIP also suppressed colony formation in soft agar and its tumorigenicity in nude mice. Taken together, our work showed that CacyBP/SIP, as a novel down-regulated gene in renal cell carcinoma, suppressed proliferation and tumorigenesis of renal cancer cells.

  18. Overexpression of the SK3 channel alters vascular remodeling during pregnancy, leading to fetal demise

    PubMed Central

    Rada, Cara C.; Pierce, Stephanie L.; Nuno, Daniel W.; Zimmerman, Kathy; Lamping, Kathryn G.; Bowdler, Noelle C.; Weiss, Robert M.

    2012-01-01

    The maternal cardiovascular system undergoes hemodynamic changes during pregnancy via angiogenesis and vasodilation to ensure adequate perfusion of the placenta. Improper vascularization at the maternal-fetal interface can cause pregnancy complications and poor fetal outcomes. Recent evidence indicates that small conductance Ca2+-activated K+ channel subtype 3 (SK3) contributes to vascular remodeling during pregnancy, and we hypothesized that abnormal SK3 channel expression would alter the ability of the maternal cardiovascular system to adapt to pregnancy demands and lead to poor fetal outcomes. We investigated this hypothesis using transgenic Kcnn3tm1Jpad/Kcnn3tm1Jpad (SK3T/T) mice that overexpress the channel. Isolated pressurized uterine arteries from nonpregnant transgenic SK3T/T mice had larger basal diameters and decreased agonist-induced constriction than those from their wild-type counterparts; however, non-receptor-mediated depolarization remained intact. In addition to vascular changes, heart rates and ejection fraction were increased, whereas end systolic volume was reduced in SK3T/T mice compared with their wild-type littermates. Uterine sonography of the fetuses on pregnancy day 14 showed a significant decrease in fetal size in SK3T/T compared with wild-type mice; thus, SK3T/T mice displayed an intrauterine growth-restricted phenotype. The SK3T/T mice showed decreased placental thicknesses and higher incidence of fetal loss, losing over half of their complement of pups by midgestation. These results establish that the SK3 channel contributes to both maternal and fetal outcomes during pregnancy and point to the importance of SK3 channel regulation in maintaining a healthy pregnancy. PMID:22785240

  19. MusaWRKY71 overexpression in banana plants leads to altered abiotic and biotic stress responses.

    PubMed

    Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2013-01-01

    WRKY transcription factors are specifically involved in the transcriptional reprogramming following incidence of abiotic or biotic stress on plants. We have previously documented a novel WRKY gene from banana, MusaWRKY71, which was inducible in response to a wide array of abiotic or biotic stress stimuli. The present work details the effects of MusaWRKY71 overexpression in transgenic banana plants. Stable integration and overexpression of MusaWRKY71 in transgenic banana plants was proved by Southern blot analysis and quantitative real time PCR. Transgenic banana plants overexpressing MusaWRKY71 displayed enhanced tolerance towards oxidative and salt stress as indicated by better photosynthesis efficiency (Fv/Fm) and lower membrane damage of the assayed leaves. Further, differential regulation of putative downstream genes of MusaWRKY71 was investigated using real-time RT-PCR expression analysis. Out of a total of 122 genes belonging to WRKY, pathogenesis-related (PR) protein genes, non-expressor of pathogenesis-related genes 1 (NPR1) and chitinase families analyzed, 10 genes (six belonging to WRKY family, three belonging to PR proteins family and one belonging to chitinase family) showed significant differential regulation in MusaWRKY71 overexpressing lines. These results indicate that MusaWRKY71 is an important constituent in the transcriptional reprogramming involved in diverse stress responses in banana. PMID:24116051

  20. MusaWRKY71 Overexpression in Banana Plants Leads to Altered Abiotic and Biotic Stress Responses

    PubMed Central

    Shekhawat, Upendra K. S.; Ganapathi, Thumballi R.

    2013-01-01

    WRKY transcription factors are specifically involved in the transcriptional reprogramming following incidence of abiotic or biotic stress on plants. We have previously documented a novel WRKY gene from banana, MusaWRKY71, which was inducible in response to a wide array of abiotic or biotic stress stimuli. The present work details the effects of MusaWRKY71 overexpression in transgenic banana plants. Stable integration and overexpression of MusaWRKY71 in transgenic banana plants was proved by Southern blot analysis and quantitative real time PCR. Transgenic banana plants overexpressing MusaWRKY71 displayed enhanced tolerance towards oxidative and salt stress as indicated by better photosynthesis efficiency (Fv/Fm) and lower membrane damage of the assayed leaves. Further, differential regulation of putative downstream genes of MusaWRKY71 was investigated using real-time RT-PCR expression analysis. Out of a total of 122 genes belonging to WRKY, pathogenesis-related (PR) protein genes, non-expressor of pathogenesis-related genes 1 (NPR1) and chitinase families analyzed, 10 genes (six belonging to WRKY family, three belonging to PR proteins family and one belonging to chitinase family) showed significant differential regulation in MusaWRKY71 overexpressing lines. These results indicate that MusaWRKY71 is an important constituent in the transcriptional reprogramming involved in diverse stress responses in banana. PMID:24116051

  1. Activin C Antagonizes Activin A in Vitro and Overexpression Leads to Pathologies in Vivo

    PubMed Central

    Gold, Elspeth; Jetly, Niti; O'Bryan, Moira K.; Meachem, Sarah; Srinivasan, Deepa; Behuria, Supreeti; Sanchez-Partida, L. Gabriel; Woodruff, Teresa; Hedwards, Shelley; Wang, Hong; McDougall, Helen; Casey, Victoria; Niranjan, Birunthi; Patella, Shane; Risbridger, Gail

    2009-01-01

    Activin A is a potent growth and differentiation factor whose synthesis and bioactivity are tightly regulated. Both follistatin binding and inhibin subunit heterodimerization block access to the activin receptor and/or receptor activation. We postulated that the activin-βC subunit provides another mechanism regulating activin bioactivity. To test our hypothesis, we examined the biological effects of activin C and produced mice that overexpress activin-βC. Activin C reduced activin A bioactivity in vitro; in LNCaP cells, activin C abrogated both activin A-induced Smad signaling and growth inhibition, and in LβT2 cells, activin C antagonized activin A-mediated activity of an follicle-stimulating hormone-β promoter. Transgenic mice that overexpress activin-βC exhibited disease in testis, liver, and prostate. Male infertility was caused by both reduced sperm production and impaired sperm motility. The livers of the transgenic mice were enlarged because of an imbalance between hepatocyte proliferation and apoptosis. Transgenic prostates showed evidence of hypertrophy and epithelial cell hyperplasia. Additionally, there was decreased evidence of nuclear Smad-2 localization in the testis, liver, and prostate, indicating that overexpression of activin-βC antagonized Smad signaling in vivo. Underlying the significance of these findings, human testis, liver, and prostate cancers expressed increased activin-βC immunoreactivity. This study provides evidence that activin-βC is an antagonist of activin A and supplies an impetus to examine its role in development and disease. PMID:19095948

  2. Overexpression of PD2 leads to increased tumorigenicity and metastasis in pancreatic ductal adenocarcinoma

    PubMed Central

    Vaz, Arokia Priyanka; Deb, Shonali; Rachagani, Satyanarayana; Dey, Parama; Muniyan, Sakthivel; Lakshmanan, Imayavaramban; Karmakar, Saswati; Smith, Lynette; Johansson, Sonny; Lele, Subodh; Ouellette, Michel; Ponnusamy, Moorthy P.; Batra, Surinder K.

    2016-01-01

    Pancreatic differentiation 2 (PD2), an important subunit of the human PAF complex, was identified after differential screening analysis of 19q13 amplicon, and its overexpression induces oncogenic transformation of NIH3T3 cells, hence raising the possibility of a role for PD2 in tumorigenesis and metastasis. To test this hypothesis, we analyzed here the functional role and clinical significance of PD2 in pancreatic ductal adenocarcinoma (PDAC) and its pathogenesis. Using immunohistochemical analysis, we found that PD2 is detected in the acini but not in the ducts in the normal pancreas. In human PDAC specimens, PD2 was instead primarily detected in the ducts (12/48 patients 25%; p-value < 0.0001), thereby showing that PDAC correlates with increased ductal expression of PD2. Consistently, PD2 expression was increased in telomerase-immortalized human pancreatic ductal cells (HPNE cells) modified to express the HPV16 E6 and E7 proteins, whose respective functions are to block p53 and RB. In addition, ectopic expression of PD2 in PDAC cells (Capan-1 and SW1990) led to increased clonogenicity and migration in vitro, and tumor growth and metastasis in vivo. Interestingly, PD2 overexpression also resulted in enrichment of cancer stem cells (CSCs) and upregulation of oncogenes such as c-Myc and cell cycle progression marker, cyclin D1. Taken together, our results support that PD2 is overexpressed in the ducts of PDAC tissues, and results in tumorigenesis and metastasis via upregulation of oncogenes such as c-Myc and cyclin hence D1 implicating PD2 upregulation in pancreatic oncogenesis with targeted therapeutic potential. PMID:26689992

  3. Overexpression of Arabidopsis Phytochelatin Synthase Paradoxically Leads to Hypersensitivity to Cadmium Stress1

    PubMed Central

    Lee, Sangman; Moon, Jae S.; Ko, Tae-Seok; Petros, David; Goldsbrough, Peter B.; Korban, Schuyler S.

    2003-01-01

    Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 μm CdCl2 stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 μm CdCl2. Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels. PMID:12586889

  4. Overexpression of ATP Sulfurylase in Indian Mustard Leads to Increased Selenate Uptake, Reduction, and Tolerance1

    PubMed Central

    Pilon-Smits, Elizabeth A.H.; Hwang, Seongbin; Mel Lytle, C.; Zhu, Yongliang; Tai, Jenny C.; Bravo, Rogelio C.; Chen, Yichang; Leustek, Tom; Terry, Norman

    1999-01-01

    In earlier studies, the assimilation of selenate by plants appeared to be limited by its reduction, a step that is thought to be mediated by ATP sulfurylase. Here, the Arabidopsis APS1 gene, encoding a plastidic ATP sulfurylase, was constitutively overexpressed in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase activity was 2- to 2.5-fold higher in shoots and roots of transgenic seedlings, and 1.5- to 2-fold higher in shoots but not roots of selenate-supplied mature ATP-sulfurylase-overexpressing (APS) plants. The APS plants showed increased selenate reduction: x-ray absorption spectroscopy showed that root and shoot tissues of mature APS plants contained mostly organic Se (possibly selenomethionine), whereas wild-type plants accumulated selenate. The APS plants were not able to reduce selenate when shoots were removed immediately before selenate was supplied. In addition, Se accumulation in APS plants was 2- to 3-fold higher in shoots and 1.5-fold higher in roots compared with wild-type plants, and Se tolerance was higher in both seedlings and mature APS plants. These studies show that ATP sulfurylase not only mediates selenate reduction in plants, but is also rate limiting for selenate uptake and assimilation. PMID:9880353

  5. Overexpression of AaWRKY1 Leads to an Enhanced Content of Artemisinin in Artemisia annua

    PubMed Central

    Jiang, Weimin; Fu, Xueqing; Pan, Qifang; Tang, Yueli; Shen, Qian; Lv, Zongyou; Yan, Tingxiang; Shi, Pu; Li, Ling; Zhang, Lida; Wang, Guofeng; Sun, Xiaofen; Tang, Kexuan

    2016-01-01

    Artemisinin is an effective component of drugs against malaria. The regulation of artemisinin biosynthesis is at the forefront of artemisinin research. Previous studies showed that AaWRKY1 can regulate the expression of ADS, which is the first key enzyme in artemisinin biosynthetic pathway. In this study, AaWRKY1 was cloned, and it activated ADSpro and CYPpro in tobacco using dual-LUC assay. To further study the function of AaWRKY1, pCAMBIA2300-AaWRKY1 construct under 35S promoter was generated. Transgenic plants containing AaWRKY1 were obtained, and four independent lines with high expression of AaWRKY1 were analyzed. The expression of ADS and CYP, the key enzymes in artemisinin biosynthetic pathway, was dramatically increased in AaWRKY1-overexpressing A. annua plants. Furthermore, the artemisinin yield increased significantly in AaWRKY1-overexpressing A. annua plants. These results showed that AaWRKY1 increased the content of artemisinin by regulating the expression of both ADS and CYP. It provides a new insight into the mechanism of regulation on artemisinin biosynthesis via transcription factors in the future. PMID:27064403

  6. Overexpression of ARGOS Genes Modifies Plant Sensitivity to Ethylene, Leading to Improved Drought Tolerance in Both Arabidopsis and Maize.

    PubMed

    Shi, Jinrui; Habben, Jeffrey E; Archibald, Rayeann L; Drummond, Bruce J; Chamberlin, Mark A; Williams, Robert W; Lafitte, H Renee; Weers, Ben P

    2015-09-01

    Lack of sufficient water is a major limiting factor to crop production worldwide, and the development of drought-tolerant germplasm is needed to improve crop productivity. The phytohormone ethylene modulates plant growth and development as well as plant response to abiotic stress. Recent research has shown that modifying ethylene biosynthesis and signaling can enhance plant drought tolerance. Here, we report novel negative regulators of ethylene signal transduction in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). These regulators are encoded by the ARGOS gene family. In Arabidopsis, overexpression of maize ARGOS1 (ZmARGOS1), ZmARGOS8, Arabidopsis ARGOS homolog ORGAN SIZE RELATED1 (AtOSR1), and AtOSR2 reduced plant sensitivity to ethylene, leading to enhanced drought tolerance. RNA profiling and genetic analysis suggested that the ZmARGOS1 transgene acts between an ethylene receptor and CONSTITUTIVE TRIPLE RESPONSE1 in the ethylene signaling pathway, affecting ethylene perception or the early stages of ethylene signaling. Overexpressed ZmARGOS1 is localized to the endoplasmic reticulum and Golgi membrane, where the ethylene receptors and the ethylene signaling protein ETHYLENE-INSENSITIVE2 and REVERSION-TO-ETHYLENE SENSITIVITY1 reside. In transgenic maize plants, overexpression of ARGOS genes also reduces ethylene sensitivity. Moreover, field testing showed that UBIQUITIN1:ZmARGOS8 maize events had a greater grain yield than nontransgenic controls under both drought stress and well-watered conditions. PMID:26220950

  7. Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity.

    PubMed

    Li, Yujing; Dhankher, Om Parkash; Carreira, Laura; Lee, David; Chen, Alice; Schroeder, Julian I; Balish, Rebecca S; Meagher, Richard B

    2004-12-01

    Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed. PMID:15653797

  8. Overexpression of AtTTP Affects ARF17 Expression and Leads to Male Sterility in Arabidopsis

    PubMed Central

    Shi, Zhi-Hao; Zhang, Cheng; Xu, Xiao-Feng; Zhu, Jun; Zhou, Que; Ma, Li-Juan; Niu, Jin; Yang, Zhong-Nan

    2015-01-01

    Callose synthesis is critical for the formation of the pollen wall pattern. CalS5 is thought to be the major synthethase for the callose wall. In the Arabidopsis anther, ARF17 regulates the expression of CalS5 and is the target of miR160. Plants expressing miR160-resistant ARF17 (35S:5mARF17 lines) with increased ARF17 mRNA levels display male sterility. Here we report a zinc finger family gene, AtTTP, which is involved in miR160 maturation and callose synthesis in Arabidopsis. AtTTP is expressed in microsporocytes, tetrads and tapetal cells in the anther. Over-expression lines of AtTTP (AtTTP-OE line) exhibited reduced male fertility. CalS5 expression was tremendously reduced and the tetrad callose wall became much thinner in the AtTTP-OE line. Northern blotting hybridization and quantitative RT-PCR analysis revealed that miR160 was decreased, while the expression of ARF17 was increased in the AtTTP-OE line. Based on these results, we propose that AtTTP associates with miR160 in order to regulate the ARF17 expression needed for callose synthesis and pollen wall formation. PMID:25822980

  9. Cardiomyocyte-specific perilipin 5 overexpression leads to myocardial steatosis and modest cardiac dysfunction1[S

    PubMed Central

    Wang, Hong; Sreenivasan, Urmila; Gong, Da-Wei; O'Connell, Kelly A.; Dabkowski, Erinne R.; Hecker, Peter A.; Ionica, Nicoleta; Konig, Manige; Mahurkar, Anup; Sun, Yezhou; Stanley, William C.; Sztalryd, Carole

    2013-01-01

    Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction. However, presence of LDs in heart is also observed in physiological conditions, such as when cellular energy needs and energy production from mitochondria fatty acid β-oxidation are high (fasting). This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function. To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific perilipin 5 (Plin5) overexpression (MHC-Plin5), a member of the perilipin protein family. Hearts from MHC-Plin5 mice expressed at least 4-fold higher levels of plin5 and exhibited a 3.5-fold increase in triglyceride content versus nontransgenic littermates. Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of peroxisome proliferator-activated receptor (PPAR)α target genes, decreased mitochondria function, and left ventricular concentric hypertrophia. Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative-induced genes via NF-E2-related factor 2 antioxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and it promotes cardiac steatosis without major heart function impairment. PMID:23345411

  10. SpMYB overexpression in tobacco plants leads to altered abiotic and biotic stress responses.

    PubMed

    Li, Jing-Bin; Luan, Yu-Shi; Yin, Ya-Li

    2014-08-15

    The MYB transcription factors are involved in various plant biochemistry and physiology processes and play a central role in plant defense response. In the present study, a full-length cDNA sequence of a MYB gene, designated as SpMYB, was isolated from tomato. SpMYB encodes the R2R3-type protein consisting of 328 amino acids. The expression level of SpMYB was strongly induced by fungal pathogens. Transgenic tobacco plants overexpressing SpMYB had an enhanced salt and drought stress tolerance compared with wild-type plants, and showed significantly improved resistance to Alternaria alternate. Further analysis revealed that transgenic tobaccos exhibited less accumulation of malondialdehyde (MDA) and more accumulation of superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) after inoculation with A. alternate. Meanwhile, changes in some photosynthetic parameters, such as photosynthetic rate (Pn), transpiration rate (Tr) and intercellular CO2 concentration (Ci) were also found in the transgenic tobaccos. Furthermore, transgenic tobaccos constitutively accumulated higher levels of pathogenesis-related (PR) gene transcripts, such as PR1 and PR2. The results suggested that the tomato SpMYB transcription factor plays an important role in responses to abiotic and biotic stress. PMID:24971506

  11. Overexpression of SKI Oncoprotein Leads to p53 Degradation through Regulation of MDM2 Protein Sumoylation*

    PubMed Central

    Ding, Boxiao; Sun, Yin; Huang, Jiaoti

    2012-01-01

    Protooncogene Ski was identified based on its ability to transform avian fibroblasts in vitro. In support of its oncogenic activity, SKI was found to be overexpressed in a variety of human cancers, although the exact molecular mechanism(s) responsible for its oncogenic activity is not fully understood. We found that SKI can negatively regulate p53 by decreasing its level through up-regulation of MDM2 activity, which is mediated by the ability of SKI to enhance sumoylation of MDM2. This stimulation of MDM2 sumoylation is accomplished through a direct interaction of SKI with SUMO-conjugating enzyme E2, Ubc9, resulting in enhanced thioester bond formation and mono-sumoylation of Ubc9. A mutant SKI defective in transformation fails to increase p53 ubiquitination and is unable to increase MDM2 levels and to increase mono-sumoylation of Ubc9, suggesting that the ability of SKI to enhance Ubc9 activity is essential for its transforming function. These results established a detailed molecular mechanism that underlies the ability of SKI to cause cellular transformation while unraveling a novel connection between sumoylation and tumorigenesis, providing potential new therapeutic targets for cancer. PMID:22411991

  12. Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide

    PubMed Central

    Dabral, Neha; Jain-Gupta, Neeta; Seleem, Mohamed N.; Sriranganathan, Nammalwar; Vemulapalli, Ramesh

    2015-01-01

    Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in mammals. Brucella strains containing the O-polysaccharide in their cell wall structure exhibit a smooth phenotype whereas the strains devoid of the polysaccharide show rough phenotype. B. abortus strain RB51 is a stable rough attenuated mutant which is used as a licensed live vaccine for bovine brucellosis. Previous studies have shown that the wboA gene, which encodes a glycosyltransferase required for the synthesis of O-polysaccharide, is disrupted in B. abortus RB51 by an IS711 element. Although complementation of strain RB51 with a functional wboA gene results in O-polysaccharide synthesis in the cytoplasm, it does not result in smooth phenotype. The aim of this study was to determine if overexpression of Brucella WbkA or WbkE, two additional putative glycosyltransferases essential for O-polysaccharide synthesis, in strain RB51 would result in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of wbkA or wbkE gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies. However, wbkA, but not wbkE, overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates. The recombinant strain was similar to strain RB51 in its attenuation characteristic and in its ability to induce protective immunity against virulent B. abortus challenge in mice. PMID:26157707

  13. Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors.

    PubMed Central

    Risma, K A; Clay, C M; Nett, T M; Wagner, T; Yun, J; Nilson, J H

    1995-01-01

    Hypersecretion of luteinizing hormone (LH) is implicated in infertility and miscarriages in women. A lack of animal models has limited progress in determining the mechanisms of LH toxicity. We have recently generated transgenic mice expressing a chimeric LH beta subunit (LH beta) in gonadotropes. The LH beta chimera contains the C-terminal peptide of the human chorionic gonadotropin beta subunit. Addition of this peptide to bovine LH beta resulted in a hormone with a longer half-life. Furthermore, targeted expression of the LH beta chimera led to elevated LH levels and infertility in female transgenics. These mice ovulated infrequently, maintained a prolonged luteal phase, and developed pathologic ovarian changes such as cyst formation, marked enlargement of ovaries, and granulosa cell tumors. Testosterone and estradiol levels were increased compared to nontransgenic littermates. An unusual extragonadal phenotype was also observed: transgenic females developed hydronephropathy and pyelonephritis. The pathology observed demonstrates a direct association between abnormal secretion of LH and infertility and underscores the utility of the transgenic model for studying how excess LH leads to cyst formation, ovarian tumorigenesis, and infertility. Images Fig. 2 Fig. 3 Fig. 5 PMID:7877975

  14. Overexpression of Protein Kinase C-ε in the Mouse Epidermis Leads to a Spontaneous Myeloproliferative-Like Disease

    PubMed Central

    Wheeler, Deric L.; Reddig, Peter J.; Ness, Kristin J.; Leith, Catherine P.; Oberley, Terry D.; Verma, Ajit K.

    2005-01-01

    , interleukin-6, and granulocyte-colony stimulating factor were up-regulated as a function of age. The transgene PKC-ε was not detected in any of the affected organs (bone marrow, liver, spleen, lung) We suggest that overexpression of PKC-ε in the epidermis may lead to the induction of specific cytokines that may, in a paracrine mechanism, perturb normal hematopoiesis in bone marrow resulting in a granulocytic skew toward that of neutrophils and eosinophils. The susceptibility of PKC-ε transgenic mice to the induction of SCC and the spontaneous development of MPD are unrelated. PMID:15632005

  15. Osteoblast-specific overexpression of amphiregulin leads to transient increase in femoral cancellous bone mass in mice.

