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Sample records for rapamycin down-regulates ldl-receptor

  1. Rapamycin down-regulates LDL-receptor expression independently of SREBP-2

    SciTech Connect

    Sharpe, Laura J.; Brown, Andrew J.

    2008-09-05

    As a key regulator of cholesterol homeostasis, sterol-regulatory element binding protein-2 (SREBP-2) up-regulates expression of genes involved in cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) Reductase) and uptake (the low density lipoprotein (LDL)-receptor). Previously, we showed that Akt, a critical kinase in cell growth and proliferation, contributes to SREBP-2 activation. However, the specific Akt target involved is unknown. A potential candidate is the mammalian target of rapamycin, mTOR. Rapamycin can cause hyperlipidaemia clinically, and we hypothesised that this may be mediated via an effect of mTOR on SREBP-2. Herein, we found that SREBP-2 activation and HMG-CoA Reductase gene expression were unaffected by rapamycin treatment. However, LDL-receptor gene expression was decreased by rapamycin, suggesting that this may contribute to the hyperlipidaemia observed in rapamycin-treated patients. Rapamycin did not affect mRNA stability, so the decrease in LDL-receptor gene expression is likely to be occurring at the transcriptional level, although independently of SREBP-2.

  2. Rapamycin, a mTOR inhibitor, induced growth inhibition in retinoblastoma Y79 cell via down-regulation of Bmi-1.

    PubMed

    Wang, Yan-Dong; Su, Yong-Jing; Li, Jian-Ying; Yao, Xiang-Chao; Liang, Guang-Jiang

    2015-01-01

    Rapamycin is useful in the treatment of certain cancers by inhibiting mTOR(mammalian target of rapamycin) pathway. Here, anticancer activity and its acting mechanisms of rapamycin were investigated in human retinoblastoma Y79 cells. CCK-8 assay showed that the IC50 value of rapamycin against human retinoblastoma Y79 cells was 0.122±0.026 μmol/L. Flow cytometry analysis indicated that rapamycin induced G1 cell cycle arrest. Western blot assay demonstrated that the mTOR pathway in Y79 cells was blocked by rapamycin. Western blot and RT-PCR assay showed that Bmi-1 was downregulated in protein and mRNA level by rapamycin treatment. Further Western blot and RNA interference assays showed that rapamycin-mediated downregulation of Bmi-1 induced decreases of cyclin E1, which accounted for rapamycin-mediated G1 cell cycle arrest in human retinoblastoma cells. Together, all these results illustrated that rapamycin induced growth inhibition of human retinoblastoma cells, and inactive of mTOR pathway and downregulation of Bmi-1 was involved in its action mechanism. PMID:26191215

  3. Rapamycin, a mTOR inhibitor, induced growth inhibition in retinoblastoma Y79 cell via down-regulation of Bmi-1

    PubMed Central

    Wang, Yan-Dong; Su, Yong-Jing; Li, Jian-Ying; Yao, Xiang-Chao; Liang, Guang-Jiang

    2015-01-01

    Rapamycin is useful in the treatment of certain cancers by inhibiting mTOR(mammalian target of rapamycin) pathway. Here, anticancer activity and its acting mechanisms of rapamycin were investigated in human retinoblastoma Y79 cells. CCK-8 assay showed that the IC50 value of rapamycin against human retinoblastoma Y79 cells was 0.122 ± 0.026 μmol/L. Flow cytometry analysis indicated that rapamycin induced G1 cell cycle arrest. Western blot assay demonstrated that the mTOR pathway in Y79 cells was blocked by rapamycin. Western blot and RT-PCR assay showed that Bmi-1 was downregulated in protein and mRNA level by rapamycin treatment. Further Western blot and RNA interference assays showed that rapamycin-mediated downregulation of Bmi-1 induced decreases of cyclin E1, which accounted for rapamycin-mediated G1 cell cycle arrest in human retinoblastoma cells. Together, all these results illustrated that rapamycin induced growth inhibition of human retinoblastoma cells, and inactive of mTOR pathway and downregulation of Bmi-1 was involved in its action mechanism. PMID:26191215

  4. Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes

    PubMed Central

    Cerrato, F; Fernández-Suárez, M E; Alonso, R; Alonso, M; Vázquez, C; Pastor, O; Mata, P; Lasunción, M A; Gómez-Coronado, D

    2015-01-01

    Background and Purpose Treatment with selective oestrogen receptor modulators (SERMs) reduces low-density lipoprotein (LDL) cholesterol levels. We assessed the effect of tamoxifen, raloxifene and toremifene and their combinations with lovastatin on LDL receptor activity in lymphocytes from normolipidaemic and familial hypercholesterolaemic (FH) subjects, and human HepG2 hepatocytes and MOLT-4 lymphoblasts. Experimental Approach Lymphocytes were isolated from peripheral blood, treated with different compounds, and 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (DiI)-labelled LDL uptake was analysed by flow cytometry. Key Results Tamoxifen, toremifene and raloxifene, in this order, stimulated DiI-LDL uptake by lymphocytes by inhibiting LDL-derived cholesterol trafficking and subsequent down-regulation of LDL receptor expression. Differently to what occurred in HepG2 and MOLT-4 cells, only tamoxifen consistently displayed a potentiating effect with lovastatin in primary lymphocytes. The SERM-mediated increase in LDL receptor activity was not altered by the anti-oestrogen ICI 182 780 nor was it reproduced by 17β-oestradiol. However, the tamoxifen-active metabolite endoxifen was equally effective as tamoxifen. The SERMs produced similar effects on LDL receptor activity in heterozygous FH lymphocytes as in normal lymphocytes, although none of them had a potentiating effect with lovastatin in heterozygous FH lymphocytes. The SERMs had no effect in homozygous FH lymphocytes. Conclusions and Implications Clinically used SERMs up-regulate LDL receptors in primary human lymphocytes. There is a mild enhancement between SERMs and lovastatin of lymphocyte LDLR activity, the potentiation being greater in HepG2 and MOLT-4 cells. The effect of SERMs is independent of oestrogen receptors but is preserved in the tamoxifen-active metabolite endoxifen. This mechanism may contribute to the cholesterol-lowering action of SERMs. PMID:25395200

  5. Iron deficiency down-regulates the Akt/TSC1-TSC2/mammalian Target of Rapamycin signaling pathway in rats and in COS-1 cells.

    PubMed

    Ndong, Moussa; Kazami, Machiko; Suzuki, Tsukasa; Uehara, Mariko; Katsumata, Shin-Ichi; Inoue, Hirohumi; Kobayashi, Ken-Ichi; Tadokoro, Tadahiro; Suzuki, Kazuharu; Yamamoto, Yuji

    2009-09-01

    Iron deficiency (ID) is one of the most commonly known forms of nutritional deficiencies. Low body iron is thought to induce neurologic defects but may also play a protective role against cancer development by cell growth arrest. Thus, ID may affect cellular pathways controlling cell growth and proliferation, the mechanism of which is still not fully understood. The serine/threonine protein kinase Akt and its downstream target, the mammalian Target of Rapamycin (mTOR), is known to play a crucial role in the regulation of cell growth and survival. Therefore, we hypothesized that Akt/mTOR pathway could be influenced by ID. Three-week-old male Wistar-strain rats were divided into 3 groups and the 2 groups had free access to a control diet (C group) or an iron-deficient diet (D group). The third group (PF group) were pair-fed the control diet to the mean intake of the D group. After 4 weeks, rats were killed and their brains were sampled. In separate experiments, COS-1 cells were cultured with or without the iron chelator deferoxamine. Western blots of brain samples and COS-1 lysates were used to analyze the expression and phosphorylation state of Akt, TSC2, mTOR, and S6 kinase proteins implicated in the Akt/mTOR pathway. Using 2 different ID models, we show for the first time that iron deficiency depresses Akt activity in rats and in COS-1 cells, leading to a decrease in mTOR activity. PMID:19854379

  6. Polymorphic DNA haplotypes at the LDL receptor locus.

    PubMed Central

    Leitersdorf, E; Chakravarti, A; Hobbs, H H

    1989-01-01

    Mutations in the low-density lipoprotein (LDL) receptor gene result in the autosomal dominant disorder familial hypercholesterolemia (FH). Many different LDL receptor mutations have been identified and characterized, demonstrating a high degree of allelic heterogeneity at this locus. The ability to identify mutant LDL receptor genes for prenatal diagnosis of homozygous FH or to study the role of the LDL receptor gene in polygenic hypercholesterolemia requires the use of closely linked RFLPs. In the present study we used 10 different RFLPs, including three newly described polymorphisms, to construct 123 independent haplotypes from 20 Caucasian American pedigrees. Our sample contained 31 different haplotypes varying in frequency from 0.8% to 29.3%; the five most common haplotypes account for 67.5% of the sample. The heterozygosity and PIC of each site were determined, and these values disclosed that eight of the RFLPs were substantially polymorphic. Linkage-disequilibrium analysis of the haplotype data revealed strong nonrandom associations among all 10 RFLPs, especially among those sites clustered in the 3' region of the gene. Evolutionary analysis suggests the occurrence of both mutational and recombinational events in the generation of the observed haplotypes. A strategy for haplotype analysis of the LDL receptor gene in individuals of Caucasian American descent is presented. Images Figure 2 Figure 3 PMID:2563635

  7. A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia.

    PubMed

    Soufi, Muhidien; Rust, Stephan; Walter, Michael; Schaefer, Juergen R

    2013-05-25

    Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone. PMID:23510778

  8. A nonsense mutation in the LDL receptor gene leads to familial hypercholesterolemia in the Druze sect

    SciTech Connect

    Landsberger, D.; Meiner, V.; Reshef, A.; Leitersdorf, E. ); Levy, Yishai ); Westhytzen, D.R. van der; Coetzee, G.A. )

    1992-02-01

    Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the LDL receptor gene. Here the authors characterize and LDL receptor mutation that is associated with a distinct haplotype and causes FH in the Druze, a small Middle Eastern Islamic sect with a high degree of inbreeding. The mutation was found in FH families from two distinct Druze villages from the Golan Heights (northern Israel). It was not found either in another Druze FH family residing in a different geographical area nor in eight Arab and four Jewish FH heterozygote index cases whose hypercholesterolemia cosegregates with an identical LDL receptor gene haplotype. The mutation, a single-base substitution, results in a termination codon in exon 4 of the LDL receptor gene that encodes for the fourth repeat of the binding domain of the mature receptor. It can be diagnosed by allele-specific oligonucleotide hybridization of PCR-amplified DNA from FH patients.

  9. Distribution of the LDL receptor within clathrin-coated pits and caveolae in rat and human liver.

    PubMed

    Ivaturi, Soumya; Wooten, Catherine J; Nguyen, Maikhanh D; Ness, Gene C; Lopez, Dayami

    2014-03-01

    Several findings suggest that the low-density lipoprotein (LDL) receptor may internalize different lipoprotein particles via diverse pathways. Using a combination of discontinuous sucrose gradients and Triton solubilization studies, we demonstrated that the LDL receptor could be located simultaneously in clathrin-coated pits and caveolae in rat and human liver and in human hepatocyte-like C3A cells. Treatment with the cholesterol biosynthesis inhibitor, zaragozic acid A, shifted the distribution of the LDL receptor to clathrin containing fractions, whereas treatment with cholesterol or LDL shifted the receptor distribution towards caveolin-1 containing fractions. The LDL-dependent shift of the LDL receptor to caveolae coincided with a reduction in internalization of Bodipy-LDL. Redistribution within plasma membrane microdomains in response to specific treatments resulting in changes in LDL receptor function represents a novel paradigm that could be exploited in the development of a new class of therapeutic drugs. PMID:24530906

  10. Effect of Genistein and L-Carnitine and Their Combination on Gene Expression of Hepatocyte HMG-COA Reductase and LDL Receptor in Experimental Nephrotic Syndrome

    PubMed Central

    YOUSEFINEJAD, Abbas; SIASSI, Fereydoon; MIRSHAFIEY, Abbas; ESHRAGHIAN, Mohammad-Reza; KOOHDANI, Fariba; JAVANBAKHT, Mohammad Hassan; SEDAGHAT, Reza; RAMEZANI, Atena; ZAREI, Mahnaz; DJALALI, Mahmoud

    2015-01-01

    Background: Nephrotic syndrome is a disorder that leads to hyperlipidemia. L-carnitine and genistein can effect on lipid metabolism and the syndrome. In the present study, we have delved into the separate and the twin-effects of L-carnitine and genistein on the gene expressions of HMG-COA reductase and LDL receptor in experimental nephrotic syndrome. Methods: In this controlled experimental study, 50 male Sprague–Dawley rats were randomly divided into five groups: NC (normal-control), PC (patient-control), LC (L-carnitine), G (genistein), LCG (L-carnitine-genistein). Adriamycin was used for inducing nephrotic syndrome and the spot urine samples and urine protein-to-creatinine ratio were measured. Hepatocytic RNA was extracted and real-time PCR was used for HMG-COA Reductase and LDL receptor gene Expression measurement. Results: The final weight of the patients groups were lower than the NC group (P=0.001), and weight gain of the NC group was higher than the other groups (P<0.001). The proteinuria and urine protein-to-creatinine ratio showed significant differences between PC group and LC, G and LCG groups at week 7 (P<0.001). The expression of HMGCOA Reductase mRNA down regulated in LC, G and LCG groups in comparison with PC group (P<0.001). ΔCT of LDLr mRNA showed significant differences between the PC group and the other patient groups (P<0.001). Conclusion: This study shows a significant decreasing (P<0.001) and non-significant increasing trend in HMG-COA Reductase and LDLr gene expression, respectively, and synergistic effect of L-carnitine and genistein on these genes in experimental nephrotic syndrome. PMID:26576346

  11. Difference in LDL receptor feedback regulation in macrophages and vascular smooth muscle cells: foam cell transformation under inflammatory stress.

    PubMed

    Ye, Qiang; Lei, Han; Fan, Zhongcai; Zheng, Wenwu; Zheng, Shuzhan

    2014-04-01

    Macrophages and vascular smooth muscle cells (VSMCs) are the major cell types involved in foam cell formation associated with atherosclerosis. The aim of this experiment was to clarify cell-specific regulation of LDL receptor in THP-1 macrophages and human VSMCs under physiological and inflammatory conditions and its potential mechanisms. Inflammatory stress was induced by adding lipopolysaccharide (LPS) to human THP-1 macrophages and human VSMCs. Intracellular total cholesterol, free cholesterol, and cholesterol ester were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein (SREBP)-2 and SREBP cleavage-activating protein (SCAP) mRNA levels using real-time quantitative polymerase chain reaction. LDL receptor, SREBP-2 and SCAP protein expression were examined by Western blotting. The translocation of SCAP from ER to Golgi was detected by confocal microscopy. LDL loading increased intracellular cholesterol level, reducing LDL receptor mRNA level in both THP-1 macrophages and VSMCs under physiological conditions. The IC50 in VSMCs was 11.25 μg/ml, which is much lower than 18.125 μg/ml in THP-1 macrophages. With the increase in concentration of LPS (0-400 ng/ml), the LDL receptor mRNA levels were upregulated in both cells, but the curve of LDL receptor mRNA in VSMCs exhibited a flatter profile than that of THP-1 macrophages. Under the treatment of 200 ng/ml of LPS, the upregulation fold of the LDL receptor mRNA in THP-1 macrophages was much higher than that of VSMCs (0.33 vs 0.04). LDL receptor blocking agent heparin decreased lipid droplets induced by LPS significantly in THP-1 macrophages and VSMCs. LDL loading reduced the SREBP2 and SCAP protein expression under physiological conditions. Exposure to LPS caused overexpression of SREBP2 and SCAP despite a high concentration of LDL in the culture

  12. LDL Receptor-Related Protein-1 (LRP1) Regulates Cholesterol Accumulation in Macrophages

    PubMed Central

    Lillis, Anna P.; Muratoglu, Selen Catania; Au, Dianaly T.; Migliorini, Mary; Lee, Mi-Jeong; Fried, Susan K.; Mikhailenko, Irina; Strickland, Dudley K.

    2015-01-01

    Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the contribution of the LDL receptor-related protein 1 (LRP1) to this process is not known. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR)-deficient background (macLRP1-/-). After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis. PMID:26061292

  13. Expression of lectin-like oxidized LDL receptor-1 in smooth muscle cells after vascular injury

    SciTech Connect

    Eto, Hideyuki; Miyata, Masaaki . E-mail: miyatam@m3.kufm.kagoshima-u.ac.jp; Kume, Noriaki; Minami, Manabu; Itabe, Hiroyuki; Orihara, Koji; Hamasaki, Shuichi; Biro, Sadatoshi; Otsuji, Yutaka; Kita, Toru; Tei, Chuwa

    2006-03-10

    Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor, and its role in restenosis after angioplasty remains unknown. We used a balloon-injury model of rabbit aorta, and reverse transcription-polymerase chain reaction revealed that LOX-1 mRNA expression was modest in the non-injured aorta, reached a peak level 2 days after injury, and remained elevated until 24 weeks after injury. Immunohistochemistry and in situ hybridization showed that LOX-1 was not detected in the media of non-injured aorta but expressed in both medial and neointimal smooth muscle cells (SMC) at 2 and 24 weeks after injury. Low concentrations of ox-LDL (10 {mu}g/mL) stimulated the cultured SMC proliferation, which was inhibited by antisense oligonucleotides of LOX-1 mRNA. Double immunofluorescense staining showed the colocalization of LOX-1 and proliferating cell nuclear antigen in human restenotic lesion. These results suggest that LOX-1 mediates ox-LDL-induced SMC proliferation and plays a role in neointimal formation after vascular injury.

  14. Cell surface expression of LDL receptor in chronic hepatitis C: correlation with viral load.

    PubMed

    Petit, Jean-Michel; Minello, Anne; Duvillard, Laurence; Jooste, Valérie; Monier, Serge; Texier, Véronique; Bour, Jean-Baptiste; Poussier, Alix; Gambert, Philippe; Verges, Bruno; Hillon, Patrick

    2007-07-01

    The LDL receptor (LDL-R) has been proposed as the viral receptor for Hepatitis C virus (HCV). This hypothesis has been based exclusively on in vitro studies. In human mononuclear cells, LDL-R gene expression has been demonstrated to be parallel and be coordinately regulated to gene expression in the human liver. The purpose of the current study was to determine the mononuclear cell surface expression of the LDL receptor in patients with HCV chronic infection according to viral load. Sixty-eight consecutive untreated chronic hepatitis C patients were studied to determine the mononuclear cell surface expression of the LDL-R. LDL-Rs were quantified at the surface of mononuclear cells in fresh blood samples taken after fasting using flow cytometry. LDL-R expression was significantly associated with LDL-cholesterol (r = -0.25; P = 0.03) and HCV-viral load (r = 0.37, P = 0.002). In multivariate analysis, the LDL-R expression was significantly associated with HCV viral load, whereas genotype, age, body mass index, and fibrosis were not. In conclusion, our data provided by a human study, suggest that the LDL-R may be one of the receptors implicated in HCV replication. PMID:17473053

  15. A Complete Backbone Assignment of the Apolipoprotein E LDL Receptor Binding Domain [Letter to the Editor

    SciTech Connect

    Xu, Chao; Sivashanmugam, Arun; Hoyt, David W.; Wang, Jianjun

    2005-06-01

    Human apolipoprotein E (apoE) is a 299-residue exchangeable apolipoprotein that was initially recognized as a major determinant in lipoprotein metabolism and cardiovascular diseases. Recent evidence has indicated that apoE also plays critical roles in several other important biological processes not directly related to its lipid transport function, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and possibly even infectious diseases. ApoE contains two structural/functional domains: A N-terminal domain spanning residues 1-191 that is responsible for apoE's LDL receptor binding activity and a C-terminal domain (residues 216-199) that is responsible for lipoprotein-binding (1). The x-ray crystal structure of the lipid-free apoE N-terminal domain was solved by Wilson et al in 1991 which represented the only high-resolution structure of this protein. This structure showed an unusually elongated four-helix bundle (2) that was organized in such 2 a way that its hydrophobic faces were directed towards the protein interior, whereas the hydrophilic faces were oriented towards the solvent. The major receptor-binding region, residues 130-150, was located on the fourth helix. The amphipathic a-helices were connected by short loops, giving rise to a compact, globular structure. However, this structure only contained residues 23-165. Recent studies have shown that residues beyond residues 23-165 are also very important to the apoE LDL receptor binding activity. For example, a mutation at position R172 reduces the receptor binding activity of apoE to only {approx}2% (3). In addition, an E3K mutant significantly increased the apoE receptor binding activity as well (4). While the x-ray crystal structure of the apoE N-terminal domain provided detailed structural information for most region of this domain, this structure does not provide an explanation of the above experimental results regarding the structural contribution to apoE's LDL receptor

  16. LRAD3, a Novel LDL Receptor Family Member that Modulates Amyloid Precursor Protein Trafficking

    PubMed Central

    Ranganathan, Sripriya; Noyes, Nathaniel C.; Migliorini, Mary; Winkles, Jeffrey A.; Battey, Frances D.; Hyman, Bradley T.; Smith, Elizabeth; Yepes, Manuel; Mikhailenko, Irina; Strickland, Dudley K.

    2011-01-01

    We have identified a novel LDL receptor family member, termed LDL receptor class A domain containing 3 (LRAD3), which is expressed in neurons. The LRAD3 gene encodes an approximately 50 kDa type I transmembrane receptor with an ectodomain containing three LDLa repeats, a transmembrane domain and a cytoplasmic domain containing a conserved dileucine internalization motif and two polyproline motifs with potential to interact with WW domain containing proteins. Immunohistochemical analysis of mouse brain reveals LRAD3 expression in the cortex and hippocampus. In the mouse hippocampal derived cell line, HT22, LRAD3 partially co-localizes with amyloid precursor protein (APP), and interacts with APP as revealed by co-immunoprecipitation experiments. To identify the portion of APP that interacts with LRAD3, we employed solid phase binding assays which demonstrated that LRAD3 failed to bind to a soluble APP fragment (sAPPα) released following α-secretase cleavage. In contrast, C99, the β-secretase product that remains cell associated, co-precipitated with LRAD3, confirming that regions within this portion of APP are important for associating with LRAD3. The association of LRAD3 with APP increases the amyloidogenic pathway of APP processing, resulting in a decrease in sAPPα production and increased Aβ peptide production. Pulse-chase experiments confirm that LRAD3 expression significantly decreases the cellular half-live of mature APP. These results reveal that LRAD3 influences APP processing and raises the possibility that LRAD3 alters APP function in neurons including its downstream signaling. PMID:21795536

  17. Postprandial dyslipidemia in men with visceral obesity: an effect of reduced LDL receptor expression?

    PubMed

    Mamo, J C; Watts, G F; Barrett, P H; Smith, D; James, A P; Pal, S

    2001-09-01

    Postprandial lipemia after an oral fat challenge was studied in middle-aged men with visceral obesity. The two groups had similar plasma cholesterol levels, but obese subjects had higher levels of plasma triglyceride and reduced amounts of high-density cholesterol. Fasting plasma insulin was fourfold greater in obese subjects because of concomitant insulin resistance, with a calculated HOMA score of 3.1 +/- 0.6 vs. 0.8 +/- 0.2, respectively. Plasma apolipoprotein B(48) (apoB(48)) and retinyl palmitate (RP) after an oral fat challenge were used to monitor chylomicron metabolism. Compared with lean subjects, the fasting concentration of apoB(48) was more than twofold greater in obese individuals, suggestive of an accumulation of posthydrolyzed particles. After the oral lipid load, the incremental areas under the apoB(48) and RP curves (IAUC) were both significantly greater in obese subjects (apoB(48): 97 +/- 17 vs. 44 +/- 12 microg.ml(-1). h; RP: 3,120 +/- 511 vs. 1,308 +/- 177 U. ml(-1). h, respectively). A delay in the conversion of chylomicrons to remnants probably contributed to postprandial dyslipidemia in viscerally obese subjects. The triglyceride IAUC was 68% greater in obese subjects (4.7 +/- 0.6 vs. 2.8 +/- 0.8 mM. h, P < 0.06). Moreover, peak postprandial triglyceride was delayed by approximately 2 h in obese subjects. The reduction in triglyceride lipolysis in vivo did not appear to reflect changes in hydrolytic enzyme activities. Postheparin plasma lipase rates were found to be similar for lean and obese subjects. In this study, low-density lipoprotein (LDL) receptor expression on monunuclear cells was used as a surrogate marker of hepatic activity. We found that, in obese subjects, the binding of LDL was reduced by one-half compared with lean controls (70.9 +/- 15.07 vs. 38.9 +/- 4.6 ng LDL bound/microg cell protein, P = 0.02). Because the LDL receptor is involved in the removal of proatherogenic chylomicron remnants, we suggest that the hepatic

  18. Combined effects of cholesterol reduction and apolipoprotein A-I expression on atherosclerosis in LDL receptor deficient mice.