    PubMed

    Vaidya, Mithila; Lehner, Diana; Handschuh, Stephan; Jay, Freya F; Erben, Reinhold G; Schneider, Marlon R

    2015-12-01

    The epidermal growth factor receptor ligand amphiregulin (AREG) has been implicated in bone physiology and in bone anabolism mediated by intermittent parathyroid hormone treatment. However, the functions of AREG in bone have been only incipiently evaluated in vivo. Here, we generated transgenic mice overexpressing AREG specifically in osteoblasts (Col1-Areg). pQCT analysis of the femoral metaphysis revealed increased trabecular bone mass at 4, 8, and 10weeks of age in Col1-Areg mice compared to control littermates. However, the high bone mass phenotype was transient and disappeared in older animals. Micro-CT analysis of the secondary spongiosa confirmed increased trabecular bone volume and trabecular number in the distal femur of 4-week-old AREG-tg mice compared to control littermates. Furthermore, μ-CT analysis of the primary spongiosa revealed unaltered production of new bone trabeculae in distal femora of Col1-Areg mice. Histomorphometric analysis revealed a reduced number of osteoclasts in 4-week-old Col1-Areg mice, but not at later time points. Cancellous bone formation rate remained unchanged in Col1-Areg mice at all time points. In addition, bone mass and bone turnover in lumbar vertebral bodies were similar in Col1-Areg and control mice at all ages examined. Proliferation and differentiation of osteoblasts isolated from neonatal calvariae did not differ between Col1-Areg and control mice. Taken together, these data suggest that AREG overexpression in osteoblasts induces a transient high bone mass phenotype in the trabecular compartment of the appendicular skeleton by a growth-related, non-cell autonomous mechanism, leading to a positive bone balance with unchanged bone formation and lowered bone resorption. PMID:26103093

  16. Overexpression of Arabidopsis thaliana ERI, the homolog of C. elegans Enhancer of RNAinterference, leads to enhanced growth

    PubMed Central

    Meyer, Rhonda C.; Hönig, Gunnar; Brandt, Ronny; Arana-Ceballos, Fernando; Neitsch, Cathleen; Reuter, Gunter; Altmann, Thomas; Kuhlmann, Markus

    2015-01-01

    Organisms adopt a wide range of strategies to adapt to change. Gene silencing describes the ability of organisms to modulate the expression of susceptible genes at certain times at the transcriptional or the translational level. In all known eukaryotic organisms 21-nt long short interfering RNAs (siRNAs) are the effector molecules of post-transcriptional gene silencing (PTGS), while 24-nt long siRNAs are involved in PTGS in plants. Mutant studies in Caenorhabditis elegans lead to the identification of the enzyme ERI (Enhancer of RNAinterference) with enhanced PTGS. Although the genes involved in growth vigor and growth rate are still unknown, it becomes clearer that the population of small RNAs plays a role in the very early phase of plant development. To pinpoint the link between growth and siRNAs, the expression of Arabidopsis uni-gene Enhancer of RNAi (ERI) homolog from C. elegans was modulated. Increased degradation of small RNAs was achieved by ectopic AtERI overexpression in planta. Based on global small RNA analysis, AtERI overexpression affects mainly the population of 21 mers, excluding miRNAs. To identify target genes, AtERI gain-of-function mutants were analyzed, and differentially abundant small RNAs were identified. Plants with an elevated level of AtERI were bigger in all three light intensities analyzed, indicating an inhibitory function of particular small RNAs in plant growth, with differences in relative growth rates depending on developmental stage and light intensity. Understanding the role of these siRNAs could open new avenues for enhancing plant growth. PMID:26257748

  17. MAL Overexpression Leads to Disturbed Expression of Genes That Influence Cytoskeletal Organization and Differentiation of Schwann Cells

    PubMed Central

    Schmid, Daniela; Zeis, Thomas; Sobrio, Monia

    2014-01-01

    In the developing peripheral nervous system, a coordinated reciprocal signaling between Schwann cells and axons is crucial for accurate myelination. The myelin and lymphocyte protein MAL is a component of lipid rafts that is important for targeting proteins and lipids to distinct domains. MAL overexpression impedes peripheral myelinogenesis, which is evident by a delayed onset of myelination and reduced expression of the myelin protein zero (Mpz/P0) and the low-affinity neurotrophin receptor p75NTR. This study shows that MAL overexpression leads to a significant reduction of Mpz and p75NTR expression in primary mouse Schwann cell cultures, which was already evident before differentiation, implicating an effect of MAL in early Schwann cell development. Their transcription was robustly reduced, despite normal expression of essential transcription factors and receptors. Further, the cAMP response element-binding protein (CREB) and phosphoinositide 3-kinase signaling pathways important for Schwann cell differentiation were correctly induced, highlighting that other so far unknown rate limiting factors do exist. We identified novel genes expressed by Schwann cells in a MAL-dependent manner in vivo and in vitro. A number of those, including S100a4, RhoU and Krt23, are implicated in cytoskeletal organization and plasma membrane dynamics. We showed that S100a4 is predominantly expressed by nonmyelinating Schwann cells, whereas RhoU was localized within myelin membranes, and Krt23 was detected in nonmyelinating as well as in myelinating Schwann cells. Their differential expression during early peripheral nerve development further underlines their possible role in influencing Schwann cell differentiation and myelination. PMID:25290060

  18. Overexpression of aflR Leads to Upregulation of Pathway Gene Transcription and Increased Aflatoxin Production in Aspergillus flavus

    PubMed Central

    Flaherty, J. E.; Payne, G. A.

    1997-01-01

    The aflatoxin biosynthetic pathway regulatory gene, aflR, encodes a putative 47-kDa protein containing a zinc cluster DNA binding motif. It is required for the transcription of all of the characterized aflatoxin pathway genes in both Aspergillus flavus and Aspergillus parasiticus. The objective of this study was to examine the effects of aflR overexpression on temporal gene expression, aflatoxin production, and nitrate inhibition of aflatoxin biosynthesis in A. flavus. An inducible expression construct was made by fusing the coding region of aflR to the promoter region of the A. flavus adh1 gene. This construct was transformed into A. flavus 656-2 (FGSC A1010), a strain mutated at the aflR locus. Strain 656-2 containing the adh1(p)::aflR construct had induced transcription of two early aflatoxin pathway genes, nor-1 and pksA, and produced wild-type concentrations of aflatoxin in a temporal pattern similar to that of wild-type strains of A. flavus. Strains 656-2 and 86-10 (FGSC A1009) an aflatoxigenic strain, were transformed with a construct containing the constitutive promoter gpdA driving aflR. Transformants of these strains constitutively expressed aflR, fas-1A, pksA, nor-1, and omtA but did not constitutively produce aflatoxin. Strain 86-10 containing the gpdA(p)::aflR construct produced 50 times more aflatoxin than 86-10, but the temporal pattern of aflatoxin production was the same as for 86-10, and aflatoxin production was also induced by sucrose. The addition of 10 g of nitrate per liter to sucrose low salts medium inhibited aflatoxin production by both strain 86-10 and a transformant of 86-10 containing the gpdA(p)::aflR construct, indicating that nitrate inhibition of aflatoxin biosynthesis does not occur solely at the level of aflR transcription. These studies show that constitutive overexpression of the pathway transcriptional regulatory gene aflR leads to higher transcript accumulation of pathway genes and increased aflatoxin production but that the

  19. Constitutive Overexpression of Cystathionine γ-Synthase in Arabidopsis Leads to Accumulation of Soluble Methionine and S-Methylmethionine1

    PubMed Central

    Kim, Jungsup; Lee, Minsang; Chalam, Radhika; Martin, Melinda Neal; Leustek, Thomas; Boerjan, Wout

    2002-01-01

    The committing step in Met and S-adenosyl-l-Met (SAM) synthesis is catalyzed by cystathionine γ-synthase (CGS). Transgenic Arabidopsis plants overexpressing CGS under control of the cauliflower mosaic virus 35S promoter show increased soluble Met and its metabolite S-methyl-Met, but only at specific stages of development. The highest level of Met and S-methyl-Met was observed in seedling tissues and in flowers, siliques, and roots of mature plants where they accumulate 8- to 20-fold above wild type, whereas the level in mature leaves and other tissues is no greater than wild type. CGS-overexpressing seedlings are resistant to ethionine, a toxic Met analog. With these properties the transgenic lines resemble mto1, an Arabidopsis, CGS-mutant inactivated in the autogenous control mechanism for Met-dependent down-regulation of CGS expression. However, wild-type CGS was overexpressed in the transgenic plants, indicating that autogenous control can be overcome by increasing the level of CGS mRNA through transcriptional control. Several of the transgenic lines show silencing of CGS resulting in deformed plants with a reduced capacity for reproductive growth. Exogenous feeding of Met to the most severely affected plants partially restores their growth. Similar morphological deformities are observed in plants cosuppressed for SAM synthetase, even though such plants accumulate 250-fold more soluble Met than wild type and they overexpress CGS. The results suggest that the abnormalities associated with CGS and SAM synthetase silencing are due in part to a reduced ability to produce SAM and that SAM may be a regulator of CGS expression. PMID:11788756

  20. Deficiency and Also Transgenic Overexpression of Timp-3 Both Lead to Compromised Bone Mass and Architecture In Vivo

    PubMed Central

    Hopkinson, Mark; Poulet, Blandine; Pollard, Andrea S.; Shefelbine, Sandra J.; Chang, Yu-Mei; Francis-West, Philippa; Bou-Gharios, George; Pitsillides, Andrew A.

    2016-01-01

    Tissue inhibitor of metalloproteinases-3 (TIMP-3) regulates extracellular matrix via its inhibition of matrix metalloproteinases and membrane-bound sheddases. Timp-3 is expressed at multiple sites of extensive tissue remodelling. This extends to bone where its role, however, remains largely unresolved. In this study, we have used Micro-CT to assess bone mass and architecture, histological and histochemical evaluation to characterise the skeletal phenotype of Timp-3 KO mice and have complemented this by also examining similar indices in mice harbouring a Timp-3 transgene driven via a Col-2a-driven promoter to specifically target overexpression to chondrocytes. Our data show that Timp-3 deficiency compromises tibial bone mass and structure in both cortical and trabecular compartments, with corresponding increases in osteoclasts. Transgenic overexpression also generates defects in tibial structure predominantly in the cortical bone along the entire shaft without significant increases in osteoclasts. These alterations in cortical mass significantly compromise predicted tibial load-bearing resistance to torsion in both genotypes. Neither Timp-3 KO nor transgenic mouse growth plates are significantly affected. The impact of Timp-3 deficiency and of transgenic overexpression extends to produce modification in craniofacial bones of both endochondral and intramembranous origins. These data indicate that the levels of Timp-3 are crucial in the attainment of functionally-appropriate bone mass and architecture and that this arises from chondrogenic and osteogenic lineages. PMID:27519049

  1. Inducible Conditional Vascular-Specific Overexpression of Peroxisome Proliferator-Activated Receptor Beta/Delta Leads to Rapid Cardiac Hypertrophy

    PubMed Central

    Wagner, Kay-Dietrich; Vukolic, Ana; Baudouy, Delphine; Michiels, Jean-François

    2016-01-01

    Peroxisome proliferator-activated receptors are nuclear receptors which function as ligand-activated transcription factors. Among them, peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in the heart and thought to have cardioprotective functions due to its beneficial effects in metabolic syndrome. As we already showed that PPARβ/δ activation resulted in an enhanced cardiac angiogenesis and growth without impairment of heart function, we were interested to determine the effects of a specific activation of PPARβ/δ in the vasculature on cardiac performance under normal and in chronic ischemic heart disease conditions. We analyzed the effects of a specific PPARβ/δ overexpression in endothelial cells on the heart using an inducible conditional vascular-specific mouse model. We demonstrate that vessel-specific overexpression of PPARβ/δ induces rapid cardiac angiogenesis and growth with an increase in cardiomyocyte size. Upon myocardial infarction, vascular overexpression of PPARβ/δ, despite the enhanced cardiac vessel formation, does not protect against chronic ischemic injury. Our results suggest that the proper balance of PPARβ/δ activation in the different cardiac cell types is required to obtain beneficial effects on the outcome in chronic ischemic heart disease. PMID:27057154

  2. Overexpression of Guanylate Cyclase Activating Protein 2 in Rod Photoreceptors In Vivo Leads to Morphological Changes at the Synaptic Ribbon

    PubMed Central

    López-Begines, Santiago; Fernández-Sánchez, Laura; Cuenca, Nicolás; Llorens, Jordi; de la Villa, Pedro; Méndez, Ana

    2012-01-01

    Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic

  3. Novel transgenic rice overexpressing anthocyanidin synthase accumulates a mixture of flavonoids leading to an increased antioxidant potential.

    PubMed

    Reddy, Ambavaram M; Reddy, Vaka S; Scheffler, Brian E; Wienand, Udo; Reddy, Arjula R

    2007-01-01

    In addition to their plant-associated functions, flavonoids act as antioxidants against harmful free radicals in animals. Genetic engineering of food crops for a mix of antioxidant flavonoids is highly beneficial in promoting human health. Anthocyanidin synthase (ANS) is one of the four dioxygenases (DOX) of the flavonoid biosynthetic pathway that catalyzes the formation of anthocyanidins from leucoanthocyanidins. To investigate whether ANS mediates different DOX reactions of the pathway and produces a mix of flavonoids, the rice ANS cDNA was cloned and overexpressed in a rice mutant Nootripathu (NP). This mutant accumulates proanthocyanidins exclusively in pericarp and absolutely no anthocyanins in any tissue. In silico sequence analysis revealed that ANS contains a double-stranded beta helix and shows high sequence similarity with other DOXs of the pathway including flavonol synthase, flavonone 3beta-hydroxylase and flavone synthase I. Bacterially expressed ANS protein converted dihydroquercetin to quercetin and Pro(35S):ANS complemented the maize a2 mutant in producing anthocyanins in aleurone, suggesting that ANS functions as a DOX with different flavonoid substrates. Similarly, transgenic NP plants overexpressing Pro(MAS):ANS channeled the proanthocaynidin precursors to the production of anthocyanins in pericarp. Transgenics showed approximately ten and four-fold increase in the ANS transcripts and enzyme activity, respectively. As a result, these plants showed an increased accumulation of a mixture of flavonoids and anthocyanins, with a concomitant decrease in proanthocyanidins, suggesting that ANS may act directly on different flavonoid substrates of DOX reactions. Thus, overexpression of ANS in a rice mutant resulted in novel transgenic rice with a mixture of flavonoids and an enhanced antioxidant potential. PMID:17157544

  4. Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

    PubMed Central

    Buchberger, Elisabeth; El Harchi, Miriam; Payrhuber, Dietmar; Zommer, Anna; Schauer, Dominic; Simonitsch-Klupp, Ingrid; Bilban, Martin; Brostjan, Christine

    2013-01-01

    Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones. PMID:24146931

  5. Transgenic overexpression of the alpha-synuclein interacting protein synphilin-1 leads to behavioral and neuropathological alterations in mice.

    PubMed

    Nuber, Silke; Franck, Thomas; Wolburg, Hartwig; Schumann, Ulrike; Casadei, Nicolas; Fischer, Kristina; Calaminus, Carsten; Pichler, Bernd J; Chanarat, Sittinan; Teismann, Peter; Schulz, Jörg B; Luft, Andreas R; Tomiuk, Jürgen; Wilbertz, Johannes; Bornemann, Antje; Krüger, Rejko; Riess, Olaf

    2010-02-01

    Synphilin-1 has been identified as an interacting protein of alpha-synuclein, Parkin, and LRRK2, proteins which are mutated in familial forms of Parkinson disease (PD). Subsequently, synphilin-1 has also been shown to be an intrinsic component of Lewy bodies in sporadic PD. In order to elucidate the role of synphilin-1 in the pathogenesis of PD, we generated transgenic mice overexpressing wild-type and mutant (R621C) synphilin-1 driven by a mouse prion protein promoter. Transgenic expression of both wild-type and the R621C variant synphilin-1 resulted in increased dopamine levels of the nigrostriatal system in 3-month-old mice. Furthermore, we found pathological ubiquitin-positive inclusions in cerebellar sections and dark-cell degeneration of Purkinje cells. Both transgenic mouse lines showed significant reduction of motor skill learning and motor performance. These findings suggest a pathological role of overexpressed synphilin-1 in vivo and will help to further elucidate the mechanisms of protein aggregation and neuronal cell death. PMID:19760259

  6. Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

    PubMed Central

    Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong

    2015-01-01

    Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the

  7. Overexpression of human ABCB1 in cancer cells leads to reduced activity of GSK461364, a specific inhibitor of polo-like kinase 1.

    PubMed

    Wu, Chung-Pu; Hsiao, Sung-Han; Luo, Shi-Yu; Tuo, Wei-Cherng; Su, Ching-Ya; Li, Yan-Qing; Huang, Yang-Hui; Hsieh, Chia-Hung

    2014-10-01

    Polo-like kinase 1 (Plk1) is a serine/threonine kinase involved in the regulation of mitosis and is overexpressed in many tumor types. Inhibition of Plk1 leads to cell cycle arrest, onset of apoptosis, and cell death, thus Plk1 has emerged as an important target for cancer treatment. GSK461364 is a potent inhibitor of Plk1 that inhibits the proliferation of multiple human cancer cell lines by promoting G2/M cell cycle arrest at low concentrations. However, as is the case for many therapeutic drugs, the risk of developing drug resistance to GSK461364 can present a therapeutic challenge to clinicians. Since the overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 is one of the most common mechanisms of drug resistance, we aimed to investigate the effect of ABCB1 on the cellular efficacy of GSK461364. In this study, we observed a significantly reduced activity of GSK461364 in cells overexpressing human ABCB1. We showed that GSK461364 stimulates the ABCB1 ATPase activity and competitively inhibits ABCB1-mediated efflux of calcein-AM in a concentration-dependent manner. Moreover, as a way to assess the impact of ABCB1 on the efficacy of GSK461364, we evaluated the G2/M cell cycle arrest and apoptosis induced by GSK461364. We discovered that, by inhibiting the function of ABCB1, the reduced G2/M cell cycle arrest, apoptosis, and sensitivity to GSK461364 treatment in ABCB1-overexpressing cells can be significantly restored. In conclusion, in order to achieve a better therapeutic outcome, combination therapy of GSK461364 with a modulator of ABCB1 should be further investigated as a potential treatment approach. PMID:25192198

  8. Overexpression of Plastidic Protoporphyrinogen IX Oxidase Leads to Resistance to the Diphenyl-Ether Herbicide Acifluorfen1

    PubMed Central

    Lermontova, Inna; Grimm, Bernhard

    2000-01-01

    The use of herbicides to control undesirable vegetation has become a universal practice. For the broad application of herbicides the risk of damage to crop plants has to be limited. We introduced a gene into the genome of tobacco (Nicotiana tabacum) plants encoding the plastid-located protoporphyrinogen oxidase of Arabidopsis, the last enzyme of the common tetrapyrrole biosynthetic pathway, under the control of the cauliflower mosaic virus 35S promoter. The transformants were screened for low protoporphyrin IX accumulation upon treatment with the diphenyl ether-type herbicide acifluorfen. Leaf disc incubation and foliar spraying with acifluorfen indicated the lower susceptibility of the transformants against the herbicide. The resistance to acifluorfen is conferred by overexpression of the plastidic isoform of protoporphyrinogen oxidase. The in vitro activity of this enzyme extracted from plastids of selected transgenic lines was at least five times higher than the control activity. Herbicide treatment that is normally inhibitory to protoporphyrinogen IX oxidase did not significantly impair the catalytic reaction in transgenic plants and, therefore, did not cause photodynamic damage in leaves. Therefore, overproduction of protoporphyrinogen oxidase neutralizes the herbicidal action, prevents the accumulation of the substrate protoporphyrinogen IX, and consequently abolishes the light-dependent phytotoxicity of acifluorfen. PMID:10631251

  9. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    PubMed

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function. PMID:15755682

  10. Genomic loss of EZH2 leads to epigenetic modifications and overexpression of the HOX gene clusters in myelodysplastic syndrome

    PubMed Central

    Chang, Chun-Kang; He, Qi; Wu, Ling-Yun; Zhang, Zheng; Shi, Wen-Hui; Guo, Juan; Zhu, Yang; Zhao, You-Shan; Gu, Shu-Cheng; Fei, Cheng-Ming; Li, Xiao

    2016-01-01

    The role of EZH2 in cancer is complex and may vary depending on cancer type or stage. We examined the effect of altered EZH2 levels on H3K27 methylation, HOX gene expression, and malignant phenotype in myelodysplastic syndrome (MDS) cell lines and an in vivo xenograft model. We also studied links between EZH2 expression and prognosis in MDS patients. Patients with high-grade MDS exhibited lower levels of EZH2 expression than those with low-grade MDS. Low EZH2 expression was associated with high percentages of blasts, shorter survival, and increased transformation of MDS into acute myeloid leukemia (AML). MDS patients frequently had reductions in EZH2 copy number. EZH2 knockdown increased tumor growth capacity and reduced H3K27me3 levels in both MDS-derived leukemia cells and in a xenograft model. H3K27me3 levels were reduced and HOX gene cluster expression was increased in MDS patients. EZH2 knockdown also increased HOX gene cluster expression by reducing H3K27me3, and H3K27 demethylating agents increased HOX gene cluster expression in MDS-derived cell lines. These findings suggest genomic loss of EZH2 contributes to overexpression of the HOX gene clusters in MDS through epigenetic modifications. PMID:26812882

  11. Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India

    PubMed Central

    2016-01-01

    Objectives The present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India. Methods 76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed. Results Out of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump. Conclusions This study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options. PMID:26866484

  12. Lead uptake increases drought tolerance of wild type and transgenic poplar (Populus tremula x P. alba) overexpressing gsh 1.

    PubMed

    Samuilov, Sladjana; Lang, Friedericke; Djukic, Matilda; Djunisijevic-Bojovic, Danijela; Rennenberg, Heinz

    2016-09-01

    Growth and development of plants largely depends on their adaptation ability in a changing climate. This is particularly true on heavy metal contaminated soils, but the interaction of heavy metal stress and climate on plant performance has not been intensively investigated. The aim of the present study was to elucidate if transgenic poplars (Populus tremula x P. alba) with enhanced glutathione content possess an enhanced tolerance to drought and lead (Pb) exposure (single and in combination) and if they are good candidates for phytoremediation of Pb contaminated soil. Lead exposure reduced growth and biomass accumulation only in above-ground tissue of wild type poplar, although most of lead accumulated in the roots. Drought caused a decline of the water content rather than reduced biomass production, while Pb counteracted this decline in the combined exposure. Apparently, metals such as Pb possess a protective function against drought, because they interact with abscisic acid dependent stomatal closure. Lead exposure decreased while drought increased glutathione content in leaves of both plant types. Lead accumulation was higher in the roots of transgenic plants, presumably as a result of chelation by glutathione. Water deprivation enhanced Pb accumulation in the roots, but Pb was subject to leakage out of the roots after re-watering. Transgenic plants showed better adaptation under mild drought plus Pb exposure partially due to improved glutathione synthesis. However, the transgenic plants cannot be considered as a good candidate for phytoremediation of Pb, due to its small translocation to the shoots and its leakage out of the roots upon re-watering. PMID:27396669

  13. Retraction: "Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells" by Bao et al.

    PubMed

    2016-08-01

    The above article, published online on April 18, 2011 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the second author that found Figures 1C and 4C to be inappropriately re-used and re-labeled. REFERENCE Bao B, Wang Z, Ali S, Kong D, Banerjee S, Ahmad A, Li Y, Azmi AS, Miele L, Sarkar FH. 2011. Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells. J Cell Biochem 112:2296-2306; doi: 10.1002/jcb.23150. PMID:27301890

  14. Epigenetic alterations leading to TMPRSS4 promoter hypomethylation and protein overexpression predict poor prognosis in squamous lung cancer patients

    PubMed Central

    Villalba, Maria; Diaz-Lagares, Angel; Redrado, Miriam; de Aberasturi, Arrate L.; Segura, Victor; Bodegas, Maria Elena; Pajares, Maria J.; Pio, Ruben; Freire, Javier; Gomez-Roman, Javier; Montuenga, Luis M.; Esteller, Manel; Sandoval, Juan; Calvo, Alfonso

    2016-01-01

    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which highlights the need of innovative therapeutic options. Although targeted therapies can be successfully used in a subset of patients with lung adenocarcinomas (ADC), they are not appropriate for patients with squamous cell carcinomas (SCC). In addition, there is an unmet need for the identification of prognostic biomarkers that can select patients at risk of relapse in early stages. Here, we have used several cohorts of NSCLC patients to analyze the prognostic value of both protein expression and DNA promoter methylation status of the prometastatic serine protease TMPRSS4. Moreover, expression and promoter methylation was evaluated in a panel of 46 lung cancer cell lines. We have demonstrated that a high TMPRSS4 expression is an independent prognostic factor in SCC. Similarly, aberrant hypomethylation in tumors, which correlates with high TMPRSS4 expression, is an independent prognostic predictor in SCC. The inverse correlation between expression and methylation status was also observed in cell lines. In vitro studies showed that treatment of cells lacking TMPRSS4 expression with a demethylating agent significantly increased TMPRSS4 levels. In conclusion, TMPRSS4 is a novel independent prognostic biomarker regulated by epigenetic changes in SCC and a potential therapeutic target in this tumor type, where targeted therapy is still underdeveloped. PMID:26989022

  15. Overexpression of dnIKK in mesenchymal stem cells leads to increased migration and decreased invasion upon TNFα stimulation.