    PubMed

    Kawashiri, Masa-aki; Zhang, YuZhen; Puré, Ellen; Rader, Daniel J

    2002-11-01

    Reduction of total and LDL cholesterol reduces atherosclerosis and clinical cardiovascular events. High density lipoprotein (HDL) cholesterol levels have a strong inverse association with atherosclerosis, and overexpression of apolipoprotein A-I (apoA-I), the major protein component of HDL, reduces atherosclerosis in hypercholesterolemic animals. However, little is known about the potential for additive or synergistic effects between cholesterol reduction and apoA-I overexpression on atherosclerosis. In the current study, we tested the hypothesis that significant reduction of plasma cholesterol combined with overexpression of apoA-I would reduce atherosclerosis to a greater extent than either one alone. We used somatic gene transfer of the LDL receptor (to induce cholesterol reduction) and apoA-I in LDL receptor deficient mice fed a Western type diet and compared the combination to expression of each gene alone and to controls. Atherosclerosis was quantitated using two independent methods, by en face analysis of the entire aorta and by cross-sectional analysis of the aortic root. Although the reduction of cholesterol was transient, expression of the LDL receptor alone significantly reduced atherosclerosis by 45% in the aorta and 44% in the aortic root compared with controls. Overexpression of human apoA-I alone reduced atherosclerosis by 42% in the aorta and 44% in the aortic root compared with controls. Co-expression of the LDL receptor with apoA-I resulted in significantly higher levels of apoA-I than expression of apoA-I alone. Although co-expression of the LDL receptor and apoA-I reduced atherosclerosis by 37% in the aorta and 32% in the aortic root compared with controls, the reduction in atherosclerosis was no different than that seen with expression of the LDL receptor alone or apoA-I alone. In summary, in this relatively short-term murine model, simultaneous reduction of cholesterol and expression of apoA-I was associated with higher levels of apoA-I than

  19. Upregulation of hepatic LDL transport by n-3 fatty acids in LDL receptor knockout mice.

    PubMed

    Vasandani, Chandna; Kafrouni, Abdallah I; Caronna, Antonella; Bashmakov, Yuriy; Gotthardt, Michael; Horton, Jay D; Spady, David K

    2002-05-01

    We determined the effects of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) on parameters of plasma lipoprotein and hepatic lipid metabolism in LDL receptor (LDLr) knockout mice. Dietary n-3 PUFA decreased the rate of appearance and increased the hepatic clearance of IDL/LDL resulting in a marked decrease in the plasma concentration of these particles. Dietary n-3 PUFA increased the hepatic clearance of IDL/LDL through a mechanism that appears to involve apolipoprotein (apo)E but is independent of the LDLr, the LDLr related protein (LRP), the scavenger receptor B1, and the VLDLr. The decreased rate of appearance of IDL/VLDL in the plasma of animals fed n-3 PUFA could be attributed to a marked decrease in the plasma concentration of precursor VLDL. Decreased plasma VLDL concentrations were due in part to decreased hepatic secretion of VLDL triglyceride and cholesteryl esters, which in turn was associated with decreased concentrations of these lipids in liver. Decreased hepatic triglyceride concentrations in animals fed n-3 PUFA were due in part to suppression of fatty acid synthesis as a result of a decrease in sterol regulatory element binding protein-1 (SREBP-1) expression and processing. In conclusion, these studies indicate that n-3 PUFA can markedly decrease the plasma concentration of apoB-containing lipoproteins and enhance hepatic LDL clearance through a mechanism that does not involve the LDLr pathway or LRP. PMID:11971949

  20. Modulation of the LDL receptor and LRP levels by HIV protease inhibitors.

    PubMed

    Tran, Huan; Robinson, Susan; Mikhailenko, Irina; Strickland, Dudley K

    2003-10-01

    Inhibitors of the human immunodeficiency virus (HIV)-1 protease have proven to be effective antiretroviral drugs. However, patients receiving these drugs develop serious metabolic abnormalities, including hypercholesterolemia. The objective of the present study was to identify mechanisms by which HIV protease inhibitors increase plasma cholesterol levels. We hypothesized that HIV protease inhibitors may affect gene regulation of certain LDL receptor (LDLR) family members, thereby altering the catabolism of cholesterol-containing lipoproteins. In this present study we investigated the effect of several HIV protease inhibitors (ABT-378, Amprenavir, Indinavir, Nelfinavir, Ritonavir, and Saquinavir) on mRNA, protein, and functional levels of LDLR family members. Our results demonstrate that one of these drugs, Nelfinavir, significantly decreases LDLR and LDLR-related protein (LRP) mRNA and protein levels, resulting in the reduced functional activity of these two receptors. Nelfinavir exerts its effect by reducing levels of active SREBP1 in the nucleus. The finding that Nelfinavir reduces the levels of two key receptors (LRP and LDLR) involved in lipoprotein catabolism and maintenance of vessel wall integrity identifies a mechanism that causes hypercholesterolemia complications in HIV patients treated with this drug and raises concerns about the atherogenic nature of Nelfinavir. PMID:12837856

  1. Conjugated linoleic acid upregulates LDL receptor gene expression in HepG2 cells.

    PubMed

    Yu-Poth, Shaomei; Yin, Dezhong; Zhao, Guixiang; Kris-Etherton, Penny M; Etherton, Terry D

    2004-01-01

    Conjugated linoleic acid (CLA) exerts anticarcinogenic and antiatherosclerotic effects in animals. The present study was conducted to examine the effects of CLA on LDL receptor (LDLr) expression in HepG2 cells, and to evaluate whether the sterol response element binding protein 1 (SREBP-1) and acyl CoA:cholesterol acyltransferase (ACAT) were involved in the regulation of LDLr expression by CLA. When HepG2 cells were cultured with serum-free DMEM for 48 h, there was a three- to fivefold (P<0.05) increase in LDLr protein and mRNA levels. Incubation of HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH, 5 mg/L) for 24 h decreased LDLr protein and mRNA by 50-70% (P<0.05) and mature SREBP-1 by 20-40% (P<0.05). CLA, but not linoleic acid, antagonized the depressive effects of 25OH and increased both LDLr protein and mRNA abundance twofold (P<0.05). LDLr protein and mRNA abundance were not different when HepG2 cells were cultured with CLA (0.4 mmol/L) plus 25OH in the presence or absence of an ACAT inhibitor (58-035, 1 mg/L). Furthermore, CLA had no effect on SREBP-1 abundance. These results suggest that CLA upregulates LDLr expression via a mechanism that is independent of ACAT and SREBP-1. PMID:14704295

  2. A novel mosaic protein containing LDL receptor elements is highly conserved in humans and chickens.

    PubMed

    Mörwald, S; Yamazaki, H; Bujo, H; Kusunoki, J; Kanaki, T; Seimiya, K; Morisaki, N; Nimpf, J; Schneider, W J; Saito, Y

    1997-05-01

    Certain receptors belonging to the LDL receptor (LDLR) gene family appear to constitute a newly identified branch whose members are expressed in brain, in addition to other tissues. In support of this concept, we have now discovered the expression and delineated the molecular structures of a representative of this emerging branch from two such diverse species as human and chicken. This membrane receptor, called LR11 and thus far only known to exist in the rabbit, is a complex seven-domain mosaic protein containing, among other structural elements, a cluster of 11 LDLR ligand-binding repeats and a domain with homology to VPS10, a yeast receptor for vacuolar protein sorting. Cytoplasmic signature sequences define the receptor as competent for endocytosis. The most striking properties of LR11s are their (1) high degree of structural conservation (>80% identity among mammals and birds), with 100% identity in the membrane-spanning and cytoplasmic domains of rabbit and human; (2) lack of regulation by cholesterol and estrogen; and (3) expression in brain. The features of LR11 suggest important roles in intercellular and intracellular ligand transport processes, some of which it may share with other brain-specific LDLR family members. PMID:9157966

  3. LRP6 Protein Regulates Low Density Lipoprotein (LDL) Receptor-mediated LDL Uptake*

    PubMed Central

    Ye, Zhi-jia; Go, Gwang-Woong; Singh, Rajvir; Liu, Wenzhong; Keramati, Ali Reza; Mani, Arya

    2012-01-01

    Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6R611C mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6WT) and LRP6R611C in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6WT was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6WT forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6R611C. These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels. PMID:22128165

  4. Myeloid Deletion of α1AMPK Exacerbates Atherosclerosis in LDL Receptor Knockout (LDLRKO) Mice.

    PubMed

    Cao, Qiang; Cui, Xin; Wu, Rui; Zha, Lin; Wang, Xianfeng; Parks, John S; Yu, Liqing; Shi, Hang; Xue, Bingzhong

    2016-06-01

    Macrophage inflammation marks all stages of atherogenesis, and AMPK is a regulator of macrophage inflammation. We therefore generated myeloid α1AMPK knockout (MAKO) mice on the LDL receptor knockout (LDLRKO) background to investigate whether myeloid deletion of α1AMPK exacerbates atherosclerosis. When fed an atherogenic diet, MAKO/LDLRKO mice displayed exacerbated atherosclerosis compared with LDLRKO mice. To determine the underlying pathophysiological pathways, we characterized macrophage inflammation/chemotaxis and lipid/cholesterol metabolism in MAKO/LDLRKO mice. Myeloid deletion of α1AMPK increased macrophage inflammatory gene expression and enhanced macrophage migration and adhesion to endothelial cells. Remarkably, MAKO/LDLRKO mice also displayed higher composition of circulating chemotaxically active Ly-6C(high) monocytes, enhanced atherosclerotic plaque chemokine expression, and monocyte recruitment into plaques, leading to increased atherosclerotic plaque macrophage content and inflammation. MAKO/LDLRKO mice also exhibited higher plasma LDL and VLDL cholesterol content, increased circulating apolipoprotein B (apoB) levels, and higher liver apoB expression. We conclude that macrophage α1AMPK deficiency promotes atherogenesis in LDLRKO mice and is associated with enhanced macrophage inflammation and hypercholesterolemia and that macrophage α1AMPK may serve as a therapeutic target for prevention and treatment of atherosclerosis. PMID:26822081

  5. In vivo gene therapy for hyperlipidemia: phenotypic correction in Watanabe rabbits by hepatic delivery of the rabbit LDL receptor gene.

    PubMed Central

    Li, J; Fang, B; Eisensmith, R C; Li, X H; Nasonkin, I; Lin-Lee, Y C; Mims, M P; Hughes, A; Montgomery, C D; Roberts, J D

    1995-01-01

    Elevations of plasma total or LDL cholesterol are major risk factors for cardiovascular disease. Efforts directed at preventing and treating cardiovascular disease have often focused on reducing the levels of these substances in the blood. The Watanabe Heritable Hyperlipidemic Rabbit, which has exceedingly high plasma cholesterol levels resulting from an LDL receptor deficiency, provides an excellent animal model for testing new treatments. A recombinant adenoviral vector containing the rabbit LDL receptor cDNA was administered to Watanabe rabbits. Plasma total cholesterol levels in the treated animals were reduced from 825.5 +/- 69.8 (mean +/- SD) to 247.3 +/- 61.5 mg/dl 6 d after infusion. These animals also demonstrated a 300-400% increase in plasma levels of HDL cholesterol and apo AI 10 d after treatment. As a result, the LDL:HDL ratio exhibited a dramatic decrease. Because only the rabbit LDL receptor gene was used for treatment, the results strongly suggest that the elevations of plasma HDL cholesterol and apo AI were secondary to a reduction in plasma total cholesterol in the treated animals. These results suggest an inverse relationship between plasma LDL and HDL cholesterol levels and imply that reduction of LDL cholesterol levels may have a beneficial effect on plasma HDL cholesterol. PMID:7860759

  6. In vivo gene therapy for hyperlipidemia: phenotypic correction in Watanabe rabbits by hepatic delivery of the rabbit LDL receptor gene.

    PubMed

    Li, J; Fang, B; Eisensmith, R C; Li, X H; Nasonkin, I; Lin-Lee, Y C; Mims, M P; Hughes, A; Montgomery, C D; Roberts, J D

    1995-02-01

    Elevations of plasma total or LDL cholesterol are major risk factors for cardiovascular disease. Efforts directed at preventing and treating cardiovascular disease have often focused on reducing the levels of these substances in the blood. The Watanabe Heritable Hyperlipidemic Rabbit, which has exceedingly high plasma cholesterol levels resulting from an LDL receptor deficiency, provides an excellent animal model for testing new treatments. A recombinant adenoviral vector containing the rabbit LDL receptor cDNA was administered to Watanabe rabbits. Plasma total cholesterol levels in the treated animals were reduced from 825.5 +/- 69.8 (mean +/- SD) to 247.3 +/- 61.5 mg/dl 6 d after infusion. These animals also demonstrated a 300-400% increase in plasma levels of HDL cholesterol and apo AI 10 d after treatment. As a result, the LDL:HDL ratio exhibited a dramatic decrease. Because only the rabbit LDL receptor gene was used for treatment, the results strongly suggest that the elevations of plasma HDL cholesterol and apo AI were secondary to a reduction in plasma total cholesterol in the treated animals. These results suggest an inverse relationship between plasma LDL and HDL cholesterol levels and imply that reduction of LDL cholesterol levels may have a beneficial effect on plasma HDL cholesterol. PMID:7860759

  7. Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

    SciTech Connect

    Ellsworth, J.L.; Brown, C.; Cooper, A.D.

    1988-05-01

    The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the apparent Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by (3H)thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; (14C)acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column.

  8. Intradomain Confinement of Disulfides in the Folding of Two Consecutive Modules of the LDL Receptor

    PubMed Central

    Martínez-Oliván, Juan; Fraga, Hugo; Arias-Moreno, Xabier; Ventura, Salvador; Sancho, Javier

    2015-01-01

    The LDL receptor internalizes circulating LDL and VLDL particles for degradation. Its extracellular binding domain contains ten (seven LA and three EGF) cysteine-rich modules, each bearing three disulfide bonds. Despite the enormous number of disulfide combinations possible, LDLR oxidative folding leads to a single native species with 30 unique intradomain disulfides. Previous folding studies of the LDLR have shown that non native disulfides are initially formed that lead to compact species. Accordingly, the folding of the LDLR has been described as a "coordinated nonvectorial” reaction, and it has been proposed that early compaction funnels the reaction toward the native structure. Here we analyze the oxidative folding of LA4 and LA5, the modules critical for ApoE binding, isolated and in the LA45 tandem. Compared to LA5, LA4 folding is slow and inefficient, resembling that of LA5 disease-linked mutants. Without Ca++, it leads to a mixture of many two-disulfide scrambled species and, with Ca++, to the native form plus two three-disulfide intermediates. The folding of the LA45 tandem seems to recapitulate that of the individual repeats. Importantly, although the folding of the LA45 tandem takes place through formation of scrambled isomers, no interdomain disulfides are detected, i.e. the two adjacent modules fold independently without the assistance of interdomain covalent interactions. Reduction of incredibly large disulfide combinatorial spaces, such as that in the LDLR, by intradomain confinement of disulfide bond formation might be also essential for the efficient folding of other homologous disulfide-rich receptors. PMID:26168158

  9. The lectin-like oxidized LDL receptor-1: a new potential molecular target in colorectal cancer

    PubMed Central

    Murdocca, Michela; Mango, Ruggiero; Pucci, Sabina; Biocca, Silvia; Testa, Barbara; Capuano, Rosamaria; Paolesse, Roberto; Sanchez, Massimo; Orlandi, Augusto; di Natale, Corrado; Novelli, Giuseppe; Sangiuolo, Federica

    2016-01-01

    The identification of new biomarkers and targets for tailored therapy in human colorectal cancer (CRC) onset and progression is an interesting challenge. CRC tissue produces an excess of ox-LDL, suggesting a close correlation between lipid dysfunction and malignant transformation. Lectin-like oxidized LDL receptor-1 (LOX-1) is involved in several mechanisms closely linked to tumorigenesis. Here we report a tumor specific LOX-1 overexpression in human colon cancers: LOX-1 results strongly increased in the 72% of carcinomas (P<0.001), and strongly overexpressed in 90% of highly aggressive and metastatic tumours (P<0.001), as compared to normal mucosa. Moreover LOX-1 results modulated since the early stage of the disease (adenomas vs normal mucosa; P<0.001) suggesting an involvement in tumor insurgence and progression. The in vitro knockdown of LOX-1 in DLD-1 and HCT-8 colon cancer cells by siRNA and anti-LOX-1 antibody triggers to an impaired proliferation rate and affects the maintenance of cell growth and tumorigenicity. The wound-healing assay reveals an evident impairment in closing the scratch. Lastly knockdown of LOX-1 delineates a specific pattern of volatile compounds characterized by the presence of a butyrate derivative, suggesting a potential role of LOX-1 in tumor-specific epigenetic regulation in neoplastic cells. The role of LOX-1 as a novel biomarker and molecular target represents a concrete opportunity to improve current therapeutic strategies for CRC. In addition, the innovative application of a technology focused to the identification of LOX-1 driven volatiles specific to colorectal cancer provides a promising diagnostic tool for CRC screening and for monitoring the response to therapy. PMID:26895376

  10. The lectin-like oxidized LDL receptor-1: a new potential molecular target in colorectal cancer.

    PubMed

    Murdocca, Michela; Mango, Ruggiero; Pucci, Sabina; Biocca, Silvia; Testa, Barbara; Capuano, Rosamaria; Paolesse, Roberto; Sanchez, Massimo; Orlandi, Augusto; di Natale, Corrado; Novelli, Giuseppe; Sangiuolo, Federica

    2016-03-22

    The identification of new biomarkers and targets for tailored therapy in human colorectal cancer (CRC) onset and progression is an interesting challenge. CRC tissue produces an excess of ox-LDL, suggesting a close correlation between lipid dysfunction and malignant transformation. Lectin-like oxidized LDL receptor-1 (LOX-1) is involved in several mechanisms closely linked to tumorigenesis. Here we report a tumor specific LOX-1 overexpression in human colon cancers: LOX-1 results strongly increased in the 72% of carcinomas (P<0.001), and strongly overexpressed in 90% of highly aggressive and metastatic tumours (P<0.001), as compared to normal mucosa. Moreover LOX-1 results modulated since the early stage of the disease (adenomas vs normal mucosa; P<0.001) suggesting an involvement in tumor insurgence and progression. The in vitro knockdown of LOX-1 in DLD-1 and HCT-8 colon cancer cells by siRNA and anti-LOX-1 antibody triggers to an impaired proliferation rate and affects the maintenance of cell growth and tumorigenicity. The wound-healing assay reveals an evident impairment in closing the scratch. Lastly knockdown of LOX-1 delineates a specific pattern of volatile compounds characterized by the presence of a butyrate derivative, suggesting a potential role of LOX-1 in tumor-specific epigenetic regulation in neoplastic cells. The role of LOX-1 as a novel biomarker and molecular target represents a concrete opportunity to improve current therapeutic strategies for CRC. In addition, the innovative application of a technology focused to the identification of LOX-1 driven volatiles specific to colorectal cancer provides a promising diagnostic tool for CRC screening and for monitoring the response to therapy. PMID:26895376

  11. Calpain Inhibition Attenuates Angiotensin II-induced Abdominal Aortic Aneurysms and Atherosclerosis in LDL Receptor Deficient Mice

    PubMed Central

    Subramanian, Venkateswaran; Uchida, Haruhito Adam; Ijaz, Talha; Moorleghen, Jessica J.; Howatt, Deborah A.; Balakrishnan, Anju

    2011-01-01

    Chronic infusion of angiotensin II (AngII) augments atherosclerosis and abdominal aortic aneurysm (AAAs) formation in hypercholesterolemic mice. AngII-induced AAAs are associated with medial macrophage accumulation and matrix metalloproteinase (MMP) activation. Inhibition of calpain, a calcium-activated neutral cysteine protease, by overexpression of its endogenous inhibitor, calpastatin, attenuates AngII-induced leukocyte infiltration, perivascular inflammation, and MMP activation in mice. The purpose of this study was to define whether pharmacological inhibition of calpain influences AngII-induced AAAs in hypercholesterolemic mice. Male LDL receptor −/− mice were fed a fat-enriched diet and administered with either vehicle or a calpain-specific inhibitor, BDA-410 (30 mg/kg/day) for 5 weeks. After 1 week of feeding, mice were infused with AngII (1,000 ng/kg/min) for 4 weeks. AngII-infusion profoundly increased aortic calpain protein and activity. BDA-410 administration had no effect on plasma cholesterol concentrations or AngII-increased systolic blood pressure. Calpain inhibition significantly attenuated AngII-induced AAA formation and atherosclerosis development. BDA-410 administration attenuated activation of MMP12, pro-inflammatory cytokines (IL-6, MCP-1) and macrophage infiltration into the aorta. BDA-410 administration significantly attenuated thioglycollate-elicited macrophage accumulation in the peritoneal cavity. We conclude that calpain inhibition using BDA-410 attenuated AngII-induced AAA formation and atherosclerosis development in LDL receptor −/− mice. PMID:21964156

  12. Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity.

    PubMed

    Pullinger, C R; Hennessy, L K; Chatterton, J E; Liu, W; Love, J A; Mendel, C M; Frost, P H; Malloy, M J; Schumaker, V N; Kane, J P

    1995-03-01

    Detection of new ligand-defective mutations of apolipoprotein B (apoB) will enable identification of sequences involved in binding to the LDL receptor. Genomic DNA from patients attending a lipid clinic was screened by single-strand conformation polymorphism analysis for novel mutations in the putative LDL receptor-binding domain of apoB-100. A 46-yr-old woman of Celtic and Native American ancestry with primary hypercholesterolemia (total cholesterol [TC] 343 mg/dl; LDL cholesterol [LDL-C] 241 mg/dl) and pronounced peripheral vascular disease was found to be heterozygous for a novel Arg3531-->Cys mutation, caused by a C-->T transition at nucleotide 10800. One unrelated 59-yr-old man of Italian ancestry was found with the same mutation after screening 1,560 individuals. He had coronary heart disease, a TC of 310 mg/dl, and an LDL-C of 212 mg/dl. A total of eight individuals were found with the defect in the families of the two patients. They had an age- and sex-adjusted TC of 240 +/- 14 mg/dl and LDL-C of 169 +/- 10 mg/dl. This compares with eight unaffected family members with age- and sex-adjusted TC of 185 +/- 12 mg/dl and LDL-C of 124 +/- 12 mg/dl. In a dual-label fibroblast binding assay, LDL from the eight subjects with the mutation had an affinity for the LDL receptor that was 63% that of control LDL. LDL from eight unaffected family members had an affinity of 91%. By way of comparison, LDL from six patients heterozygous for the Arg3500-->Gln mutation had an affinity of 36%. The percentage mass ratio of the defective Cys3531 LDL to normal LDL was 59:41, as determined using the mAb MB19 and dynamic laser light scattering. Thus, the defective LDL had accumulated in the plasma of these patients. Using this mass ratio, it was calculated that the defective Cys3531 LDL particles bound with 27% of normal affinity. Deduced haplotypes using 10 apoB gene markers showed the Arg3531-->Cys alleles to be different in the two kindreds and indicates that the mutations arose

  13. Hematopoietic G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor-knockout mice

    PubMed Central

    Otten, Jeroen J. T.; de Jager, Saskia C. A.; Kavelaars, Annemieke; Seijkens, Tom; Bot, Ilze; Wijnands, Erwin; Beckers, Linda; Westra, Marijke M.; Bot, Martine; Busch, Matthias; Bermudez, Beatriz; van Berkel, Theo J. C.; Heijnen, Cobi J.; Biessen, Erik A. L.

    2013-01-01

    Leukocyte chemotaxis is deemed instrumental in initiation and progression of atherosclerosis. It is mediated by G-protein-coupled receptors (e.g., CCR2 and CCR5), the activity of which is controlled by G-protein-coupled receptor kinases (GRKs). In this study, we analyzed the effect of hematopoietic deficiency of a potent regulator kinase of chemotaxis (GRK2) on atherogenesis. LDL receptor-deficient (LDLr−/−) mice with heterozygous hematopoietic GRK2 deficiency, generated by bone marrow transplantation (n=15), displayed a dramatic attenuation of plaque development, with 79% reduction in necrotic core and increased macrophage content. Circulating monocytes decreased and granulocytes increased in GRK2+/− chimeras, which could be attributed to diminished granulocyte colony-forming units in bone marrow. Collectively, these data pointed to myeloid cells as major mediators of the impaired atherogenic response in GRK2+/− chimeras. LDLr−/− mice with macrophage/granulocyte-specific GRK2 deficiency (LysM-Cre GRK2flox/flox; n=8) failed to mimic the aforementioned phenotype, acquitting these cells as major responsible subsets for GRK2 deficiency-associated atheroprotection. To conclude, even partial hematopoietic GRK2 deficiency prevents atherosclerotic lesion progression beyond the fatty streak stage, identifying hematopoietic GRK2 as a potential target for intervention in atherosclerosis.—Otten, J. J. T., de Jager, S. C. A., Kavelaars, A., Seijkens, T., Bot, I., Wijnands, E., Beckers, L., Westra, M. M., Bot, M., Busch, M., Bermudez, B., van Berkel, T. J. C., Heijnen, C. J., Biessen, E. A. L. Hematopoietic G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor-knockout mice. PMID:23047899

  14. Effect of squalene synthase inhibition on the expression of hepatic cholesterol biosynthetic enzymes, LDL receptor, and cholesterol 7 alpha hydroxylase.