    PubMed

    Haasters, Florian; Prall, Wolf Christian; Westphal, Ines; Böcker, Wolfgang; Padula, Daniela; Mutschler, Wolf; Docheva, Denitsa; Schieker, Matthias

    2013-06-28

    IκB kinase 2 (IKK-2) mediates tumor necrosis-factor α (TNFα) induced invasion of human mesenchymal stem cell (hMSC) to sites of tissue injury. Suppressing IKK-2 activity leads to reduced expression of proteolytic enzymes and impaired invasive capacity. In order to further reveal mechanisms of hMSC recruitment, we here aimed to analyse the impact of IKK-2 on two-dimensional migration upon TNFα stimulation in contrast to three-dimensional invasion. An immortalized hMSC line (SCP-1) was transduced with a dominant-negative mutant of IκB kinase 2 (SCP-1 dnIKK). Migration was assessed using a linear-gradient chemotaxis chambers by time-lapse analysis. Invasive capacity through human extracellular matrix was analysed using transwell invasion assays. RT-PCR confirmed increased IKK-2 expression levels in SCP-1 dnIKK cells, while TNFα receptor I and II expression was not altered. Invasion upon TNFα stimulation was significantly reduced by 78% in SCP-1 dnIKK. In contrast, migration was significantly increased, represented by a 60% elevated forward migration index and a 2.1-fold higher mean dislocation of the center of mass towards TNFα. In conclusion, our data confirms the impact of IKK-2 in TNFα dependent hMSC recruitment. Interestingly, reducing IKK-2 function increases two-dimensional migration towards TNFα, while invasive capacity is impaired. These findings contribute to a deeper understanding of MSC's biological properties orchestrating the complex processes of stem cell recruitment and homing. PMID:23743204

  16. Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer.

    PubMed

    Apostolopoulou, K; Pateras, I S; Evangelou, K; Tsantoulis, P K; Liontos, M; Kittas, C; Tiniakos, D G; Kotsinas, A; Cordon-Cardo, C; Gorgoulis, V G

    2007-11-01

    ZBTB7A (Pokemon) is a member of the POK family of transcriptional repressors. Its main function is the suppression of the p14ARF tumour suppressor gene. Although ZBTB7A expression has been found to be increased in various types of lymphoma, there are no reports dealing with its expression in solid tumours. Given that p14(ARF) inhibits MDM2, the main negative regulator of p53, we hypothesized that overexpression of ZBTB7A could lead indirectly to p53 inactivation. To this end, we examined the status of ZBTB7A and its relationship with tumour kinetics (proliferation and apoptosis) and nodal members of the p53 network in a panel of 83 non-small cell lung carcinomas (NSCLCs). We observed, in the majority of the samples, prominent expression of ZBTB7A in the cancerous areas compared to negligible presence in the adjacent normal tissue elements. Gene amplification (two- to five-fold) was found in 27.7% of the cases, denoting its significance as a mechanism driving ZBTB7A overproduction in NSCLCs. In the remaining non-amplified group of carcinomas, analysis of the mRNA and protein expression patterns suggested that deregulation at the transcriptional and post-translational level accounts for ZBTB7A overexpression. Proliferation was associated with ZBTB7A expression (p = 0.033) but not apoptosis. The association with proliferation was reflected in the positive correlation between ZBTB7A expression and tumour size (p = 0.018). The overexpression of ZBTB7A in both p53 mutant and p53 wild-type cases, implies either a synergistic effect or that ZBTB7A exerts its oncogenic properties independently of the p14(ARF)-MDM2-p53 axis. The concomitant expression of ZBTB7A with p14(ARF) (p = 0.039), instead of the anticipated inverse relation, supports the latter notion. In conclusion, regardless of the pathway followed, the distinct expression of ZBTB7A in cancerous areas and the association with proliferation and tumour size pinpoints a role for this novel cell cycle regulator in the

  17. Overexpression of PrfA Leads to Growth Inhibition of Listeria monocytogenes in Glucose-Containing Culture Media by Interfering with Glucose Uptake

    PubMed Central

    Marr, A. K.; Joseph, B.; Mertins, S.; Ecke, R.; Müller-Altrock, S.; Goebel, W.

    2006-01-01

    Listeria monocytogenes strains expressing high levels of the virulence regulator PrfA (mutant PrfA* or wild-type PrfA) show strong growth inhibition in minimal media when they are supplemented with glucose but not when they are supplemented with glucose-6-phosphate compared to the growth of isogenic strains expressing low levels of PrfA. A significantly reduced rate of glucose uptake was observed in a PrfA*-overexpressing strain growing in LB supplemented with glucose. Comparative transcriptome analyses were performed with RNA isolated from a prfA mutant and an isogenic strain carrying multiple copies of prfA or prfA* on a plasmid. These analyses revealed that in addition to high transcriptional up-regulation of the known PrfA-regulated virulence genes (group I), there was less pronounced up-regulation of the expression of several phage and metabolic genes (group II) and there was strong down-regulation of several genes involved mainly in carbon and nitrogen metabolism in the PrfA*-overexpressing strain (group III). Among the latter genes are the nrgAB, gltAB, and glnRA operons (involved in nitrogen metabolism), the ilvB operon (involved in biosynthesis of the branched-chain amino acids), and genes for some ABC transporters. Most of the down-regulated genes have been shown previously to belong to a class of genes in Bacillus subtilis whose expression is negatively affected by impaired glucose uptake. Our results lead to the conclusion that excess PrfA (or PrfA*) interferes with a component(s) essential for phosphotransferase system-mediated glucose transport. PMID:16707681

  18. Downregulation of the Musca domestica peptidoglycan recognition protein SC (PGRP-SC) leads to overexpression of antimicrobial peptides and tardy pupation.

    PubMed

    Gao, Yifu; Tang, Ting; Gu, Jihai; Sun, Lingling; Gao, Xiaobin; Ma, Xianyong; Wang, Xiaochun; Liu, Fengsong; Wang, Jianhui

    2015-10-01

    PGRP (peptidoglycan recognition protein) is a conserved protein family that recognizes the peptidoglycan in bacterial cell wall and causes the activation of various innate immune responses. Previous studies have reported that PGRP-SCs in Drosophila dampen the activation of Immune Deficiency (Imd) pathway to microbial infection, and participate in the lifespan extension of the insects. To facilitate understanding the function of PGRP-SCs from an evolutionary angle, we identified and functionally characterized the PGRP-SC gene in the housefly Musca domestica, a species that has adapted to a septic environment much harsher than the natural habitat of Drosophila. The gene designated as MdPGRP-SC was found most abundantly expressed in the 3rd instar larvae, and is expressed at this developmental stage predominantly in the gut. MdPGRP-SC was virtually unchanged in whole larvae after a septic injury at the second larval instar, while two antimicrobial peptides (AMPs), diptericin and attacin, were upregulated in the first 24h but not later. Through dsRNA microinjection, MdPGRP-SC was knocked down by RNA interference (RNAi), and caused the significant increased expression of diptericin and attacin. The pupation of MdPGRP-SC-depleted larvae was severely suppressed compared to controls. Opposite to the expression trend of MdPGRP-SC, a spontaneous active expression of diptericin and attacin was found in pre-pupae but not in third instar larvae. Taken together, our study reveals that downregulation of MdPGRP-SC leads to the overexpression of the AMPs, and is involved in the larvae-to-pupa transition of housefly. PMID:26296288

  19. Lead

    MedlinePlus

    ... Lead Share Facebook Twitter Google+ Pinterest Contact Us Lead Poisoning is Preventable If your home was built before ... of the RRP rule. Read more . Learn about Lead Poisoning Prevention Week . Report Uncertified Contractors and Environmental Violations ...

  20. Lead

    MedlinePlus

    ... obvious symptoms, it frequently goes unrecognized. CDC’s Childhood Lead Poisoning Prevention Program is committed to the Healthy People ... Lead Levels Information for Parents Tips for preventing lead poisoning About Us Overview of CDC’s Childhood Lead Poisoning ...

  1. Overexpression of flavodoxin in bacteroids induces changes in antioxidant metabolism leading to delayed senescence and starch accumulation in alfalfa root nodules.

    PubMed

    Redondo, Francisco J; de la Peña, Teodoro Coba; Morcillo, César N; Lucas, M Mercedes; Pueyo, José J

    2009-02-01

    Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity. PMID:19098093

  2. Nonsymbiotic hemoglobin-2 leads to an elevated energy state and to a combined increase in polyunsaturated fatty acids and total oil content when overexpressed in developing seeds of transgenic Arabidopsis plants.

    PubMed

    Vigeolas, Helene; Hühn, Daniela; Geigenberger, Peter

    2011-03-01

    Nonsymbiotic hemoglobins are ubiquitously expressed in plants and divided into two different classes based on gene expression pattern and oxygen-binding properties. Most of the published research has been on the function of class 1 hemoglobins. To investigate the role of class 2 hemoglobins, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated overexpressing Arabidopsis hemoglobin-2 (AHb2) under the control of a seed-specific promoter. Overexpression of AHb2 led to a 40% increase in the total fatty acid content of developing and mature seeds in three subsequent generations. This was mainly due to an increase in the polyunsaturated C18:2 (ω-6) linoleic and C18:3 (ω-3) α-linolenic acids. Moreover, AHb2 overexpression led to an increase in the C18:2/C18:1 and C18:3/C18:2 ratios as well as in the C18:3 content in mol % of total fatty acids and in the unsaturation/saturation index of total seed lipids. The increase in fatty acid content was mainly due to a stimulation of the rate of triacylglycerol synthesis, which was attributable to a 3-fold higher energy state and a 2-fold higher sucrose content of the seeds. Under low external oxygen, AHb2 overexpression maintained an up to 5-fold higher energy state and prevented fermentation. This is consistent with AHb2 overexpression results in improved oxygen availability within developing seeds. In contrast to this, overexpression of class 1 hemoglobin did not lead to any significant increase in the metabolic performance of the seeds. These results provide evidence for a specific function of class 2 hemoglobin in seed oil production and in promoting the accumulation of polyunsaturated fatty acids by facilitating oxygen supply in developing seeds. PMID:21205621

  3. Over-expression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1.

    PubMed

    Chen, Yingnan; Fan, Xiaorong; Song, Wenjing; Zhang, Yali; Xu, Guohua

    2012-02-01

    Crop architecture parameters such as tiller number, angle and plant height are important agronomic traits that have been considered for breeding programmes. Auxin distribution within the plant has long been recognized to alter architecture. The rice (Oryza sativa L.) genome contains 12 putative PIN genes encoding auxin efflux transporters, including four PIN1 and one PIN2 genes. Here, we report that over-expression of OsPIN2 through a transgenic approach in rice (Japonica cv. Nipponbare) led to a shorter plant height, more tillers and a larger tiller angle when compared with wild type (WT). The expression patterns of the auxin reporter DR5::GUS and quantification of auxin distribution showed that OsPIN2 over-expression increased auxin transport from the shoot to the root-shoot junction, resulting in a non-tissue-specific accumulation of more free auxin at the root-shoot junction relative to WT. Over-expression of OsPIN2 enhanced auxin transport from shoots to roots, but did not alter the polar auxin pattern in the roots. Transgenic plants were less sensitive to N-1-naphthylphthalamic acid, an auxin transport inhibitor, than WT in their root growth. OsPIN2-over-expressing plants had suppressed the expression of a gravitropism-related gene OsLazy1 in the shoots, but unaltered expression of OsPIN1b and OsTAC1, which were reported as tiller angle controllers in rice. The data suggest that OsPIN2 has a distinct auxin-dependent regulation pathway together with OsPIN1b and OsTAC1 controlling rice shoot architecture. Altering OsPIN2 expression by genetic transformation can be directly used for modifying rice architecture. PMID:21777365

  4. Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations.

    PubMed

    Sillers, Ryan; Al-Hinai, Mohab Ali; Papoutsakis, Eleftherios T

    2009-01-01

    Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity. PMID:18726959

  5. Overexpressions of xylA and xylB in Klebsiella pneumoniae Lead to Enhanced 1,3-Propanediol Production by Cofermentation of Glycerol and Xylose.

    PubMed

    Lu, Xinyao; Fu, Xiaomeng; Zong, Hong; Zhuge, Bin

    2016-07-28

    1,3-Propanediol (1,3-PD) is a valuable platform compound. Many studies have shown that the supplement of NADH plays a key role in the bioproduction of 1,3-PD from Klebsiella pneumoniae. In this study, the xylA and xylB genes from Escherichia coli were overexpressed individually or simultaneously in K. pneumoniae to improve the production of 1,3-PD by cofermentation of glycerol and xylose. Compared with the parent strain, the xylose consumption was significantly increased by the introduction of these two genes. The 1,3-PD titers were raised from 17.9 g/l to 23.5, 23.9, and 24.4 g/l, respectively, by the overexpression of xylA and xylB as well as their coexpression. The glycerol conversion rate (mol/mol) was enhanced from 54.1% to 73.8%. The concentration of 2,3-butanediol was increased by 50% at the middle stage but drastically decreased after that. The NADH and NADH/NAD(+) ratio were improved. This report suggests that overexpression of xylA or xylB is an effective strategy to improve the xylose assimilation rate to provide abundant reducing power for the biosynthesis of 1,3-PD in K. pneumoniae. PMID:27056473

  6. Forebrain overexpression of CK1δ leads to down-regulation of dopamine receptors and altered locomotor activity reminiscent of ADHD

    PubMed Central

    Zhou, Mingming; Rebholz, Heike; Brocia, Christine; Warner-Schmidt, Jennifer L.; Fienberg, Allen A.; Nairn, Angus C.; Greengard, Paul; Flajolet, Marc

    2010-01-01

    Dopamine neurotransmission controls motor and perseverative behavior, is mediated by protein phosphorylation, and may be perturbed in disorders of attention and hyperactivity. To assess the role of casein kinase I (CK1) in the regulation of dopamine signaling, we generated a genetically modified mouse line that overexpresses CK1δ (CK1δ OE) specifically in the forebrain. Overexpression was confirmed both at the mRNA and at the protein levels. Under basal conditions, CK1δ OE mice exhibited horizontal and vertical hyperactivity, reduced anxiety, and nesting behavior deficiencies. The CK1δ OE mice also presented paradoxical responses to dopamine receptor stimulation, showing hypoactivity following injection of d-amphetamine or methylphenidate, indicating that CK1 activity has a profound effect on dopamine signaling in vivo. Interestingly, CK1δ overexpression led to significantly reduced D1R and D2R dopamine receptor levels. All together, under basal conditions and in response to drug stimulation, the behavioral phenotype of CK1δ OE mice is reminiscent of the symptoms and drug responses observed in attention-deficit/hyperactivity disorder and therefore the CK1δ OE mice appear to be a model for this disorder. PMID:20145109

  7. Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta-gonad-mapharsen hematopoiesis

    SciTech Connect

    Ohtsu, Naoki; Nobuhisa, Ikuo; Mochita, Miyuki; Taga, Tetsuya . E-mail: taga@kaiju.medic.kumamoto-u.ac.jp

    2007-01-01

    Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45{sup +} hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45{sup low}c-Kit{sup +} cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.

  8. Overexpression of Fkbp11, a feature of lupus B cells, leads to B cell tolerance breakdown and initiates plasma cell differentiation

    PubMed Central

    Ruer-Laventie, Julie; Simoni, Léa; Schickel, Jean-Nicolas; Soley, Anne; Duval, Monique; Knapp, Anne-Marie; Marcellin, Luc; Lamon, Delphine; Korganow, Anne-Sophie; Martin, Thierry; Pasquali, Jean-Louis; Soulas-Sprauel, Pauline

    2015-01-01

    Systemic Lupus Erythematosus (SLE) is a severe systemic autoimmune disease, characterized by multi-organ damages, triggered by an autoantibody-mediated inflammation, and with a complex genetic influence. It is today accepted that adult SLE arises from the building up of many subtle gene variations, each one adding a new brick on the SLE susceptibility and contributing to a phenotypic trait to the disease. One of the ways to find these gene variations consists in comprehensive analysis of gene expression variation in a precise cell type, which can constitute a good complementary strategy to genome wide association studies. Using this strategy, and considering the central role of B cells in SLE, we analyzed the B cell transcriptome of quiescent SLE patients, and identified an overexpression of FKBP11, coding for a cytoplasmic putative peptidyl-prolyl cis/trans isomerase and chaperone enzyme. To understand the consequences of FKBP11 overexpression on B cell function and on autoimmunity's development, we created lentiviral transgenic mice reproducing this gene expression variation. We showed that high expression of Fkbp11 reproduces by itself two phenotypic traits of SLE in mice: breakdown of B cell tolerance against DNA and initiation of plasma cell differentiation by acting upstream of Pax5 master regulator gene. PMID:26417441

  9. Overexpression of Fkbp11, a feature of lupus B cells, leads to B cell tolerance breakdown and initiates plasma cell differentiation.

    PubMed

    Ruer-Laventie, Julie; Simoni, Léa; Schickel, Jean-Nicolas; Soley, Anne; Duval, Monique; Knapp, Anne-Marie; Marcellin, Luc; Lamon, Delphine; Korganow, Anne-Sophie; Martin, Thierry; Pasquali, Jean-Louis; Soulas-Sprauel, Pauline

    2015-09-01

    Systemic Lupus Erythematosus (SLE) is a severe systemic autoimmune disease, characterized by multi-organ damages, triggered by an autoantibody-mediated inflammation, and with a complex genetic influence. It is today accepted that adult SLE arises from the building up of many subtle gene variations, each one adding a new brick on the SLE susceptibility and contributing to a phenotypic trait to the disease. One of the ways to find these gene variations consists in comprehensive analysis of gene expression variation in a precise cell type, which can constitute a good complementary strategy to genome wide association studies. Using this strategy, and considering the central role of B cells in SLE, we analyzed the B cell transcriptome of quiescent SLE patients, and identified an overexpression of FKBP11, coding for a cytoplasmic putative peptidyl-prolyl cis/trans isomerase and chaperone enzyme. To understand the consequences of FKBP11 overexpression on B cell function and on autoimmunity's development, we created lentiviral transgenic mice reproducing this gene expression variation. We showed that high expression of Fkbp11 reproduces by itself two phenotypic traits of SLE in mice: breakdown of B cell tolerance against DNA and initiation of plasma cell differentiation by acting upstream of Pax5 master regulator gene. PMID:26417441

  10. Overexpression of an Isoprenyl Diphosphate Synthase in Spruce Leads to Unexpected Terpene Diversion Products That Function in Plant Defense1[W][OPEN

    PubMed Central

    Nagel, Raimund; Berasategui, Aileen; Paetz, Christian; Gershenzon, Jonathan; Schmidt, Axel

    2014-01-01

    Spruce (Picea spp.) and other conifers employ terpenoid-based oleoresin as part of their defense against herbivores and pathogens. The short-chain isoprenyl diphosphate synthases (IDS) are situated at critical branch points in terpene biosynthesis, producing the precursors of the different terpenoid classes. To determine the role of IDS and to create altered terpene phenotypes for assessing the defensive role of terpenoids, we overexpressed a bifunctional spruce IDS, a geranyl diphosphate and geranylgeranyl diphosphate synthase in white spruce (Picea glauca) saplings. While transcript level (350-fold), enzyme activity level (7-fold), and in planta geranyl diphosphate and geranylgeranyl diphosphate levels (4- to 8-fold) were significantly increased in the needles of transgenic plants, there was no increase in the major monoterpenes and diterpene acids of the resin and no change in primary isoprenoids, such as sterols, chlorophylls, and carotenoids. Instead, large amounts of geranylgeranyl fatty acid esters, known from various gymnosperm and angiosperm plant species, accumulated in needles and were shown to act defensively in reducing the performance of larvae of the nun moth (Lymantria monacha), a conifer pest in Eurasia. These results show the impact of overexpression of an IDS and the defensive role of an unexpected accumulation product of terpenoid biosynthesis with the potential for a broader function in plant protection. PMID:24346420

  11. Overexpression of the Aspergillus niger GatA transporter leads to preferential use of D-galacturonic acid over D-xylose

    PubMed Central

    2014-01-01

    Pectin is a structural heteropolysaccharide of the primary cell walls of plants and as such is a significant fraction of agricultural waste residues that is currently insufficiently used. Its main component, D-galacturonic acid, is an attractive substrate for bioconversion. The complete metabolic pathway is present in the genome of Aspergillus niger, that is used in this study. The objective was to identify the D-galacturonic acid transporter in A. niger and to use this transporter to study D-galacturonic acid metabolism. We have functionally characterized the gene An14g04280 that encodes the D-galacturonic acid transporter in A. niger. In a mixed sugar fermentation it was found that the An14g04280 overexpression strain, in contrast to the parent control strain, has a preference for D-galacturonic acid over D-xylose as substrate. Overexpression of this transporter in A. niger resulted in a strong increase of D-galacturonic acid uptake and induction of the D-galacturonic acid reductase activity, suggesting a metabolite controlled regulation of the endogenous D-galacturonic acid catabolic pathway. PMID:25177540

  12. The over-expression of an Arabidopsis B3 transcription factor, ABS2/NGAL1, leads to the loss of flower petals.

    PubMed

    Shao, Jingxia; Liu, Xiayan; Wang, Rui; Zhang, Gaisheng; Yu, Fei

    2012-01-01

    Transcriptional regulations are involved in many aspects of plant development and are mainly achieved through the actions of transcription factors (TF). To investigate the mechanisms of plant development, we carried out genetic screens for mutants with abnormal shoot development. Taking an activation tagging approach, we isolated a gain-of-function mutant abs2-1D (abnormal shoot 2-1D). abs2-1D showed pleiotropic growth defects at both the vegetative and reproductive developmental stages. We cloned ABS2 and it encodes a RAV sub-family of plant B3 type of transcriptional factors. Phylogenetic analysis showed that ABS2 was closely related to NGATHA (NGA) genes that are involved in flower development and was previously named NGATHA-Like 1 (NGAL1). NGAL1 was expressed mainly in the root and the filament of the stamen in flower tissues and sub-cellular localization assay revealed that NGAL1 accumulated in the nucleus. Interestingly, over-expression of NGAL1 driven by the constitutive 35S promoter led to transgenic plants with conspicuous flower defects, particularly a loss-of-petal phenotype. A loss-of-function ngal1-1 mutant did not show obvious phenotype, suggesting the existence of redundant activities and also the utility of gain-of-function genetic screens. Our results show that the over-expression of NGAL1 is capable of altering flower petal development, as well as shoot development. PMID:23185464

  13. Overexpression of BDNF Increases Excitability of the Lumbar Spinal Network and Leads to Robust Early Locomotor Recovery in Completely Spinalized Rats

    PubMed Central

    Ziemlińska, Ewelina; Kügler, Sebastian; Schachner, Melitta; Wewiór, Iwona; Czarkowska-Bauch, Julita; Skup, Małgorzata

    2014-01-01

    Strategies to induce recovery from lesions of the spinal cord have not fully resulted in clinical applications. This is a consequence of a number of impediments that axons encounter when trying to regrow beyond the lesion site, and that intraspinal rearrangements are subjected to. In the present study we evaluated (1) the possibility to improve locomotor recovery after complete transection of the spinal cord by means of an adeno-associated (AAV) viral vector expressing the neurotrophin brain-derived neurotrophic factor (BDNF) in lumbar spinal neurons caudal to the lesion site and (2) how the spinal cord transection and BDNF treatment affected neurotransmission in the segments caudal to the lesion site. BDNF overexpression resulted in clear increases in expression levels of molecules involved in glutamatergic (VGluT2) and GABAergic (GABA, GAD65, GAD67) neurotransmission in parallel with a reduction of the potassium-chloride co-transporter (KCC2) which contributes to an inhibitory neurotransmission. BDNF treated animals showed significant improvements in assisted locomotor performance, and performed locomotor movements with body weight support and plantar foot placement on a moving treadmill. These positive effects of BDNF local overexpression were detectable as early as two weeks after spinal cord transection and viral vector application and lasted for at least 7 weeks. Gradually increasing frequencies of clonic movements at the end of the experiment attenuated the quality of treadmill walking. These data indicate that BDNF has the potential to enhance the functionality of isolated lumbar circuits, but also that BDNF levels have to be tightly controlled to prevent hyperexcitability. PMID:24551172

  14. Maintenance of Chloroplast Structure and Function by Overexpression of the Rice MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE Gene Leads to Enhanced Salt Tolerance in Tobacco1[W

    PubMed Central

    Wang, Shiwen; Uddin, M. Imtiaz; Tanaka, Kiyoshi; Yin, Lina; Shi, Zhonghui; Qi, Yanhua; Mano, Jun’ichi; Matsui, Kenji; Shimomura, Norihiro; Sakaki, Takeshi; Deng, Xiping; Zhang, Suiqi

    2014-01-01

    In plants, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactodiacylglycerol (DGDG) are major constituents of photosynthetic membranes in chloroplasts. One of the key enzymes for the biosynthesis of these galactolipids is MGDG synthase (MGD). To investigate the role of MGD in the plant’s response to salt stress, we cloned an MGD gene from rice (Oryza sativa) and generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD. The MGD activity in OsMGD transgenic plants was confirmed to be higher than that in the wild-type tobacco cultivar SR1. Immunoblot analysis indicated that OsMGD was enriched in the outer envelope membrane of the tobacco chloroplast. Under salt stress, the transgenic plants exhibited rapid shoot growth and high photosynthetic rate as compared with the wild type. Transmission electron microscopy observation showed that the chloroplasts from salt-stressed transgenic plants had well-developed thylakoid membranes and properly stacked grana lamellae, whereas the chloroplasts from salt-stressed wild-type plants were fairly disorganized and had large membrane-free areas. Under salt stress, the transgenic plants also maintained higher chlorophyll levels. Lipid composition analysis showed that leaves of transgenic plants consistently contained significantly higher MGDG (including 18:3-16:3 and 18:3-18:3 species) and DGDG (including 18:3-16:3, 18:3-16:0, and 18:3-18:3 species) contents and higher DGDG-MGDG ratios than the wild type did under both control and salt stress conditions. These results show that overexpression of OsMGD improves salt tolerance in tobacco and that the galactolipids MGDG and DGDG play an important role in the regulation of chloroplast structure and function in the plant salt stress response. PMID:24843077

  15. Overexpression of Plasmodium berghei ATG8 by Liver Forms Leads to Cumulative Defects in Organelle Dynamics and to Generation of Noninfectious Merozoites

    PubMed Central

    Voss, Christiane; Ehrenman, Karen; Mlambo, Godfree; Mishra, Satish; Kumar, Kota Arun; Sacci, John B.; Sinnis, Photini

    2016-01-01

    ABSTRACT Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity. PMID:27353755

  16. Hepatitis B virus (HBV) receptors: Deficiency in tumor results in scant HBV infection and overexpression in peritumor leads to higher recurrence risk

    PubMed Central

    Ye, Fei; Fan, Qing-Min; Yu, Guo-Feng; Yu, Dan-Dan; Gao, Lu; Sun, Kai; Han, Zhi-Peng; Li, Rong; Yang, Yang; Zhao, Qiu-Dong; Wu, Meng-Chao; Wang, Hong-Yang; Wei, Li-Xin

    2015-01-01

    Hepatitis B virus (HBV) infection is a risk factor for hepatocarcinogenesis and recurrence. Here, we sought to characterize intratumoral and peritumoral expression of HBsAg and its specific receptors in HBsAg-positive hepatocellular carcinoma (HCC) patients and further examined their correlation with the recurrence-free survival (RFS). HCC tissue and adjacent normal tissue specimens were acquired from HBsAg-positive patients. The presence of HBsAg and receptors, as well as hepatic progenitor cells (HPCs) were detected by tissue microassay and immunohistochemistry. Necroinflammatory activity was evaluated by HE staining. The mean IOD of HBsAg and HBV DNA in the intratumoral tissues was markedly lower than that in the peritumoral tissues (P < 0.001). Pearson correlation analysis further showed a significant correlation between the expression of HBsAg and NTCP (r = 0.461, P < 0.001) or ASGPR (r = 0.506, P < 0.001) in peritumoral tissues. And the peritumoral HBsAg and receptors presented a positive association with necroinflammatory activity (P < 0.05). Inflammation induced by HBV infection presented a positive association with HPCs activation (P < 0.05). Additionally, due to lack of HBV receptors, HPCs was not preferentially infected with HBV, but activated HPCs had a significant correlation with HBsAg expression in peritumoral tissues, and the peritumoral HPCs activation was associated with RFS of HCC patients, therefore, the overexpression of HBsAg and receptors in peritumor were also with higher recurrence risk (P < 0.05). In conclusion, lack of HBV receptors resulted in scant HBV infection in tumor cells, and overexpression of HBsAg and receptors in peritumor was strongly associated with higher recurrence risk in HCC patients. PMID:26515593

  17. Overexpression of smooth muscle myosin heavy chain leads to activation of the unfolded protein response and autophagic turnover of thick filament-associated proteins in vascular smooth muscle cells.

    PubMed

    Kwartler, Callie S; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V; Walker, Lori; Hill, Joseph A; Epstein, Henry F; Taegtmeyer, Heinrich; Milewicz, Dianna M

    2014-05-16

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications. PMID:24711452

  18. A Novel Function for the nm23-Hl Gene: Overexpression in Human Breast Carcinoma Cells Leads to the Formation of Basement Membrane and Growth Arrest

    SciTech Connect

    Howlett, Anthony R; Petersen, Ole W; Steeg, Patricia S; Bissell, Mina J

    1994-01-01

    We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia. Our purpose was to use this assay to investigate the role of the putative metastasis suppressor gene nm23-H1 in mammary development and differentiation. The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest. In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship. The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene

  19. Over-Expression of the Pikh Gene with a CaMV 35S Promoter Leads to Improved Blast Disease (Magnaporthe oryzae) Tolerance in Rice

    PubMed Central

    Azizi, Parisa; Rafii, Mohd Y.; Abdullah, Siti N. A.; Hanafi, Mohamed M.; Maziah, M.; Sahebi, Mahbod; Ashkani, Sadegh; Taheri, Sima; Jahromi, Mohammad F.

    2016-01-01

    Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g−1 in transgenic plants. The M. oryzae population was constant at 31, 48

  20. The Over-Expression of Two Transcription Factors, ABS5/bHLH30 and ABS7/MYB101, Leads to Upwardly Curly Leaves

    PubMed Central

    Wang, Rui; Wu, Haicui; Liang, Shuang; Shao, Jingxia; Qi, Yafei; An, Lijun; Yu, Fei

    2014-01-01

    Proper leaf development is essential for plant growth and development, and leaf morphogenesis is under the control of intricate networks of genetic and environmental cues. We are interested in dissecting these regulatory circuits genetically and report here the isolation of two Arabidopsis dominant mutants, abnormal shoot5-1D (abs5-1D) and abs7-1D identified through activation tagging screens. Both abs5-1D and abs7-1D display an intriguing upwardly curly leaf phenotype. Molecular cloning showed that the elevated expression of a bHLH transcription factor ABS5/T5L1/bHLH30 or a MYB transcription factor ABS7/MYB101 is the cause for the abnormal leaf phenotypes found in abs5-1D or abs7-1D, respectively. Protoplast transient expression assays confirmed that both ABS5/T5L1 and ABS7/MYB101 are targeted to the nucleus. Interestingly, the expression domains of auxin response reporter DR5::GUS were abnormal in leaves of abs5-1D and ABS5/T5L1 over-expression lines. Moreover, cotyledon venation analysis showed that more areoles and free-ending veins are formed in abs5-1D. We found that the epidermis-specific expressions of ABS5/T5L1 or ABS7/MYB101 driven by the Arabidopsis Meristem Layer 1 promoter (PAtML1) were sufficient to recapitulate the curly leaf phenotype of abs5-1D or abs7-1D. In addition, PAtML1::ABS5 lines exhibited similar changes in DR5::GUS expression patterns as those found in 35S-driven ABS5/T5L1 over-expression lines. Our work demonstrated that enhanced expressions of two transcription factors, ABS5/T5L1 and ABS7/MYB101, are able to alter leaf lamina development and reinforce the notion that leaf epidermis plays critical roles in regulating plant organ morphogenesis. PMID:25268707

  1. Over-Expression of the Pikh Gene with a CaMV 35S Promoter Leads to Improved Blast Disease (Magnaporthe oryzae) Tolerance in Rice.

    PubMed

    Azizi, Parisa; Rafii, Mohd Y; Abdullah, Siti N A; Hanafi, Mohamed M; Maziah, M; Sahebi, Mahbod; Ashkani, Sadegh; Taheri, Sima; Jahromi, Mohammad F

    2016-01-01

    Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g(-1) in transgenic plants. The M. oryzae population was constant at 31, 48

  2. Over-expression of F5H in COMT-deficient Arabidopsis leads to enrichment of an unusual lignin and disruption of pollen wall formation.

    PubMed

    Weng, Jing-Ke; Mo, Huaping; Chapple, Clint

    2010-12-01

    The presence of the phenylpropanoid polymer lignin in plant cell walls impedes breakdown of polysaccharides to the fermentable sugars that are used in biofuel production. Genetically modified plants with altered lignin properties hold great promise to improve biomass degradability. Here, we describe the generation of a new type of lignin enriched in 5-hydroxy-guaiacyl units by over-expressing ferulate 5-hydroxylase in a line of Arabidopsis lacking caffeic acid O-methyltransferase. The lignin modification strategy had a profound impact on plant growth and development and cell-wall properties, and resulted in male sterility due to complete disruption of formation of the pollen wall. The modified plants showed significantly improved cell-wall enzymatic saccharification efficiency without a reduction in post-harvest biomass yield despite the alterations in the overall growth morphology. This study demonstrated the plasticity of lignin polymerization in terms of incorporation of unusual monomers that chemically resemble conventional monomers, and also revealed the link between the biosynthetic pathways of lignin and the pollen wall-forming sporopollenin. PMID:21143672

  3. ALOX5AP Overexpression in Adipose Tissue Leads to LXA4 Production and Protection Against Diet-Induced Obesity and Insulin Resistance.

    PubMed

    Elias, Ivet; Ferré, Tura; Vilà, Laia; Muñoz, Sergio; Casellas, Alba; Garcia, Miquel; Molas, Maria; Agudo, Judith; Roca, Carles; Ruberte, Jesús; Bosch, Fatima; Franckhauser, Sylvie

    2016-08-01

    Eicosanoids, such as leukotriene B4 (LTB4) and lipoxin A4 (LXA4), may play a key role during obesity. While LTB4 is involved in adipose tissue inflammation and insulin resistance, LXA4 may exert anti-inflammatory effects and alleviate hepatic steatosis. Both lipid mediators derive from the same pathway, in which arachidonate 5-lipoxygenase (ALOX5) and its partner, arachidonate 5-lipoxygenase-activating protein (ALOX5AP), are involved. ALOX5 and ALOX5AP expression is increased in humans and rodents with obesity and insulin resistance. We found that transgenic mice overexpressing ALOX5AP in adipose tissue had higher LXA4 rather than higher LTB4 levels, were leaner, and showed increased energy expenditure, partly due to browning of white adipose tissue (WAT). Upregulation of hepatic LXR and Cyp7a1 led to higher bile acid synthesis, which may have contributed to increased thermogenesis. In addition, transgenic mice were protected against diet-induced obesity, insulin resistance, and inflammation. Finally, treatment of C57BL/6J mice with LXA4, which showed browning of WAT, strongly suggests that LXA4 is responsible for the transgenic mice phenotype. Thus, our data support that LXA4 may hold great potential for the future development of therapeutic strategies for obesity and related diseases. PMID:27207555

  4. Overexpression of an endo-1,4-β-glucanase V gene (EGV) from Trichoderma reesei leads to the accumulation of cellulase activity in transgenic rice.

    PubMed

    Li, X Y; Liu, F; Hu, Y F; Xia, M; Cheng, B J; Zhu, S W; Ma, Q

    2015-01-01

    The ectopic expression of cellulase in biomass can reduce the cost of biofuel conversion. This trait modification technique is highly beneficial for biofuel production. In this study, we isolated an endo-1,4-beta-glucanase gene (EGV) from Trichoderma reesei and inserted this gene downstream of a fragment encoding the signal peptide Apo-SP in a modified pCAMBIA1301 vector to obtain an Apo-SP and AsRed fusion protein. Transient expression of this fusion protein in onion epidermal cells showed that the Apo-SP signal was localized to the plastids. EGV transgenic rice plants that did not carry screening marker genes were obtained through overexpression of the pDTB double T-DNA vector. Western blotting showed that EGV was expressed in the dry straw of T0 generation transgenic rice plants and in fresh leaves of the T1 generation. More importantly, our results also showed that the peptide product of EGV in the transgenic plants folded correctly and was capable of digesting the cellulase substrate CMC. Additionally, cellulase activity remained stable in the straw that had been dried at room temperature for three months. This study presents an important technical approach for the development of transgenic rice straw that has stable cellulase activity and can be used for biofuel conversion. PMID:26782396

  5. Variant Rett syndrome in a girl with a pericentric X-chromosome inversion leading to epigenetic changes and overexpression of the MECP2 gene.

    PubMed

    Vieira, José Pedro; Lopes, Fátima; Silva-Fernandes, Anabela; Sousa, Maria Vânia; Moura, Sofia; Sousa, Susana; Costa, Bruno M; Barbosa, Mafalda; Ylstra, Bauke; Temudo, Teresa; Lourenço, Teresa; Maciel, Patrícia

    2015-11-01

    Rett syndrome is a neurodevelopmental disorder caused by mutations in the MECP2 gene. We investigated the genetic basis of disease in a female patient with a Rett-like clinical. Karyotype analysis revealed a pericentric inversion in the X chromosome -46,X,inv(X)(p22.1q28), with breakpoints in the cytobands where the MECP2 and CDKL5 genes are located. FISH analysis revealed that the MECP2 gene is not dislocated by the inversion. However, and in spite of a balanced pattern of X inactivation, this patient displayed hypomethylation and an overexpression of the MECP2 gene at the mRNA level in the lymphocytes (mean fold change: 2.55±0.38) in comparison to a group of control individuals; the expression of the CDKL5 gene was similar to that of controls (mean fold change: 0.98±0.10). No gains or losses were detected in the breakpoint regions encompassing known or suspected transcription regulatory elements. We propose that the de-regulation of MECP2 expression in this patient may be due to alterations in long-range genomic interactions caused by the inversion and hypothesize that this type of epigenetic de-regulation of the MECP2 may be present in other RTT-like patients. PMID:26287660

  6. Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice

    PubMed Central

    Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

    2015-01-01

    Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity. PMID:26305245

  7. Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

    PubMed

    Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

    2015-01-01

    Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity. PMID:26305245

  8. Stable knock-down of efflux transporters leads to reduced glucuronidation in UGT1A1-overexpressing HeLa cells: the evidence for glucuronidation-transport interplay.

    PubMed

    Zhang, Xingwang; Dong, Dong; Wang, Huailing; Ma, Zhiguo; Wang, Yifei; Wu, Baojian

    2015-04-01

    Efflux of glucuronide is facilitated by the membrane transporters including BCRP and MRPs. In this study, we aimed to determine the effects of transporter expression on glucuronide efflux and cellular glucuronidation. Single efflux transporter (i.e., BCRP, MRP1, MRP3, or MRP4) was stably knocked-down in UGT1A1-overexpressing HeLa cells. Knock-down of transporters was performed by stable transfection of short-hairpin RNA (shRNA) using lentiviral vectors. Glucuronidation and glucuronide transport in the cells were characterized using three different aglycones (i.e., genistein, apigenin, and emodin) with distinct metabolic activities. BCRP knock-down resulted in significant reductions in excretion of glucuronides (42.9% for genistein glucuronide (GG), 21.1% for apigenin glucuronide (AG) , and 33.7% for emodin glucuronide (EG); p < 0.01) and in cellular glucuronidation (38.3% for genistein, 38.6% for apigenin, and 34.7% for emodin; p < 0.01). Knock-down of a MRP transporter led to substantial decreases in excretion of GG (32.3% for MRP1, 36.7% for MRP3, and 36.6% for MRP4; p < 0.01) and AG (59.3% for MRP1, 24.7% for MRP3, and 34.1% for MRP4; p < 0.01). Also, cellular glucuronidation of genistein (38.3% for MRP1, 32.3% for MRP3, and 31.1% for MRP4; p < 0.01) and apigenin (40.6% for MRP1, 32.4% for MRP3, and 34.6% for MRP4; p < 0.001) was markedly suppressed. By contrast, silencing of MRPs did not cause any changes in either excretion of EG or cellular glucuronidation of emodin. In conclusion, cellular glucuronidation was significantly altered by decreasing expression of efflux transporters, revealing a strong interplay of glucuronidation with efflux transport. PMID:25741749

  9. Overexpression of H1 Calponin in Osteoblast Lineage Cells Leads to a Decrease in Bone Mass by Disrupting Osteoblast Function and Promoting Osteoclast Formation

    PubMed Central

    Su, Nan; Chen, Maomao; Chen, Siyu; Li, Can; Xie, Yangli; Zhu, Ying; Zhang, Yaozong; Zhao, Ling; He, Qifen; Du, Xiaolan; Chen, Di; Chen, Lin

    2013-01-01

    H1 calponin (CNN1) is known as a smooth muscle-specific, actin-binding protein which regulates smooth muscle contractive activity. Although previous studies have shown that CNN1 has effect on bone, the mechanism is not well defined. To investigate the role of CNN1 in maintaining bone homeostasis, we generated transgenic mice overexpressing Cnn1 under the control of the osteoblast-specific 3.6-kb Col1a1 promoter. Col1a1-Cnn1 transgenic mice showed delayed bone formation at embryonic stage and decreased bone mass at adult stage. Morphology analyses showed reduced trabecular number, thickness and defects in bone formation. The proliferation and migration of osteoblasts were decreased in Col1a1-Cnn1 mice due to alterations in cytoskeleton. The early osteoblast differentiation of Col1a1-Cnn1 mice was increased, but the late stage differentiation and mineralization of osteoblasts derived from Col1a1-Cnn1 mice were significantly decreased. In addition to impaired bone formation, the decreased bone mass was also associated with enhanced osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) staining revealed increased osteoclast numbers in tibias of 2-month-old Col1a1-Cnn1 mice, and increased numbers of osteoclasts co-cultured with Col1a1-Cnn1 osteoblasts. The ratio of RANKL to OPG was significantly increased in Col1a1-Cnn1 osteoblasts. These findings reveal a novel function of CNN1 in maintaining bone homeostasis by coupling bone formation to bone resorption. PMID:23044709

  10. Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress.

    PubMed

    Rey, P; Gillet, B; Römer, S; Eymery, F; Massimino, J; Peltier, G; Kuntz, M

    2000-03-01

    Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints. PMID:10758499

  11. Combined exposure to protons and 56Fe leads to overexpression of Il13 and reactivation of repetitive elements in the mouse lung

    NASA Astrophysics Data System (ADS)

    Nzabarushimana, Etienne; Prior, Sara; Miousse, Isabelle R.; Pathak, Rupak; Allen, Antiño R.; Latendresse, John; Olsen, Reid H. J.; Raber, Jacob; Hauer-Jensen, Martin; Nelson, Gregory A.; Koturbash, Igor

    2015-11-01

    Interest in deep space exploration underlines the needs to investigate the effects of exposure to combined sources of space radiation. The lung is a target organ for radiation, and exposure to protons and heavy ions as radiation sources may lead to the development of degenerative disease and cancer. In this study, we evaluated the pro-fibrotic and epigenetic effects of exposure to protons (150 MeV/nucleon, 0.1 Gy) and heavy iron ions (56Fe, 600 MeV/nucleon, 0.5 Gy) alone or in combination (protons on Day 1 and 56Fe on Day 2) in C57BL/6 male mice 4 weeks after irradiation. Exposure to 56Fe, proton or in combination, did not result in histopathological changes in the murine lung. At the same time, combined exposure to protons and 56Fe resulted in pronounced molecular alterations in comparison with either source of radiation alone. Specifically, we observed a substantial increase in the expression of cytokine Il13, loss of expression of DNA methyltransferase Dnmt1, and reactivation of LINE-1, SINE B1 retrotransposons, and major and minor satellites. Given the deleterious potential of the observed effects that may lead to development of chronic lung injury, pulmonary fibrosis, and cancer, future studies devoted to the investigation of the long-term effects of combined exposures to proton and heavy ions are clearly needed.

  12. Combined exposure to protons and (56)Fe leads to overexpression of Il13 and reactivation of repetitive elements in the mouse lung.

    PubMed

    Nzabarushimana, Etienne; Prior, Sara; Miousse, Isabelle R; Pathak, Rupak; Allen, Antiño R; Latendresse, John; Olsen, Reid H J; Raber, Jacob; Hauer-Jensen, Martin; Nelson, Gregory A; Koturbash, Igor

    2015-11-01

    Interest in deep space exploration underlines the needs to investigate the effects of exposure to combined sources of space radiation. The lung is a target organ for radiation, and exposure to protons and heavy ions as radiation sources may lead to the development of degenerative disease and cancer. In this study, we evaluated the pro-fibrotic and epigenetic effects of exposure to protons (150 MeV/nucleon, 0.1 Gy) and heavy iron ions ((56)Fe, 600 MeV/nucleon, 0.5 Gy) alone or in combination (protons on Day 1 and (56)Fe on Day 2) in C57BL/6 male mice 4 weeks after irradiation. Exposure to (56)Fe, proton or in combination, did not result in histopathological changes in the murine lung. At the same time, combined exposure to protons and (56)Fe resulted in pronounced molecular alterations in comparison with either source of radiation alone. Specifically, we observed a substantial increase in the expression of cytokine Il13, loss of expression of DNA methyltransferase Dnmt1, and reactivation of LINE-1, SINE B1 retrotransposons, and major and minor satellites. Given the deleterious potential of the observed effects that may lead to development of chronic lung injury, pulmonary fibrosis, and cancer, future studies devoted to the investigation of the long-term effects of combined exposures to proton and heavy ions are clearly needed. PMID:26553631

  13. Depletion of the Polyamines Spermidine and Spermine by Overexpression of Spermidine/spermine N1-Acetyltransferase1 (SAT1) Leads to Mitochondria-Mediated Apoptosis in Mammalian Cells

    PubMed Central

    Mandal, Swati; Mandal, Ajeet; Park, Myung Hee

    2015-01-01

    The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of HEK293T cells with an adenovirus (AdSAT1) encoding a key polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase1 (SAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/ propidium iodide Fluorescent Activated Cell Sorter (FACS) analyses, Terminal Uridine Nucleotide End- Labeling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1 transduced cells (at 24–72 h), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in the polyamine-depleted cells occurs by the mitochondrial intrinsic pathway, as evidenced by loss of mitochondrial membrane potential, increase in proapoptotic Bax, decrease in anti-apoptotic Bcl-xl, Bcl2, and Mcl-1 and release of cytochrome c from mitochondria, upon transduction with AdSAT1. Moreover, transmission electron microscopy images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial alteration, vacuolization and membrane blebbing. The apoptosis appears to result largely from depletion of the polyamines, spermidine and spermine, as polyamine analogs, α-methylspermidine and N1,N12-dimethylspermine that are not substrates for SAT1 could partially restore growth and prevent apoptosis of AdSAT1-transduced cells. Inhibition of polyamine oxidases did not restore the growth of AdSAT1-transduced cells or block apoptosis, suggesting that the growth arrest and apoptosis were not induced by oxidative stress resulting from accelerated polyamine catabolism. Taken together, these data provide strong evidence that the depletion of polyamines spermidine and spermine leads to mitochondria-mediated apoptosis. PMID:25849284

  14. Characterization of mutations in regulatory genes of Tyl cluster leading to overexpression of tylosin in mutant γ-1 of Streptomyces fradiae NRRL-2702.