    PubMed

    Ness, G C; Zhao, Z; Keller, R K

    1994-06-01

    Squalene synthase catalyzes the committed step in the biosynthesis of sterols. Treating rats with zaragozic acid A, a potent inhibitor of squalene synthase, caused marked increases in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, squalene synthase, and LDL receptor mRNA levels. The increase in HMG-CoA reductase mRNA fully accounted for the increases seen in enzyme protein and activity. Farnesyl pyrophosphate synthase mRNA and activity were only slightly increased by zaragozic acid A, while cholesterol 7 alpha hydroxylase mRNA levels were decreased substantially. When rats were pretreated with zaragozic acid A, there was no change in mRNA levels for the cholesterol biosynthetic enzymes or cholesterol 7 alpha hydroxylase upon subsequent treatment with mevalonolactone. Under these same conditions, the enzymatic activity of HMG-CoA reductase was also unaffected. Mevalonolactone treatment reduced the zaragozic acid A-mediated increase in hepatic LDL receptor mRNA levels. Feeding cholesterol eliminated the zaragozic acid A-induced increase in HMG-CoA reductase mRNA levels. These results suggest that inhibition of squalene synthase decreases the level of a squalene-derived regulatory product, resulting in altered amounts of several mRNAs and coordinate increases in HMG-CoA reductase mRNA, protein, and activity. The increase in HMG-CoA reductase gene expression was closely related to the degree of inhibition of cholesterol synthesis caused by zaragozic acid A. PMID:7911291

  15. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    SciTech Connect

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  16. Reduction in Dietary Omega-6 Polyunsaturated Fatty Acids: Eicosapentaenoic Acid plus Docosahexaenoic Acid Ratio Minimizes Atherosclerotic Lesion Formation and Inflammatory Response in the LDL Receptor Null Mouse

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dietary very long chain omega-3 polyunsaturated fatty acids (PUFA) have been associated with reduced CVD risk. LDL receptor null mice (LDLr-/-) were used to assess different dietary ratios of omega-6 PUFA to eicosapentaenoic acid plus docosahexaenoic acid (omega-6:EPA+DHA) on atherogenesis and infl...

  17. Genetic variation at the LDL receptor and HMG-CoA reductase gene loci, lipid levels, statin response, and cardiovascular disease incidence in PROSPER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our purpose was to evaluate associations of single nucleotide polymorphisms (SNPs) at the low density lipoprotein (LDL) receptor (LDLRC44857T, minor allele frequency (MAF) 0.26, and A44964G, MAF 0.25, both in the untranslated region) and HMG-CoA reductase (HMGCRi18 T >G, MAF 0.019) gene loci with ba...

  18. Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes*

    PubMed Central

    Tate, Jennifer J.; Cooper, Terrance G.

    2013-01-01

    Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc13 localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc13 localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal. PMID:23935103

  19. A lipidomics study reveals hepatic lipid signatures associating with deficiency of the LDL receptor in a rat model

    PubMed Central

    Quan, Chao; Hu, Chunxiu; Xie, Bingxian; Du, Yinan; Chen, Liang; Yang, Wei; Yang, Liu; Chen, Qiaoli; Shen, Bin; Hu, Bian; Zheng, Zhihong; Zhu, Haibo; Huang, Xingxu; Xu, Guowang; Chen, Shuai

    2016-01-01

    ABSTRACT The low-density lipoprotein receptor (LDLR) plays a critical role in the liver for the clearance of plasma low-density lipoprotein (LDL). Its deficiency causes hypercholesterolemia in many models. To facilitate the usage of rats as animal models for the discovery of cholesterol-lowering drugs, we took a genetic approach to delete the LDLR in rats aiming to increase plasma LDL cholesterol (LDL-C). An LDLR knockout rat was generated via zinc-finger nuclease technology, which harbors a 19-basepair deletion in the seventh exon of the ldlr gene. As expected, deletion of the LDLR elevated total cholesterol and total triglyceride in the plasma, and caused a tenfold increase of plasma LDL-C and a fourfold increase of plasma very low-density lipoprotein (VLDL-C). A lipidomics analysis revealed that deletion of the LDLR affected hepatic lipid metabolism, particularly lysophosphatidylcholines, free fatty acids and sphingolipids in the liver. Cholesterol ester (CE) 20:4 also displayed a significant increase in the LDLR knockout rats. Taken together, the LDLR knockout rat offers a new model of hypercholesterolemia, and the lipidomics analysis reveals hepatic lipid signatures associating with deficiency of the LDL receptor. PMID:27378433

  20. Hypolipidemic action of the SERM acolbifene is associated with decreased liver MTP and increased SR-BI and LDL receptors.

    PubMed

    Lemieux, Christian; Gélinas, Yves; Lalonde, Josée; Labrie, Fernand; Cianflone, Katherine; Deshaies, Yves

    2005-06-01

    This study aimed to identify the mechanisms of the hypolipidemic action of the selective estrogen receptor modulator (SERM) acolbifene (ACOL). Four weeks of treatment with ACOL reduced fasting and postprandial plasma triglycerides (TGs), an effect associated with lower VLDL-TG secretion rate (-25%), and decreased mRNA of microsomal triglyceride transfer protein (MTP; -29%). ACOL increased liver TG concentration (+100%) and amplified the feeding-induced increase in the master lipogenic regulators sterol-regulatory element binding protein-1a (SREBP-1a) and SREBP-1c. ACOL decreased total, HDL, and non-HDL cholesterol (CHOL) by 50%. SREBP-2 mRNA and HMG-CoA reductase activity were minimally affected by ACOL. However, in the fasted state, liver concentration of scavenger receptor class B type I (SR-BI) protein, but not mRNA, was 3-fold higher in ACOL-treated than in control animals and correlated with plasma HDL-CHOL levels (r = 0.80, P < 0.002). Liver LDL receptor (LDLR) protein, but not mRNA, was increased 2-fold by ACOL, independently of the nutritional status. This study demonstrates that ACOL possesses the unique ability among SERMs to reduce VLDL-TG secretion, likely by reducing MTP expression, and strongly suggests that the robust hypocholesterolemic action of ACOL is related to increased removal of CHOL from the circulation as a consequence of enhanced liver SR-BI and LDLR abundance. PMID:15741653

  1. LDL immune complexes stimulate LDL receptor expression in U937 histiocytes via extracellular signal-regulated kinase and AP-1.

    PubMed

    Fu, Yuchang; Huang, Yan; Bandyopadhyay, Sumita; Virella, Gabriel; Lopes-Virella, Maria F

    2003-07-01

    We have previously shown that LDL-containing immune complexes (LDL-ICs) induce up-regulation of LDL receptor (LDLR) expression in human macrophages. The present study further investigated the molecular mechanisms leading to LDLR up-regulation by LDL-ICs as well as the signaling pathways involved. Results showed that treatment of U937 histiocytes with LDL-ICs did not increase the precursors and the cleaved forms of sterol-regulatory element binding proteins (SREBPs) 1a and 2, suggesting that SREBPs may not be involved in LDLR up-regulation by LDL-ICs. Promoter deletion and mutation studies showed that the AP-1 binding sites were essential for LDL-IC-stimulated LDLR expression. Electrophoretic mobility shift assays further demonstrated that LDL-ICs stimulated transcription factor AP-1 activity. Studies assessing the signaling pathways involved in LDLR up-regulation by LDL-ICs showed that the up-regulation of LDLR was extracellular signal-regulated kinase (ERK) dependent. In conclusion, the present study shows that LDL-ICs up-regulate LDLR expression via the ERK signaling pathway and the AP-1 motif-dependent transcriptional activation. PMID:12730303

  2. LDL Receptor-related Protein 1 Regulates the Abundance of Diverse Cell-signaling Proteins in the Plasma Membrane Proteome

    PubMed Central

    Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F.; Gonias, Steven L.

    2010-01-01

    LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, which are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 co-immunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not co-immunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome. PMID:20919742

  3. A lipidomics study reveals hepatic lipid signatures associating with deficiency of the LDL receptor in a rat model.

    PubMed

    Wang, Hong Yu; Quan, Chao; Hu, Chunxiu; Xie, Bingxian; Du, Yinan; Chen, Liang; Yang, Wei; Yang, Liu; Chen, Qiaoli; Shen, Bin; Hu, Bian; Zheng, Zhihong; Zhu, Haibo; Huang, Xingxu; Xu, Guowang; Chen, Shuai

    2016-01-01

    The low-density lipoprotein receptor (LDLR) plays a critical role in the liver for the clearance of plasma low-density lipoprotein (LDL). Its deficiency causes hypercholesterolemia in many models. To facilitate the usage of rats as animal models for the discovery of cholesterol-lowering drugs, we took a genetic approach to delete the LDLR in rats aiming to increase plasma LDL cholesterol (LDL-C). An LDLR knockout rat was generated via zinc-finger nuclease technology, which harbors a 19-basepair deletion in the seventh exon of the ldlr gene. As expected, deletion of the LDLR elevated total cholesterol and total triglyceride in the plasma, and caused a tenfold increase of plasma LDL-C and a fourfold increase of plasma very low-density lipoprotein (VLDL-C). A lipidomics analysis revealed that deletion of the LDLR affected hepatic lipid metabolism, particularly lysophosphatidylcholines, free fatty acids and sphingolipids in the liver. Cholesterol ester (CE) 20:4 also displayed a significant increase in the LDLR knockout rats. Taken together, the LDLR knockout rat offers a new model of hypercholesterolemia, and the lipidomics analysis reveals hepatic lipid signatures associating with deficiency of the LDL receptor. PMID:27378433

  4. Site-specific influence of polyunsaturated fatty acids on atherosclerosis in immune incompetent LDL receptor deficient mice.

    PubMed

    Reardon, Catherine A; Blachowicz, Lydia; Gupta, Gaorav; Lukens, John; Nissenbaum, Michael; Getz, Godfrey S

    2006-08-01

    Polyunsaturated fatty acids (PUFA) are thought to influence plasma lipid levels, atherosclerosis, and the immune system. In this study, we fed male LDL receptor deficient (LDLR(-/-)) mice and immune incompetent LDLR(-/-) RAG2(-/-) mice diets containing predominantly saturated fats (milk fat) or PUFA (safflower oil) to determine if the response to diet was influenced by immune status. Relative to milk fat diet, plasma lipid and VLDL levels in both the LDLR(-/-) and LDLR(-/-) RAG2(-/-) mice fed safflower oil diet were lower, suggesting that the primary effect of PUFA on plasma lipids was not due to its inhibition of the immune system. Neither diet nor immune status influenced hepatic triglyceride production and post-heparin lipase activity, suggesting that the differences in triglyceride levels are due to differences in rates of catabolism of triglyceride-rich lipoproteins. While both diets promoted atherogenesis, both aortic root and innominate artery atherosclerosis in LDLR(-/-) mice was less in safflower oil fed animals. In contrast, a site-specific effect of PUFA was observed in the immune incompetent LDLR(-/-) RAG2(-/-). In these mice, aortic root atherosclerosis, but not innominate artery atherosclerosis, was less in PUFA fed animal. These results suggest that PUFA and the immune system may influence innominate artery atherosclerosis by some overlapping mechanisms. PMID:16280127

  5. Technetium-99m labelled LDL as a tracer for quantitative LDL scintigraphy. II. In vivo validation, LDL receptor-dependent and unspecific hepatic uptake and scintigraphic results.

    PubMed

    Leitha, T; Staudenherz, A; Gmeiner, B; Hermann, M; Hüttinger, M; Dudczak, R

    1993-08-01

    The purpose of this study was to determine whether the hepatic uptake of dialysed technetium-99m labelled low-density lipoprotein (99mTc-LDL) reflects the hepatic LDL receptor activity and to what extent the non-LDL receptor-dependent 99mTc-LDL uptake by non-parenchymal cells relates to the diagnostic utility of quantitative 99mTc-LDL scintigraphy of the liver. New Zealand White rabbits and Watanabe Heritable Hyperlipidaemic rabbits, which were sacrificed 24 h after simultaneous injection of 99mTc-LDL and iodine-125 labelled LDL, were clearly discriminated by their hepatic 99mTc-LDL uptake according to their genetically different hepatic LDL receptor activity. Yet the hepatic 99mTc-LDL uptake exceeded the 125I-LDL uptake in all animals. The different hepatic uptake of the tracers was elucidated in the isolated perfused rat liver and was due to rapid intracellular degradation and the release of low molecular catabolites of 125I-LDL. In contrast, 99mTc activity was trapped in the liver. Analysis of biliary 99mTc activity provided evidence for the excretion of 99mTc-labelled apolipoprotein B. The amount of biliary excreted protein-bound 99mTc was linked to total hepatic 99mTc-LDL uptake and presumably reflected LDL receptor-mediated apolipoprotein excretion. Collagenase liver perfusion in Sprague-Dawley rats 90 min following simultaneous injection of 99mTc- and 125I-LDL and subsequent cell separation by gradient centrifugation revealed that 99mTc-LDL and 125I-LDL had a comparably low uptake into non-parenchymal cells; thus its contribution can be neglected for scintigraphic purposes. Planar scintigraphy was performed in New Zealand White and Watanabe Heritable Hyperlipidaemic rabbits.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8404953

  6. Lectin-like ox-LDL receptor-1 (LOX-1)-Toll-like receptor 4 (TLR4) interaction and autophagy in CATH.a differentiated cells exposed to angiotensin II.

    PubMed

    Ding, Zufeng; Liu, Shijie; Wang, Xianwei; Khaidakov, Magomed; Dai, Yao; Deng, Xiaoyan; Fan, Yubo; Xiang, David; Mehta, Jawahar L

    2015-04-01

    Toll-like receptors (TLRs) play an essential role in innate immune response. Expression of TLRs has also been linked to autophagy. As the main receptor for oxidized low-density lipoprotein (ox-LDL) on the cell surface, lectin-like ox-LDL receptor-1 (LOX-1) is upregulated by proinflammatory cytokines and has been linked to the development of autophagy. However, the relationship between LOX-1, autophagy, and TLR4 in neurons has not been defined. Here, we show that Angiotensin II (Ang II) treatment of CATH.a differentiated neuronal cells resulted in the expression of TLR4 (and associated signals MyD88 and Toll/interleukin-1 receptor domain-containing adapter-inducing interferon (TRIF)), LOX-1 autophagy. LOX-1 knockdown (transfection with specific small interfering RNA (siRNA)) resulted in reduced expression of TLR4 (and associated signals MyD88 and TRIF) and P-P38 mitogen-activated protein kinase (MAPK) and autophagy. TLR4 knockdown with siRNA resulted in reduced LOX-1 expression and autophagy, indicating a positive feedback between LOX-1 and TLR4. Knockdown of TRIF as well as MyD88 or inhibition of P38 MAPK also inhibited the expression of LOX-1 and TLR4 and autophagy. Importantly, pretreatment with 3-methyladenine (autophagy inhibitor) enhanced while rapamycin (autophagy inducer) decreased the expression of LOX-1, TLR4, and P-P38 MAPK. These studies suggest the presence of a bidirectional link between LOX-1and TLR4 in cultured CATH.a differentiated cells exposed to Ang II with an important role for autophagy in this link. PMID:24902807

  7. Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages.

    PubMed

    Hossain, Ekhtear; Ota, Akinobu; Karnan, Sivasundaram; Takahashi, Miyuki; Mannan, Shahnewaj B; Konishi, Hiroyuki; Hosokawa, Yoshitaka

    2015-02-01

    There is a growing body of evidence supporting an intimate association of immune activation with the pathogenesis of cardiovascular diseases, including atherosclerosis. Uptake of oxidized low-density lipoprotein (oxLDL) through scavenging receptors promotes the formation of mature lipid-laden macrophages, which subsequently leads to exacerbation of regional inflammation and atherosclerotic plaque formation. In this study, we first examined changes in the mRNA level of the lectin-like oxLDL receptor-1 (LOX-1) in the mouse macrophage cell line RAW264.7 and the human PMA-induced macrophage cell line THP-1 after LPS stimulation. LPS significantly up-regulated LOX-1 mRNA in RAW264.7 cells; LOX-1 cell-surface protein expression was also increased. Flow cytometry and fluorescence microscopy analyses showed that cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with LPS stimulation. The augmented uptake of Dil-oxLDL was almost completely abrogated by treatment with an anti-LOX-1 antibody. Of note, knockdown of Erk1/2 resulted in a significant reduction of LPS-induced LOX-1 up-regulation. Treatment with U0126, a specific inhibitor of MEK, significantly suppressed LPS-induced expression of LOX-1 at both the mRNA and protein levels. Furthermore, LOX-1 promoter activity was significantly augmented by LPS stimulation; this augmentation was prevented by U0126 treatment. Similar results were also observed in human PMA-induced THP-1 macrophages. Taken together, our results indicate that LPS up-regulates LOX-1, at least in part through activation of the Erk1/2 signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of TLR4-mediated aberrant LOX-1 signaling in the pathogenesis of atherosclerosis. PMID:25348362

  8. Long-chain polyunsaturated fatty acids upregulate LDL receptor protein expression in fibroblasts and HepG2 cells.

    PubMed

    Yu-Poth, Shaomei; Yin, Dezhong; Kris-Etherton, Penny M; Zhao, Guixiang; Etherton, Terry D

    2005-11-01

    The objective of this study was to investigate the effect of individual PUFAs on LDL receptor (LDLr) expression in human fibroblasts and HepG2 cells, and to evaluate whether acyl CoA:cholesterol acyltransferase (ACAT) and sterol regulatory element-binding protein 1 (SREBP-1) were involved in the regulation of LDLr expression by fatty acids. When fibroblasts and HepG2 cells were cultured with serum-free defined medium for 48 h, there was a 3- to 5-fold (P < 0.05) increase in LDLr protein and mRNA levels. Incubation of fibroblasts and HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH-cholesterol, 5 mg/L) for 24 h decreased LDLr protein and mRNA levels by 50-90% (P < 0.05). Arachidonic acid [AA, 20:4(n-6)], EPA [20:5(n-3)], and DHA [22:6(n-3)] antagonized the depression of LDLr gene expression by 25OH-cholesterol and increased LDLr protein abundance 1- to 3-fold (P < 0.05), but had no significant effects on LDLr mRNA levels. Oleic (18:1), linoleic (18:2), and alpha-linolenic acids [18:3(n-3)] did not significantly affect LDLr expression. ACAT inhibitor (58-035, 1 mg/L) attenuated the regulatory effect of AA on LDLr protein abundance by approximately 40% (P < 0.05), but did not modify the regulatory effects of other unsaturated fatty acids in HepG2 cells. The present results suggest that AA, EPA, and DHA increase LDLr protein levels, and that ACAT plays a role in modulating the effects of AA on LDLr protein levels. Furthermore, the effects of the fatty acids appeared to be independent of any change in SREBP-1 protein. PMID:16251608

  9. Down-regulation of PERK enhances resistance to ionizing radiation

    SciTech Connect

    Oommen, Deepu Prise, Kevin M.

    2013-11-08

    Highlights: •PERK enhances the sensitivity of cancer cells to ionizing radiation. •Down-regulation of PERK results in enhanced DNA repair. •Ionizing radiation-induced apoptosis is inhibited in PERK-down regulated cancer cells. -- Abstract: Although, ionizing radiation (IR) has been implicated to cause stress in endoplasmic reticulum (ER), how ER stress signaling and major ER stress sensors modulate cellular response to IR is unclear. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein which initiates unfolded protein response (UPR) or ER stress signaling when ER homeostasis is disturbed. Here, we report that down-regulation of PERK resulted in increased clonogenic survival, enhanced DNA repair and reduced apoptosis in irradiated cancer cells. Our study demonstrated that PERK has a role in sensitizing cancer cells to IR.

  10. Effects of D-4F on Vasodilation and Vessel Wall Thickness in Hypercholesterolemic LDL Receptor Null and LDL receptor/ApoA-I Double Knockout Mice on Western Diet

    PubMed Central

    Ou, Jingsong; Wang, Jingli; Xu, Hao; Ou, Zhijun; Sorci-Thomas, Mary G.; Jones, Deron W.; Signorino, Paul; Densmore, John C.; Kaul, Sushma; Oldham, Keith T.; Pritchard, Kirkwood A.

    2005-01-01

    Previously we showed L-4F, a novel apolipoprotein A-I (apoA-I) mimetic, improved vasodilation in two dissimilar models of vascular disease; hypercholesterolemic low-density lipoprotein (LDL) receptor null (Ldlr −/−) mice and transgenic sickle cell disease mice. Here we determine the mechanisms by which D-4F improves vasodilation and arterial wall thickness in hypercholesterolemic Ldlr −/− mice and Ldlr −/−/apoA-I null (apoA-I −/−), double knockout mice. Ldlr −/− and Ldlr −/−/apoA-I −/− mice were fed western diet (WD) ± D-4F. Oral D-4F restored endothelium- and eNOS-dependent vasodilation in direct relationship to duration of treatments and reduced wall thickness in as little as 2 weeks in vessels with pre-existing disease in Ldlr −/− mice. D-4F had no effect on total or HDL cholesterol concentrations but reduced proinflammatory HDL levels. D-4F had no effect on plasma myeloperoxidase (MPO) concentrations but reduced MPO association with apoA-I as well as 3-nitrotyrosine in apoA-I. D-4F increased endothelium- and eNOS-dependent vasodilation in Ldlr −/−/apoA-I −/− mice but did not reduce wall thickness as it had in Ldlr −/− mice. Vascular endothelial cells were treated with 22-hydroxycholesterol (22-OHC) ± L-4F. 22-OHC decreased nitric oxide (•NO) and increased superoxide anion (O2 •−) production and increased ABCA-1 and collagen expression. L-4F restored •NO and O2 •− balance, had little effect on ABCA-1 expression but reduced collagen expression. These data demonstrate that although D-4F restores vascular endothelial cell and eNOS function to increase vasodilation, HDL containing apoA-I, or at least some critical concentration of the anti-atherogenic lipoprotein, is required for D-4F to decrease vessel wall thickness. PMID:16224061

  11. Biotic Stress Globally Down-Regulates Photosynthesis Genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Upon herbivore and pathogen attacks, plants switch from processes supporting growth and reproduction to defense by inducing a set of defense genes and down-regulating most of the nuclear encoded photosynthetic genes. To determine if this transcriptional response is universal we used transcriptome da...

  12. Delayed Correlation of mRNA and Protein Expression in Rapamycin-treated Cells and a Role for Ggc1 in Cellular Sensitivity to Rapamycin*

    PubMed Central

    Fournier, Marjorie L.; Paulson, Ariel; Pavelka, Norman; Mosley, Amber L.; Gaudenz, Karin; Bradford, William D.; Glynn, Earl; Li, Hua; Sardiu, Mihaela E.; Fleharty, Brian; Seidel, Christopher; Florens, Laurence; Washburn, Michael P.

    2010-01-01

    To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Δggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression

  13. Disruption of LDL receptor gene in transgenic SREBP-1a mice unmasks hyperlipidemia resulting from production of lipid-rich VLDL.