    PubMed

    Khaliq, Shazia; Ghauri, Muhammad A; Akhtar, Kalsoom

    2014-01-01

    Tylosin is a veterinary antibiotic and is commercially produced using Streptomyces fradiae. Previously, we developed a mutant γ-1 of S. fradiae NRRL-2702 with a 6.87-fold increase in tylosin yield as compared with the wild-type strain through irradiation mutagenesis. The present studies were conducted to explore mutational changes in regulatory genes (TylQ, TylP, TylS, TylR, and TylT) of Tyl cluster that may lead to an enhanced expression of tylosin. Expression analysis by RT-PCR revealed that TylQ was switched off earlier in mutant γ-1 while no change in expression pattern of TylP was observed between the wild-type and mutant γ-1 strains. However, a point mutation with a substitution of T to A was recorded at position 214 in the 420-bp product of TylP from mutant γ-1 that resulted in a change of one amino acid (serine to threonine) at position 72. Moreover, no mutation in the nucleotide sequence of TylS, TylR, and TylT genes was detected. PMID:24270892

  15. Epithelial-mesenchymal interaction mechanisms leading to the over-expression of neprilysin are involved in the UVB-induced formation of wrinkles in the skin.

    PubMed

    Imokawa, Genji

    2016-08-01

    In clinical studies, the formation of facial wrinkles has been closely linked to the loss of elastic properties of the skin. Repetitive UVB irradiation of animal skin at suberythemal doses significantly reduces its elastic properties, resulting in the formation of wrinkles. That also elicits a marked alteration in the three-dimensional structure of elastic fibres, which is closely associated with a subsequent reduction in the elastic properties of the skin. While UVB irradiation stimulates the activity of skin fibroblast-derived elastase in the dermis, a synthetic inhibitor specific for skin fibroblast-derived elastase as well as an extract of Zingiber officinale (L.) Rose capable of inhibiting skin fibroblast-derived elastase, but not neutrophil elastase, prevented wrinkle formation in our studies of animal and human facial skin, respectively. The close interrelationship among wrinkle formation, elastic properties and elastic fibre linearity is revealed by the effects of different concentrations of the elastase inhibitor, which indicates that enhanced elastase activity by dermal fibroblasts plays a pivotal role in the UVB wrinkling mechanism. Fortunately, we were able to identify human skin fibroblast-derived elastase as the previously known enzyme neprilysin/neutral endopeptidase. Using both a UVB-conditioned medium assay and a co-culture system, we characterized the epithelial-mesenchymal interaction between keratinocytes and fibroblasts which leads to increased expression of neprilysin at the transcriptional, translational and enzymatic levels. Our results demonstrate that interleukin-1α and granulocyte-macrophage colony-stimulating factor are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by skin fibroblasts. PMID:27539896

  16. OsTIR1 and OsAFB2 Downregulation via OsmiR393 Overexpression Leads to More Tillers, Early Flowering and Less Tolerance to Salt and Drought in Rice

    PubMed Central

    Ou, Xiaojin; Fang, Zhongming; Tian, Changen; Duan, Jun; Wang, Yaqin; Zhang, Mingyong

    2012-01-01

    The microRNA miR393 has been shown to play a role in plant development and in the stress response by targeting mRNAs that code for the auxin receptors in Arabidopsis. In this study, we verified that two rice auxin receptor gene homologs (OsTIR1 and OsAFB2) could be targeted by OsmiR393 (Os for Oryza sativa). Two new phenotypes (increased tillers and early flowering) and two previously observed phenotypes (reduced tolerance to salt and drought and hyposensitivity to auxin) were observed in the OsmiR393-overexpressing rice plants. The OsmiR393-overexpressing rice demonstrated hyposensitivity to synthetic auxin-analog treatments. These data indicated that the phenotypes of OsmiR393-overexpressing rice may be caused through hyposensitivity to the auxin signal by reduced expression of two auxin receptor genes (OsTIR1 and OsAFB2). The expression of an auxin transporter (OsAUX1) and a tillering inhibitor (OsTB1) were downregulated by overexpression of OsmiR393, which suggested that a gene chain from OsmiR393 to rice tillering may be from OsTIR1 and OsAFB2 to OsAUX1, which affected the transportation of auxin, then to OsTB1, which finally controlled tillering. The positive phenotypes (increased tillers and early flowering) and negative phenotypes (reduced tolerance to salt and hyposensitivity to auxin) of OsmiR393-overexpressing rice present a dilemma for molecular breeding. PMID:22253868

  17. Overexpression of the transcription factor RAP2.6 leads to enhanced callose deposition in syncytia and enhanced resistance against the beet cyst nematode Heterodera schachtii in Arabidopsis roots

    PubMed Central

    2013-01-01

    Background Cyst nematodes invade the roots of their host plants as second stage juveniles and induce a syncytium which is their source of nutrients throughout their life. A transcriptome analysis of syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots has shown that gene expression in the syncytium is different from that of the root with thousands of genes upregulated or downregulated. Among the downregulated genes are many which code for defense-related proteins. One gene which is strongly downregulated codes for the ethylene response transcription factor RAP2.6. The genome of Arabidopsis contains 122 ERF transcription factor genes which are involved in a variety of developmental and stress responses. Results Expression of RAP2.6 was studied with RT-PCR and a promoter::GUS line. During normal growth conditions the gene was expressed especially in roots and stems. It was inducible by Pseudomonas syringae but downregulated in syncytia from a very early time point on. Overexpression of the gene enhanced the resistance against H. schachtii which was seen by a lower number of nematodes developing on these plants as well as smaller syncytia and smaller female nematodes. A T-DNA mutant had a reduced RAP2.6 transcript level but this did not further increase the susceptibility against H. schachtii. Neither overexpression lines nor mutants had an effect on P. syringae. Overexpression of RAP2.6 led to an elevated expression of JA-responsive genes during early time points after infection by H. schachtii. Syncytia developing on overexpression lines showed enhanced deposition of callose. Conclusions Our results showed that H. schachtii infection is accompanied by a downregulation of RAP2.6. It seems likely that the nematodes use effectors to actively downregulate the expression of this and other defense-related genes to avoid resistance responses of the host plant. Enhanced resistance of RAP2.6 overexpression lines seemed to be due to enhanced

  18. Quantitative nature of overexpression experiments

    PubMed Central

    Moriya, Hisao

    2015-01-01

    Overexpression experiments are sometimes considered as qualitative experiments designed to identify novel proteins and study their function. However, in order to draw conclusions regarding protein overexpression through association analyses using large-scale biological data sets, we need to recognize the quantitative nature of overexpression experiments. Here I discuss the quantitative features of two different types of overexpression experiment: absolute and relative. I also introduce the four primary mechanisms involved in growth defects caused by protein overexpression: resource overload, stoichiometric imbalance, promiscuous interactions, and pathway modulation associated with the degree of overexpression. PMID:26543202

  19. let-7 Overexpression Leads to an Increased Fraction of Cells in G2/M, Direct Down-regulation of Cdc34, and Stabilization of Wee1 Kinase in Primary Fibroblasts*S⃞

    PubMed Central

    Legesse-Miller, Aster; Elemento, Olivier; Pfau, Sarah J.; Forman, Joshua J.; Tavazoie, Saeed; Coller, Hilary A.

    2009-01-01

    microRNAs play a critically important role in a wide array of biological processes including those implicated in cancer, neuro-degenerative and metabolic disorders, and viral infection. Although we have begun to understand microRNA biogenesis and function, experimental demonstration of their functional effects and the molecular mechanisms by which they function remains a challenge. Members of the let-7/miR-98 family play a critical role in cell cycle control with respect to differentiation and tumorigenesis. In this study, we show that exogenous addition of pre-let-7 in primary human fibroblasts results in a decrease in cell number and an increased fraction of cells in the G2/M cell cycle phase. Combining microarray techniques with DNA sequence analysis to identify potential let-7 targets, we discovered 838 genes with a let-7 binding site in their 3′-untranslated region that were down-regulated upon overexpression of let-7b. Among these genes is cdc34, the ubiquitin-conjugating enzyme of the Skp1/cullin/F-box (SCF) complex. Cdc34 protein levels are strongly down-regulated by let-7 overexpression. Reporter assays demonstrated direct regulation of the cdc34 3′-untranslated region by let-7. We hypothesized that low Cdc34 levels would result in decreased SCF activity, stabilization of the SCF target Wee1, and G2/M accumulation. Consistent with this hypothesis, small interfering RNA-mediated down-regulation of Wee1 reversed the G2/M phenotype induced by let-7 overexpression. We conclude that Cdc34 is a functional target of let-7 and that let-7 induces down-regulation of Cdc34, stabilization of the Wee1 kinase, and an increased fraction of cells in G2/M in primary fibroblasts. PMID:19126550

  20. Knocking down p53 with siRNA does not affect the overexpression of p21WAF-1 after exposure of IMR-90 hTERT fibroblasts to a sublethal concentration of H2O2 leading to premature senescence.

    PubMed

    Zdanov, Stephanie; Debacq-Chainiaux, Florence; Toussaint, Olivier

    2007-04-01

    Premature senescence of IMR-90 human diploid fibroblasts (HDFs) expressing telomerase was induced by exposure to sublethal concentration of H(2)O(2), with appearance of several biomarkers of cellular senescence like enlarged cell shape, senescence-associated beta-galactosidase (SA ss-gal) activity, and cell cycle arrest. The induction of stress-induced premature senescence (SIPS) was associated with a transient increase in DNA-binding activity of p53 and an increased expression of p21(WAF-1). p53 small interferent RNA (siRNA) affected the basal level of p21(WAF-1) mRNA but did not affect the overexpression of p21(WAF-1) after stress. This siRNA approach confirms previous results obtained with other methods. PMID:17460194

  1. Overexpression of Poplar PtrWRKY89 in Transgenic Arabidopsis Leads to a Reduction of Disease Resistance by Regulating Defense-Related Genes in Salicylate- and Jasmonate-Dependent Signaling

    PubMed Central

    Liu, Rui; Jiao, Bo; Zhao, Xin; Ling, Zhengyi; Luo, Keming

    2016-01-01

    The plant hormones jasmonic acid (JA) and salicylic acid (SA) play key roles in plant defenses against pathogens and several WRKY transcription factors have been shown to have a role in SA/JA crosstalk. In a previous study, overexpression of the poplar WRKY gene PtrWRKY89 enhanced resistance to pathogens in transgenic poplars. In this study, the promoter of PtrWRKY89 (ProPtrWRKY89) was isolated and used to drive GUS reporter gene. High GUS activity was observed in old leaves of transgenic Arabidopsis containing ProPtrWRKY89-GUS construct and GUS expression was extremely induced by SA solution and SA+MeJA mixture but not by MeJA treatment. Subcellular localization and transactivation assays showed that PtrWRKY89 acted as a transcription activator in the nucleus. Constitutive expression of PtrWRKY89 in Arabidopsis resulted in more susceptible to Pseudomonas syringae and Botrytis cinerea compared to wild-type plants. Quantitative real-time PCR (qRT-PCR) analysis confirmed that marker genes of SA and JA pathways were down-regulated in transgenic Arabidopsis after pathogen inoculations. Overall, our results indicated that PtrWRKY89 modulates a cross talk in resistance to P. syringe and B. cinerea by negatively regulating both SA and JA pathways in Arabidopsis. PMID:27019084

  2. Drug target identification using a trypanosome overexpression library.

    PubMed

    Begolo, Daniela; Erben, Esteban; Clayton, Christine

    2014-10-01

    Elucidation of molecular targets is very important for lead optimization during the drug development process. We describe a direct method to find targets of antitrypanosomal compounds against Trypanosoma brucei using a trypanosome overexpression library. As proof of concept, we treated the library with difluoromethylornithine and DDD85646 and identified their respective targets, ornithine decarboxylase and N-myristoyltransferase. The overexpression library could be a useful tool to study the modes of action of novel antitrypanosomal drug candidates. PMID:25049244

  3. Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

    PubMed

    Akaike, Y; Kuwano, Y; Nishida, K; Kurokawa, K; Kajita, K; Kano, S; Masuda, K; Rokutan, K

    2015-06-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent

  4. Perinatal asphyxia leads to PARP-1 overactivity, p65 translocation, IL-1β and TNF-α overexpression, and apoptotic-like cell death in mesencephalon of neonatal rats: prevention by systemic neonatal nicotinamide administration.

    PubMed

    Neira-Peña, T; Rojas-Mancilla, E; Munoz-Vio, V; Perez, R; Gutierrez-Hernandez, M; Bustamante, D; Morales, P; Hermoso, M A; Gebicke-Haerter, P; Herrera-Marschitz, M

    2015-05-01

    Perinatal asphyxia (PA) is a leading cause of neuronal damage in newborns, resulting in long-term neurological and cognitive deficits, in part due to impairment of mesostriatal and mesolimbic neurocircuitries. The insult can be as severe as to menace the integrity of the genome, triggering the overactivation of sentinel proteins, including poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 overactivation implies increased energy demands, worsening the metabolic failure and depleting further NAD(+) availability. Using a global PA rat model, we report here evidence that hypoxia increases PARP-1 activity, triggering a signalling cascade leading to nuclear translocation of the NF-κB subunit p65, modulating the expression of IL-1β and TNF-α, pro-inflammatory molecules, increasing apoptotic-like cell death in mesencephalon of neonate rats, monitored with Western blots, qPCR, TUNEL and ELISA. PARP-1 activity increased immediately after PA, reaching a maximum 1-8 h after the insult, while activation of the NF-κB signalling pathway was observed 8 h after the insult, with a >twofold increase of p65 nuclear translocation. IL-1β and TNF-α mRNA levels were increased 24 h after the insult, together with a >twofold increase in apoptotic-like cell death. A single dose of the PARP-1 inhibitor nicotinamide (0.8 mmol/kg, i.p.), 1 h post delivery, prevented the effect of PA on PARP-1 activity, p65 translocation, pro-inflammatory cytokine expression and apoptotic-like cell death. The present study demonstrates that PA leads to PARP-1 overactivation, increasing the expression of pro-inflammatory cytokines and cell death in mesencephalon, effects prevented by systemic neonatal nicotinamide administration, supporting the idea that PARP-1 inhibition represents a therapeutic target against the effects of PA. PMID:25835215

  5. Roscovitine-activated HIP2 kinase induces phosphorylation of wt p53 at Ser-46 in human MCF-7 breast cancer cells.

    PubMed

    Wesierska-Gadek, Józefa; Schmitz, M Lienhard; Ranftler, Carmen

    2007-03-01

    Human MCF-7 breast cancer cells are relatively resistant to conventional chemotherapy due to the lack of caspase-3 activity. We reported recently that roscovitine (ROSC), a potent cyclin-dependent kinase 2 inhibitor, arrests human MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis. Exposure of MCF-7 cells to ROSC also strongly activates the wt p53 tumor suppressor protein in a time- and dose-dependent manner. The p53 level increased despite upregulation of Hdm-2 protein and was attributable to the site-specific phosphorylation at Ser-46. The p53 protein phosphorylated at serine 46 causes the up-regulation of the p53AIP1 protein, a component of mitochondria. In the present study we identified the pathway mediating ROSC-induced p53 activation. Exposure of MCF-7 cells to ROSC activated homeodomain-intereacting protein kinase-2 (HIPK2). The overexpression of wild-type but not kinase inactive HIPK2 increased the basal and ROSC-induced level of p53 phosphorylation at Ser-46 and strongly enhanced the rate of apoptosis in cells exposed to ROSC. We show that HIPK2 is activated by ROSC and mediates ROSC-induced P-Ser-46-p53, thereby stabilizing wt p53 and increasing the efficacy of drug-induced apoptosis in MCF-7 cells. These results identify HIPK2 as a component of the ROSC-induced signaling pathway leading to the stabilization and activation of wt p53 protein. PMID:17203463

  6. Overexpression of CD45RA isoforms in carriers of the C77G mutation leads to hyporeactivity of CD4+CD25highFoxp3+ regulatory T cells.

    PubMed

    Pokoyski, C; Lienen, T; Rother, S; Schock, E; Plege-Fleck, A; Geffers, R; Schwinzer, R

    2015-12-01

    Disorders in regulatory T-cell (T(reg)) function can result in the breakdown of immunological self-tolerance. Thus, the identification of mechanisms controlling the activity of T(reg) is of great relevance. We used T(reg) from individuals carrying the C77G polymorphism as models to study the role of CD45 molecules in humans. C77G prevents splicing of CD45 exon A thereby leading to an aberrant expression pattern of CD45 isoforms in affected individuals. Resting and in vitro expanded/activated CD4(+)CD25(high)Foxp3(+) T(reg) from carriers of C77G strongly expressed CD45RA isoforms whereas these isoforms were almost absent in cells from individuals with wild-type CD45. C77G T(reg) showed diminished upregulation of activation markers, lower phosphorylation of p56(lck)(Y505) and a reduced proliferative potential when stimulated with anti-TcR or anti-TcR plus CD28 mAb suggesting decreased responsiveness to activating stimuli. In addition, the capacity to suppress proliferation of conventional CD4(+) T cells was impaired in C77G T(reg). Furthermore, microarray studies revealed distinct gene expression patterns in T(reg) from C77G carriers. These data suggest that the changes in CD45 isoform combination resulting from the C77G mutation alter the responsiveness of T(reg) to TcR-mediated signaling. Targeting CD45 isoform expression might be a useful approach to modulate T(reg) function. PMID:26355564

  7. Zinc supplementation augments in vivo antitumor effect of chemotherapy by restoring p53 function.

    PubMed

    Margalit, Ofer; Simon, Amos J; Yakubov, Eduard; Puca, Rosa; Yosepovich, Ady; Avivi, Camila; Jacob-Hirsch, Jasmine; Gelernter, Ilana; Harmelin, Alon; Barshack, Iris; Rechavi, Gideon; D'Orazi, Gabriella; Givol, David; Amariglio, Ninette

    2012-08-15

    Activated p53 is necessary for tumor suppression. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of functional p53. HIPK2 modulates wild-type p53 activity toward proapoptotic transcription and tumor suppression by the phosphorylation of serine 46. Knock-down of HIPK2 interferes with tumor suppression and sensitivity to chemotherapy. Combined administration of adriamycin and zinc restores activity of misfolded p53 and enables the induction of its proapoptotic and tumor suppressor functions in vitro and in vivo. We therefore looked for a cancer model where HIPK2 expression is low. MMTV-neu transgenic mice overexpressing HER2/neu, develop mammary tumors at puberty with a long latency, showing very low expression of HIPK2. Here we show that whereas these tumors are resistant to adriamycin treatment, a combination of adriamycin and zinc suppresses tumor growth in vivo in these mice, an effect evidenced by the histological features of the mammary tumors. The combined treatment of adriamycin and zinc also restores wild-type p53 conformation and induces proapoptotic transcription activity. These findings may open up new possibilities for the treatment of human cancers via the combination of zinc with chemotherapeutic agents, for a selected group of patients expressing low levels of HIPK2, with an intact p53. In addition, HIPK2 may serve as a new biomarker for tumor aggressiveness. PMID:21932419

  8. Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage.

    PubMed

    de la Vega, Laureano; Hornung, Juliane; Kremmer, Elisabeth; Milanovic, Maja; Schmitz, M Lienhard

    2013-06-01

    Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis. PMID:23620283

  9. Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage

    PubMed Central

    de la Vega, Laureano; Hornung, Juliane; Kremmer, Elisabeth; Milanovic, Maja; Schmitz, M. Lienhard

    2013-01-01

    Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis. PMID:23620283

  10. Overexpression of kinesins mediates docetaxel resistance in breast cancer cells.

    PubMed

    De, Sarmishtha; Cipriano, Rocky; Jackson, Mark W; Stark, George R

    2009-10-15

    Resistance to chemotherapy remains a major barrier to the successful treatment of cancer. To understand mechanisms underlying docetaxel resistance in breast cancer, we used an insertional mutagenesis strategy to identify proteins whose overexpression confers resistance. A strong promoter was inserted approximately randomly into the genomes of tumor-derived breast cancer cells, using a novel lentiviral vector. We isolated a docetaxel-resistant clone in which the level of the kinesin KIFC3 was elevated. When KIFC3 or the additional kinesins KIFC1, KIF1A, or KIF5A were overexpressed in the breast cancer cell lines MDA-MB231 and MDA-MB 468, the cells became more resistant to docetaxel. The binding of kinesins to microtubules opposes the stabilizing effect of docetaxel that prevents cytokinesis and leads to apoptosis. Our finding that kinesins can mediate docetaxel resistance might lead to novel therapeutic approaches in which kinesin inhibitors are paired with taxanes. PMID:19789344

  11. Where Will LEAD Lead?

    ERIC Educational Resources Information Center

    Wildman, Louis

    After setting forth eight assumptions concerning the education of educational administrators, findings about the Leadership in Educational Administration Development (LEAD) program are discussed. The analysis is based on the first-year applications, telephone conversations with staff at a majority of the project sites, and additional material…

  12. LEADING WITH LEADING INDICATORS

    SciTech Connect

    PREVETTE, S.S.

    2005-01-27

    This paper documents Fluor Hanford's use of Leading Indicators, management leadership, and statistical methodology in order to improve safe performance of work. By applying these methods, Fluor Hanford achieved a significant reduction in injury rates in 2003 and 2004, and the improvement continues today. The integration of data, leadership, and teamwork pays off with improved safety performance and credibility with the customer. The use of Statistical Process Control, Pareto Charts, and Systems Thinking and their effect on management decisions and employee involvement are discussed. Included are practical examples of choosing leading indicators. A statistically based color coded dashboard presentation system methodology is provided. These tools, management theories and methods, coupled with involved leadership and employee efforts, directly led to significant improvements in worker safety and health, and environmental protection and restoration at one of the nation's largest nuclear cleanup sites.

  13. NF-E2 Overexpression Delays Erythroid Maturation and Increases Erythrocyte Production

    PubMed Central

    Mutschler, Manuel; Magin, Angela S.; Buerge, Martina; Roelz, Roland; Schanne, Daniel H.; Will, Britta; Pilz, Ingo H.; Migliaccio, Anna Rita; Pahl, Heike L.

    2009-01-01

    Summary The transcription factor Nuclear Factor-Erythroid 2 (NF-E2) is overexpressed in the vast majority of patients with polycythaemia vera (PV). In murine models, NF-E2 overexpression increases proliferation and promotes cellular viability in the absence of erythropoietin (EPO). EPO-independent growth is a hallmark of PV. We therefore hypothesized that NF-E2 overexpression contributes to erythrocytosis, the pathognomonic feature of PV. Consequently, we investigated the effect of NF-E2 overexpression in healthy CD34+ cells. NF-E2 overexpression led to a delay in erythroid maturation, manifested by a belated appearance of glycophorin A-positive erythroid precursors. Maturation delay was similarly observed in primary PV patient erythroid cultures compared to healthy controls. Protracted maturation led to a significant increase in the accumulated number of erythroid cells both in PV cultures and in CD34+ cells overexpressing NF-E2. Similarly, NF-E2 overexpression altered erythroid colony formation, leading to an increase in BFU-E formation. These data indicate that NF-E2 overexpression delays the early phase of erythroid maturation, resulting in an expansion of erythroid progenitors, thereby increasing the number of erythrocytes derived from one CD34+ cell. These data propose a role for NF-E2 in mediating the erythrocytosis of PV. PMID:19466964

  14. The effect of aquaporin 5 overexpression on the Ras signaling pathway

    SciTech Connect

    Woo, Janghee; Lee, Juna; Kim, Myoung Sook; Jang, Se Jin; Sidransky, David; Moon, Chulso

    2008-03-07

    Human aquaporin 5 (AQP5) has been shown to be overexpressed in multiple cancers, such as pancreatic cancer and colon cancer. Furthermore, it has been reported that ectopic expression of AQP5 leads to many phenotypic changes characteristic of transformation. However, the biochemical mechanism leading to transformation in AQP5-overexpressing cells has not been clearly elucidated. In this report, the overexpression of AQP5 in NIH3T3 cells demonstrated a significant effect on Ras activity and, thus, cell proliferation. Furthermore, this influence was shown to be mediated by phosphorylation of the PKA consensus site of AQP5. This is the first evidence demonstrating an association between AQP5 and a signaling pathway, namely the Ras signal transduction pathway, which may be the basis of the oncogenic properties seen in AQP-overexpressing cells.