    PubMed

    Horton, J D; Shimano, H; Hamilton, R L; Brown, M S; Goldstein, J L

    1999-04-01

    Transgenic mice that overexpress the nuclear form of sterol regulatory element binding protein-1a (SREBP-1a) in liver (TgBP-1a mice) were shown previously to overproduce cholesterol and fatty acids and to accumulate massive amounts of cholesterol and triglycerides in hepatocytes. Despite the hepatic overproduction of lipids, the plasma levels of cholesterol ( approximately 45 mg/dl) and triglycerides ( approximately 55 mg/dl) were not elevated, perhaps owing to degradation of lipid-enriched particles by low-density lipoprotein (LDL) receptors. To test this hypothesis, in the current studies we bred TgBP-1a mice with LDL receptor knockout mice. As reported previously, LDLR-/- mice manifested a moderate elevation in plasma cholesterol ( approximately 215 mg/dl) and triglycerides ( approximately 155 mg/dl). In contrast, the doubly mutant TgBP-1a;LDLR-/- mice exhibited marked increases in plasma cholesterol ( approximately 1,050 mg/dl) and triglycerides ( approximately 900 mg/dl). These lipids were contained predominantly within large very-low-density lipoprotein (VLDL) particles that were relatively enriched in cholesterol and apolipoprotein E. Freshly isolated hepatocytes from TgBP-1a and TgBP-1a;LDLR-/- mice overproduced cholesterol and fatty acids and secreted increased amounts of these lipids into the medium. Electron micrographs of livers from TgBP-1a mice showed large amounts of enlarged lipoproteins within the secretory pathway. We conclude that the TgBP-1a mice produce large lipid-rich lipoproteins, but these particles do not accumulate in plasma because they are degraded through the action of LDL receptors. PMID:10194480

  14. p65 down-regulates DEPTOR expression in response to LPS stimulation in hepatocytes.

    PubMed

    Yu, Xiaoling; Jin, Dan; Yu, An; Sun, Jun; Chen, Xiaodong; Yang, Zaiqing

    2016-09-01

    DEPTOR, a novel endogenous inhibitor of mTOR, plays an important role in regulating the inflammatory response in vascular endothelial cells (ECs) and in mouse skeletal muscle. However, the regulatory mechanism of DEPTOR transcription and its effects on liver inflammation are unknown presently. Here we reported the role of DEPTOR in regulating inflammatory response in mouse liver-derived Hepa1-6 cells and in a mouse model with LPS-induced hepatic inflammation. The results revealed that DEPTOR over-expression in Hepa1-6 liver cells increased the mRNA levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1). Contrasting results were observed in Hepa1-6 cells with DEPTOR interference. Treatment Hepa1-6 cells with rapamycin, a specific inhibitor of mTORC1, increased MCP-1 mRNA, but have no significant effect on IL-6 mRNA. DEPTOR expression was down-regulated in Hepa1-6 cells with the treatment of inflammatory stimuli LPS or the over-expression of p65/NF-κB, a key inflammatory transcription factor. NF-κB antagonist (PDTC) and inhibitor (IκBα) blocked the effect of LPS on DEPTOR expression. The study in vivo showed that DEPTOR mRNA and protein were significantly reduced in a mouse model with LPS-induced hepatic inflammation, which was accompanied by a concurrent activation of the mTOR signaling pathway. Further, the transcriptional regulation of DEPTOR was explored, which revealed that DEPTOR promoter activity was significantly down-regulated by NF-κB. The progressive deletions and mutations demonstrated that the NF-κB binding motif situated at -145/-127 region is an essential component required for the DEPTOR promoter activity. Chromatin immunoprecipitation (ChIP) assays determined that p65 can directly interact with the DEPTOR promoter DNA. Those results indicate DEPTOR regulates liver inflammation at least partially via mTORC1 pathway, and is down-regulated by LPS through p65. PMID:27179948

  15. Down-regulation of SIRT3 promotes ovarian carcinoma metastasis.

    PubMed

    Dong, Xue-Cai; Jing, Li-Min; Wang, Wen-Xiang; Gao, Yu-Xia

    2016-07-01

    Distant metastasis and local recurrence are still the major causes for failure of treatment in patients with ovarian carcinoma (OC), making it urgent to further elicit the molecular mechanisms of OC metastasis. Sirtuin-3 (SIRT3), a member of the NAD(+)-dependent Class III histone deacetylases, may function as different role depending on the cell-type and tumor-type. However, the function and mechanism of SIRT3 has been not explored in OC metastasis. Here, we found that SIRT3 was significantly down-regulated in the metastatic tissues and highly metastatic cell line of ovarian cancer. In addition, knockdown of SIRT3 enhanced the migration and invasion in vitro and the liver metastasis in vivo of ovarian cancer cell. By contrast, ectopic overexpression of SIRT3 dramatically suppressed cancer cell metastatic capability. Mechanistically, SIRT3 inhibits epithelial-to-mesenchymal transition (EMT) by down-regulating Twist in ovarian cancer cells. Furthermore, an interaction between SIRT3 and Twist was detected. In conclusion, our results demonstrated that SIRT3 plays a crucial suppressive role in the metastasis of ovarian cancer by down-regulating Twist, and that this novel SIRT3/Twist axis may be valuable to develop new strategies for treating OC patients with metastasis. PMID:27216459

  16. The role of a conserved acidic residue in calcium-dependent protein folding for a low density lipoprotein (LDL)-A module: implications in structure and function for the LDL receptor superfamily.

    PubMed

    Guo, Ying; Yu, Xuemei; Rihani, Kayla; Wang, Qing-Yin; Rong, Lijun

    2004-04-16

    One common feature of the more than 1,000 complement-type repeats (or low density lipoprotein (LDL)-A modules) found in LDL receptor and the other members of the LDL receptor superfamily is a cluster of five highly conserved acidic residues in the C-terminal region, DXXXDXXDXXDE. However, the role of the third conserved aspartate of these LDL-A modules in protein folding and ligand recognition has not been elucidated. In this report, using a model LDL-A module and several experimental approaches, we demonstrate that this acidic residue, like the other four conserved acidic residues, is involved in calcium-dependent protein folding. These results suggest an alternative calcium coordination conformation for the LDL-A modules. The proposed model provides a plausible explanation for the conservation of this acidic residue among the LDL-A modules. Furthermore, the model can explain why mutations of this residue in human LDL receptor cause familial hypercholesterolemia. PMID:14749324

  17. Metformin potentiates rapamycin and cisplatin in gastric cancer in mice

    PubMed Central

    Gao, Yunshu; Jiao, Xiaodong; Huang, Suyun; Wang, Jiejun; Li, Zhaosheng; Xie, Keping

    2015-01-01

    Here we showed that pAMPKα and PTEN were down-regulated and p-mTOR, p-S6, p-4EBP1, MMP7, and DCN1 were up-regulated in human gastric cancer tissue samples as compared to that in the noncancerous tissues. Metformin inhibited tumor growth in mice. Also it enhanced cisplatin- or rapamycin-induced reduction of tumor growth as compared with treatment of either drug alone. In addition to activation of AMPK and suppression of the mTOR pathway, a series of increased and decreased genes expression were induced by metformin, including PTEN, MMP7, and FN1. We suggest that metformin could potentially be used for the treatment of gastric cancer especially in combination with cisplatin or rapamycin. PMID:25909163

  18. The closed conformation of the LDL receptor is destabilized by the low Ca(++) concentration but favored by the high Mg(++) concentration in the endosome.

    PubMed

    Martínez-Oliván, Juan; Arias-Moreno, Xabier; Hurtado-Guerrero, Ramón; Carrodeguas, José Alberto; Miguel-Romero, Laura; Marina, Alberto; Bruscolini, Pierpaolo; Sancho, Javier

    2015-11-30

    The LDL receptor (LDLR) internalizes LDL and VLDL particles. In the endosomes, it adopts a closed conformation important for recycling, by interaction of two modules of the ligand binding domain (LR4-5) and a β-propeller motif. Here, we investigate by SPR the interactions between those two modules and the β-propeller. Our results indicate that the two modules cooperate to bind the β-propeller. The binding is favored by low pH and by high [Ca(++)]. Our data show that Mg(++), at high concentration in the endosome, favors the formation of the closed conformation by replacing the structuring effect of Ca(++) in LR5. We propose a sequential model of LDL release where formation of the close conformation follows LDL release. PMID:26526611

  19. Red wine polyphenolics increase LDL receptor expression and activity and suppress the secretion of ApoB100 from human HepG2 cells.

    PubMed

    Pal, Sebely; Ho, Nerissa; Santos, Carlos; Dubois, Paul; Mamo, John; Croft, Kevin; Allister, Emma

    2003-03-01

    Epidemiologic studies suggest that the consumption of red wine may lower the risk of cardiovascular disease. The cardioprotective effect of red wine has been attributed to the polyphenols present in red wine, particularly resveratrol (a stilbene, with estrogen-like activity), and the flavonoids, catechin, epicatechin, quercetin and phenolic acids such as gallic acid. At present, very little is known about the mechanisms by which red wine phenolic compounds benefit the cardiovascular system. Therefore, the aim of this study was to elucidate whether red wine polyphenolics reduce lipoprotein production and clearance by the liver. Cultured HepG2 cells were incubated in the presence of dealcoholized red wine, alcohol-containing red wine and atorvastatin for 24 h. The apolipoprotien B100 (apoB100) protein (marker of hepatic lipoproteins) was quantified on Western blots with an anti-apoB100 antibody and the enhanced chemiluminescence detection system. Apolipoprotein B100 levels in the cells and that secreted into the media were significantly reduced by 50% in liver cells incubated with alcohol-stripped red wine compared with control cells. This effect of dealcoholized red wine on apoB100 production in HepG2 cells was similar to the effect of atorvastatin. Apo B100 production was significantly attenuated by 30% in cells incubated with alcoholized red wine, suggesting that the alcohol was masking the effect of red wine polyphenolics. Apo B100 production was significantly attenuated by 45% with the polyphenolic compounds resveratrol and quercertin. In addition, dealcoholized and alcoholized red wine and atorvastatin significantly increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA and LDL receptor binding activity relative to controls. Dealcoholized red wine also increased LDL receptor gene expression. Collectively, this study suggests that red wine polyphenolics regulate major pathways involved in lipoprotein metabolism. PMID:12612140

  20. Down-regulation of laminin-5 in breast carcinoma cells.

    PubMed Central

    Martin, K. J.; Kwan, C. P.; Nagasaki, K.; Zhang, X.; O'Hare, M. J.; Kaelin, C. M.; Burgeson, R. E.; Pardee, A. B.; Sager, R.

    1998-01-01

    BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of

  1. Impact of Omega-6 Polyunsaturated Fatty Acid:Eicosapentaenoic acid (EPA)+Docosahexaenoic Acid (DHA) Ratios in LDL Receptor Knockout (LDLr-/-) Mice on Atherosclerotic Lesion Formation and Elicited Peritoneal Macrophage Inflamm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Very long chain omega-3 fatty acids have been associated with decreased risk of CVD. LDL receptor knockout mice were used to assess the effect of different omega-6:EPA+DHA ratios on atherosclerotic lesion formation and elicited peritoneal macrophage inflammatory response. Methods and R...

  2. Rapamycin Inhibits Cardiac Hypertrophy by Promoting Autophagy via the MEK/ERK/Beclin-1 Pathway

    PubMed Central

    Gu, Jun; Hu, Wei; Song, Zhi-Ping; Chen, Yue-Guang; Zhang, Da-Dong; Wang, Chang-Qian

    2016-01-01

    Rapamycin, also known as sirolimus, is an antifungal agent and immunosuppressant drug used to prevent organ rejection in transplantation. However, little is known about the role of rapamycin in cardiac hypertrophy and the signaling pathways involved. Here, the effect of rapamycin was examined using phenylephrine (PE) induced cardiomyocyte hypertrophy in vitro and in a rat model of aortic banding (AB) - induced hypertrophy in vivo. Inhibition of MEK/ERK signaling reversed the effect of rapamycin on the up-regulation of LC3-II, Beclin-1 and Noxa, and the down-regulation of Mcl-1 and p62. Silencing of Noxa or Beclin-1 suppressed rapamycin-induced autophagy, and co-immunoprecipitation experiments showed that Noxa abolishes the inhibitory effect of Mcl-1 on Beclin-1, promoting autophagy. In vivo experiments showed that rapamycin decreased AB-induced cardiac hypertrophy in a MEK/ERK dependent manner. Taken together, our results indicate that rapamycin attenuates cardiac hypertrophy by promoting autophagy through a mechanism involving the modulation of Noxa and Beclin-1 expression by the MEK/ERK signaling pathway. PMID:27047390

  3. The Enigma of Rapamycin Dosage.

    PubMed

    Mukhopadhyay, Suman; Frias, Maria A; Chatterjee, Amrita; Yellen, Paige; Foster, David A

    2016-03-01

    The mTOR pathway is a critical regulator of cell growth, proliferation, metabolism, and survival. Dysregulation of mTOR signaling has been observed in most cancers and, thus, the mTOR pathway has been extensively studied for therapeutic intervention. Rapamycin is a natural product that inhibits mTOR with high specificity. However, its efficacy varies by dose in several contexts. First, different doses of rapamycin are needed to suppress mTOR in different cell lines; second, different doses of rapamycin are needed to suppress the phosphorylation of different mTOR substrates; and third, there is a differential sensitivity of the two mTOR complexes mTORC1 and mTORC2 to rapamycin. Intriguingly, the enigmatic properties of rapamycin dosage can be explained in large part by the competition between rapamycin and phosphatidic acid (PA) for mTOR. Rapamycin and PA have opposite effects on mTOR whereby rapamycin destabilizes and PA stabilizes both mTOR complexes. In this review, we discuss the properties of rapamycin dosage in the context of anticancer therapeutics. Mol Cancer Ther; 15(3); 347-53. ©2016 AACR. PMID:26916116

  4. Tryptanthrin ameliorates atopic dermatitis through down-regulation of TSLP.

    PubMed

    Han, Na-Ra; Moon, Phil-Dong; Kim, Hyung-Min; Jeong, Hyun-Ja

    2014-01-15

    Atopic dermatitis (AD) is a common skin disease that greatly worsens quality of life. Thymic stromal lymphopoietin (TSLP) plays a decisive role in the development of AD. The purpose of this study is to examine whether tryptanthrin (TR) would suppress AD through the regulation of TSLP. We analyzed the effect of TR on the level of TSLP from phorbol myristate acetate/calcium ionophore A23187-activated human mast cell line, HMC-1 cells, in 2,4-dinitrofluorobenzene-induced AD-like skin lesions of NC/Nga mice, and in anti-CD3/anti-CD28-stimulated splenocytes. TR significantly suppressed the level of intracellular calcium and the production and mRNA expression of TSLP through the blockade of receptor-interacting protein 2/caspase-1/nuclear factor-κB pathway in the activated HMC-1 cells. TR also significantly suppressed the levels of histidine decarboxylase and IL-1β. Furthermore, TR ameliorated clinical symptoms in the AD model. TR significantly reduced the levels of TSLP, IL-4, IFN-γ, IL-6, TNF-α, thymus and activation-regulated chemokine, and caspase-1 in AD skin lesions. Also, TR significantly reduced the serum levels of histamine and IL-4 in the AD model. Finally, TR significantly inhibited the production of IL-4, IFN-γ, and TNF-α from the stimulated splenocytes. Taken together, TR exhibits the potential to be a therapeutic agent for AD through down-regulation of TSLP. PMID:24295961

  5. Simulated weightlessness down-regulated antioxidant defense system in rats

    NASA Astrophysics Data System (ADS)

    Li, Qi; Qu, Lina; Li, Yingxian; Bi, Lei; Huang, Zengming; Wang, Bo

    A variety of experiments suggest that space flight is associated with an increase in oxidative stress in organism The aim of the present study is to investigate whether or not simulated weightlessness by tail-suspension can affect the antioxidant defense system in rats and the possible protection effects of Chinese medicine named Liu Wei Di Huang Wan LWDHW Blood plasma of rats was taken after 21 days - tail-suspension for the assessment of the change of antioxidant defense system The total antioxidant capacity T-AOC was significantly decreased and the content of malondialdehyde MDA was increased after simulated weightlessness Activities of antioxidant enzymes such as superoxide dismutase and catalase were lower than those in the controlled groups However the activity of glutathione peroxidase was increased in comparison with the controlled groups Adequate dosage of LWDHW could inhibit the production of MAD and improve T-AOC in tail-suspension rats These results suggested that tail-suspension might break the oxidative antioxidative balance and down-regulate antioxidant defense system and Chinese medicine LWDHW was shown to protect rats from oxidative damage during simulated weightlessness Key words Simulated weightlessness Tail-suspension Antioxidant defense system Rats

  6. Chronic rapamycin treatment on the nutrient utilization and metabolism of juvenile turbot (Psetta maxima)

    PubMed Central

    Wang, Qingchao; He, Gen; Mai, Kangsen; Xu, Wei; Zhou, Huihui; Wang, Xuan; Mei, Lin

    2016-01-01

    High dietary protein inclusion is necessary in fish feeds and also represents a major cost in the aquaculture industry, which demands improved dietary conversion into body proteins in fish. In mammals, the target of rapamycin (TOR) is a key nutritionally responsive molecule governing postprandial anabolism. However, its physiological significance in teleosts has not been fully examined. In the present study, we examined the nutritional physiology of turbot after chronic rapamycin inhibition. Our results showed that a 6-week inhibition of TOR using dietary rapamycin inclusion (30 mg/kg diet) reduced growth performance and feed utilization. The rapamycin treatment inhibited TOR signaling and reduced expression of key enzymes in glycolysis, lipogenesis, cholesterol biosynthesis, while increasing the expression of enzymes involved in gluconeogenesis. Furthermore, rapamycin treatment increased intestinal goblet cell number in turbot, while the expressions of Notch and Hes1 were down regulated. It was possible that stimulated goblet cell differentiation by rapamycin was mediated through Notch-Hes1 pathway. Therefore, our results demonstrate the important role of TOR signaling in fish nutritional physiology. PMID:27305975

  7. Chronic rapamycin treatment on the nutrient utilization and metabolism of juvenile turbot (Psetta maxima).

    PubMed

    Wang, Qingchao; He, Gen; Mai, Kangsen; Xu, Wei; Zhou, Huihui; Wang, Xuan; Mei, Lin

    2016-01-01

    High dietary protein inclusion is necessary in fish feeds and also represents a major cost in the aquaculture industry, which demands improved dietary conversion into body proteins in fish. In mammals, the target of rapamycin (TOR) is a key nutritionally responsive molecule governing postprandial anabolism. However, its physiological significance in teleosts has not been fully examined. In the present study, we examined the nutritional physiology of turbot after chronic rapamycin inhibition. Our results showed that a 6-week inhibition of TOR using dietary rapamycin inclusion (30 mg/kg diet) reduced growth performance and feed utilization. The rapamycin treatment inhibited TOR signaling and reduced expression of key enzymes in glycolysis, lipogenesis, cholesterol biosynthesis, while increasing the expression of enzymes involved in gluconeogenesis. Furthermore, rapamycin treatment increased intestinal goblet cell number in turbot, while the expressions of Notch and Hes1 were down regulated. It was possible that stimulated goblet cell differentiation by rapamycin was mediated through Notch-Hes1 pathway. Therefore, our results demonstrate the important role of TOR signaling in fish nutritional physiology. PMID:27305975

  8. Enhanced hypocholesterolemic effects of interesterified oils are mediated by upregulating LDL receptor and cholesterol 7-α- hydroxylase gene expression in rats.

    PubMed

    Reena, Malongil B; Gowda, Lalitha R; Lokesh, Belur R

    2011-01-01

    The concentration of LDL cholesterol in plasma is strongly influenced by the amount and type of lipid in the diet. Our studies have shown that positional changes in the fatty acids in blended oil introduced using lipase-catalyzed interesterification differentially modulate circulating LDL levels in rats compared with those observed in rats given a physical blend of oils. To investigate the molecular basis of these differences, transcriptional profiling of genes involved in cholesterol homeostasis was studied after feeding rats with a semipurified diet containing 10% fat from native oils; coconut oil (CNO), rice bran oil (RBO), or sesame oil (SESO); blended (B); CNO+RBO(B) or CNO+SESO(B) and interesterified oil (I); CNO+RBO(I) or CNO+SESO(I) for 60 d. Hepatic LDL receptor (LDL-R) expression significantly increased in rats fed interesterified oils by 100-200% compared with rats fed blended oils and by 400-500% compared with rats fed CNO. Positional alteration in fatty acids of oils used in the diet induced changes in LDL-R expression, which was accompanied by parallel changes in cholesterol-7α-hydroxylase (CYP7A1) and SREBP-2 genes. This suggested that not only the fatty acid type but also its position in the TG of dietary lipids play an important role in maintaining plasma cholesterol levels by suitably modulating gene expression for LDL-R in rat liver. PMID:21106933

  9. Inducible Apoe Gene Repair in Hypomorphic ApoE Mice Deficient in the LDL Receptor Promotes Atheroma Stabilization with a Human-like Lipoprotein Profile

    PubMed Central

    Eberlé, Delphine; Luk, Fu Sang; Kim, Roy Y.; Olivas, Victor R.; Kumar, Nikit; Posada, Jessica M.; Li, Kang; Gaudreault, Nathalie; Rapp, Joseph H.; Raffai, Robert L.

    2013-01-01

    Objective To study atherosclerosis regression in mice following plasma lipid reduction to moderately elevated apolipoprotein B (apoB)-lipoprotein levels. Approach and Results Chow-fed hypomorphic Apoe mice deficient in LDL receptor expression (Apoeh/hLdlr−/−Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non-HDL:HDL-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6Chi monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extra-cellular matrix remodeling and cell migration were changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. Conclusions Restoring apoE expression in Apoeh/hLdlr−/−Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non-HDL:HDL-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6Chi monocytes levels and genetic reprogramming of lesional macrophages. PMID:23788760

  10. Effects of High Fat Feeding and Diabetes on Regression of Atherosclerosis Induced by Low-Density Lipoprotein Receptor Gene Therapy in LDL Receptor-Deficient Mice

    PubMed Central

    Willecke, Florian; Yuan, Chujun; Oka, Kazuhiro; Chan, Lawrence; Hu, Yunying; Barnhart, Shelley; Bornfeldt, Karin E.; Goldberg, Ira J.; Fisher, Edward A.

    2015-01-01

    We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr-/-) mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR). After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr-/- mice in different metabolic states. PMID:26046657

  11. Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor.

    PubMed

    Yamamoto, Koichi; Kakino, Akemi; Takeshita, Hikari; Hayashi, Norihiro; Li, Lei; Nakano, Atsushi; Hanasaki-Yamamoto, Hiroko; Fujita, Yoshiko; Imaizumi, Yuki; Toyama-Yokoyama, Serina; Nakama, Chikako; Kawai, Tatsuo; Takeda, Masao; Hongyo, Kazuhiro; Oguro, Ryosuke; Maekawa, Yoshihiro; Itoh, Norihisa; Takami, Yoichi; Onishi, Miyuki; Takeya, Yasushi; Sugimoto, Ken; Kamide, Kei; Nakagami, Hironori; Ohishi, Mitsuru; Kurtz, Theodore W; Sawamura, Tatsuya; Rakugi, Hiromi

    2015-08-01

    The angiotensin II type 1 receptor (AT1) is a 7-transmembrane domain GPCR that when activated by its ligand angiotensin II, generates signaling events promoting vascular dysfunction and the development of cardiovascular disease. Here, we show that the single-transmembrane oxidized LDL (oxLDL) receptor (LOX-1) resides in proximity to AT1 on cell-surface membranes and that binding of oxLDL to LOX-1 can allosterically activate AT1-dependent signaling events. oxLDL-induced signaling events in human vascular endothelial cells were abolished by knockdown of AT1 and inhibited by AT1 blockade (ARB). oxLDL increased cytosolic G protein by 350% in Chinese hamster ovary (CHO) cells with genetically induced expression of AT1 and LOX-1, whereas little increase was observed in CHO cells expressing only LOX-1. Immunoprecipitation and in situ proximity ligation assay (PLA) assays in CHO cells revealed the presence of cell-surface complexes involving LOX-1 and AT1. Chimeric analysis showed that oxLDL-induced AT1 signaling events are mediated via interactions between the intracellular domain of LOX-1 and AT1 that activate AT1. oxLDL-induced impairment of endothelium-dependent vascular relaxation of vascular ring from mouse thoracic aorta was abolished by ARB or genetic deletion of AT1. These findings reveal a novel pathway for AT1 activation and suggest a new mechanism whereby oxLDL may be promoting risk for cardiovascular disease. PMID:25877213

  12. Reduced VLDL clearance in Apoe(-/-)Npc1(-/-) mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels.

    PubMed

    Ishibashi, Minako; Masson, David; Westerterp, Marit; Wang, Nan; Sayers, Scott; Li, Rong; Welch, Carrie L; Tall, Alan R

    2010-09-01

    Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe(-/-)Npc1(-/-) mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe(-/-)Npc1(-/-) liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe(-/-)Npc1(-/-) liver was unexpected. However, several other LXR target genes also increased in Apoe(-/-)Npc1(-/-) liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe(-/-) mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe(-/-)Npc1(-/-) mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL. PMID:20562239

  13. Rapamycin interacts synergistically with idarubicin to induce T-leukemia cell apoptosis in vitro and in a mesenchymal stem cell simulated drug-resistant microenvironment via Akt/mammalian target of rapamycin and extracellular signal-related kinase signaling pathways.