  15. Negative regulation of beta4 integrin transcription by homeodomain-interacting protein kinase 2 and p53 impairs tumor progression.

    PubMed

    Bon, Giulia; Di Carlo, Selene E; Folgiero, Valentina; Avetrani, Paolo; Lazzari, Chiara; D'Orazi, Gabriella; Brizzi, Maria Felice; Sacchi, Ada; Soddu, Silvia; Blandino, Giovanni; Mottolese, Marcella; Falcioni, Rita

    2009-07-15

    Increased expression of alpha(6)beta(4) integrin in several epithelial cancers promotes tumor progression; however, the mechanism underlying its transcriptional regulation remains unclear. Here, we show that depletion of homeodomain-interacting protein kinase 2 (HIPK2) activates beta(4) transcription that results in a strong increase of beta(4)-dependent mitogen-activated protein kinase and Akt phosphorylation, anchorage-independent growth, and invasion. In contrast, stabilization of HIPK2 represses beta(4) expression in wild-type p53 (wtp53)-expressing cells but not in p53-null cells or cells expressing mutant p53, indicating that HIPK2 requires a wtp53 to inhibit beta(4) transcription. Consistent with our in vitro findings, a strong correlation between beta(4) overexpression and HIPK2 inactivation by cytoplasmic relocalization was observed in wtp53-expressing human breast carcinomas. Under loss of function of HIPK2 or p53, the p53 family members TAp63 and TAp73 strongly activate beta(4) transcription. These data, by revealing that beta(4) expression is transcriptionally repressed in tumors by HIPK2 and p53 to impair beta(4)-dependent tumor progression, suggest that loss of p53 function favors the formation of coactivator complex with the TA members of the p53 family to allow beta(4) transcription. PMID:19567674

  16. ZIC1 Overexpression Is Oncogenic in Liposarcoma

    PubMed Central

    Brill, Elliott; Gobble, Ryan; Angeles, Christina; Lagos-Quintana, Mariana; Crago, Aimee; Laxa, Bernadette; DeCarolis, Penelope; Zhang, Lei; Antonescu, Cristina; Socci, Nicholas D.; Taylor, Barry S.; Sander, Chris; Koff, Andrew; Singer, Samuel

    2012-01-01

    Liposarcomas are aggressive mesenchymal cancers with poor outcomes that exhibit remarkable histologic diversity, with five recognized subtypes. Currently, the mainstay of therapy for liposarcoma is surgical excision since liposarcomas are often resistant to traditional chemotherapy. In light of the high mortality associated with liposarcoma and the lack of effective systemic therapy, we sought novel genomic alterations driving liposarcomagenesis that might serve as therapeutic targets. ZIC1, a critical transcription factor for neuronal development, is overexpressed in all five subtypes of liposarcoma compared with normal fat and in liposarcoma cell lines compared with adipose-derived stem cells (ASC). Here we show that ZIC1 contributes to the pathogenesis of liposarcoma. ZIC1 knockdown inhibits proliferation, reduces invasion, and induces apoptosis in dedifferentiated and myxoid/round cell liposarcoma cell lines, but not in either ASC or a lung cancer cell line with low ZIC1 expression. ZIC1 knockdown is associated with increased nuclear expression of p27 protein, and the down-regulation of pro-survival target genes: BCL2L13, JunD, Fam57A, and EIF3M. Our results demonstrate that ZIC1 expression is essential for liposarcomagenesis and that targeting ZIC1 or its downstream targets may lead to novel therapy for liposarcoma. PMID:20713527

  17. Lead Poisoning

    MedlinePlus

    ... Experiments Stories Lessons Topics Games Activities Lessons MENU Lead Poisoning Kids Homepage Topics Pollution Lead Poisoning What is ... you can avoid contact with it! Sources of Lead Poisoning HOUSE PAINTS: Before1950, lead-based paint was used ...

  18. Lead Test

    MedlinePlus

    ... to determine lead sources, educating family members about lead poisoning , and instituting follow-up testing to monitor the ... high levels of lead, see the article on Lead Poisoning . The Occupational Safety and Health Administration (OSHA) has ...

  19. Lead Poisoning

    MedlinePlus

    Lead Poisoning What is it and who is affected? Lead is a highly toxic substance, exposure to which ... and children can suffer from the effects of lead poisoning, but childhood lead poisoning is much more frequent. ...

  20. Overexpression of Mafb in Podocytes Protects against Diabetic Nephropathy

    PubMed Central

    Yoh, Keigyou; Ojima, Masami; Okamura, Midori; Nakamura, Megumi; Hamada, Michito; Shimohata, Homare; Moriguchi, Takashi; Yamagata, Kunihiro; Takahashi, Satoru

    2014-01-01

    We previously showed that the transcription factor Mafb is essential for podocyte differentiation and foot process formation. Podocytes are susceptible to injury in diabetes, and this injury leads to progression of diabetic nephropathy. In this study, we generated transgenic mice that overexpress Mafb in podocytes using the nephrin promoter/enhancer. To examine a potential pathogenetic role for Mafb in diabetic nephropathy, Mafb transgenic mice were treated with either streptozotocin or saline solution. Diabetic nephropathy was assessed by renal histology and biochemical analyses of urine and serum. Podocyte-specific overexpression of Mafb had no effect on body weight or blood glucose levels in either diabetic or control mice. Notably, albuminuria and changes in BUN levels and renal histology observed in diabetic wild-type animals were ameliorated in diabetic Mafb transgenic mice. Moreover, hyperglycemia-induced downregulation of Nephrin was mitigated in diabetic Mafb transgenic mice, and reporter assay results suggested that Mafb regulates Nephrin directly. Mafb transgenic glomeruli also overexpressed glutathione peroxidase, an antioxidative stress enzyme, and levels of the oxidative stress marker 8-hydroxydeoxyguanosine decreased in the urine of diabetic Mafb transgenic mice. Finally, Notch2 expression increased in diabetic glomeruli, and this effect was enhanced in diabetic Mafb transgenic glomeruli. These data indicate Mafb has a protective role in diabetic nephropathy through regulation of slit diaphragm proteins, antioxidative enzymes, and Notch pathways in podocytes and suggest that Mafb could be a therapeutic target. PMID:24722438

  1. Skeletal overexpression of gremlin impairs bone formation and causes osteopenia.

    PubMed

    Gazzerro, Elisabetta; Pereira, Renata C; Jorgetti, Vanda; Olson, Sarah; Economides, Aris N; Canalis, Ernesto

    2005-02-01

    Skeletal cells synthesize bone morphogenetic proteins (BMPs) and BMP antagonists. Gremlin, a BMP antagonist, is expressed in osteoblasts and opposes BMP effects on osteoblastic differentiation and function in vitro. However, its effects in vivo are not known. To investigate the actions of gremlin on bone remodeling in vivo, we generated transgenic mice overexpressing gremlin under the control of the osteocalcin promoter. Gremlin transgenics exhibited bone fractures and reduced bone mineral density by 20-30%, compared with controls. Static and dynamic histomorphometry of femurs revealed that gremlin overexpression caused reduced trabecular bone volume and the appearance of woven bone. Polarized light microscopy revealed disorganized collagen bundles at the endosteal cortical surface. Gremlin transgenic mice displayed a 70% decrease in the number of osteoblasts/trabecular area and reduced mineral apposition and bone formation rates. In vivo bromodeoxyuridine labeling and marrow stromal cell cultures demonstrated an inhibitory effect of gremlin on osteoblastic cell replication, but no change on apoptosis was detected. Marrow stromal cells from gremlin transgenics displayed a reduced response to BMP on phosphorylated mothers against decapentaplegic 1/5/8 phosphorylation and reduced free cytosolic beta-catenin levels. In conclusion, transgenic mice overexpressing gremlin in the bone microenvironment have decreased osteoblast number and function leading to osteopenia and spontaneous fractures. PMID:15539560

  2. Lead Poisoning

    MedlinePlus

    Lead is a metal that occurs naturally in the earth's crust. Lead can be found in all parts of our ... from human activities such as mining and manufacturing. Lead used to be in paint; older houses may ...

  3. Lead poisoning

    MedlinePlus

    ... swallows a lead object or breathes in lead dust, some of the poison can stay in the ... a health problem. Lead is everywhere, including dirt, dust, new toys, and old house paint. Unfortunately, you ...

  4. Lead Toxicity

    MedlinePlus

    ... homes. • Most people, especially children, who suffer from lead poisoning are exposed through lead-contaminated household dust or ... and six if they are at risk of lead poisoning (see: ). Who can I call to get more ...

  5. Lead Poisoning

    MedlinePlus

    ... our environment. Much of it comes from human activities such as mining and manufacturing. Lead used to be in paint; older houses may still have lead paint. You could be exposed to lead by Eating food or drinking water that contains lead. Water pipes in older homes ...

  6. Lead poisoning

    SciTech Connect

    Rekus, J.F.

    1992-08-01

    Construction workers who weld, cut or blast structural steel coated with lead-based paint are at significant risk of lead poisoning. Although technology to control these exposures may not have existed when the lead standard was promulgated, it is available today. Employers who do not take steps to protect their employees from lead exposure may be cited and fined severely for their failure.

  7. Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migration

    PubMed Central

    2013-01-01

    stromal thickness and collagen deposition, and stromal cells isolated from Cdc42 overexpressing mammary glands exhibited elevated mRNA expression of extracellular matrix proteins and remodeling enzymes. Conclusions These data suggest that Cdc42 overexpression disrupts mammary gland branching morphogenesis by altering Rho GTPase and MAPK signaling, leading to increased MEC contractility and migration in association with stromal alterations. Our studies provide insight into how aberrant Cdc42 expression may contribute to mammary tumorigenesis. PMID:24074261

  8. Homeodomain-interacting protein kinases (Hipks) promote Wnt/Wg signaling through stabilization of beta-catenin/Arm and stimulation of target gene expression.

    PubMed

    Lee, Wendy; Swarup, Sharan; Chen, Joanna; Ishitani, Tohru; Verheyen, Esther M

    2009-01-01

    The Wnt/Wingless (Wg) pathway represents a conserved signaling cascade involved in diverse biological processes. Misregulation of Wnt/Wg signal transduction has profound effects on development. Homeodomain-interacting protein kinases (Hipks) represent a novel family of serine/threonine kinases. Members of this group (in particular Hipk2) are implicated as important factors in transcriptional regulation to control cell growth, apoptosis and development. Here, we provide genetic and phenotypic evidence that the sole Drosophila member of this family, Hipk, functions as a positive regulator in the Wg pathway. Expression of hipk in the wing rescues loss of the Wg signal, whereas loss of hipk can enhance decreased wg signaling phenotypes. Furthermore, loss of hipk leads to diminished Arm protein levels, whereas overexpression of hipk promotes the Wg signal by stabilizing Arm, resulting in activation of Wg responsive targets. In Wg transcriptional assays, Hipk enhanced Tcf/Arm-mediated gene expression in a kinase-dependent manner. In addition, Hipk can bind to Arm and Drosophila Tcf, and phosphorylate Arm. Using both in vitro and in vivo assays, Hipk was found to promote the stabilization of Arm. We observe similar molecular interactions between Lef1/beta-catenin and vertebrate Hipk2, suggesting a direct and conserved role for Hipk proteins in promoting Wnt signaling. PMID:19088090

  9. MicroRNA overexpression increases cortical neuronal vulnerability to injury

    PubMed Central

    Truettner, Jessie S.; Motti, Dario; Dietrich, W. Dalton

    2013-01-01

    Previously we reported that several microRNAs (miRNA) are upregulated following experimentally induced traumatic brain injury (TBI) using both in vivo and in vitro approaches. Specific miRNAs were found to be sensitive to therapeutic hypothermia and may therefore be important targets for neuroprotective strategies. In this study we developed plasmid constructs that overexpress temperature sensitive miRNAs: miR-34a, miR-451, and miR-874. These constructs were transfected into cultured cortical neurons that were subjected to stretch injury using a cell injury controller device. Levels of expression of genes associated with stress, inflammation, apoptosis and transcriptional regulation were measured by qRT-PCR. mRNA levels of cytokines interleukin 1-β (IL1-β) and tumor necrosis factor alpha (TNF-α) as well as heat shock protein 70 (HSP70) and Caspase 11 were found to be increased up to 24 fold higher than controls in cells overexpressing these miRNAs. After moderate stretch injury, the expression of IL1-β, TNF-α, HSP70 and Caspase 11 all increased over control levels found in uninjured cells suggesting that overexpression of these miRNAs increases cellular vulnerability. miR-34a directly inhibits Bcl2 and XIAP, both anti-apoptotic proteins. The observed increase in Caspase 11 with over-expression of miR-34a indicates that miR-34a may be inducing apoptosis by reducing the levels of antiapoptotic proteins. miR-34a is predicted to inhibit Jun, which was seen to decrease in cells overexpressing this miRNA along with Fos. Over expression of several miRNAs found to be induced by TBI in vivo (miR-34a, miR-451 and miR-874) leads to increased vulnerability in transfected neurons. Therapeutic hypothermia blunts the expression of these miRNAs in vivo and antisense silencing could be a potential therapeutic approach to targeting the consequences of TBI. PMID:23948100

  10. Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas

    PubMed Central

    Faria, Márcia; Capinha, Liliana; Simões-Pereira, Joana; Bugalho, Maria João; Silva, Ana Luísa

    2016-01-01

    RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC) is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs) and 33 follicular thyroid adenomas (FTAs). RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01) and poorer clinical outcome (P = 0.01) suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions. PMID:27127508

  11. Inhibition of laminin-5 production in breast epithelial cells by overexpression of p300.

    PubMed

    Miller, K A; Chung, J; Lo, D; Jones, J C; Thimmapaya, B; Weitzman, S A

    2000-03-17

    The transcriptional coactivator p300 is essential for normal embryonic development and cellular differentiation. We have been studying the role of p300 in the transcription of a variety of genes, and we became interested in the role of this coactivator in the transcription of genes important in breast epithelial cell biology. From MCF-10A cells (spontaneously immortalized, nontransformed human breast epithelial cells), we developed cell lines that stably overexpress p300. These p300-overexpressing cells displayed reduced adhesion to culture dishes and were found to secrete an extracellular matrix deficient in laminin-5. Laminin-5 is the major extracellular matrix component produced by breast epithelium. Immunofluorescence studies, as well as experiments using normal matrix, confirmed that the decreased adhesion of p300-overexpressing cells is due to laminin-5-deficient extracellular matrix and not due to loss of laminin-5 receptors. Northern blots revealed markedly decreased levels of expression of two of the genes (designated LAMA3 and LAMC2) encoding the alpha3 and gamma2 chains of the laminin-5 heterotrimer in the cells that overexpress p300, whereas LAMB3 mRNA, encoding the third or beta3 chain of laminin-5, was not markedly reduced. Transient transfection experiments with a vector containing a murine LAMA3 promoter demonstrate that overexpressing p300 down-regulates the LAMA3 promoter. In summary, overexpression of p300 leads to down-regulation of laminin-5 production in breast epithelial cells, resulting in decreased adhesion. PMID:10713141

  12. Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas.

    PubMed

    Faria, Márcia; Capinha, Liliana; Simões-Pereira, Joana; Bugalho, Maria João; Silva, Ana Luísa

    2016-01-01

    RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC) is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs) and 33 follicular thyroid adenomas (FTAs). RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01) and poorer clinical outcome (P = 0.01) suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions. PMID:27127508

  13. Lead poisoning

    MedlinePlus

    ... lead is still found in some modern faucets. Soil contaminated by decades of car exhaust or years ... house paint scrapings. Lead is more common in soil near highways and houses. Hobbies involving soldering, stained ...

  14. Kif14 overexpression accelerates murine retinoblastoma development.

    PubMed

    O'Hare, Michael; Shadmand, Mehdi; Sulaiman, Rania S; Sishtla, Kamakshi; Sakisaka, Toshiaki; Corson, Timothy W

    2016-10-15

    The mitotic kinesin KIF14 has an essential role in the recruitment of proteins required for the final stages of cytokinesis. Genomic gain and/or overexpression of KIF14 has been documented in retinoblastoma and a number of other cancers, such as breast, lung and ovarian carcinomas, strongly suggesting its role as an oncogene. Despite evidence of oncogenic properties in vitro and in xenografts, Kif14's role in tumor progression has not previously been studied in a transgenic cancer model. Using a novel Kif14 overexpressing, simian virus 40 large T-antigen retinoblastoma (TAg-RB) double transgenic mouse model, we aimed to determine Kif14's role in promoting retinal tumor formation. Tumor initiation and development in double transgenics and control TAg-RB littermates were documented in vivo over a time course by optical coherence tomography, with subsequent ex vivo quantification of tumor burden. Kif14 overexpression led to an accelerated initiation of tumor formation in the TAg-RB model and a significantly decreased tumor doubling time (1.8 vs. 2.9 weeks). Moreover, overall percentage tumor burden was also increased by Kif14 overexpression. These data provide the first evidence that Kif14 can promote tumor formation in susceptible cells in vivo. PMID:27270502

  15. Overexpression of OsDof12 affects plant architecture in rice (Oryza sativa L.)

    PubMed Central

    Wu, Qi; Li, Dayong; Li, Dejun; Liu, Xue; Zhao, Xianfeng; Li, Xiaobing; Li, Shigui; Zhu, Lihuang

    2015-01-01

    Dof (DNA binding with one finger) proteins, a class of plant-specific transcription factors, are involved in plant growth and developmental processes and stress responses. However, their biological functions remain to be elucidated, especially in rice (Oryza sativa L.). Previously, we have reported that OsDof12 can promote rice flowering under long-day conditions. Here, we further investigated the other important agronomical traits of the transgenic plants overexpressing OsDof12 and found that overexpressing OsDof12 could lead to reduced plant height, erected leaf, shortened leaf blade, and smaller panicle resulted from decreased primary and secondary branches number. These results implied that OsDof12 is involved in rice plant architecture formation. Furthermore, we performed a series of Brassinosteroid (BR)-responsive tests and found that overexpression of OsDof12 could also result in BR hyposensitivity. Of note, in WT plants the expression of OsDof12 was found up-regulated by BR treatment while in OsDof12 overexpression plants two positive BR signaling regulators, OsBRI1 and OsBZR1, were significantly down-regulated, indicating that OsDof12 may act as a negative BR regulator in rice. Taken together, our results suggested that overexpression of OsDof12 could lead to altered plant architecture by suppressing BR signaling. Thus, OsDof12 might be used as a new potential genetic regulator for future rice molecular breeding. PMID:26500670

  16. Phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2.

    PubMed

    Hikasa, Hiroki; Sokol, Sergei Y

    2011-04-01

    Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos. PMID:21285352

  17. Overexpression of Protochlorophyllide Oxidoreductase C Regulates Oxidative Stress in Arabidopsis

    PubMed Central

    Pattanayak, Gopal K.; Tripathy, Baishnab C.

    2011-01-01

    Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen (1O2). As there is no enzymatic detoxification mechanism available in plants to destroy 1O2, its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the 1O2 generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of 1O2 and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to 1O2-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5

  18. Dimethylarginine dimethylaminohydrolase (DDAH) overexpression attenuates agricultural organic dust extract-induced inflammation

    PubMed Central

    Bailey, KL; Wyatt, TA; Wells, SM; Klein, EB; Robinson, JE; Romberger, DJ; Poole, JA

    2013-01-01

    Modern, industrialized farming practices have lead to working conditions that include high levels of airborne dust. Agricultural workers inhale these complex organic dusts on a daily basis, leading to airway inflammation and higher risk for developing chronic obstructive pulmonary disease. The mechanisms regulating the organic dust-induced airway inflammatory response are not well-defined. We investigated whether overexpression of dimethylarginine dimethylaminohydrolase (DDAH) would lead to diminished pulmonary inflammation in an animal model of organic dust extract exposure. We instilled wild-type (WT) and DDAH overexpressing mice with an aqueous organic dust extract (ODE) collected from a swine confinement building. We found that inflammatory indices such as neutrophil influx and inflammatory cytokine production was lower in the DDAH overexpressing mice compared to WT after organic dust extract (ODE) instillation. We went on to determine how DDAH was mediating the decrease in inflammation induced by ODE. PKCα and PKCε play an essential role in the ODE inflammatory response. In a model of lung slices from WT and DDAH overexpressing mice, we demonstrated an increase in PKCα and PKCε in the WT mice exposed to ODE. This increase was diminished in the DDAH overexpressing mice exposed to ODE. We also tested an important component of the ODE, peptidoglycan (PGN). We noted a similar decrease in neutrophils and inflammatory cytokines in the DDAH overexpressing animals instilled with PGN compared to WT. In conclusion, our studies found a role for DDAH in regulating the ODE-triggered activation of epithelial PKCα and PKCε, a previously unrecognized mechanism of action. This ultimately results in diminished pulmonary inflammation. PMID:25221746

  19. Lead Pencils

    NASA Technical Reports Server (NTRS)

    Gray, L. B.

    1971-01-01

    A study, undertaken to determine the lead content of paint on various pencils in the Goddard supply system, is reported. The survey found that lead content varied from .04 mg per pencil for carmine colored pencils to approximately 43 mg per pencil for yellow colored pencils. Results also show that yellow pencils had higher lead content than other colors analyzed. More detailed results are given in tabular form.

  20. An apple rootstock overexpressing a peach CBF gene alters growth and flowering in the scion but does not impact cold hardiness or dormancy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The C-repeat Binding Factor (CBF) transcription factor is involved in responses to low temperature and water deficit in many plant species. Overexpression of CBF genes leads to enhanced freezing tolerance and growth inhibition in many species. The overexpression of a peach CBF (PpCBF1) gene in a t...