    PubMed

    Wu, Kang-Ni; Zhao, Yan-Min; He, Ying; Wang, Bin-Sheng; Du, Kai-Li; Fu, Shan; Hu, Kai-Min; Zhang, Li-Fei; Liu, Li-Zhen; Hu, Yong-Xian; Wang, Ying-Jia; Huang, He

    2014-03-01

    T-cell acute lymphoblastic leukemias (T-ALLs) are clonal lymphoid malignancies with a poor prognosis, and still a lack of effective treatment. Here we examined the interactions between the mammalian target of rapamycin (mTOR) inhibitor rapamycin and idarubicin (IDA) in a series of human T-ALL cell lines Molt-4, Jurkat, CCRF-CEM and CEM/C1. Co-exposure of cells to rapamycin and IDA synergistically induced T-ALL cell growth inhibition and apoptosis mediated by caspase activation via the intrinsic mitochondrial pathway and extrinsic pathway. Combined treatment with rapamycin and IDA down-regulated Bcl-2 and Mcl-1, and inhibited the activation of phosphoinositide 3-kinase (PI3K)/mTOR and extracellular signal-related kinase (ERK). They also played synergistic pro-apoptotic roles in the drug-resistant microenvironment simulated by mesenchymal stem cells (MSCs) as a feeder layer. In addition, MSCs protected T-ALL cells from IDA cytotoxicity by up-regulating ERK phosphorylation, while rapamycin efficiently reversed this protective effect. Taken together, we confirm the synergistic antitumor effects of rapamycin and IDA, and provide an insight into the potential future clinical applications of combined rapamycin-IDA regimens for treating T-cell malignancies. PMID:23741975

  14. Myeloid Cell-Specific ABCA1 Deletion Has Minimal Impact on Atherogenesis in Atherogenic Diet-Fed LDL Receptor Knockout Mice

    PubMed Central

    Bi, Xin; Zhu, Xuewei; Gao, Chuan; Shewale, Swapnil; Cao, Qiang; Liu, Mingxia; Boudyguina, Elena; Gebre, Abraham K.; Wilson, Martha D.; Brown, Amanda L.; Parks, John S.

    2014-01-01

    Objective Transplantation studies suggest that bone marrow (BM) cell ABCA1 protects against atherosclerosis development. However, the in vivo impact of macrophage ABCA1 expression on atherogenesis is not fully understood because BM contains other leukocytes and hematopoietic stem and progenitor cells. Myeloid-specific ABCA1 knockout (MSKO) mice in the LDL receptor knockout (LDLrKO) C57BL/6 background were developed to address this question. Approach and Results Chow-fed MSKO/LDLrKO (DKO) vs. LDLrKO (SKO) mice had similar plasma lipid concentrations, but atherogenic diet (AD)-fed DKO mice had reduced plasma VLDL/LDL concentrations resulting from decreased hepatic VLDL triglyceride secretion. Resident peritoneal macrophages from AD-fed DKO vs. SKO mice had significantly higher cholesterol content, but similar proinflammatory gene expression. Atherosclerosis extent was similar between genotypes after 10–16 wks of AD, but increased modestly in DKO mice by 24 wks of AD. Lesional macrophage content was similar, likely due to higher monocyte flux through aortic root lesions in DKO vs. SKO mice. After transplantation of DKO or SKO BM into SKO mice and 16 wk of AD feeding, atherosclerosis extent was similar and plasma apoB lipoproteins was reduced in mice receiving DKO BM. When differences in plasma VLDL/LDL concentrations were minimized by maintaining mice on chow for 24 wks, DKO mice had modest, but significantly more, atherosclerosis compared to SKO mice. Conclusions Myeloid cell ABCA1 increases hepatic VLDL triglyceride secretion and plasma VLDL/LDL concentrations in AD-fed LDLrKO mice, offsetting its atheroprotective role in decreasing macrophage cholesterol content, resulting in minimal increase in atherosclerosis. PMID:24833800

  15. PCSK9 inhibition-mediated reduction in Lp(a) with evolocumab: an analysis of 10 clinical trials and the LDL receptor's role.

    PubMed

    Raal, Frederick J; Giugliano, Robert P; Sabatine, Marc S; Koren, Michael J; Blom, Dirk; Seidah, Nabil G; Honarpour, Narimon; Lira, Armando; Xue, Allen; Chiruvolu, Padmaja; Jackson, Simon; Di, Mei; Peach, Matthew; Somaratne, Ransi; Wasserman, Scott M; Scott, Rob; Stein, Evan A

    2016-06-01

    Lipoprotein (a) [Lp(a)] is independently associated with CVD risk. Evolocumab, a monoclonal antibody (mAb) to proprotein convertase subtilisin/kexin type 9 (PCSK9), decreases Lp(a). The potential mechanisms were assessed. A pooled analysis of Lp(a) and LDL cholesterol (LDL-C) in 3,278 patients from 10 clinical trials (eight phase 2/3; two extensions) was conducted. Within each parent study, biweekly and monthly doses of evolocumab statistically significantly reduced Lp(a) at week 12 versus control (P < 0.001 within each study); pooled median (quartile 1, quartile 3) percent reductions were 24.7% (40.0, 3.6) and 21.7% (39.9, 4.2), respectively. Reductions were maintained through week 52 of the open-label extension, and correlated with LDL-C reductions [with and without correction for Lp(a)-cholesterol] at both time points (P < 0.0001). The effect of LDL and LDL receptor (LDLR) availability on Lp(a) cell-association was measured in HepG2 cells: cell-associated LDL fluorescence was reversed by unlabeled LDL and Lp(a). Lp(a) cell-association was reduced by coincubation with LDL and PCSK9 and reversed by adding PCSK9 mAb. These studies support that reductions in Lp(a) with PCSK9 inhibition are partly due to increased LDLR-mediated uptake. In most situations, Lp(a) appears to compete poorly with LDL for LDLR binding and internalization, but when LDLR expression is increased with evolocumab, particularly in the setting of low circulating LDL, Lp(a) is reduced. PMID:27102113

  16. Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

    SciTech Connect

    Hossain, Ekhtear; Ota, Akinobu; Karnan, Sivasundaram; Damdindorj, Lkhagvasuren; Takahashi, Miyuki; Konishi, Yuko; Konishi, Hiroyuki; Hosokawa, Yoshitaka

    2013-12-15

    Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. - Highlights: • Sodium arsenite (SA) increases LOX-1 expression in mouse aortic endothelial cells. • SA enhances cellular uptake of oxidized LDL in dose-dependent manner. • SA-induced ROS generation enhances phosphorylation of NF-κB. • SA upregulates LOX-1 expression through ROS-activated NF-κB signaling pathway.

  17. LDL receptor-related protein-1 regulates NFκB and microRNA-155 in macrophages to control the inflammatory response.

    PubMed

    Mantuano, Elisabetta; Brifault, Coralie; Lam, Michael S; Azmoon, Pardis; Gilder, Andrew S; Gonias, Steven L

    2016-02-01

    LDL receptor-related protein-1 (LRP1) is an endocytic and cell-signaling receptor. In mice in which LRP1 is deleted in myeloid cells, the response to lipopolysaccharide (LPS) was greatly exacerbated. LRP1 deletion in macrophages in vitro, under the control of tamoxifen-activated Cre-ER(T) fusion protein, robustly increased expression of proinflammatory cytokines and chemokines. In LRP1-expressing macrophages, proinflammatory mediator expression was regulated by LRP1 ligands in a ligand-specific manner. The LRP1 agonists, α2-macroglobulin and tissue-type plasminogen activator, attenuated expression of inflammatory mediators, even in the presence of LPS. The antagonists, receptor-associated protein (RAP) and lactoferrin (LF), and LRP1-specific antibody had the entirely opposite effect, promoting inflammatory mediator expression and mimicking LRP1 deletion. NFκB was rapidly activated in response to RAP and LF and responsible for the initial increase in expression of proinflammatory mediators. RAP and LF also significantly increased expression of microRNA-155 (miR-155) after a lag phase of about 4 h. miR-155 expression reflected, at least in part, activation of secondary cell-signaling pathways downstream of TNFα. Although miR-155 was not involved in the initial induction of cytokine expression in response to LRP1 antagonists, miR-155 was essential for sustaining the proinflammatory response. We conclude that LRP1, NFκB, and miR-155 function as members of a previously unidentified system that has the potential to inhibit or sustain inflammation, depending on the continuum of LRP1 ligands present in the macrophage microenvironment. PMID:26787872

  18. Pregnancy-associated osteoporosis with a heterozygous deactivating LDL receptor-related protein 5 (LRP5) mutation and a homozygous methylenetetrahydrofolate reductase (MTHFR) polymorphism.

    PubMed

    Cook, Fiona J; Mumm, Steven; Whyte, Michael P; Wenkert, Deborah

    2014-04-01

    Pregnancy-associated osteoporosis (PAO) is a rare, idiopathic disorder that usually presents with vertebral compression fractures (VCFs) within 6 months of a first pregnancy and delivery. Spontaneous improvement is typical. There is no known genetic basis for PAO. A 26-year-old primagravida with a neonatal history of unilateral blindness attributable to hyperplastic primary vitreous sustained postpartum VCFs consistent with PAO. Her low bone mineral density (BMD) seemed to respond to vitamin D and calcium therapy, with no fractures after her next successful pregnancy. Investigation of subsequent fetal losses revealed homozygosity for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism associated both with fetal loss and with osteoporosis (OP). Because her neonatal unilateral blindness and OP were suggestive of loss-of-function mutation(s) in the gene that encodes LDL receptor-related protein 5 (LRP5), LRP5 exon and splice site sequencing was also performed. This revealed a unique heterozygous 12-bp deletion in exon 21 (c.4454_4465del, p.1485_1488del SSSS) in the patient, her mother and sons, but not her father or brother. Her mother had a normal BMD, no history of fractures, PAO, ophthalmopathy, or fetal loss. Her two sons had no ophthalmopathy and no skeletal issues. Her osteoporotic father (with a family history of blindness) and brother had low BMDs first documented at ages ∼40 and 32 years, respectively. Serum biochemical and bone turnover studies were unremarkable in all subjects. We postulate that our patient's heterozygous LRP5 mutation together with her homozygous MTHFR polymorphism likely predisposed her to low peak BMD. However, OP did not cosegregate in her family with the LRP5 mutation, the homozygous MTHFR polymorphism, or even the combination of the two, implicating additional genetic or nongenetic factors in her PAO. Nevertheless, exploration for potential genetic contributions to PAO may explain part of the pathogenesis of this

  19. High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis, Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide

    PubMed Central

    Ghosh, Siddhartha S.; Righi, Samuel; Krieg, Richard; Kang, Le; Carl, Daniel; Wang, Jing; Massey, H. Davis; Sica, Domenic A.; Gehr, Todd W. B.; Ghosh, Shobha

    2015-01-01

    A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency. PMID:26580567

  20. Involvement of Sp1 and SREBP-1a in transcriptional activation of the LDL receptor gene by insulin and LH in cultured porcine granulosa-luteal cells.

    PubMed

    Sekar, Natesampillai; Veldhuis, Johannes D

    2004-07-01

    Luteinizing hormone (LH) and insulin stimulate transcriptional activity of the porcine low-density lipoprotein (LDL) receptor (LDLR) promoter supra-additively in primary cultures of granulosa-luteal cells. The mechanistic basis of this bihormonal interaction is unknown. The pig LDLR gene promoter includes three putative Sp1/Sp3-binding sites and one sterol response element (SRE) site 5' upstream to the transcriptional start site. To assess the role of SRE-binding protein (SREBP) in LDLR gene regulation, swine granulosa-luteal cells were cotransfected with CMV/SREBP-1a or SREBP-2 and the pLDLR1076/luc promoter. SREBP-1a and SREBP-2 stimulated LDLR gene transcription eight- and fourfold, respectively. LH alone augmented stimulation by SREBP-1 twofold. Conversely, cotransfection of a dominant-negative mutant form of SREBP-1a repressed basal and hormonally stimulated LDLR promoter activity by >80% (P < 0.01). Mutation of the SRE -167 ATCACCCCATG -157 to -167 ATCACCgCATG -157 bp decreased basal expression by 50% and LH + insulin- and LH + IGF-I-stimulated transcriptional activity by 80% and >90%, respectively (both P < 0.01). Mutations within each of the three flanking putative Sp1/Sp3 sites at -216/-211, -201/-196, and -151/-146 bp in the LDLR gene promoter also reduced basal activity (by >85%) and hormonal responsiveness (>95%, P < 0.05). EMSA confirmed that presumptive SRE-1 and Sp1/Sp3 elements bind respective peptides. Mithramycin, an inhibitor of Sp1/Sp3 protein(s) binding, blocked hormonally induced LDLR promoter expression by 80%. Basal transcription and supra-additive stimulation of porcine LDLR gene transcription by LH and insulin in granulosa-luteal cells require SREBP-1a and Sp1/Sp3-binding elements. PMID:14998783

  1. D-4F reduces EO6 immunoreactivity, SREBP-1c mRNA levels, and renal inflammation in LDL receptor-null mice fed a Western diet.

    PubMed

    Buga, Georgette M; Frank, Joy S; Mottino, Giuliano A; Hakhamian, Ashkan; Narasimha, Ajay; Watson, Andrew D; Yekta, Babak; Navab, Mohamad; Reddy, Srinivasa T; Anantharamaiah, G M; Fogelman, Alan M

    2008-01-01

    LDL receptor-null (LDLR(-/-)) mice on a Western diet (WD) develop endothelial dysfunction and atherosclerosis, which are improved by the apolipoprotein A-I (apoA-I) mimetic peptide D-4F. Focusing on the kidney, LDLR(-/-)mice were fed a WD with D-4F or the inactive control peptide scrambled D-4F (ScD-4F) added to their drinking water. The control mice (ScD-4F) developed glomerular changes, increased immunostaining for MCP-1/CCL2 chemokine, increased macrophage CD68 and F4/80 antigens, and increased oxidized phospholipids recognized by the EO6 monoclonal antibody in both glomerular and tublo-interstitial areas. All of these parameters were significantly reduced by D-4F treatment, approaching levels found in wild-type C57BL/6J or LDLR(-/-) mice fed a chow diet. Sterol-regulatory element binding protein-1c (SREBP-1c) mRNA levels and triglyceride levels were elevated in the kidneys of the control mice (ScD-4F) fed the WD compared with C57BL/6J and LDLR(-/-) mice on chow (P < 0.001 and P < 0.001, respectively) and compared with D-4F-treated mice on the WD (P < 0.01). There was no significant difference in plasma lipids, lipoproteins, glucose, blood pressure, or renal apoB levels between D-4F- and ScD-4F-treated mice. We conclude that D-4F reduced renal oxidized phospholipids, resulting in lower expression of SREBP-1c, which, in turn, resulted in lower triglyceride content and reduced renal inflammation. PMID:17925450

  2. High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis, Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide.

    PubMed

    Ghosh, Siddhartha S; Righi, Samuel; Krieg, Richard; Kang, Le; Carl, Daniel; Wang, Jing; Massey, H Davis; Sica, Domenic A; Gehr, Todd W B; Ghosh, Shobha

    2015-01-01

    A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency. PMID:26580567

  3. Effect of long-term ingestion of weakly oxidised flaxseed oil on biomarkers of oxidative stress in LDL-receptor knockout mice.

    PubMed

    Nogueira, M S; Kessuane, M C; Lobo Ladd, A A B; Lobo Ladd, F V; Cogliati, B; Castro, I A

    2016-07-01

    The effect of oxidised fatty acids on atherosclerosis progression is controversial. Thus, our objective was to evaluate the effect of long-term consumption of weakly oxidised PUFA from flaxseed oil on oxidative stress biomarkers of LDL-receptor(-/-) mice. To test our hypothesis, mice were separated into three groups. The first group received a high-fat diet containing fresh flaxseed oil (CONT-), the second was fed the same diet prepared using heated flaxseed oil (OXID), and the third group received the same diet containing fresh flaxseed oil and had diabetes induced by streptozotocin (CONT+). Oxidative stress, aortic parameters and non-alcoholic fatty liver disease were assessed. After 3 months, plasma lipid profile, glucose levels, body weight, energy intake and dietary intake did not differ among groups. Likewise, oxidative stress, plasma malondialdehyde (MDA), hepatic MDA expressed as nmol/mg portion (ptn) and antioxidant enzymes did not differ among the groups. Hepatic linoleic acid, α-linolenic acid, arachidonic acid and EPA acid declined in the OXID and CONT+ groups. Aortic wall thickness, lumen and diameter increased only in the OXID group. OXID and CONT+ groups exhibited higher concentrations of MDA, expressed as μmol/mg ptn per %PUFA, when compared with the CONT- group. Our results suggest that ingestion of oxidised flaxseed oil increases hepatic MDA concentration and is potentially pro-atherogenic. In addition, the mean MDA value observed in all groups was similar to those reported in other studies that used xenobiotics as oxidative stress inducers. Thus, the diet applied in this study represents an interesting model for further research involving antioxidants. PMID:27197628

  4. CETP inhibitors downregulate hepatic LDL receptor and PCSK9 expression in vitro and in vivo through a SREBP2 dependent mechanism

    PubMed Central

    Dong, Bin; Singh, Amar Bahadur; Kelvin Kan, Chin Fung; Liu, Jingwen

    2015-01-01

    Background CETP inhibitors block the transfer of cholesteryl ester from HDL-C to VLDL-C and LDL-C, thereby raising HDL-C and lowering LDL-C. In this study, we explored the effect of CETP inhibitors on hepatic LDL receptor (LDLR) and PCSK9 expression and further elucidated the underlying regulatory mechanism. Results We first examined the effect of anacetrapib (ANA) and dalcetrapib (DAL) on LDLR and PCSK9 expression in hepatic cells in vitro. ANA exhibited a dose-dependent inhibition on both LDLR and PCSK9 expression in CETP-positive HepG2 cells and human primary hepatocytes as well as CETP-negative mouse primary hepatocytes (MPH). Moreover, the induction of LDLR protein expression by rosuvastatin in MPH was blunted by cotreatment with ANA. In both HepG2 and MPH ANA treatment reduced the amount of mature form of SREBP2 (SREBP2-M). In vivo, oral administration of ANA to dyslipidemic C57BL/6J mice at a daily dose of 50 mg/kg for 1 week elevated serum total cholesterol by approximately 24.5% (p<0.05%) and VLDL-C by 70% (p<0.05%) with concomitant reductions of serum PCSK9 and liver LDLR/SREBP2-M protein. Finally, we examined the in vitro effect of two other strong CETP inhibitors evacetrapib and torcetrapib on LDLR/PCSK9 expression and observed a similar inhibitory effect as ANA in a concentration range of 1–10 µM. Conclusion Our study revealed an unexpected off-target effect of CETP inhibitors that reduce the mature form of SREBP2, leading to attenuated transcription of hepatic LDLR and PCSK9. This negative regulation of SREBP pathway by ANA manifested in mice where CETP activity was absent and affected serum cholesterol metabolism. PMID:24950000

  5. High-fructose feeding promotes accelerated degradation of hepatic LDL receptor and hypercholesterolemia in hamsters via elevated circulating PCSK9 levels

    PubMed Central

    Dong, Bin; Singh, Amar Bahadur; Azhar, Salman; Seidah, Nabil G.; Liu, Jingwen

    2015-01-01

    Background High fructose diet (HFD) induces dyslipidemia and insulin resistance in experimental animals and humans with incomplete mechanistic understanding. By utilizing mice and hamsters as in vivo models, we investigated whether high fructose consumption affects serum PCSK9 and liver LDL receptor (LDLR) protein levels. Results Feeding mice with a HFD increased serum cholesterol and reduced serum PCSK9 levels as compared with the mice fed a normal chow diet (NCD). In contrast to the inverse relationship in mice, serum PCSK9 and cholesterol levels were co-elevated in HFD-fed hamsters. Liver tissue analysis revealed that PCSK9 mRNA and protein levels were both reduced in mice and hamsters by HFD feeding, however, liver LDLR protein levels were markedly reduced by HFD in hamsters but not in mice. We further showed that circulating PCSK9 clearance rates were significantly lower in hamsters fed a HFD as compared with the hamsters fed NCD, providing additional evidence for the reduced hepatic LDLR function by HFD consumption. The majority of PCSK9 in hamster serum was detected as a 53 kDa N-terminus cleaved protein. By conducting in vitro studies, we demonstrate that this 53 kDa truncated hamster PCSK9 is functionally active in promoting hepatic LDLR degradation. Conclusion Our studies for the first time demonstrate that high fructose consumption increases serum PCSK9 concentrations and reduces liver LDLR protein levels in hyperlipidemic hamsters. The positive correlation between circulating cholesterol and PCSK9 and the reduction of liver LDLR protein in HFD-fed hamsters suggest that hamster is a better animal model than mouse to study the modulation of PCSK9/LDLR pathway by atherogenic diets. PMID:25682035

  6. Immunization with malondialdehyde-modified low-density lipoprotein (LDL) reduces atherosclerosis in LDL receptor-deficient mice challenged with Porphyromonas gingivalis.

    PubMed

    Turunen, S Pauliina; Kummu, Outi; Wang, Chunguang; Harila, Kirsi; Mattila, Riikka; Sahlman, Marjo; Pussinen, Pirkko J; Hörkkö, Sohvi

    2015-05-01

    Periodontal infections increase the risk of atherosclerotic vascular disease via partly unresolved mechanisms. Of the natural IgM Abs that recognize molecular mimicry on bacterial epitopes and modified lipid and protein structures, IgM directed against oxidized low-density lipoprotein (LDL) is associated with atheroprotective properties. Here, the effect of natural immune responses to malondialdehyde-modified LDL (MDA-LDL) in conferring protection against atherosclerosis, which was accelerated by the major periodontopathogen Porphyromonas gingivalis, was investigated. LDL receptor-deficient (LDLR(-/-)) mice were immunized with mouse MDA-LDL without adjuvant before topical application challenge with live P. gingivalis. Atherosclerosis was analyzed after a high-fat diet, and plasma IgG and IgM Ab levels were measured throughout the study, and the secretion of IL-5, IL-10 and IFN-γ in splenocytes stimulated with MDA-LDL was determined. LDLR(-/-) mice immunized with MDA-LDL had elevated IgM and IgG levels to MDA-LDL compared with saline-treated controls. MDA-LDL immunization diminished aortic lipid depositions after challenge with P. gingivalis compared with mice receiving only P. gingivalis challenge. Immunization of LDLR(-/-) mice with homologous MDA-LDL stimulated the production of IL-5, implicating general activation of B-1 cells. Immune responses to MDA-LDL protected from the P. gingivalis-accelerated atherosclerosis. Thus, the linkage between bacterial infectious burden and atherogenesis is suggested to be modulated via natural IgM directed against cross-reactive epitopes on bacteria and modified LDL. PMID:25134521

  7. mTORC1 Down-Regulates Cyclin-Dependent Kinase 8 (CDK8) and Cyclin C (CycC)

    PubMed Central

    Feng, Daorong; Youn, Dou Yeon; Zhao, Xiaoping; Gao, Yanguang; Quinn, William J.; Xiaoli, Alus M.; Sun, Yan; Birnbaum, Morris J.; Pessin, Jeffrey E.; Yang, Fajun

    2015-01-01

    In non-alcoholic fatty liver disease (NAFLD) and insulin resistance, hepatic de novo lipogenesis is often elevated, but the underlying mechanisms remain poorly understood. Recently, we show that CDK8 functions to suppress de novo lipogenesis. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a critical regulator of CDK8 and its activating partner CycC. Using pharmacologic and genetic approaches, we show that increased mTORC1 activation causes the reduction of the CDK8-CycC complex in vitro and in mouse liver in vivo. In addition, mTORC1 is more active in three mouse models of NAFLD, correlated with the lower abundance of the CDK8-CycC complex. Consistent with the inhibitory role of CDK8 on de novo lipogenesis, nuclear SREBP-1c proteins and lipogenic enzymes are accumulated in NAFLD models. Thus, our results suggest that mTORC1 activation in NAFLD and insulin resistance results in down-regulation of the CDK8-CycC complex and elevation of lipogenic protein expression. PMID:26042770

  8. Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain

    PubMed Central

    Zaliauskiene, Lolita; Kang, Sunghyun; Brouillette, Christie G.; Lebowitz, Jacob; Arani, Ramin B.; Collawn, James F.

    2000-01-01

    How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the “signal” within the TM necessary and sufficient for down-regulation is Thr11Gln17Thr19 (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well (∼79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals. PMID:10930460

  9. Induction of Fatal Inflammation in LDL Receptor and ApoA-I Double-Knockout Mice Fed Dietary Fat and Cholesterol

    PubMed Central

    Zabalawi, Manal; Bhat, Shaila; Loughlin, Tara; Thomas, Michael J.; Alexander, Eric; Cline, Mark; Bullock, Bill; Willingham, Mark; Sorci-Thomas, Mary G.

    2003-01-01

    Atherogenic response to dietary fat and cholesterol challenge was evaluated in mice lacking both the LDL receptor (LDLr−/−) and apoA-I (apoA-I−/−) gene, LDLr−/−/apoA-I−/− or double-knockout mice. Gender- and age-matched LDLr−/−/apoA-I−/− mice were fed a diet consisting of 0.1% cholesterol and 10% palm oil for 16 weeks and compared to LDLr−/− mice or single-knockout mice. The LDLr−/− mice showed a 6- to 7-fold increase in total plasma cholesterol (TPC) compared to their chow-fed mice counterparts, while LDLr−/−/apoA-I−/− mice showed only a 2- to 3-fold increase in TPC compared to their chow-fed controls. This differential response to the atherogenic diet was unanticipated, since chow-fed LDLr−/− and LDLr−/−/apoA-I−/− mice began the study with similar LDL levels and differed primarily in their HDL concentration. The 6-fold diet-induced increase in TPC observed in the LDLr−/− mice occurred mainly in VLDL/LDL and not in HDL. Mid-study plasma samples taken after 8 weeks of diet feeding showed that LDLr−/− mice had TPC concentrations approximately 60% of their 16-week level, while the LDLr−/−/apoA-I−/− mice had reached 100% of their 16-week TPC concentration after only 8 weeks of diet. Male LDLr−/− mice showed similar aortic cholesterol levels to male LDLr−/−/apoA-I−/− mice despite a 4-fold higher VLDL/LDL concentration in the LDLr−/− mice. A direct comparison of the severity of aortic atherosclerosis between female LDLr−/− and LDLr−/−/apoA-I−/− mice was compromised due to the loss of female LDLr−/−/apoA-I−/− mice between 10 and 14 weeks into the study. Diet-fed female and, with time, male LDLr−/−/apoA-I−/− mice suffered from severe ulcerated cutaneous xanthomatosis. This condition, combined with a complete depletion of adrenal cholesterol, manifested in fatal wasting of the affected mice. In conclusion, LDLr−/− and LDLr−/−/apoA-I−/− mice showed

  10. Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors.

    PubMed

    Chaturvedi, K; Bandari, P; Chinen, N; Howells, R D

    2001-04-13

    This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors. PMID:11152677

  11. Cytokine-mediated down-regulation of CYP1A1 in Hepa1 cells.

    PubMed

    Paton, T E; Renton, K W

    1998-06-01

    The activation of host defense mechanisms down-regulates microsomal cytochrome P450 in cell culture, humans, and animals. Investigation into various aspects of this effect using in vivo models has yet to define clearly the role that cytokines play in this phenomenon. The mechanism of down-regulation by immunostimulants, such as lipopolysaccharide (LPS), is explored with an in vitro model, utilizing a murine hepatoma (Hepa1) and a murine macrophage (IC-21) cell line. It is hypothesized that down-regulation of P450 activity by immunostimulants involves the activation of immune cells and the subsequent release of cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). The effects of immunostimulation on P450 activity are assessed by ethoxyresorufin O-dealkylase, an assay that measures CYP1A activity in Hepa1 cells. Initial studies demonstrated that LPS added directly to hepatoma cells had no effect on the levels of CYP1A1 activity. In contrast, a significant down-regulation in CYP1A1 activity occurred when hepatoma cells were incubated with monocyte conditioned medium obtained by incubating LPS with IC-21 cells. When pentoxifylline, a TNF-alpha synthesis inhibitor, was co-administered with LPS to macrophages, the down-regulation of CYP1A1 activity was prevented. The direct administration of murine recombinant TNF-alpha to hepatoma cells resulted in a down-regulation of CYP1A1 activity. These results implicated the release of TNF-alpha from macrophages as an important step in the down-regulation of CYP1A1 by LPS. PMID:9714297

  12. Serendipity in splendid isolation: rapamycin.

    PubMed

    Rao, V Koneti

    2016-01-01

    In this issue of Blood, Bride et al report results of the first prospective multi-institutional trial of a long-term single-agent therapy for refractory cytopenias using rapamycin in 30 patients and show remarkable efficacy in children with autoimmune lymphoproliferative syndrome (ALPS). PMID:26744433

  13. CDK14 expression is down-regulated by cigarette smoke in vivo and in vitro

    PubMed Central

    Pollack, Daniel; Xiao, Yuxuan; Shrivasatava, Vibha; Levy, Avi; Andrusier, Miriam; D’Armiento, Jeanine; Holz, Marina K.; Vigodner, Margarita

    2016-01-01

    In this study, DNA arrays have been employed to monitor gene expression patterns in testis of mice exposed to tobacco smoke for 24 weeks and compared to control animals. The results of the analysis revealed significant changes in expression of several genes that may have a role in spermatogenesis. Cdk14 was chosen for further characterization because of a suggested role in the testis and in regulation of Wnt signaling. RT-PCR analysis confirmed down regulation of Cdk14 in mice exposed to cigarette smoke (CS). Cdk14 is expressed in all testicular cells; spermatogonia- and Sertoli-derived cell lines treated with cigarette smoke extract (CSE) in vitro showed down-regulation of CDK14 mRNA and protein levels as well as down-regulation of β-catenin levels. CS-induced down-regulation of CDK14 mRNA and protein levels was also observed in several lung epithelium-derived cell lines including primary normal human bronchial epithelial cells (NHBE), suggesting that the effect is not restricted to the testis. Similar to testicular cells, CS-induced down-regulation of CDK14 in lung cells correlated with decreased levels of β-catenin, a finding suggesting impaired Wnt signaling. In the lungs, CDK14 was localized to the alveolar and bronchial epithelium. PMID:25680692

  14. Down-regulation of phospholipase C-beta1 following chronic muscarinic receptor activation.

    PubMed

    Sorensen, S D; Linseman, D A; Fisher, S K

    1998-04-01

    To determine whether prolonged activation of a phospholipase C-coupled receptor can lead to a down-regulation of its effector enzyme, SH-SY5Y neuroblastoma cells were incubated for 24 h with the muscarinic receptor agonist, oxotremorine-M. Under these conditions, significant reductions (46-53%) in muscarinic cholinergic receptor density, G(alphaq/11) and phospholipase C-beta1 (but not the beta3-or gamma1 isoforms) were observed. These results suggest that a selective down-regulation of phospholipase C-beta1 may play a role in adaptation to chronic muscarinic receptor activation. PMID:9617763

  15. An 8-gene signature, including methylated and down-regulated glutathione peroxidase 3, of gastric cancer.

    PubMed

    Zhang, Xianglan; Yang, Jung-Jin; Kim, Yoon Sun; Kim, Ki-Yeol; Ahn, Woong Shick; Yang, Sanghwa

    2010-02-01

    We have identified an 8-gene signature with significant expression differences between gastric cancer and normal gastric tissues. This 8-gene set can predict the normal and cancer status of gastric tissues with more than 96% accuracy in a totally independent microarray dataset. The 8 genes are composed of down-regulated KLF4, GPX3, SST and LIPF, together with up-regulated SERPINH1, THY1 and INHBA in gastric cancer. To corroborate the differential gene expression pattern, we chose GPX3 and examined its expression pattern in detail. A comparison of GPX3 expression pattern shows a broader down-regulated pattern in multiple types of cancers, including cervical, thyroid, head and neck, lung cancers and melanoma than in healthy controls. An immuno-histostaining analysis in tissue microarrays confirms GPX3 down-regulation in gastric cancer. Mechanism-wise GPX3 down-regulation in gastric cancer is due to promoter hypermethylation. Collectively, these results show a correct identification of 8 genes as gastric cancer biomarkers. PMID:20043075

  16. Alpha-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase.

    PubMed

    Manna, Sunil K; Sarkar, Abira; Sreenivasan, Yashin

    2006-03-01

    Considering the role of interleukin-8 (IL-8) in a large number of acute and chronic inflammatory diseases, the regulation of IL-8-mediated biological responses is important. Alpha-melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that alpha-MSH interacts predominantly with melanocortin-1 receptors and inhibits several IL-8-induced biological responses in macrophages and neutrophils. It down-regulated receptors for IL-8 but not for TNF, IL-4, IL-13 or TNF-related apoptosis-inducing ligand (TRAIL) in neutrophils. It down-regulated CXCR type 1 and 2 but not mRNA levels. alpha-MSH did not inhibit IL-8 binding in purified cell membrane or affinity-purified CXCR. IL-8 or anti-CXCR Ab protected against alpha-MSH-mediated inhibition of IL-8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in alpha-MSH-treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in alpha-MSH-induced down-regulation of CXCR. These studies suggest that alpha-MSH inhibits IL-8-mediated biological responses by down-regulating CXCR through induction of serine protease and that alpha-MSH acts as a potent immunomodulator in neutrophil-driven inflammatory distress. PMID:16479540

  17. Rapamycin inhibits the growth of glioblastoma.

    PubMed

    Arcella, Antonietta; Biagioni, Francesca; Antonietta Oliva, Maria; Bucci, Domenico; Frati, Alessandro; Esposito, Vincenzo; Cantore, Giampaolo; Giangaspero, Felice; Fornai, Francesco

    2013-02-01

    The molecular target of rapamycin (mTOR) is up-regulated in glioblastoma (GBM) and this is associated with the rate of cell growth, stem cell proliferation and disease relapse. Rapamycin is a powerful mTOR inhibitor and strong autophagy inducer. Previous studies analyzed the effects of rapamycin in GBM cell lines. However, to our knowledge, no experiment was carried out to evaluate the effects of rapamycin neither in primary cells derived from GBM patients nor in vivo in brain GBM xenograft. These data are critical to get a deeper insight into the effects of such adjuvant therapy in GBM patients. In the present study, various doses of rapamycin were tested in primary cell cultures from GBM patients. These effects were compared with that obtained by the same doses of rapamycin in GBM cell lines (U87Mg). The effects of rapamycin were also evaluated in vivo, in brain tumors developed from mouse xenografts. Rapamycin, starting at the dose of 10nm inhibited cell growth both in U87Mg cell line and primary cell cultures derived from various GBM patients. When administered in vivo to brain xenografts in nude mice rapamycin almost doubled the survival time of mice and inhibited by more than 95% of tumor volume. PMID:23261661

  18. Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system.

    PubMed

    Arriola Apelo, Sebastian I; Neuman, Joshua C; Baar, Emma L; Syed, Faizan A; Cummings, Nicole E; Brar, Harpreet K; Pumper, Cassidy P; Kimple, Michelle E; Lamming, Dudley W

    2016-02-01

    Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA-approved drug rapamycin has been shown to promote lifespan and delay age-related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long-term prophylactic use of rapamycin as a therapy for age-related diseases. While the beneficial effects of rapamycin are largely mediated by the inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by the inhibition of a second mTOR-containing complex, mTORC2, which is much less sensitive to rapamycin. We hypothesized that different rapamycin dosing schedules or the use of FDA-approved rapamycin analogs with different pharmacokinetics might expand the therapeutic window of rapamycin by more specifically targeting mTORC1. Here, we identified an intermittent rapamycin dosing schedule with minimal effects on glucose tolerance, and we find that this schedule has a reduced impact on pyruvate tolerance, fasting glucose and insulin levels, beta cell function, and the immune system compared to daily rapamycin treatment. Further, we find that the FDA-approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 while having a reduced impact on glucose and pyruvate tolerance. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs. PMID:26463117

  19. SAMHD1 is down regulated in lung cancer by methylation and inhibits tumor cell proliferation

    SciTech Connect

    Wang, Jia-lei; Lu, Fan-zhen; Shen, Xiao-Yong; Wu, Yun; Zhao, Li-ting

    2014-12-12

    Highlights: • SAMHD1 expression level is down regulated in lung adenocarcinoma. • The promoter of SAMHD1 is methylated in lung adenocarcinoma. • Over expression of SAMHD1 inhibits the proliferation of lung cancer cells. - Abstract: The function of dNTP hydrolase SAMHD1 as a viral restriction factor to inhibit the replication of several viruses in human immune cells was well established. However, its regulation and function in lung cancer have been elusive. Here, we report that SAMHD1 is down regulated both on protein and mRNA levels in lung adenocarcinoma compared to adjacent normal tissue. We also found that SAMHD1 promoter is highly methylated in lung adenocarcinoma, which may inhibit its gene expression. Furthermore, over expression of the SAMHD1 reduces dNTP level and inhibits the proliferation of lung tumor cells. These results reveal the regulation and function of SAMHD1 in lung cancer, which is important for the proliferation of lung tumor cells.

  20. Down-regulation of the desmosomal cadherin desmocollin 3 in human breast cancer.

    PubMed

    Klus, G T; Rokaeus, N; Bittner, M L; Chen, Y; Korz, D M; Sukumar, S; Schick, A; Szallasi, Z

    2001-07-01

    In previous studies using cDNA microarray analysis, we have identified an expressed sequence tag which is consistently down-regulated in six human breast tumor cell lines. In the current study, we have determined this tag to be part of the mRNA sequence of human desmocollin 3, a member of the cadherin superfamily of proteins and an integral component of desmosomes. Desmosomes are sites of adhesion between adjacent cells in layers of epithelia, as well as in some non-epithelial tissues, and play an important role in the maintenance of tissue structure. Northern analysis, quantitative real-time polymerase chain reaction assay and Western blot analysis showed that desmocollin 3 is present in normal and immortalized human mammary epithelial cells, but consistently exhibits a significant, and often complete, down-regulation in breast cancer cell lines and primary breast tumors, both at the mRNA and protein levels. PMID:11408939

  1. Down-regulation of lipoprotein lipase increases glucose uptake in L6 muscle cells

    SciTech Connect

    Lopez, Veronica; Saraff, Kumuda; Medh, Jheem D.

    2009-11-06

    Thiazolidinediones (TZDs) are synthetic hypoglycemic agents used to treat type 2 diabetes. TZDs target the peroxisome proliferator activated receptor-gamma (PPAR-{gamma}) and improve systemic insulin sensitivity. The contributions of specific tissues to TZD action, or the downstream effects of PPAR-{gamma} activation, are not very clear. We have used a rat skeletal muscle cell line (L6 cells) to demonstrate that TZDs directly target PPAR-{gamma} in muscle cells. TZD treatment resulted in a significant repression of lipoprotein lipase (LPL) expression in L6 cells. This repression correlated with an increase in glucose uptake. Down-regulation of LPL message and protein levels using siRNA resulted in a similar increase in insulin-dependent glucose uptake. Thus, LPL down-regulation improved insulin sensitivity independent of TZDs. This finding provides a novel method for the management of insulin resistance.

  2. Antigenic modulation and down regulation-their similarities suggest they may have a common origin.

    PubMed

    Cohen, E P

    1980-01-01

    Antigenic modulation, the antibody-induced disappearance of membrane-associated determinants of neoplastic cells, has a fundamental similarity to the down regulation of receptors for hormones and other ligands affecting the physiology of various nonneoplastic cell types. After hormone stimulation, cells previously responsive become refractory to further stimulation. Diminution in responsiveness correlates with a quantitative reduction in the numbers of specific complementary hormone receptors present. Murine leukemia cells, as one example "escape" antibody-mediated destruction through the mechanism of antigenic modulation. Resistance of the cells correlates with a reduction of the numbers of specific antigens present. In both down regulation and modulation, continued cellular metabolism in the absence of ligand leads to a re-expression of the hormone receptor/membrane antigen involved. It is postulated that the two phenomena have evolved from similar underlying cellular mechanisms. PMID:6256059

  3. Kanglaite attenuates UVB-induced down-regulation of aquaporin-3 in cultured human skin keratinocytes

    PubMed Central

    SHAN, SHI-JUN; XIAO, TING; CHEN, JOHN; GENG, SHI-LING; LI, CHANG-PING; XU, XUEGANG; HONG, YUXIAO; JI, CHAO; GUO, YING; WEI, HUACHEN; LIU, WEI; LI, DAPENG; CHEN, HONG-DUO

    2012-01-01

    Ultraviolet (UV) radiation plays an important role in the pathogenesis of skin photoaging. Depending on the wavelength of UV, the epidermis is affected primarily by UVB. One major characteristic of photoaging is the dehydration of the skin. Membrane-inserted water channels (aquaporins) are involved in this process. In this study we demonstrated that UVB radiation induced aquaporin-3 (AQP3) down-regulation in cultured human skin keratinocytes. Kanglaite is a mixture consisting of extractions of Coix Seed, which is an effective anti-neoplastic agent and can inhibit the activities of protein kinase C and NF-κB. We demonstrated that Kanglaite inhibited UVB-induced AQP3 down-regulation of cultured human skin keratinocytes. Our findings provide a potential new agent for anti-photoaging. The related molecular mechanisms remain to be further elucidated. PMID:22211241

  4. Dipeptides Inhibit Melanin Synthesis in Mel-Ab Cells through Down-Regulation of Tyrosinase

    PubMed Central

    Lee, Hyun-e; Kim, Eun-Hyun; Choi, Hye-Ryung; Sohn, Uy Dong; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Park, Kyoung-Chan

    2012-01-01

    This study investigated the effects of proline-serine (PS) and valine-serine (VS) dipeptides on melanogenesis in Mel-Ab cells. Proline-serine and VS significantly inhibited melanin synthesis in a concentration-dependent manner, though neither dipeptide directly inhibited tyrosinase activity in a cell-free system. Both PS and VS down-regulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. In a follow-up study also described here, the effects of these dipeptides on melanogenesis-related signal transduction were quantified. Specifically, PS and VS induced ERK phosphorylation, though they had no effect on phosphorylation of the cAMP response element binding protein (CREB). These data suggest that PS and VS inhibit melanogenesis through ERK phosphorylation and subsequent down-regulation of MITF and tyrosinase. Properties of these dipeptides are compatible with application as skin-whitening agents. PMID:22915995

  5. Down-regulation of microRNA-144 in air pollution-related lung cancer

    PubMed Central

    Pan, Hong-Li; Wen, Zhe-Sheng; Huang, Yun-Chao; Cheng, Xin; Wang, Gui-Zhen; Zhou, Yong-Chun; Wang, Zai-Yong; Guo, Yong-Qing; Cao, Yi; Zhou, Guang-Biao

    2015-01-01

    Air pollution has been classified as a group 1 carcinogen in humans, but the underlying tumourigenic mechanisms remain unclear. In Xuanwei city of Yunnan Province, the lung cancer incidence is among the highest in China, owing to severe air pollution generated by the combustion of smoky coal, providing a unique opportunity to dissect lung carcinogenesis. To identify abnormal miRNAs critical for air pollution-related tumourigenesis, we performed microRNA microarray analysis in 6 Xuanwei non-small cell lung cancers (NSCLCs) and 4 NSCLCs from control regions where smoky coal was not used. We found 13 down-regulated and 2 up-regulated miRNAs in Xuanwei NSCLCs. Among them, miR-144 was one of the most significantly down-regulated miRNAs. The expanded experiments showed that miR-144 was down-regulated in 45/51 (88.2%) Xuanwei NSCLCs and 34/54 (63%) control region NSCLCs (p = 0.016). MiR-144 interacted with the oncogene Zeb1 at 2 sites in its 3′ untranslated region, and a decrease in miR-144 resulted in increased Zeb1 expression and an epithelial mesenchymal transition phenotype. Ectopic expression of miR-144 suppressed NSCLCs in vitro and in vivo by targeting Zeb1. These results indicate that down-regulation of miR-144 is critical for air pollution-related lung cancer, and the miR-144-Zeb1 signalling pathway could represent a potential therapeutic target. PMID:26395400

  6. Photosynthesis down-regulation precedes carbohydrate accumulation under sink limitation in Citrus.

    PubMed

    Nebauer, Sergio G; Renau-Morata, Begoña; Guardiola, José Luis; Molina, Rosa-Victoria

    2011-02-01

    Photosynthesis down-regulation due to an imbalance between sources and sinks in Citrus leaves could be mediated by excessive accumulation of carbohydrates. However, there is limited understanding of the physiological role of soluble and insoluble carbohydrates in photosynthesis regulation and the elements triggering the down-regulation process. In this work, the role of non-structural carbohydrates in the regulation of photosynthesis under a broad spectrum of source-sink relationships has been investigated in the Salustiana sweet orange. Soluble sugar and starch accumulation in leaves, induced by girdling experiments, did not induce down-regulation of the photosynthetic rate in the presence of sinks (fruits). The leaf-to-fruit ratio did not modulate photosynthesis but allocation of photoassimilates to the fruits. The lack of strong sink activity led to a decrease in the photosynthetic rate and starch accumulation in leaves. However, photosynthesis down-regulation due to an excess of total soluble sugars or starch was discarded because photosynthesis and stomatal conductance reduction occurred prior to any significant accumulation of these carbohydrates. Gas exchange and fluorescence parameters suggested biochemical limitations to photosynthesis. In addition, the expression of carbon metabolism-related genes was altered within 24 h when strong sinks were removed. Sucrose synthesis and export genes were inhibited, whereas the expression of ADP-glucose pyrophosphorylase was increased to cope with the excess of assimilates. In conclusion, changes in starch and soluble sugar turnover, but not sugar content per se, could provide the signal for photosynthesis regulation. In these conditions, non-stomatal limitations strongly inhibited the photosynthetic rate prior to any significant increase in carbohydrate levels. PMID:21367744

  7. Prodigiosin down-regulates survivin to facilitate paclitaxel sensitization in human breast carcinoma cell lines

    SciTech Connect

    Ho, T.-F.; Peng, Y.-T.; Chuang, S.-M.; Lin, S.-C.; Feng, B.-L.; Lu, C.-H.; Yu, W.-J.; Chang, J.-S. Chang, C.-C.

    2009-03-01

    Prodigiosin is a bacterial metabolite with potent anticancer activity, which is attributed to its proapoptotic effect selectively active in malignant cells. Still, the molecular mechanisms whereby prodigiosin induces apoptosis remain largely unknown. In particular, the role of survivin, a vital inhibitor of apoptosis, in prodigiosin-induced apoptosis has never been addressed before and hence was the primary goal of this study. Our results showed that prodigiosin dose-dependently induced down-regulation of survivin in multiple breast carcinoma cell lines, including MCF-7, T-47D and MDA-MB-231. This down-regulation is mainly regulated at the level of transcription, as prodigiosin reduced the levels of both survivin mRNA and survivin promoter activity but failed to rescue survivin expression when proteasome-mediated degradation is abolished. Importantly, overexpression of survivin rendered cells more resistant to prodigiosin, indicating an essential role of survivin down-regulation in prodigiosin-induced apoptosis. In addition, we found that prodigiosin synergistically enhanced cell death induced by paclitaxel, a chemotherapy drug known to up-regulate survivin that in turn confers its own resistance. This paclitaxel sensitization effect of prodigiosin is ascribed to the lowering of survivin expression, because prodigiosin was shown to counteract survivin induction by paclitaxel and, notably, the sensitization effect was severely abrogated in cells that overexpress survivin. Taken together, our results argue that down-regulation of survivin is an integral component mediating prodigiosin-induced apoptosis in human breast cancer cells, and further suggest the potential of prodigiosin to sensitize anticancer drugs, including paclitaxel, in the treatment of breast cancer.

  8. Tannic acid down-regulates the angiotensin type 1 receptor through a MAPK-dependent mechanism.

    PubMed

    Yesudas, Rekha; Gumaste, Upendra; Snyder, Russell; Thekkumkara, Thomas

    2012-03-01

    In the present study, we investigated the effects of tannic acid (TA), a hydrolysable polyphenol, on angiotensin type 1 receptor (AT1R) expression in continuously passaged rat liver epithelial cells. Under normal conditions, exposure of cells to TA resulted in the down-regulation of AT1R-specific binding in concentrations ranging from 12.5-100 μg/ml (7.34-58.78 μm) over a time period of 2-24 h with no change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA on AT1R was specific and reversible. In TA-treated cells, we observed a significant reduction in AngII-mediated intracellular calcium signaling, a finding consistent with receptor down-regulation. Under similar conditions, TA down-regulated AT1R mRNA expression without changing the rate of mRNA degradation, suggesting that TA's effect is mediated through transcriptional inhibition. Cells expressing recombinant AT1R without the native promoter show no change in receptor expression, whereas a pCAT reporter construct possessing the rat AT1R promoter was significantly reduced in activity. Furthermore, TA induced the phosphorylation of MAPK p42/p44. Pretreatment of the cells with a MAPK kinase (MEK)-specific inhibitor PD98059 prevented TA-induced MAPK phosphorylation and down-regulation of the AT1R. Moreover, there was no reduction in AngII-mediated intracellular calcium release upon MEK inhibition, suggesting that TA's observed inhibitory effect is mediated through MEK/MAPK signaling. Our findings demonstrate, for the first time, that TA inhibits AT1R gene expression and cellular response, suggesting the observed protective effects of dietary polyphenols on cardiovascular conditions may be, in part, through inhibition of AT1R expression. PMID:22322600

  9. Inhibition of the mechanistic target of rapamycin (mTOR) - Rapamycin and beyond

    PubMed Central

    Lamming, Dudley W.