  1. Overexpression of neurofilament H disrupts normal cell structure and function

    NASA Technical Reports Server (NTRS)

    Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

    2002-01-01

    Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

  2. Leading Democratically

    ERIC Educational Resources Information Center

    Brookfield, Stephen

    2010-01-01

    Democracy is the most venerated of American ideas, the one for which wars are fought and people die. So most people would probably agree that leaders should be able to lead well in a democratic society. Yet, genuinely democratic leadership is a relative rarity. Leading democratically means viewing leadership as a function or process, rather than…

  3. Hand1 overexpression inhibits medulloblastoma metastasis.

    PubMed

    Asuthkar, Swapna; Guda, Maheedhara R; Martin, Sarah E; Antony, Reuben; Fernandez, Karen; Lin, Julian; Tsung, Andrew J; Velpula, Kiran K

    2016-08-19

    Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis. PMID:27297109

  4. Transition metals activate TFEB in overexpressing cells

    PubMed Central

    Peña, Karina A.; Kiselyov, Kirill

    2015-01-01

    Transition metal toxicity is an important factor in the pathogenesis of numerous human disorders, including neurodegenerative diseases. Lysosomes have emerged as important factors in transition metal toxicity because they handle transition metals via endocytosis, autophagy, absorption from the cytoplasm and exocytosis. Transcription factor EB (TFEB) regulates lysosomal biogenesis and the expression of lysosomal proteins in response to lysosomal and/or metabolic stresses. Since transition metals cause lysosomal dysfunction, we proposed that TFEB may be activated to drive gene expression in response to transition metal exposure and that such activation may influence transition metal toxicity. We found that transition metals copper (Cu) and iron (Fe) activate recombinant TFEB and stimulate the expression of TFEB-dependent genes in TFEB-overexpressing cells. In cells that show robust lysosomal exocytosis, TFEB was cytoprotective at moderate levels of Cu exposure, decreasing oxidative stress as reported by the expression of heme oxygenase-1 (HMOX1) gene. However, at high levels of Cu exposure, particularly in cells with low levels of lysosomal exocytosis, activation of overexpressed TFEB was toxic, increasing oxidative stress and mitochondrial damage. Based on these data, we conclude that TFEB-driven gene network is a component of the cellular response to transition metals. These data suggest limitations and disadvantages of TFEB overexpression as a therapeutic approach. PMID:26251447

  5. Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications

    PubMed Central

    Iqbal, Nida; Iqbal, Naveed

    2014-01-01

    Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family having tyrosine kinase activity. Dimerization of the receptor results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways leading to cell proliferation and tumorigenesis. Amplification or overexpression of HER2 occurs in approximately 15–30% of breast cancers and 10–30% of gastric/gastroesophageal cancers and serves as a prognostic and predictive biomarker. HER2 overexpression has also been seen in other cancers like ovary, endometrium, bladder, lung, colon, and head and neck. The introduction of HER2 directed therapies has dramatically influenced the outcome of patients with HER2 positive breast and gastric/gastroesophageal cancers; however, the results have been proved disappointing in other HER2 overexpressing cancers. This review discusses the role of HER2 in various cancers and therapeutic modalities available targeting HER2. PMID:25276427

  6. Plumbagin induces apoptosis in Her2-overexpressing breast cancer cells through the mitochondrial-mediated pathway.

    PubMed

    Kawiak, Anna; Zawacka-Pankau, Joanna; Lojkowska, Ewa

    2012-04-27

    Breast cancer is the leading cause of death-related cancers in women. Approximately 30% of breast cancers overexpress the Her2 oncogene, which is associated with a poor prognosis and increased resistance to chemotherapy. Plumbagin (1), a constituent of species in the plant genera Drosera and Plumbago, displays antineoplastic activity toward various cancers. The present study was aimed at determining the anticancer potential of 1 toward Her2-overexpressing breast cancer cells and defining the mode of cell death induced in these cells. The results showed that 1 exhibited high antiproliferative activity toward the Her2-overexpressing cell lines SKBR3 and BT474. The antiproliferative activity of 1 was associated with apoptosis-mediated cell death, as revealed by caspase activation and an increase in the sub-G1 fraction of the cell cycle. Compound 1 increased the levels of the proapoptotic Bcl-2 family of proteins and decreased the level of the antiapoptotic Bcl-2 protein in SKBR3 and BT474 cells. Thus, these findings indicate that 1 induces apoptosis in Her2-overexpressing breast cancers through the mitochondrial-mediated pathway and suggest its potential for further investigation for the treatment of Her2-overexpressing breast cancer. PMID:22512718

  7. Overexpression of MMP-7 increases collagen 1A2 in the aging kidney

    PubMed Central

    Ślusarz, Anna; Nichols, LaNita A; Grunz-Borgmann, Elizabeth A; Chen, Gang; Akintola, Adebayo D; Catania, Jeffery M; Burghardt, Robert C; Trzeciakowski, Jerome P; Parrish, Alan R

    2013-01-01

    The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis that leads to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however, it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, with over a 500-fold upregulation in 2-year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of protein kinase A (PKA), src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 upregulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src, and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD. PMID:24273653

  8. Overexpression of MMP-7 Increases Collagen 1A2 in the Aging Kidney.

    PubMed

    Oelusarz, Anna; Nichols, Lanita A; Grunz-Borgmann, Elizabeth A; Chen, Gang; Akintola, Adebayo D; Catania, Jeffery M; Burghardt, Robert C; Trzeciakowski, Jerome P; Parrish, Alan R

    2013-10-01

    The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis, that lead to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, and over a 500 fold up-regulation in 2 year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of PKA, src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 up-regulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD. PMID:24273653

  9. Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

    SciTech Connect

    Chuang, Jian-Ying; Hung, Jan-Jong

    2011-04-15

    Highlights: {yields} Overexpression of HDAC1 induces Sp1 deacetylation and raises Sp1/p300 complex formation to bind to PP2Ac promoter. {yields} Overexpression of HDAC1 strongly inhibits the phosphorylation of pRb through up-regulation of PP2A. {yields} Overexpressed HDAC1 restrains cell proliferaction and induces cell senescence though a novel Sp1/PP2A/pRb pathway. -- Abstract: Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.

  10. Ecotoxicology: Lead

    USGS Publications Warehouse

    Scheuhammer, A.M.; Beyer, W.N.; Schmitt, C.J.

    2008-01-01

    Lead (Pb) is a naturally occurring metallic element; trace concentrations are found in all environmental media and in all living things. However, certain human activities, especially base metal mining and smelting; combustion of leaded gasoline; the use of Pb in hunting, target shooting, and recreational angling; the use of Pb-based paints; and the uncontrolled disposal of Pb-containing products such as old vehicle batteries and electronic devices have resulted in increased environmental levels of Pb, and have created risks for Pb exposure and toxicity in invertebrates, fish, and wildlife in some ecosystems.

  11. Tetraethyl lead

    Integrated Risk Information System (IRIS)

    Tetraethyl lead ; CASRN 78 - 00 - 2 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  12. In vivo clonal overexpression of neuroligin 3 and neuroligin 2 in neurons of the rat cerebral cortex. Differential effects on GABAergic synapses and neuronal migration

    PubMed Central

    Fekete, Christopher D.; Chiou, Tzu-Ting; Miralles, Celia P.; Harris, Rachel S.; Fiondella, Christopher G.; LoTurco, Joseph J.; De Blas, Angel L.

    2015-01-01

    We have studied the effect of clonal overexpression of neuroligin 3 (NL3) or neuroligin 2 (NL2) in the adult rat cerebral cortex following in utero electroporation (IUEP) at embryonic stage E14. Overexpression of NL3 leads to a large increase in vGAT and GAD65 in the GABAergic contacts that the overexpressing neurons receive. Overexpression of NL2 produced a similar effect but to a lesser extent. In contrast, overexpression of NL3 or NL2 after IUEP, does not affect vGlut1 in the glutamatergic contacts that the NL3 or NL2 overexpressing neurons receive. The NL3 or NL2 overexpressing neurons do not show increased innervation by parvalbumin-containing GABAergic terminals or increased parvalbumin in the same terminals that show increased vGAT. These results indicate that the observed increase in vGAT and GAD65 is not due to increased GABAergic innervation but to increased expression of vGAT and GAD65 in the GABAergic contacts that NL3 or NL2 overexpressing neurons receive. The majority of bright vGAT puncta contacting the NL3 overexpressing neurons have no gephyrin juxtaposed to them indicating that many of these contacts are non-synaptic. This contrasts with the majority of the NL2 overexpressing neurons, which show plenty of synaptic gephyrin clusters juxtaposed to vGAT. Besides having an effect on GABAergic contacts, overexpression of NL3 interferes with the neuronal radial migration, in the cerebral cortex, of the neurons overexpressing NL3. PMID:25565602

  13. Capsule depolymerase overexpression reduces Bacillus anthracis virulence.

    PubMed

    Scorpio, Angelo; Chabot, Donald J; Day, William A; Hoover, Timothy A; Friedlander, Arthur M

    2010-05-01

    Capsule depolymerase (CapD) is a gamma-glutamyl transpeptidase and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and to capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-type B. anthracis Ames but was detected in strains engineered with a consensus ribosome-binding site for capD. Strains overexpressing capD at amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence. PMID:20110296

  14. Nucleophosmin is overexpressed in thyroid tumors

    SciTech Connect

    Pianta, Annalisa; Puppin, Cinzia; Franzoni, Alessandra; Fabbro, Dora; Di Loreto, Carla; Bulotta, Stefania; Deganuto, Marta; Paron, Igor; Tell, Gianluca; Puxeddu, Efisio; Filetti, Sebastiano; Russo, Diego; Damante, Giuseppe

    2010-07-02

    Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed in tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.

  15. Statins Reduce Melanoma Development and Metastasis through MICA Overexpression.

    PubMed

    Pich, Christine; Teiti, Iotefa; Rochaix, Philippe; Mariamé, Bernard; Couderc, Bettina; Favre, Gilles; Tilkin-Mariamé, Anne-Françoise

    2013-01-01

    Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MHC class I Chain-related protein A (MICA) membrane expression. Peroxisome Proliferator-Activated Receptor gamma is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments. PMID:23493799

  16. Paraoxonase-1 overexpression prevents experimental abdominal aortic aneurysm progression.

    PubMed

    Burillo, Elena; Tarin, Carlos; Torres-Fonseca, Monica-Maria; Fernandez-García, Carlos-Ernesto; Martinez-Pinna, Roxana; Martinez-Lopez, Diego; Llamas-Granda, Patricia; Camafeita, Emilio; Lopez, Juan Antonio; Vega de Ceniga, Melina; Aviram, Michael; Egido, Jesus; Blanco-Colio, Luis-Miguel; Martín-Ventura, Jose-Luis

    2016-06-01

    Abdominal aortic aneurysm (AAA) is a permanent dilation of the aorta due to excessive proteolytic, oxidative and inflammatory injury of the aortic wall. We aimed to identify novel mediators involved in AAA pathophysiology, which could lead to novel therapeutic approaches. For that purpose, plasma from four AAA patients and four controls were analysed by a label-free proteomic approach. Among identified proteins, paraoxonase-1 (PON1) was decreased in plasma of AAA patients compared with controls, which was further validated in a bigger cohort of samples by ELISA. The phenylesterase enzymatic activity of PON1 was also decreased in serum of AAA patients compared with controls. To address the potential role of PON1 as a mediator of AAA, experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and human transgenic PON1 (HuTgPON1) mice. Similar to humans, PON1 activity was also decreased in serum of elastase-induced AAA mice compared with healthy mice. Interestingly, overexpression of PON1 was accompanied by smaller aortic dilation and higher elastin and vascular smooth muscle cell (VSMC) content in the AAA of HuTgPON1 compared with WT mice. Moreover, HuTgPON1 mice display decreased oxidative stress and apoptosis, as well as macrophage infiltration and monocyte chemoattractant protein-1 (MCP1) expression, in elastase-induced AAA. In conclusion, decreased circulating PON1 activity is associated with human and experimental AAA. PON1 overexpression in mice protects against AAA progression by reducing oxidative stress, apoptosis and inflammation, suggesting that strategies aimed at increasing PON1 activity could prevent AAA. PMID:26993251

  17. Increasing sulphite formation in Saccharomyces cerevisiae by overexpression of MET14 and SSU1.

    PubMed

    Donalies, Ute E B; Stahl, Ulf

    2002-04-01

    Saccharomyces cerevisiae produces sulphite as an intermediate product during the assimilatory reduction of sulphate to sulphide. Three genes, MET3, MET14 and MET16, are essential for this reduction. We investigated the level of transcription of these genes in strains of S. cerevisiae with high, medium and low sulphite formation. The level of MET14- and MET16-mRNA varied with sulphite production, whereas the level of MET3-mRNA was very weak in almost all strains. We also analysed the effect of overexpression of MET14 and MET16 on sulphite formation. Two strains with low sulphite production were transformed with high-copy plasmids containing either or both MET14 and MET16. The overexpression of these two genes leads to a two- to three-fold sulphite formation. In addition, inactivation of MET10, encoding a subunit of the sulphite reductase, also leads to a distinct increase in sulphite formation; however, the cells became methionine auxotroph. The overexpression of SSU1, a gene encoding a putative sulphite pump, yields a slight increase in sulphite accumulation, whereas overexpression of SSU1, together with MET14, increases sulphite formation up to 10-fold. Furthermore, sulphite formation strongly depends on growth conditions, e.g. yeast transformants growing in wort produce much higher amounts of sulphite when compared to growth in minimal media. The addition of glucose can also increase the sulphite formation in strains overexpressing MET14 and/or SSU1 under oxygen-limiting conditions, while the addition of glucose has no significant effect under aerobic conditions. PMID:11921096

  18. Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy

    PubMed Central

    Li, Ran; Bai, Jian; Ding, Liang; Gu, Rong; Wang, Lian; Xu, Biao

    2016-01-01

    Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure. PMID:26914935

  19. Overexpressed oncogenic tumor-self antigens

    PubMed Central

    Bright, Robert K; Bright, Jennifer D; Byrne, Jennifer A

    2014-01-01

    Overexpressed tumor-self antigens represent the largest group of candidate vaccine targets. Those exhibiting a role in oncogenesis may be some of the least studied but perhaps most promising. This review considers this subset of self antigens by highlighting vaccine efforts for some of the better known members and focusing on TPD52, a new promising vaccine target. We shed light on the importance of both preclinical and clinical vaccine studies demonstrating that tolerance and autoimmunity (presumed to preclude this class of antigens from vaccine development) can be overcome and do not present the obstacle that might have been expected. The potential of this class of antigens for broad application is considered, possibly in the context of low tumor burden or adjuvant therapy, as is the need to understand mechanisms of tolerance that are relatively understudied. PMID:25483660

  20. bcl-2 overexpression promotes myocyte proliferation

    PubMed Central

    Limana, Federica; Urbanek, Konrad; Chimenti, Stefano; Quaini, Federico; Leri, Annarosa; Kajstura, Jan; Nadal-Ginard, Bernardo; Izumo, Seigo; Anversa, Piero

    2002-01-01

    To determine the influence of Bcl-2 on the developmental biology of myocytes, we analyzed the population dynamics of this cell type in the heart of transgenic (TG) mice overexpressing Bcl-2 under the control of the α-myosin heavy chain promoter. TG mice and non-TG (wild type, WT) mice were studied at 24 days, 2 months, and 4 months after birth. Bcl-2 overexpression produced a significant increase in the percentage of cycling myocytes and their mitotic index. These effects were strictly connected to the expression of the transgene, as demonstrated in isolated myocytes. The formation of mitotic spindle and contractile ring was identified in replicating cells. These typical aspects of mitosis were complemented with the demonstration of karyokinesis and cytokinesis to provide structural evidence of cell division. Apoptosis was low at all ages and was not affected by Bcl-2. The higher cell replication rate in TG was conditioned by a decrease in the expression of the cell-cycle inhibitors, p21WAF1 and p16INK4a, and by an increase in Mdm2-p53 complexes. In comparison with WT, TG had 0.4 × 106, 0.74 × 106, and 1.2 × 106 more myocytes in the left ventricle at 24 days, 2 months, and 4 months, respectively. Binucleated myocytes were 12% and 25% larger in WT than in TG mice at 2 and 4 months of age. Taken together, these observations reveal a previously uncharacterized replication-enhancing function of Bcl-2 in myocytes in vivo in the absence of stressful conditions. PMID:11983915

  1. N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts

    PubMed Central

    Rinne, M. L.; He, Y.; Pachkowski, B. F.; Nakamura, J.; Kelley, M. R.

    2005-01-01

    Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO2(CH2)2-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents. PMID:15905475

  2. Cyclin E overexpression impairs progression through mitosis by inhibiting APCCdh1

    PubMed Central

    Keck, Jamie M.; Summers, Matthew K.; Tedesco, Donato; Ekholm-Reed, Susanna; Chuang, Li-Chiou; Jackson, Peter K.; Reed, Steven I.

    2007-01-01

    Overexpression of cyclin E, an activator of cyclin-dependent kinase 2, has been linked to human cancer. In cell culture models, the forced expression of cyclin E leads to aneuploidy and polyploidy, which is consistent with a direct role of cyclin E overexpression in tumorigenesis. In this study, we show that the overexpression of cyclin E has a direct effect on progression through the latter stages of mitotic prometaphase before the complete alignment of chromosomes at the metaphase plate. In some cases, such cells fail to divide chromosomes, resulting in polyploidy. In others, cells proceed to anaphase without the complete alignment of chromosomes. These phenotypes can be explained by an ability of overexpressed cyclin E to inhibit residual anaphase-promoting complex (APCCdh1) activity that persists as cells progress up to and through the early stages of mitosis, resulting in the abnormal accumulation of APCCdh1 substrates as cells enter mitosis. We further show that the accumulation of securin and cyclin B1 can account for the cyclin E–mediated mitotic phenotype. PMID:17664332

  3. Over-expression of Topoisomerase II Enhances Salt Stress Tolerance in Tobacco

    PubMed Central

    John, Riffat; Ganeshan, Uma; Singh, Badri N.; Kaul, Tanushri; Reddy, Malireddy K.; Sopory, Sudhir K.; Rajam, Manchikatla V.

    2016-01-01

    Topoisomerases are unique enzymes having an ability to remove or add DNA supercoils and untangle the snarled DNA. They can cut, shuffle, and religate DNA strands and remove the torsional stress during DNA replication, transcription or recombination events. In the present study, we over-expressed topoisomerase II (TopoII) in tobacco (Nicotiana tabaccum) and examined its role in growth and development as well as salt (NaCl) stress tolerance. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis respectively. Percent seed germination, shoot growth, and chlorophyll content revealed that transgenic lines over-expressing the NtTopoIIα-1 gene exhibited enhanced tolerance to salt (150 and 200 mM NaCl) stress. Moreover, over-expression of TopoII lead to the elevation in proline and glycine betaine levels in response to both concentrations of NaCl as compared to wild-type. In response to NaCl stress, TopoII over-expressing lines showed reduced lipid peroxidation derived malondialdehyde (MDA) generation. These results suggest that TopoII plays a pivotal role in salt stress tolerance in plants.

  4. Notch intracellular domain overexpression in adipocytes confers lipodystrophy in mice

    PubMed Central

    Chartoumpekis, Dionysios V.; Palliyaguru, Dushani L.; Wakabayashi, Nobunao; Khoo, Nicholas K.H.; Schoiswohl, Gabriele; O'Doherty, Robert M.; Kensler, Thomas W.

    2015-01-01

    Objective The Notch family of intermembrane receptors is highly conserved across species and is involved in cell fate and lineage control. Previous in vitro studies have shown that Notch may inhibit adipogenesis. Here we describe the role of Notch in adipose tissue by employing an in vivo murine model which overexpresses Notch in adipose tissue. Methods Albino C57BL/6J RosaNICD/NICD::Adipoq-Cre (Ad-NICD) male mice were generated to overexpress the Notch intracellular domain (NICD) specifically in adipocytes. Male RosaNICD/NICD mice were used as controls. Mice were evaluated metabolically at the ages of 1 and 3 months by assessing body weights, serum metabolites, body composition (EchoMRI), glucose tolerance and insulin tolerance. Histological sections of adipose tissue depots as well as of liver were examined. The mRNA expression profile of genes involved in adipogenesis was analyzed by quantitative real-time PCR. Results The Ad-NICD mice were heavier with significantly lower body fat mass compared to the controls. Small amounts of white adipose tissue could be seen in the 1-month old Ad-NICD mice, but was almost absent in the 3-months old mice. The Ad-NICD mice also had higher serum levels of glucose, insulin, triglyceride and non-esterified fatty acids. These differences were more prominent in the older (3-months) than in the younger (1-month) mice. The Ad-NICD mice also showed severe insulin resistance along with a steatotic liver. Gene expression analysis in the adipose tissue depots showed a significant repression of lipogenic (Fasn, Acacb) and adipogenic pathways (C/ebpα, C/ebpβ, Pparγ2, Srebf1). Conclusions Increased Notch signaling in adipocytes in mice results in blocked expansion of white adipose tissue which leads to ectopic accumulation of lipids and insulin resistance, thus to a lipodystrophic phenotype. These results suggest that further investigation of the role of Notch signaling in adipocytes could lead to the manipulation of this pathway for

  5. Geminin Overexpression Promotes Imatinib Sensitive Breast Cancer: A Novel Treatment Approach for Aggressive Breast Cancers, Including a Subset of Triple Negative

    PubMed Central

    Blanchard, Zannel; Mullins, Nicole; Ellipeddi, Pavani; Lage, Janice M.; McKinney, Shawn; El-Etriby, Rana; Zhang, Xu; Isokpehi, Raphael; Hernandez, Brenda; ElShamy, Wael M.

    2014-01-01

    Breast cancer is the second leading cause of cancer-related deaths in women. Triple negative breast cancer (TNBC) is an aggressive subtype that affects 10–25% mostly African American women. TNBC has the poorest prognosis of all subtypes with rapid progression leading to mortality in younger patients. So far, there is no targeted treatment for TNBC. To that end, here we show that c-Abl is one of several tyrosine kinases that phosphorylate and activate geminin’s ability to promote TNBC. Analysis of >800 breast tumor samples showed that geminin is overexpressed in ∼50% of all tumors. Although c-Abl is overexpressed in ∼90% of all tumors, it is only nuclear in geminin overexpressing tumors. In geminin-negative tumors, c-Abl is only cytoplasmic. Inhibiting c-Abl expression or activity (using imatinib or nilotinib) prevented geminin Y150 phosphorylation, inactivated the protein, and most importantly converted overexpressed geminin from an oncogene to an apoptosis inducer. In pre-clinical orthotopic breast tumor models, geminin-overexpressing cells developed aneuploid and invasive tumors, which were suppressed when c-Abl expression was blocked. Moreover, established geminin overexpressing orthotopic tumors regressed when treated with imatinib or nilotinib. Our studies support imatinib/nilotonib as a novel treatment option for patients with aggressive breast cancer (including a subset of TNBCs)-overexpressing geminin and nuclear c-Abl. PMID:24789045

  6. Overexpression of Glycolate Oxidase Confers Improved Photosynthesis under High Light and High Temperature in Rice.

    PubMed

    Cui, Li-Li; Lu, Yu-Sheng; Li, Yong; Yang, Chengwei; Peng, Xin-Xiang

    2016-01-01

    While glycolate oxidase (GLO) is well known as a key enzyme for the photorespiratory metabolism in plants, its physiological function and mechanism remains to be further clarified. Our previous studies have shown that suppression of GLO in rice leads to stunted growth and inhibited photosynthesis (Pn) which is positively and linearly correlated with decreased GLO activities. It is, therefore, of interest to further understand whether Pn can be improved when GLO is up-regulated? In this study, four independent overexpression rice lines, with gradient increases in GLO activity, were generated and functionally analyzed. Phenotypic observations showed that the growth could be improved when GLO activities were increased by 60 or 100%, whereas reduced growth was noticed when the activity was further increased by 150 or 210%. As compared with WT plants, all the overexpression plants exhibited significantly improved Pn under conditions of high light and high temperature, but not under normal conditions. In addition, the overexpression plants were more resistant to the MV-induced photooxidative stress. It was further demonstrated that the antioxidant enzymes, and the antioxidant metabolite glutathione was not significantly altered in the overexpression plants. In contrast, H2O2 and salicylic acid (SA) were correspondingly induced upon the GLO overexpression. Taken together, the results suggest that GLO may play an important role for plants to cope with high light and high temperature, and that H2O2 and SA may serve as signaling molecules to trigger stress defense responses but antioxidant reactions appear not to be involved in the defense. PMID:27540387

  7. Overexpression of Glycolate Oxidase Confers Improved Photosynthesis under High Light and High Temperature in Rice

    PubMed Central

    Cui, Li-Li; Lu, Yu-sheng; Li, Yong; Yang, Chengwei; Peng, Xin-Xiang

    2016-01-01

    While glycolate oxidase (GLO) is well known as a key enzyme for the photorespiratory metabolism in plants, its physiological function and mechanism remains to be further clarified. Our previous studies have shown that suppression of GLO in rice leads to stunted growth and inhibited photosynthesis (Pn) which is positively and linearly correlated with decreased GLO activities. It is, therefore, of interest to further understand whether Pn can be improved when GLO is up-regulated? In this study, four independent overexpression rice lines, with gradient increases in GLO activity, were generated and functionally analyzed. Phenotypic observations showed that the growth could be improved when GLO activities were increased by 60 or 100%, whereas reduced growth was noticed when the activity was further increased by 150 or 210%. As compared with WT plants, all the overexpression plants exhibited significantly improved Pn under conditions of high light and high temperature, but not under normal conditions. In addition, the overexpression plants were more resistant to the MV-induced photooxidative stress. It was further demonstrated that the antioxidant enzymes, and the antioxidant metabolite glutathione was not significantly altered in the overexpression plants. In contrast, H2O2 and salicylic acid (SA) were correspondingly induced upon the GLO overexpression. Taken together, the results suggest that GLO may play an important role for plants to cope with high light and high temperature, and that H2O2 and SA may serve as signaling molecules to trigger stress defense responses but antioxidant reactions appear not to be involved in the defense. PMID:27540387

  8. Heterologous Overexpression of Poplar SnRK2 Genes Enhanced Salt Stress Tolerance in Arabidopsis thaliana

    PubMed Central

    Song, Xueqing; Yu, Xiang; Hori, Chiaki; Demura, Taku; Ohtani, Misato; Zhuge, Qiang