    2016-01-01

    Rapamycin is an FDA-approved immunosuppressant and anti-cancer agent discovered in the soil of Easter Island in the early 1970s. Rapamycin is a potent and selective inhibitor of the mTOR (mechanistic Target Of Rapamycin) protein kinase, which acts as a central integrator of nutrient signaling pathways. During the last decade, genetic and pharmaceutical inhibition of mTOR pathway signaling has been found to promote longevity in yeast, worms, flies and mice. In this chapter, we will discuss the molecular biology underlying the effects of rapamycin and its physiological effects; evidence for rapamycin as an anti-aging compound; mechanisms by which rapamycin may extend lifespan; and the potential limitations of rapamycin as an anti-aging molecule. Finally, we will discuss possible strategies that may allow us to inhibit mTOR signaling safely while minimizing side effects, and reap the health, social and economic benefits from slowing the aging process. PMID:27048303

  10. Inhibition of the Mechanistic Target of Rapamycin (mTOR)-Rapamycin and Beyond.

    PubMed

    Lamming, Dudley W

    2016-01-01

    Rapamycin is a Food and Drug Administration (FDA)-approved immunosuppressant and anticancer agent discovered in the soil of Easter Island in the early 1970s. Rapamycin is a potent and selective inhibitor of the mechanistic target of rapamycin (mTOR) protein kinase, which acts as a central integrator of nutrient signaling pathways. During the last decade, genetic and pharmaceutical inhibition of mTOR pathway signaling has been found to promote longevity in yeast, worms, flies, and mice. In this article, we will discuss the molecular biology underlying the effects of rapamycin and its physiological effects, evidence for rapamycin as an antiaging compound, mechanisms by which rapamycin may extend life span, and the potential limitations of rapamycin as an antiaging molecule. Finally, we will discuss possible strategies that may allow us to inhibit mTOR signaling safely while minimizing side effects, and reap the health, social, and economic benefits from slowing the aging process. PMID:27048303

  11. Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar.

    PubMed Central

    Baucher, M.; Chabbert, B.; Pilate, G.; Van Doorsselaere, J.; Tollier, M. T.; Petit-Conil, M.; Cornu, D.; Monties, B.; Van Montagu, M.; Inze, D.; Jouanin, L.; Boerjan, W.

    1996-01-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry. PMID:12226459

  12. Down-regulation of telomerase activity by anticancer drugs in human ovarian cancer cells.

    PubMed

    Kunifuji, Yasumasa; Gotoh, Sadao; Abe, Tetsuya; Miura, Masayoshi; Karasaki, Yuji

    2002-07-01

    Maintenance of telomere length is crucial for survival of cells. Telomerase, an enzyme that is responsible for elongation of shortened telomeres, is active in human germ cells as well as most tumor tissues and experimentally immortalized cells. In contrast, most mature somatic cells in human tissues express undetectable or low telomerase activity, implying the existence of a stringent and negative regulatory mechanism. In this study we report the effects of anticancer drugs on telomerase activity in human cancer cells. In assaying for telomerase activity, we basically followed the original TRAP assay system, but with some modifications. A down-regulation of telomerase activity was found when cells of a human ovarian cancer cell line, A2780, were treated with;cis-diamminedichloroplatinum(II) (CDDP; cisplatin). However, down-regulation of telomerase activity was not found in cells of a cisplatin-resistant cell line, A2780CP, treated with cisplatin. On the other hand, telomerase activity in both the cell lines A2780 and A2780CP was reduced when A2780 or A2780CP was treated with adriamycin, an anthracycline antibiotic having a broad spectrum of antineoplastic activity. The different effects on the telomerase activity of the two types of anticancer drugs may be due the distinct chemical functions of these drugs. The present results may indicate a positive relationship between anticancer effects and down-regulation of telomerase activity by anticancer drugs. PMID:12172504

  13. CHIP mediates down-regulation of nucleobindin-1 in preosteoblast cell line models.

    PubMed

    Xue, Fuying; Wu, Yanping; Zhao, Xinghui; Zhao, Taoran; Meng, Ying; Zhao, Zhanzhong; Guo, Junwei; Chen, Wei

    2016-08-01

    Nucleobindin-1 (NUCB1), also known as Calnuc, is a highly conserved, multifunctional protein widely expressed in tissues and cells. It contains two EF-hand motifs which have been shown to play a crucial role in binding Ca(2+) ions. In this study, we applied comparative two-dimensional gel electrophoresis to characterize differentially expressed proteins in HA-CHIP over-expressed and endogenous CHIP depleted MC3T3-E1 stable cell lines, identifying NUCB1 as a novel CHIP/Stub1 targeted protein. NUCB1 interacts with and is down-regulated by CHIP by both proteasomal dependent and independent pathways, suggesting that CHIP-mediated down-regulation of nucleobindin-1 might play a role in osteoblast differentiation. The chaperone protein Hsp70 was found to be important for CHIP and NUCB1 interaction as well as CHIP-mediated NUCB1 down-regulation. Our findings provide new insights into understanding the stability regulation of NUCB1. PMID:27178152

  14. Rapamycin preserves gut homeostasis during Drosophila aging.

    PubMed

    Fan, Xiaolan; Liang, Qing; Lian, Ting; Wu, Qi; Gaur, Uma; Li, Diyan; Yang, Deying; Mao, Xueping; Jin, Zhihua; Li, Ying; Yang, Mingyao

    2015-11-01

    Gut homeostasis plays an important role in maintaining the overall body health during aging. Rapamycin, a specific inhibitor of mTOR, exerts prolongevity effects in evolutionarily diverse species. However, its impact on the intestinal homeostasis remains poorly understood. Here, we demonstrate that rapamycin can slow down the proliferation rate of intestinal stem cells (ISCs) in the aging guts and induce autophagy in the intestinal epithelium in Drosophila. Rapamycin can also significantly affect the FOXO associated genes in intestine and up-regulate the negative regulators of IMD/Rel pathway, consequently delaying the microbial expansion in the aging guts. Collectively, these findings reveal that rapamycin can delay the intestinal aging by inhibiting mTOR and thus keeping stem cell proliferation in check. These results will further explain the mechanism of healthspan and lifespan extension by rapamycin in Drosophila. PMID:26431326

  15. Rapamycin preserves gut homeostasis during Drosophila aging

    PubMed Central

    Lian, Ting; Wu, Qi; Gaur, Uma; Li, Diyan; Yang, Deying; Mao, Xueping; Jin, Zhihua; Li, Ying; Yang, Mingyao

    2015-01-01

    Gut homeostasis plays an important role in maintaining the overall body health during aging. Rapamycin, a specific inhibitor of mTOR, exerts prolongevity effects in evolutionarily diverse species. However, its impact on the intestinal homeostasis remains poorly understood. Here, we demonstrate that rapamycin can slow down the proliferation rate of intestinal stem cells (ISCs) in the aging guts and induce autophagy in the intestinal epithelium in Drosophila. Rapamycin can also significantly affect the FOXO associated genes in intestine and up-regulate the negative regulators of IMD/Rel pathway, consequently delaying the microbial expansion in the aging guts. Collectively, these findings reveal that rapamycin can delay the intestinal aging by inhibiting mTOR and thus keeping stem cell proliferation in check. These results will further explain the mechanism of healthspan and lifespan extension by rapamycin in Drosophila. PMID:26431326

  16. Role of EGF receptor ligands in TCDD-induced EGFR down-regulation and cellular proliferation.

    PubMed

    Campion, Christina M; Leon Carrion, Sandra; Mamidanna, Gayatri; Sutter, Carrie Hayes; Sutter, Thomas R; Cole, Judith A

    2016-06-25

    In cultures of normal human epidermal keratinocytes (NHEKs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces the expression of the epidermal growth factor receptor ligands transforming growth factor-α (TGF-α) and epiregulin (EREG). TCDD also down-regulates EGF receptors (EGFR), suggesting that decreases in signaling contribute to the effects of TCDD. In this study, we treated post-confluent NHEKs with 10 nM TCDD and assessed its effects on EGFR binding, EGFR ligand secretion, basal ERK activity, and proliferation. TCDD caused time-dependent deceases in [(125)I]-EGF binding to levels 78% of basal cell values at 72 h. Amphiregulin (AREG) levels increased with time in culture in basal and TCDD-treated cells, while TGF-α and epiregulin (EREG) secretion were stimulated by TCDD. Inhibiting EGFR ligand release with the metalloproteinase inhibitor batimastat prevented EGFR down-regulation and neutralizing antibodies for AREG and EREG relieved receptor down-regulation. In contrast, neutralizing TGF-α intensified EGFR down-regulation. Treating NHEKs with AREG or TGF-α caused rapid internalization of receptors with TGF-α promoting recycling within 90 min. EREG had limited effects on rapid internalization or recycling. TCDD treatment increased ERK activity, a response reduced by batimastat and the neutralization of all three ligands indicating that the EGFR and its ligands maintain ERK activity. All three EGFR ligands were required for the maintenance of total cell number in basal and TCDD-treated cultures. The EGFR inhibitor PD1530305 blocked basal and TCDD-induced increases in the number of cells labeled by 5-ethynyl-2'-deoxyuridine, identifying an EGFR-dependent pool of proliferating cells that is larger in TCDD-treated cultures. Overall, these data indicate that TCDD-induced EGFR down-regulation in NHEKs is caused by AREG, TGF-α, and EREG, while TGF-α enhances receptor recycling to maintain a pool of EGFR at the cell surface. These receptors are required for

  17. Mechanisms of allele-selective down-regulation of HLA class I in Burkitt's lymphoma.

    PubMed

    Imreh, M P; Zhang, Q J; de Campos-Lima, P O; Imreh, S; Krausa, P; Browning, M; Klein, G; Masucci, M G

    1995-07-01

    Burkitt lymphomas (BL) that arise in HLA-AII-positive individuals are characterized by selective loss/down-regulation of the HLA AII polypeptide. We have investigated the molecular basis of such down-regulation by comparing 5 pairs of BL lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the normal B cells of the same individuals. The presence of apparently intact HLA AII genes was confirmed in all 5 BL/LCL pairs by polymerase chain reaction (PCR) typing and by Southern-blot hybridization with HLA A locus-specific probes. Northern-blot analysis with locus- and allele-specific probes revealed a significantly lower expression or absence of AII-specific mRNA in all 5 BL lines compared to the corresponding LCLs. Up-regulation of AII-specific mRNA was achieved by IFN alpha treatment of 2 BL lines with low HLA AII expression (BL-28 and BL-72) while the treatment had no effect in 3 BL lines (WWI-BL, WW2-BL and BL41) that did not express the endogenous gene. HLA AII expression was restored by transfection of the gene in WWI-BL whereas transfectants of BL-41 remained AII-negative. An HLA-AII-promoter-driven chloramphenicol acetyl transferase reporter gene (pAIICAT) was active in WWI-BL but not in BL-41. HLA-AII was expressed in hybrids of BL-41 with an AII-positive LCL, while expression of the endogenous HLA AII gene could not be restored by fusion of BL-41 with an AII-negative LCL, although an adequate set of transcription factors was present in the hybrid. Our results suggest that genetic defects and lack of transcription factors may contribute to the selective down-regulation of HLA AII in BL cells. PMID:7601573

  18. Keratin 8 absence down-regulates colonocyte HMGCS2 and modulates colonic ketogenesis and energy metabolism.

    PubMed

    Helenius, Terhi O; Misiorek, Julia O; Nyström, Joel H; Fortelius, Lina E; Habtezion, Aida; Liao, Jian; Asghar, M Nadeem; Zhang, Haiyan; Azhar, Salman; Omary, M Bishr; Toivola, Diana M

    2015-06-15

    Simple-type epithelial keratins are intermediate filament proteins important for mechanical stability and stress protection. Keratin mutations predispose to human liver disorders, whereas their roles in intestinal diseases are unclear. Absence of keratin 8 (K8) in mice leads to colitis, decreased Na/Cl uptake, protein mistargeting, and longer crypts, suggesting that keratins contribute to intestinal homeostasis. We describe the rate-limiting enzyme of the ketogenic energy metabolism pathway, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), as a major down-regulated protein in the K8-knockout (K8(-/-)) colon. K8 absence leads to decreased quantity and activity of HMGCS2, and the down-regulation is not dependent on the inflammatory state, since HMGCS2 is not decreased in dextran sulfate sodium-induced colitis. Peroxisome proliferator-activated receptor α, a transcriptional activator of HMGCS2, is similarly down-regulated. Ketogenic conditions-starvation or ketogenic diet-increase K8(+/+) HMGCS2, whereas this response is blunted in the K8(-/-) colon. Microbiota-produced short-chain fatty acids (SCFAs), substrates in the colonic ketone body pathway, are increased in stool, which correlates with decreased levels of their main transporter, monocarboxylate transporter 1 (MCT1). Microbial populations, including the main SCFA-butyrate producers in the colon, were not altered in the K8(-/-). In summary, the regulation of the SCFA-MCT1-HMGCS2 axis is disrupted in K8(-/-) colonocytes, suggesting a role for keratins in colonocyte energy metabolism and homeostasis. PMID:25904331

  19. Keratin 8 absence down-regulates colonocyte HMGCS2 and modulates colonic ketogenesis and energy metabolism

    PubMed Central

    Helenius, Terhi O.; Misiorek, Julia O.; Nyström, Joel H.; Fortelius, Lina E.; Habtezion, Aida; Liao, Jian; Asghar, M. Nadeem; Zhang, Haiyan; Azhar, Salman; Omary, M. Bishr; Toivola, Diana M.

    2015-01-01

    Simple-type epithelial keratins are intermediate filament proteins important for mechanical stability and stress protection. Keratin mutations predispose to human liver disorders, whereas their roles in intestinal diseases are unclear. Absence of keratin 8 (K8) in mice leads to colitis, decreased Na/Cl uptake, protein mistargeting, and longer crypts, suggesting that keratins contribute to intestinal homeostasis. We describe the rate-limiting enzyme of the ketogenic energy metabolism pathway, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), as a major down-regulated protein in the K8-knockout (K8−/−) colon. K8 absence leads to decreased quantity and activity of HMGCS2, and the down-regulation is not dependent on the inflammatory state, since HMGCS2 is not decreased in dextran sulfate sodium-induced colitis. Peroxisome proliferator–activated receptor α, a transcriptional activator of HMGCS2, is similarly down-regulated. Ketogenic conditions—starvation or ketogenic diet—increase K8+/+ HMGCS2, whereas this response is blunted in the K8−/− colon. Microbiota-produced short-chain fatty acids (SCFAs), substrates in the colonic ketone body pathway, are increased in stool, which correlates with decreased levels of their main transporter, monocarboxylate transporter 1 (MCT1). Microbial populations, including the main SCFA-butyrate producers in the colon, were not altered in the K8−/−. In summary, the regulation of the SCFA-MCT1-HMGCS2 axis is disrupted in K8−/− colonocytes, suggesting a role for keratins in colonocyte energy metabolism and homeostasis. PMID:25904331

  20. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    SciTech Connect

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa; Shin, Incheol

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  1. Requirements for Cell Rounding and Surface Protein Down-Regulation by Ebola Virus Glycoprotein

    PubMed Central

    Francica, Joseph R.; Matukonis, Meghan K.; Bates, Paul

    2009-01-01

    Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of β1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects. PMID:19013626

  2. Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer

    PubMed Central

    Wendt, Michael K.; Taylor, Molly A.; Schiemann, Barbara J.; Schiemann, William P.

    2011-01-01

    Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-β (TGF-β) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-β. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated β1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating β1 integrin expression necessary for metastatic outgrowth. PMID:21613543

  3. Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket

    PubMed Central

    Descarpentries, Clotilde; Frisan, Emilie; Adam, Kevin; Verdier, Frederique; Floquet, Célia; Dubreuil, Patrice; Lacombe, Catherine; Fontenay, Michaela; Mayeux, Patrick; Kosmider, Olivier

    2013-01-01

    The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket. PMID:23637779

  4. Down-regulation of Wnt10a affects odontogenesis and proliferation in mesenchymal cells

    SciTech Connect

    Liu, Yang Han, Dong Wang, Lei Feng, Hailan

    2013-05-17

    Highlights: •Down-regulation of Wnt10a in dental mesenchymal cells impairs odontogenesis of reassociated tooth germs. •Dspp is down- and up-regulated after Wnt10a-knockdown and overexpression in dental mesenchymal cells. •Down-regulation of Wnt10a inhibits proliferation of dental mesenchymal cells. -- Abstract: The WNT10a mutation has been found in patients with abnormal odontogenesis. In mice, Wnt10a expression is found in the tooth germ, but its role has not yet been elucidated. We aimed to investigate the role of Wnt10a in odontogenesis. Mesenchymal cells of the first mandibular molar germ at the bell stage were isolated, transfected with Wnt10a SiRNA or plasmid, and reassociated with epithelial part of the molar germ. Scrambled SiRNA or empty vector was used in the control group. The reassociated tooth germs were transplanted into mice subrenal capsules. After gene modification, dental mesenchymal cells cultured in vitro were checked for cell proliferation and the expression of Dspp was examined. All 12 reassociated tooth germs in the control group resumed odontogenesis, while only 5 of 12 in the Wnt10a knockdown group developed into teeth. After Wnt10a knockdown, the mesenchymal cells cultured in vitro presented repressed proliferation. Wnt10a knockdown and overexpression led to both down- and up-regulation of Dspp. We conclude that the down-regulation of Wnt10a impairs odontogensis and cell proliferation, and that Wnt10a regulates Dspp expression in mesenchymal cells. These findings help to elucidate the mechanism of abnormal tooth development in patients with the WNT10A mutation.

  5. Cell surface modulation of gene expression in brain cells by down regulation of glucocorticoid receptors

    SciTech Connect

    McGinnis, J.F.; de Vellis, J.

    1981-02-01

    The concentration of glycerol-3-phosphate dehydrogenase (GPDH; sn-glycerol-3-phosphate:NAD/sup +/ 2-oxidoreductase, EC 1.1.1.8) had previously been determined to be regulated by glucocorticoids in rat brain cells in vivo and in cell culture. We now demonstrate that concanavalin A (Con A) can inhibit the induction of GPDH in a dose-dependent manner in C6 rat glioma cells and in primary cultures of rat brain oligodendrocytes. The inhibition specifically prevents the appearance of new molecules of GPDH, although Con A does not significantly inhibit protein synthesis in these cells, nor does it affect the activity of another solube enzyme, lactate dehydrogenase. The ability to block enzyme induction is not limited to Con A, because other lectins also inhibit induction. The molecular mechanism by which Con A inhibits GPDH induction appears to be by the down regulation of the cytoplasmic glucocorticoid receptors, because exposure to Con A results in the loss of more than 90% of the receptor activity. Con A does not inhibit the receptor assay and no direct interaction between the receptor and Con A could be demonstrated. This down regulation is not tumor cell specific and appears to be a general phenomenon, because it occurs in normal oligodendrocytes and even in normal astrocytes (a cell type in which the gene for GPDH is not expressed). The down regulation of glucocorticoid receptors in normal brain cells suggests two important corollaries. First, it demonstrates the existence of a rate-limiting step controlling the glucocorticoid-dependent gene expression in brain cells and possibly represents a regulatory site common to all glucocorticoid target cells. Second, it suggests that the response to glucocorticoids of oligodendrocytes and astrocytes can be regulated in vivo by cell surface contact with endogenous lectins, neighboring cells, or both.

  6. Curcumin and Emodin Down-Regulate TGF-β Signaling Pathway in Human Cervical Cancer Cells

    PubMed Central

    Thacker, Pooja Chandrakant; Karunagaran, Devarajan

    2015-01-01

    Cervical cancer is the major cause of cancer related deaths in women, especially in developing countries and Human Papilloma Virus infection in conjunction with multiple deregulated signaling pathways leads to cervical carcinogenesis. TGF-β signaling in later stages of cancer is known to induce epithelial to mesenchymal transition promoting tumor growth. Phytochemicals, curcumin and emodin, are effective as chemopreventive and chemotherapeutic compounds against several cancers including cervical cancer. The main objective of this work was to study the effect of curcumin and emodin on TGF-β signaling pathway and its functional relevance to growth, migration and invasion in two cervical cancer cell lines, SiHa and HeLa. Since TGF-β and Wnt/β-catenin signaling pathways are known to cross talk having common downstream targets, we analyzed the effect of TGF-β on β-catenin (an important player in Wnt/β-catenin signaling) and also studied whether curcumin and emodin modulate them. We observed that curcumin and emodin effectively down regulate TGF-β signaling pathway by decreasing the expression of TGF-β Receptor II, P-Smad3 and Smad4, and also counterbalance the tumorigenic effects of TGF-β by inhibiting the TGF-β-induced migration and invasion. Expression of downstream effectors of TGF-β signaling pathway, cyclinD1, p21 and Pin1, was inhibited along with the down regulation of key mesenchymal markers (Snail and Slug) upon curcumin and emodin treatment. Curcumin and emodin were also found to synergistically inhibit cell population and migration in SiHa and HeLa cells. Moreover, we found that TGF-β activates Wnt/β-catenin signaling pathway in HeLa cells, and curcumin and emodin down regulate the pathway by inhibiting β-catenin. Taken together our data provide a mechanistic basis for the use of curcumin and emodin in the treatment of cervical cancer. PMID:25786122

  7. Photoactivation of hypericin down-regulates glutathione S-transferase activity in nasopharyngeal cancer cells.

    PubMed

    Du, H Y; Olivo, M; Tan, B K H; Bay, B H

    2004-04-30

    Photodynamic therapy (PDT) is a new modality of treatment for cancer. Hypericin is a photosensitizer, which is known to generate reactive oxygen species upon activation with light. We observed that photoactivated hypericin induces the generation of reactive oxygen intermediates in nasopharyngeal cancer (NPC) cells in vitro. There was also significant reduction of Glutathione S-transferase (GST) activity in HK1 and CNE-2 NPC cells and in tumor tissues from the NPC/HK1 murine tumor model by hypericin-mediated PDT. As antioxidants protect cells against phototoxicity, down-regulation of GST activity would potentiate the efficacy of hypericin-PDT treatment. PMID:15072826

  8. Do we live in a largely top-down regulated world?

    PubMed

    Banse, Karl

    2007-06-01

    Based on a review of mostly recent literature for a public lecture, the question is discussed whether we live in a largely "top-down" regulated world rather than one formed "bottom-up" by the resources for plant and animal growth. Of course, the top-down mechanism is predicated by bottom-up production, especially by the plants. Examples for the effects of grazing and predation for the land and the open sea, but including coral reefs, are discussed. The answer to the question posed by the title is affirmative. Ecosystems altered by man and urgent needs for marine conservation are briefly treated. PMID:17762153

  9. Down-regulation of insulin receptors is related to insulin internalization

    SciTech Connect

    Geiger, D.; Carpentier, J.L.; Gorden, P.; Orci, L. )

    1989-11-01

    In the present study, we have tested the influence of inhibition of endocytosis by hypertonic medium on the regulation of cell surface insulin receptors. We show that active internalization of {sup 125}I-insulin is markedly inhibited by hypertonic media and that, in parallel, cell surface invaginations are significantly diminished. These two events are accompanied by a marked inhibition of cell surface insulin receptor down-regulation. These data provide further strong evidence that receptor-mediated endocytosis is the major mechanism by which insulin receptors are regulated at the surface of target cells.