    2016-01-01

    Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress responses as a global positive regulator of abscisic acid signaling. In the genome of the model tree Populus trichocarpa, 12 SnRK2 genes have been identified, and some are upregulated by abiotic stresses. In this study, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis thaliana and found that overexpression of PtSnRK2.5 and PtSnRK2.7 genes enhanced stress tolerance. In the PtSnRK2.5 and PtSnRK2.7 overexpressors, chlorophyll content, and root elongation were maintained under salt stress conditions, leading to higher survival rates under salt stress compared with those in the wild type. Transcriptomic analysis revealed that PtSnRK2.7 overexpression affected stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, even under normal growth conditions. However, the stress response genes reported to be upregulated in Arabidopsis SRK2C/SnRK2.6 and wheat SnRK2.8 overexpressors were not changed by PtSnRK2.7 overexpression. Furthermore, PtSnRK2.7 overexpression widely and largely influenced the transcriptome in response to salt stress; genes related to transport activity, including anion transport-related genes, were characteristically upregulated, and a variety of metabolic genes were specifically downregulated. We also found that the salt stress response genes were greatly upregulated in the PtSnRK2.7 overexpressor. Taken together, poplar subclass 2 PtSnRK2 genes can modulate salt stress tolerance in Arabidopsis, through the activation of cellular signaling pathways in a different manner from that by herbal subclass 2 SnRK2 genes. PMID:27242819

  9. Negative regulation of DNMT3A de novo DNA methylation by frequently overexpressed UHRF family proteins as a mechanism for widespread DNA hypomethylation in cancer

    PubMed Central

    Jia, Yuanhui; Li, Pishun; Fang, Lan; Zhu, Haijun; Xu, Liangliang; Cheng, Hao; Zhang, Junying; Li, Fei; Feng, Yan; Li, Yan; Li, Jialun; Wang, Ruiping; Du, James X; Li, Jiwen; Chen, Taiping; Ji, Hongbin; Han, Jackie; Yu, Wenqiang; Wu, Qihan; Wong, Jiemin

    2016-01-01

    Global DNA hypomethylation is a most common epigenetic alteration in cancer, but the mechanism remains elusive. Previous studies demonstrate that UHRF1 but not UHRF2 is required for mediating DNA maintenance methylation by DNMT1. Here we report unexpectedly a conserved function for UHRF1 and UHRF2: inhibiting de novo DNA methylation by functioning as E3 ligases promoting DNMT3A degradation. UHRF1/2 are frequently overexpressed in cancers and we present evidence that UHRF1/2 overexpression downregulates DNMT3A proteins and consequently leads to DNA hypomethylation. Abrogating this negative regulation on DNMT3A or overexpression of DNMT3A leads to increased DNA methylation and impaired tumor growth. We propose a working model that UHRF1/2 safeguards the fidelity of DNA methylation and suggests that UHRF1/2 overexpression is likely a causal factor for widespread DNA hypomethylation in cancer via suppressing DNMT3A. PMID:27462454

  10. Overexpression of Akt1 Enhances Adipogenesis and Leads to Lipoma Formation in Zebrafish

    PubMed Central

    Rajendran, R. Samuel; Shen, Chia-Ning; Chen, Te-Hao; Yen, Chueh-Chuan; Chuang, Chih-Kuang; Lin, Dar-Shong; Hsiao, Chung-Der

    2012-01-01

    Background Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the Akt1 gene. Methodology/Principal Findings Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1)cy18 displays severely obese phenotypes at the adult stage. In Tg(krt4:Hsa.myrAkt1)cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3α/β, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(krt4:Hsa.myrAkt1)cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(krt4:Hsa.myrAkt1)cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(krt4:Hsa.myrAkt1)cy18, suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues. Conclusion/Significance Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an important role in balancing normal levels of fat tissue in vivo. The obese zebrafish examined in this study could be a new powerful model to screen novel drugs for the treatment of human obesity. PMID:22623957

  11. Overexpression of protein disulfide isomerase in Aspergillus.

    PubMed

    El-Adawi, H; Khanh, N Q; Gassen, H

    2000-10-01

    One of the major problems with the production of biotechnologically valuable proteins has been the purification of the product. For Escherichia coli and Saccharomyces cerevisiae, there are several techniques for the purification of intracellular proteins, but these are time consuming and often result in poor yields. Purification can be considerably facilitated, if the product is secreted from the host cell. In the work presented, we have constructed an expression vector (pSGNH2) for the secretion of protein disulfide isomerase (PDI; EC 5.3.4.1) from Aspergillus niger, in which the retention signal His-Asp-Glu-Leu (H-D-E-L) was modified to Ala-Leu-Glu-Gln (A-L-E-Q) via the polymerase chain reaction (PCR) method. The PDI gene was placed under the control of the A. oryzae alpha-amylase promoter. This expression vector was transformed into A. niger NRRL3, resulting in PDI secretion into the medium. The catalytic activity of overexpressed PDI from A. niger was indistinguishable from that of PDI isolated from bovine liver. With further strain improvement and optimization of culture conditions, it could be possible to raise the PDI production to the bioprocessing scale. PMID:10977899

  12. Frequent Nek1 overexpression in human gliomas.

    PubMed

    Zhu, Jun; Cai, Yu; Liu, Pin; Zhao, Weiguo

    2016-08-01

    Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients' poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. PMID:27251576

  13. RCAN1 Overexpression Exacerbates Calcium Overloading-Induced Neuronal Apoptosis

    PubMed Central

    Herculano, Bruno; Song, Weihong

    2014-01-01

    Down Syndrome (DS) patients develop characteristic Alzheimer's Disease (AD) neuropathology after their middle age. Prominent neuronal loss has been observed in the cortical regions of AD brains. However, the underlying mechanism leading to this neuronal loss in both DS and AD remains to be elucidated. Calcium overloading and oxidative stress have been implicated in AD pathogenesis. Two major isoforms of regulator of calcineurin 1 (RCAN1), RCAN1.1 and RCAN1.4, are detected in human brains. In this report we defined the transcriptional regulation of RCAN1.1 and RCAN1.4 by two alternative promoters. Calcium overloading upregulated RCAN1.4 expression by activating RCAN1.4 promoter through calcineurin-NFAT signaling pathway, thus forming a negative feedback loop in isoform 4 regulation. Furthermore, RCAN1.4 overexpression exacerbated calcium overloading-induced neuronal apoptosis, which was mediated by caspase-3 apoptotic pathway. Our results suggest that downregulating RCAN1.4 expression in neurons could be beneficial to AD patients. PMID:24751678

  14. An immunological renal disease in transgenic mice that overexpress Fli-1, a member of the ets family of transcription factor genes

    SciTech Connect

    Zhang, Liqun; Teng, Yen-Tung; Melet, F.

    1995-12-01

    This report describes how expression of the proto-oncogene Fli-1 is involved in the regulation of lymphopoiesis. Transgenic mice were generated which overexpressed the oncogene, leading to a high incidence of immunological renal diseases and death. The data suggests that overexpression of Fli-1 perturbs normal lymphoid cell function and programmed cell death, making these transgenic mice suitable as a biological model for autoimmune disease in humans. 35 refs., 6 figs., 4 tabs.

  15. Peripheral T-cell Lymphoma with Cyclin D1 overexpression: a case report

    PubMed Central

    2012-01-01

    Peripheral T-cell lymphomas not otherwise specified are generally considered aggressive non-Hodgkin lymphomas, because of poor natural outcome and response to therapy. They show a complex karyotype without any specific genetic hallmark. We report a case of peripheral T-cell lymphoma not otherwise specified with heterogeneous nuclear Cyclin D1 immunohistochemical overexpression, due to gene copy gain, a phenomenon similar to that observed in Mantle Cell Lymphoma characterized by t(11;14)(q13;q32). In this case report we underline the diagnostic pitfall rapresented by Cyclin D1 immunoistochemical overexpression in a T-cell lymphoma. Several pitfalls could lead to misinterpretation of diagnosis, therefore, we underlined the need to integrate the classical histology and immunohistochemistry with molecular tests as clonality or Fluorescence in situ hybridization. Virtual slide The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1117747619703769 PMID:22770229

  16. Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth

    SciTech Connect

    Wang, Jiying; Rao, Qing; Wang, Min; Wei, Hui; Xing, Haiyan; Liu, Hang; Wang, Yanzhong; Tang, Kejing; Peng, Leiwen; Tian, Zheng; Wang, Jianxiang

    2009-09-04

    Rac1 belongs to the Rho family that act as critical mediators of signaling pathways controlling cell migration and proliferation and contributes to the interactions of hematopoietic stem cells with their microenvironment. Alteration of Rac1 might result in unbalanced interactions and ultimately lead to leukemogenesis. In this study, we analyze the expression of Rac1 protein in leukemia patients and determine its role in the abnormal behaviours of leukemic cells. Rac1 protein is overexpressed in primary acute myeloid leukemia cells as compared to normal bone marrow mononuclear cells. siRNA-mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation, and colony formation. Additionally, blocking Rac1 activity by an inhibitor of Rac1-GTPase, NSC23766, suppressed cell migration and growth. We conclude that overexpression of Rac1 contributes to the accelerated migration and high proliferation potential of leukemia cells, which could be implicated in leukemia development and progression.

  17. Overexpression of Superoxide Dismutase Protects Plants from Oxidative Stress (Induction of Ascorbate Peroxidase in Superoxide Dismutase-Overexpressing Plants).

    PubMed Central

    Gupta, A. S.; Webb, R. P.; Holaday, A. S.; Allen, R. D.

    1993-01-01

    Photosynthesis of leaf discs from transgenic tobacco plants (Nicotiana tabacum) that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD+) was protected from oxidative stress caused by exposure to high light intensity and low temperature. Under the same conditions, leaf discs of plants that did not express the pea SOD isoform (SOD-) had substantially lower photosynthetic rates. Young plants of both genotypes were more sensitive to oxidative stress than mature plants, but SOD+ plants retained higher photosynthetic rates than SOD- plants at all developmental stages tested. Not surprisingly, SOD+ plants had approximately 3-fold higher SOD specific activity than SOD- plants. However, SOD+ plants also exhibited a 3- to 4-fold increase in ascorbate peroxidase (APX) specific activity and had a corresponding increase in levels of APX mRNA. Dehydroascorbate reductase and glutathione reductase specific activities were the same in both SOD+ and SOD- plants. These results indicate that transgenic tobacco plants that overexpress pea Cu/Zn SOD II can compensate for the increased levels of SOD with increased expression of the H2O2-scavenging enzyme APX. Therefore, the enhancement of the active oxygen-scavenging system that leads to increased oxidative stress protection in SOD+ plants could result not only from increased SOD levels but from the combined increases in SOD and APX activity. PMID:12232001

  18. IL-17 induces type V collagen overexpression and EMT via TGF-β-dependent pathways in obliterative bronchiolitis

    PubMed Central

    Vittal, Ragini; Fan, Lin; Greenspan, Daniel S.; Mickler, Elizabeth A.; Gopalakrishnan, Bagavathi; Gu, Hongmei; Benson, Heather L.; Zhang, Chen; Burlingham, William; Cummings, Oscar W.

    2013-01-01

    Obliterative bronchiolitis (OB), a fibrotic airway lesion, is the leading cause of death after lung transplantation. Type V collagen [col(V)] overexpression and IL-17-mediated anti-col(V) immunity are key contributors to OB pathogenesis. Here, we report a previously undefined role of IL-17 in inducing col(V) overexpression, leading to epithelial mesenchymal transition (EMT) and subsequent OB. We observed IL-17-mediated induction of col(V) α1 chains [α1 (V)] in normal airway epithelial cells in vitro and detected α1 (V)-specific antibodies in bronchoalveolar lavage fluid of lung transplant patients. Overexpression of IL-17 and col(V) was detected in OB lesions in patient lung biopsies and in a murine OB model. IL-17 is shown to induce EMT, TGF-β mRNA expression, and SMAD3 activation, whereas downregulating SMAD7 expression in vitro. Pharmacological inhibition of TGF-βRI tyrosine kinase, p38 MAPK, or focal adhesion kinase prevented col(V) overexpression and EMT. In murine orthotopic lung transplants, neutralizing IL-17 significantly decreased TGF-β mRNA and protein expression and prevented epithelial repair/OB. Our findings highlight a feed-forward loop between IL-17 and TGF-β, leading to induction of col(V) and associated epithelial repair, thus providing one possible link between autoimmunity and OB after lung transplantation. PMID:23262228

  19. 100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy

    PubMed Central

    Yue, Yongping; Wasala, Nalinda B; Bostick, Brian; Duan, Dongsheng

    2016-01-01

    Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity. PMID:27419194

  20. 100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy.

    PubMed

    Yue, Yongping; Wasala, Nalinda B; Bostick, Brian; Duan, Dongsheng

    2016-01-01

    Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity. PMID:27419194

  1. [Overexpression of FKS1 to improve yeast autolysis-stress].

    PubMed

    Li, Jia; Wang, Jinjing; Li, Qi

    2015-09-01

    With the development of high gravity brewing, yeast cells are exposed to multiple brewing-associated stresses, such as increased osmotic pressure, enhanced alcohol concentration and nutritional imbalance. These will speed up yeast autolysis, which seriously influence beer flavor and quality. To increase yeast anti-autolytic ability, FKS1 overexpression strain was constructed by 18S rDNA. The concentration of β-1,3-glucan of overexpression strain was 62% higher than that of wild type strain. Meantime, FKS1 overexpression strain increased anti-stress ability at 8% ethanol, 0.4 mol/L NaCl and starvation stress. Under simulated autolysis, FKS1 showed good anti-autolytic ability by slower autolysis. These results confirms the potential of FKS1 overexpression to tackle yeast autolysis in high-gravity brewing. PMID:26955712

  2. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Melén, Karin; Blomberg, Anders; von Heijne, Gunnar

    2006-07-01

    Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies. caffeine | paraquat | salt tolerance | yeast

  3. Regulation of mir-196b by MLL and its overexpression by MLL fusions contributes to immortalization

    PubMed Central

    Popovic, Relja; Riesbeck, Laurie E.; Velu, Chinavenmeni S.; Chaubey, Aditya; Zhang, Jiwang; Achille, Nicholas J.; Erfurth, Frank E.; Eaton, Katherine; Lu, Jun; Grimes, H. Leighton; Chen, Jianjun; Rowley, Janet D.

    2009-01-01

    Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here, we show that MLL normally regulates expression of mir-196b, a hematopoietic microRNA located within the HoxA cluster, in a pattern similar to that of the surrounding 5′ Hox genes, Hoxa9 and Hoxa10, during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage, mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b, while treatment of MLL-AF9 transformed bone marrow cells with mir-196–specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore, overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival, as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development. PMID:19188669

  4. Overexpressed homeobox B9 regulates oncogenic activities by transforming growth factor-β1 in gliomas

    SciTech Connect

    Fang, Liping; Xu, Yinghui; Zou, Lijuan

    2014-03-28

    Highlights: • HOXB9 is overexpressed in gliomas. • HOXB9 over expression had shorter survival time than down expression in gliomas. • HOXB9 stimulated the proliferation, migration and sphere formation of glioma cells. • Activation of TGF-β1 contributed to HOXB9-induced oncogenic activities. - Abstract: Glioma is the leading cause of deaths related to tumors in the central nervous system. The mechanisms of gliomagenesis remain elusive to date. Homeobox B9 (HOXB9) has a crucial function in the regulation of gene expression and cell survival, but its functions in glioma formation and development have yet to be elucidated. This study showed that HOXB9 expression in glioma tissues was significantly higher than that in nontumor tissues. Higher HOXB9 expression was also significantly associated with advanced clinical stage in glioma patients. HOXB9 overexpression stimulated the proliferation, migration, and sphere formation of glioma cells, whereas HOXB9 knockdown elicited an opposite effect. HOXB9 overexpression also increased the tumorigenicity of glioma cells in vivo. Moreover, the activation of transforming growth factor-β1 contributed to HOXB9-induced oncogenic activities. HOXB9 could be used as a predictable biomarker to be detected in different pathological and histological subtypes in glioma for diagnosis or prognosis.

  5. Overexpressed HDAC4 is associated with poor survival and promotes tumor progression in esophageal carcinoma

    PubMed Central

    Mai, Shi-Juan; Wang, Meng-He; Zhang, Mei-Yin; Zheng, X.F. Steven; Wang, Hui-Yun

    2016-01-01

    Histone deacetylases (HDACs) mediate histone deacetylation, leading to transcriptional repression, which is involved in many diseases, including age-related tissue degeneration, heart failure and cancer. In this study, we were aimed to investigate the expression, clinical significance and biological function of HDAC4 in esophageal carcinoma (EC). We found that HDAC4 mRNA and protein are overexpressed in esophageal squamous cell carcinoma (ESCC) tissues and cell lines. HDAC4 overexpression is associated with higher tumor grade, advanced clinical stage and poor survival. Mechanistically, HDAC4 promotes proliferation and G1/S cell cycle progression in EC cells by inhibiting cyclin-dependent kinase (CDK) inhibitors p21 and p27 and up-regulating CDK2/4 and CDK-dependent Rb phosphorylation. HDAC4 also enhances ESCC cell migration. Furthermore, HDAC4 positively regulates epithelial-mesenchymal transition (EMT) by increasing the expression of Vimentin and decreasing the expression of E-Cadherin/α-Catenin. Together, our study shows that HDAC4 overexpression is important for the oncogenesis of EC, which may serve as a useful prognostic biomarker and therapeutic target for this malignancy. PMID:27295551

  6. MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers

    PubMed Central

    Mohseni, Morassa; Cidado, Justin; Croessmann, Sarah; Cravero, Karen; Cimino-Mathews, Ashley; Wong, Hong Yuen; Scharpf, Rob; Zabransky, Daniel J.; Abukhdeir, Abde M.; Garay, Joseph P.; Wang, Grace M.; Beaver, Julia A.; Cochran, Rory L.; Blair, Brian G.; Rosen, D. Marc; Erlanger, Bracha; Argani, Pedram; Hurley, Paula J.; Lauring, Josh; Park, Ben Ho

    2014-01-01

    Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy. PMID:25422431

  7. Overexpression of the anti-apoptotic protein AVEN contributes to increased malignancy in hematopoietic neoplasms.

    PubMed

    Eißmann, M; Melzer, I M; Fernández, S B M; Michel, G; Hrabě de Angelis, M; Hoefler, G; Finkenwirth, P; Jauch, A; Schoell, B; Grez, M; Schmidt, M; Bartholomae, C C; Newrzela, S; Haetscher, N; Rieger, M A; Zachskorn, C; Mittelbronn, M; Zörnig, M

    2013-05-16

    AVEN has been identified as an inhibitor of apoptosis, which binds to the adaptor protein, APAF-1, and thereby prevents apoptosome formation and mitochondrial apoptosis. Recent data have demonstrated high expression levels of AVEN messenger RNA in acute leukemias as well as a positive correlation between AVEN mRNA overexpression and poor prognosis in childhood acute lymphoblastic leukemia. On the basis of these data, we investigated the potential involvement of AVEN in tumorigenesis. First, we confirmed the overexpression of AVEN in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) patient samples. We then established a transgenic mouse model with T-cell-specific overexpression of AVEN, with which we demonstrated the oncogenic cooperation of AVEN with heterozygous loss of p53. Finally, we used a subcutaneous xenograft mouse model to show that AVEN knockdown in the T-ALL cell lines, MOLT-4 and CCRF-CEM, and in the acute myeloblastic leukemia cell line, Kasumi-1, leads to a halt in tumor growth owing to the increased apoptosis and decreased proliferation of tumor cells. Collectively, our data demonstrate that the anti-apoptotic molecule, AVEN, functions as an oncoprotein in hematopoietic neoplasms. PMID:22751129

  8. Overexpression of Methyl-CpG Binding Protein 2 Impairs TH1 Responses

    PubMed Central

    Yang, Tianshu; Ramocki, Melissa B.; Neul, Jeffrey L.; Lu, Wen; Roberts, Luz; Knight, John; Ward, Christopher S.; Zoghbi, Huda Y.; Kheradmand, Farrah; Corry, David B.

    2013-01-01

    The DNA binding protein methyl-CpG binding protein 2 (MeCP2) critically influences neuronal and brain function by modulating gene expression, and children with overexpression of the MECP2 gene exhibit postnatal neurological syndromes. We demonstrate that some children with MECP2 duplication also display variable immunological abnormalities that include reductions in memory T and B cells and natural killer cells and immunoglobulin assay responses. Moreover, whereas mice with MeCP2 overexpression were unable to control infection with the intra-macrophage parasite Leishmania major and secrete interferon-γ (IFN-γ) from involved lymph nodes, they were able to control airway fungal infection by Aspergillus niger and mount protective T helper cell type 2 (TH2)–dependent allergic responses. Relative to normal T cells, TH cells from children and mice with MECP2 duplication displayed similar impairments in IFN-γ secretion and TH1 responses that were due to both MeCP2-dependent suppression of IFN-γ transcription and sequestration of the IFN-γ locus as assessed by chromatin immunoprecipitation assay. Thus, overexpressed MeCP2 aberrantly suppresses IFN-γ secretion from TH cells, potentially leading to a partially immunodeficient state. Our findings establish a rational basis for identifying, treating, and preventing infectious complications potentially affecting children with MECP2 duplication. PMID:23220634

  9. NDRG1 overexpressing gliomas are characterized by reduced tumor vascularization and resistance to antiangiogenic treatment.

    PubMed

    Broggini, Thomas; Wüstner, Marie; Harms, Christoph; Stange, Lena; Blaes, Jonas; Thomé, Carina; Harms, Ulrike; Mueller, Susanne; Weiler, Markus; Wick, Wolfgang; Vajkoczy, Peter; Czabanka, Marcus

    2016-10-01

    Hypoxia-regulated molecules play an important role in vascular resistance to antiangiogenic treatment. N-myc downstream-regulated-gene 1 (NDRG1) is significantly upregulated during hypoxia in glioma. It was the aim of the present study to analyze the role of NDRG1 on glioma angiogenesis and on antiangiogenic treatment. Orthotopically implanted NDRG1 glioma showed reduced tumor growth and vessel density compared to controls. RT-PCR gene array analysis revealed a 30-fold TNFSF15 increase in NDRG1 tumors. Consequently, the supernatant from NDRG1 transfected U87MG glioma cells resulted in reduced HUVEC proliferation, migration and angiogenic response in tube formation assays in vitro. This effect was provoked by increased TNFSF15 promoter activity in NDRG1 cells. Mutations in NF-κB and AP-1 promoter response elements suppressed TNFSF15 promoter activity. Moreover, U87MG glioma NDRG1 knockdown supernatant contained multiple proangiogenic proteins and increased HUVEC spheroid sprouting. Sunitinib treatment of orhotopically implanted mice reduced tumor volume and vessel density in controls; in NDRG1 overexpressing cells no reduction of tumor volume or vessel density was observed. NDRG1 overexpression leads to reduced tumor growth and angiogenesis in experimental glioma via upregulation of TNFSF15. In NDRG1 overexpressing glioma antiangiogenic treatment does not yield a therapeutic response. PMID:26297987

  10. Calpastatin overexpression limits calpain-mediated proteolysis and behavioral deficits following traumatic brain injury

    PubMed Central

    Schoch, Kathleen M.; Evans, Heather N.; Brelsfoard, Jennifer M.; Madathil, Sindhu K.; Takano, Jiro; Saido, Takaomi C.; Saatman, Kathryn E.

    2012-01-01

    Traumatic brain injury (TBI) results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Although the specific inhibitor of calpains, calpastatin, is present within neurons, normal levels of calpastatin are unable to fully prevent the damaging proteolytic activity of calpains after injury. In this study, increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a calcium-calmodulin dependent kinase II α promoter. Naïve hCAST transgenic mice exhibited enhanced neuronal calpastatin expression and significantly reduced protease activity. Acute calpain-mediated spectrin proteolysis in the cortex and hippocampus induced by controlled cortical impact brain injury was significantly attenuated in calpastatin overexpressing mice. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points. Partial hippocampal neuroprotection observed at a moderate injury severity was lost after severe TBI. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. In addition, the reduction in spectrin proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury. PMID:22572592