  10. Availability of chemosensory receptors is down-regulated by habituation of larvae to a morphogenetic signal.

    PubMed Central

    Trapido-Rosenthal, H G; Morse, D E

    1986-01-01

    Larvae of the gastropod mollusc Haliotis rufescens are induced to settle from the plankton and metamorphose in response to exogenous gamma-aminobutyric acid (GABA) and a number of GABA-mimetic compounds, including a GABA-mimetic inducer uniquely associated with the surfaces of the naturally recruiting algae. Previous evidence has shown that recognition of these inducers is mediated by specialized chemosensory receptors on the larval epithelium and that transduction of the morphogenetic signal then is mediated by cAMP and excitatory depolarization. We demonstrate here the specific and saturable labeling of a population of larval receptors with the GABA analog beta-(p-chlorophenyl)-[3H]GABA ([3H]baclofen); identification of these labeled receptors with those controlling metamorphosis is suggested by four independent criteria: the effectiveness of GABA and its close structural analogs to induce metamorphosis is closely correlated with the effectiveness of these compounds to compete for binding to this receptor; the natural inducer purified from the recruiting algae competes for binding to this receptor; (-)-[3H]baclofen specifically bound to the receptors is shed from the larvae after approximately 20 hr, at the time corresponding to the metamorphic abscission and shedding of sensory cilia and other structures from the larvae; and the availability of the receptors for labeling and the ability of the larvae to respond to GABA and GABA analogs can be down-regulated in parallel by habituation of the larvae early in their development. These down-regulated larvae are fully capable of settlement and metamorphosis in response to agents that elevate intracellular cAMP or depolarize the chemosensory membrane, confirming that down-regulation is confined to the receptors, with no effect on the postreceptor pathway. The results reported here thus suggest that the sensitivity of marine invertebrate larvae to morphogenetic stimuli from the environment can be down-regulated by

  11. p53 and rapamycin are additive

    PubMed Central

    Campisi, Judith; Huang, Jing; Jones, Diane; Dodds, Sherry G.; Williams, Charnae; Hubbard, Gene; Livi, Carolina B.; Gao, Xiaoli; Weintraub, Susan; Curiel, Tyler; Sharp, Z. Dave; Hasty, Paul

    2015-01-01

    Mechanistic target of rapamycin (mTOR) is a kinase found in a complex (mTORC1) that enables macromolecular synthesis and cell growth and is implicated in cancer etiology. The rapamycin-FK506 binding protein 12 (FKBP12) complex allosterically inhibits mTORC1. In response to stress, p53 inhibits mTORC1 through a separate pathway involving cell signaling and amino acid sensing. Thus, these different mechanisms could be additive. Here we show that p53 improved the ability of rapamycin to: 1) extend mouse life span, 2) suppress ionizing radiation (IR)-induced senescence-associated secretory phenotype (SASP) and 3) increase the levels of amino acids and citric acid in mouse embryonic stem (ES) cells. This additive effect could have implications for cancer treatment since rapamycin and p53 are anti-oncogenic. PMID:26158292

  12. Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel.

    PubMed

    Sonnemann, Jürgen; Gekeler, Volker; Ahlbrecht, Katrin; Brischwein, Klaus; Liu, Chao; Bader, Peter; Müller, Cornelia; Niethammer, Dietrich; Beck, James F

    2004-06-25

    Previous studies point to protein kinase C (PKC) isozyme eta as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCeta with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCeta and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCeta or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCeta down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCeta might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy. PMID:15159020

  13. Down-regulation of endothelin binding sites in rat vascular smooth muscle cells

    SciTech Connect

    Roubert, P.; Gillard, V.; Plas, P.; Chabrier, P.E.; Braquet, P. )

    1990-04-01

    In cultured rat aortic smooth muscle cells, ({sup 125}I)endothelin (ET-1) bound to an apparent single class of high affinity recognition sites with a dissociation constant of 1.84 +/- 0.29 nmol/L and a maximum binding of 62 +/- 10.5 fmol/10(6) cells. The binding was not affected by calcium antagonists or vasoactive substances, including angiotensin II, arginine vasopressin, atrial natriuretic factor and bradykinin. Exposure of the cells to ET-1 (0.01 nmol/L to 10 nmol/L) resulted in an apparent dose-dependent reduction of the number of endothelin binding sites with no significant modification of its binding affinity. The time course of the down-regulation of ET-1 binding sites showed that this effect was present after 30 min incubation and persisted after 18 h. This indicates that down-regulation of ET-1 binding sites can modulate the activity of ET-1 and suggests a rapid internalization of ET-1 in vascular cells.

  14. Terpene down-regulation triggers defense responses in transgenic orange leading to resistance against fungal pathogens.

    PubMed

    Rodríguez, Ana; Shimada, Takehiko; Cervera, Magdalena; Alquézar, Berta; Gadea, José; Gómez-Cadenas, Aurelio; De Ollas, Carlos José; Rodrigo, María Jesús; Zacarías, Lorenzo; Peña, Leandro

    2014-01-01

    Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores. PMID:24192451

  15. Glucosamine Modulates T Cell Differentiation through Down-regulating N-Linked Glycosylation of CD25.

    PubMed

    Chien, Ming-Wei; Lin, Ming-Hong; Huang, Shing-Hwa; Fu, Shin-Huei; Hsu, Chao-Yuan; Yen, B Lin-Ju; Chen, Jiann-Torng; Chang, Deh-Ming; Sytwu, Huey-Kang

    2015-12-01

    Glucosamine has immunomodulatory effects on autoimmune diseases. However, the mechanism(s) through which glucosamine modulates different T cell subsets and diseases remain unclear. We demonstrate that glucosamine impedes Th1, Th2, and iTreg but promotes Th17 differentiation through down-regulating N-linked glycosylation of CD25 and subsequently inhibiting its downstream Stat5 signaling in a dose-dependent manner. The effect of glucosamine on T helper cell differentiation was similar to that induced by anti-IL-2 treatment, further supporting an IL-2 signaling-dependent modulation. Interestingly, excess glucose rescued this glucosamine-mediated regulation, suggesting a functional competition between glucose and glucosamine. High-dose glucosamine significantly decreased Glut1 N-glycosylation in Th1-polarized cells. This finding suggests that both down-regulated IL-2 signaling and Glut1-dependent glycolytic metabolism contribute to the inhibition of Th1 differentiation by glucosamine. Finally, glucosamine treatment inhibited Th1 cells in vivo, prolonged the survival of islet grafts in diabetic recipients, and exacerbated the severity of EAE. Taken together, our results indicate that glucosamine interferes with N-glycosylation of CD25, and thereby attenuates IL-2 downstream signaling. These effects suggest that glucosamine may be an important modulator of T cell differentiation and immune homeostasis. PMID:26468284

  16. High glucose induces dysfunction of airway epithelial barrier through down-regulation of connexin 43.

    PubMed

    Yu, Hongmei; Yang, Juan; Zhou, Xiangdong; Xiao, Qian; Lü, Yang; Xia, Li

    2016-03-01

    The airway epithelium is a barrier to the inhaled antigens and pathogens. Connexin 43 (Cx43) has been found to play critical role in maintaining the function of airway epithelial barrier and be involved in the pathogenesis of the diabetic retinal vasculature, diabetes nephropathy and diabetes skin. Hyperglycemia has been shown to be an independent risk factor for respiratory infections. We hypothesize that the down-regulation of Cx43 induced by HG alters the expression of tight junctions (zonula occludens-1 (ZO-1) and occludin) and contributes to dysfunction of airway epithelial barrier, and Cx43 plays a critical role in the process in human airway epithelial cells (16 HBE). We show that high glucose (HG) decreased the expression of ZO-1 and occludin, disassociated interaction between Cx43 and tight junctions, and then increased airway epithelial transepithelial electrical resistance (TER) and permeability by down-regulation of Cx43 in human airway epithelial cells. These observations demonstrate an important role for Cx43 in regulating HG-induced dysfunction of airway epithelial barrier. These findings may bring new insights into the molecular pathogenesis of pulmonary infection related to diabetes mellitus and lead to novel therapeutic intervention for the dysfunction of airway epithelial barrier in chronic inflammatory airway diseases. PMID:26902399

  17. Down-regulation of tissue N:P ratios in terrestrial plants by elevated CO2

    NASA Astrophysics Data System (ADS)

    Deng, Q.; Hui, D.; Luo, Y.; Elser, J. J.; Wang, Y.; Loladze, I.; Zhang, Q.; Dennis, S.

    2015-12-01

    Increasing atmospheric CO2 concentrations generally alter element stoichiometry in plants. However, a comprehensive evaluation of the elevated CO2 impact on plant nitrogen:phosphorus (N:P) ratios and the underlying mechanism has not been conducted. We synthesized the results from 112 previously published studies using meta-analysis to evaluate the effects of elevated CO2 on the N:P ratio of terrestrial plants and to explore the underlying mechanism based on plant growth and soil P dynamics. Our results show that terrestrial plants grown under elevated CO2 had lower N:P ratios in both above- and below-ground biomass across different ecosystem types. The response ratio for plant N:P was negatively correlated with the response ratio for plant growth in croplands and grasslands, and showed a stronger relationship for P than for N. In addition, the CO2-induced down-regulation of plant N:P was accompanied by 19.3% and 4.2% increases in soil phosphatase activity and labile P, respectively, and a 10.1% decrease in total soil P. Our results show that down-regulation of plant N:P under elevated CO2 corresponds with accelerated soil P cycling. These findings should be useful for better understanding of terrestrial plant stoichiometry in response to elevated CO2 and of the underlying mechanisms affecting nutrient dynamics under climate change.

  18. Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

    PubMed

    Werner, Ernst R; Bahrami, Soheyl; Heller, Regine; Werner-Felmayer, Gabriele

    2002-03-22

    GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine. PMID:11799107

  19. Serotonin-induced down-regulation of cell surface serotonin transporter

    PubMed Central

    Jørgensen, Trine Nygaard; Christensen, Peter Møller; Gether, Ulrik

    2014-01-01

    The serotonin transporter (SERT) terminates serotonergic signaling and enables refilling of synaptic vesicles by mediating reuptake of serotonin (5-HT) released into the synaptic cleft. The molecular and cellular mechanisms controlling SERT activity and surface expression are not fully understood. Here we demonstrate that the substrate 5-HT itself causes acute down-regulation of SERT cell surface expression. To assess surface SERT expression by ELISA, we used a SERT variant (TacSERT) where the N-terminus of SERT was fused to the intracellular tail of the extracellularly FLAG-tagged single-membrane spanning protein Tac. In stably transfected HEK293 cells, 5-HT caused a dose-dependent reduction in TacSERT surface signal with an EC50 value equivalent to the Km value observed for 5-HT uptake. The 5-HT-induced reduction in surface signal reached maximum within 40-60 min and was blocked by the selective SERT inhibitor S-citalopram. 5-HT-induced reduction in SERT expression was further supported by surface biotinylation experiments showing 5-HT-induced reduction in wild type SERT plasma membrane levels. Moreover, preincubation with 5-HT lowered the Vmax for 5-HT uptake in cultured raphe serotonergic neurons, indicting that endogenous cell-surface resident SERT likewise is down-regulated in the presence of substrate. PMID:24462583

  20. Potent anti-prostate cancer agents derived from a novel androgen receptor down-regulating agent.

    PubMed

    Purushottamachar, Puranik; Khandelwal, Aakanksha; Vasaitis, Tadas S; Bruno, Robert D; Gediya, Lalji K; Njar, Vincent C O

    2008-04-01

    The search for novel androgen receptor (AR) down-regulating agents by catalyst HipHop pharmacophore modeling led to the discovery of some lead molecules. Unexpectedly, the effect of these leads on human prostate cancer LNCaP cell viability did not correlate with the ability of the compounds to cause down-regulation of AR protein expression. Through rational synthetic optimization of the lead compound (BTB01434), we have discovered a series of novel substituted diaryl molecules as potent anti-prostate cancer agents. Some compounds (1-6) were shown to be extremely potent inhibitors of LNCaP cell viability with GI(50) values in the nanomolar range (1.45-83 nM). The most potent compound (4-methylphenyl)[(4-methylphenyl)sulfonyl]amine (5) with a GI(50) value of 1.45 nM is 27,000 times more potent than our lead compound BTB01434 (GI(50)=39.8 microM). In addition, some of the compounds exhibited modest anti-androgenic activities and one was also a potent inhibitor (GI(50)=850 nM) of PC-3 (AR-null) cell growth. A clear structure-activity relationship (SAR) has been established for activity against LNCaP cells, where potent molecules possess two substituted/unsubstituted aromatic rings connected through a sulfonamide linker. These novel compounds are strong candidates for development for the treatment of hormone-sensitive and importantly hormone-refractory prostate cancers in humans. PMID:18316193

  1. Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

    SciTech Connect

    Grovdal, Lene Melsaether; Johannessen, Lene E.; Rodland, Marianne Skeie; Madshus, Inger Helene; Stang, Espen

    2008-04-01

    The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of {sup 125}I-EGF internalization, whereas internalization of {sup 125}I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized {sup 125}I-EGF was increased, while degradation of {sup 125}I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

  2. Inhibition of CK2α down-regulates Notch1 signalling in lung cancer cells

    PubMed Central

    Zhang, Shulin; Long, Hao; Yang, Yi-Lin; Wang, Yucheng; Hsieh, David; Li, Weiming; Au, Alfred; Stoppler, Hubert J; Xu, Zhidong; Jablons, David M; You, Liang

    2013-01-01

    Protein kinase CK2 is frequently elevated in a variety of human cancers. The Notch1 signalling pathway has been implicated in stem cell maintenance and its aberrant activation has been shown in several types of cancer including lung cancer. Here, we show, for the first time, that CK2α is a positive regulator of Notch1 signalling in lung cancer cell lines A549 and H1299. We found that Notch1 protein level was reduced after CK2α silencing. Down-regulation of Notch1 transcriptional activity was demonstrated after the silencing of CK2α in lung cancer cells. Furthermore, small-molecule CK2α inhibitor CX-4945 led to a dose-dependent inhibition of Notch1 transcriptional activity. Conversely, forced overexpression of CK2α resulted in an increase in Notch1 transcriptional activity. Finally, the inhibition of CK2α led to a reduced proportion of stem-like CD44 + /CD24− cell population. Thus, we report that the inhibition of CK2α down-regulates Notch1 signalling and subsequently reduces a cancer stem-like cell population in human lung cancer cells. Our data suggest that CK2α inhibitors may be beneficial to the lung cancer patients with activated Notch1 signalling. PMID:23651443

  3. CIP2A down regulation enhances the sensitivity of pancreatic cancer cells to gemcitabine

    PubMed Central

    He, Jin; Zhao, Long; Wang, Xiaodong; Li, Zhennan; Qian, Jianjun

    2016-01-01

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein which participates in inhibiting tumor apoptosis in pancreatic cancer cells. Using immunohistochemical staining, we investigated the expression of CIP2A protein in 72 cases of human pancreatic ductal adenocarcinoma (PDAC) tissue and 27 cases of adjacent normal pancreatic tissue. The positive rate of CIP2A protein expression in pancreatic cancer tissue was70.83 %, which was significantly higher than that in adjacent non- cancerous pancreatic tissue (11.11%). The expression of CIP2A was found to be correlated with TNM stage, but not correlated with age, gender, tumor location, smoking status, alcohol consumption, diabetes, high blood pressure, BMI, tumor size, lymph node metastasis or distant metastases. Kaplan- Meier survival analysis showed that patients with positive CIP2A protein expression had a lower overall survival rate than patients without CIP2A expression. COX regression analysis indicated that expression of CIP2A was an independent prognostic factor for pancreatic ductal adenocarcinoma. In addition, down-regulation of CIP2A inhibited cell proliferation and increased sensitivity to gemcitabine in pancreatic cancer cells by decreasing AKT signaling pathway. Our results indicated that down-regulation of CIP2A could be a novel therapeutic strategy for pancreatic cancer. PMID:26894380

  4. Arctiin induces cell growth inhibition through the down-regulation of cyclin D1 expression.

    PubMed

    Matsuzaki, Youichirou; Koyama, Makoto; Hitomi, Toshiaki; Yokota, Tomoya; Kawanaka, Mayumi; Nishikawa, Akiyoshi; Germain, Doris; Sakai, Toshiyuki

    2008-03-01

    Arctiin is a major lignan constituent of Arctium lappa and has anti-cancer properties in animal models. It was recently reported that arctiin induces growth inhibition in human prostate cancer PC-3 cells. However, the growth inhibitory mechanism of arctiin remains unknown. Herein we report that arctiin induces growth inhibition and dephosphorylates the tumor-suppressor retinoblastoma protein in human immortalized keratinocyte HaCaT cells. We also show that the growth inhibition caused by arctiin is associated with the down-regulation of cyclin D1 protein expression. Furthermore, the arctiin-induced suppression of cyclin D1 protein expression occurs in various types of human tumor cells, including osteosarcoma, lung, colorectal, cervical and breast cancer, melanoma, transformed renal cells and prostate cancer. Depletion of the cyclin D1 protein using small interfering RNA-rendered human breast cancer MCF-7 cells insensitive to the growth inhibitory effects of arctiin, implicates cyclin D1 as an important target of arctiin. Taken together, these results suggest that arctiin down-regulates cyclin D1 protein expression and that this at least partially contributes to the anti-proliferative effect of arctiin. PMID:18288407

  5. [Down-regulation of TIPE2 promotes the proliferation and immune activity of T lymphocytes].

    PubMed

    Huang, Lihong; Chen, Jiangyong; Hong, Bin

    2016-07-01

    Objective To utilize specific small interfering RNA (siRNA) to silence the expression of tumor necrosis factor α-induced protein 8 like-2 (TIPE2) gene of T lymphocytes and investigate the effect of TIPE2 targeting siRNA on T lymphocyte proliferation and immune function. Methods Mouse spleen T lymphocytes were sorted by magnetic beads. Western blotting was used to screen and validate an effective siRNA to silence the TIPE2 gene expression of T lymphocytes. Twenty-four hours after transfection with the siRNA into T lymphocytes, the expression of CD69 in each group was detected by flow cytometry. Seventy-two hours after transfection, the proliferation of the T lymphocytes was measured with CCK-8 assay; meanwhile, the secretion levels of interleukin 2 (IL-2) and interferon γ (IFN-γ) in each group were measured by ELISA. Results We obtained TIPE2 targeting siRNA sequences and effectively silenced the expression of TIPE2 gene. After TIPE2 gene expression was down-regulated, the expression of the CD69 on T lymphocytes increased, and the proliferation of T lymphocytes and the secretion of IL-2 and IFN-γ were enhanced. Conclusion Down-regulation of TIPE2 gene expression can promote the T lymphocyte proliferation and immune activity. PMID:27363266

  6. The siRNA-Mediated Down-Regulation of Vascular Endothelial Growth Factor Receptor1

    PubMed Central

    Jafari Sani, Moslem; Yazdi, Foad; Masoomi Karimi, Masoomeh; Alizadeh, Javad; Rahmati, Majid; Zarei Mahmudabadi, Ali

    2016-01-01

    Background Angiogenesis is an important biological process involved in the proliferation of endothelial cells, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is considered as a prominent regulator of angiogenesis which exerts the aforementioned effect(s) through its respective receptors (VEGFR1 and VEGFR2). VEGF receptors are targeted as a therapeutic candidate for cancer growth inhibition. RNAi as a new and promising strategy has provided a useful means to specifically suppress gene expression in cancer cells. Objectives The current study aimed to down-regulate expression of the VEGFR1 using siRNA. Materials and Methods This experimental study designed specific siRNAs against VEGFR1. Total RNA was extracted from human umbilical vain endothelial cell (HUVEC) and subsequently cDNA was synthetized. PCR was performed using specific primers to amplify the target gene. After double digestion and purification, the gene was cloned into pEFGP-N1 expression vector. Then, AGS cells were transfected with recombinant pEGFP-N1 using lipofectamin. The gene expression and down-regulation were evaluated by fluorescence scanning, reverse transcription PCR (RT-PCR) and Western blot techniques. Results Fluorescent scanning, RT-PCR (27.68%) and western blot analysis (31.06%) showed that the expression of VEGFR1 was suppressed effectively. Conclusions The results of the current study showed that specifically designed siRNA can be considered as an appropriate strategy to suppress gene expression and might be a promising tool to prevent angiogenesis. PMID:27275397

  7. Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

    SciTech Connect

    Silva, Patricio; Soto, Nicolás; Díaz, Jorge; Mendoza, Pablo; Díaz, Natalia; Quest, Andrew F.G.; Torres, Vicente A.

    2015-08-21

    The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5 is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo.

  8. Density enhanced phosphatase-1 down-regulates urokinase receptor surface expression in confluent endothelial cells

    PubMed Central

    Brunner, Patrick M.; Heier, Patricia C.; Mihaly-Bison, Judit; Priglinger, Ute; Binder, Bernd R.

    2011-01-01

    VEGF165, the major angiogenic growth factor, is known to activate various steps in proangiogenic endothelial cell behavior, such as endothelial cell migration and invasion, or endothelial cell survival. Thereby, the urokinase-type plasminogen activator (uPA) system has been shown to play an essential role not only by its proteolytic capacities, but also by induction of intracellular signal transduction. Therefore, expression of its cell surface receptor uPAR is thought to be an essential regulatory mechanism in angiogenesis. We found that uPAR expression on the surface of confluent endothelial cells was down-regulated compared with subconfluent proliferating endothelial cells. Regulation of uPAR expression was most probably affected by extracellular signal-regulated kinase 1/2 (ERK1/2) activation, a downstream signaling event of the VEGF/VEGF-receptor system. Consistently, the receptor-like protein tyrosine phosphatase DEP-1 (density enhanced phosphatase-1/CD148), which is abundantly expressed in confluent endothelial cells, inhibited the VEGF-dependent activation of ERK1/2, leading to down-regulation of uPAR expression. Overexpression of active ERK1 rescued the DEP-1 effect on uPAR. That DEP-1 plays a biologic role in angiogenic endothelial cell behavior was demonstrated in endothelial cell migration, proliferation, and capillary-like tube formation assays in vitro. PMID:21304107

  9. Down-Regulation of Rad51 and Decreased Homologous Recombination in Hypoxic Cancer Cells

    PubMed Central

    Bindra, Ranjit S.; Schaffer, Paul J.; Meng, Alice; Woo, Jennifer; Måseide, Kårstein; Roth, Matt E.; Lizardi, Paul; Hedley, David W.; Bristow, Robert G.; Glazer, Peter M.

    2004-01-01

    There is an emerging concept that acquired genetic instability in cancer cells can arise from the dysregulation of critical DNA repair pathways due to cell stresses such as inflammation and hypoxia. Here we report that hypoxia specifically down-regulates the expression of RAD51, a key mediator of homologous recombination in mammalian cells. Decreased levels of Rad51 were observed in multiple cancer cell types during hypoxic exposure and were not associated with the cell cycle profile or with expression of hypoxia-inducible factor. Analyses of RAD51 gene promoter activity, as well as mRNA and protein stability, indicate that the hypoxia-mediated regulation of this gene occurs via transcriptional repression. Decreased expression of Rad51 was also observed to persist in posthypoxic cells for as long as 48 h following reoxygenation. Correspondingly, we found reduced levels of homologous recombination in both hypoxic and posthypoxic cells, suggesting that the hypoxia-associated reduction in Rad51 expression has functional consequences for DNA repair. In addition, hypoxia-mediated down-regulation of Rad51 was confirmed in vivo via immunofluorescent image analysis of experimental tumors in mice. Based on these findings, we propose a novel mechanism of genetic instability in the tumor microenvironment mediated by hypoxia-induced suppression of the homologous recombination pathway in cancer cells. The aberrant regulation of Rad51 expression may also create heterogeneity in the DNA damage response among cells within tumors, with implications for the response to cancer therapies. PMID:15367671

  10. Cryptotanshinone targets tumor-initiating cells through down-regulation of stemness genes expression

    PubMed Central

    ZHANG, YING; CABARCAS, STEPHANIE M.; ZHENG, JI; SUN, LEI; MATHEWS, LESLEY A.; ZHANG, XIAOHU; LIN, HONGSHENG; FARRAR, WILLIAM L.

    2016-01-01

    Recent evidence indicates that tumor-initiating cells (TICs), also called cancer stem cells (CSCs), are responsible for tumor initiation and progression, therefore representing an important cell population that may be used as a target for the development of future anticancer therapies. In the present study, Cryptotanshinone (CT), a traditional Chinese herbal medicine, was demonstrated to regulate the behaviors of LNCaP prostate cells and prostate LNCaP TICs. The results demonstrate that treatment with CT alters cellular proliferation, cell cycle status, migration, viability, colony formation and notably, sphere formation and down-regulation of stemness genes (Nanog, OCT4, SOX2, β-catenin, CXCR4) in TICs. The present study demonstrates that CT targets the LNCaP CD44+CD24- population that is representative of prostate TICs and also affects total LNCaP cells as well via down-regulation of stemness genes. The strong effect with which CT has on prostate TICs suggests that CT may potentially function as a novel natural anticancer agent that specifically targets TICs. PMID:27313698

  11. Curcumin down-regulates AR gene expression and activation in prostate cancer cell lines.

    PubMed

    Nakamura, Keiichiro; Yasunaga, Yutaka; Segawa, Takehiko; Ko, Daejin; Moul, Judd W; Srivastava, Shiv; Rhim, Johng S

    2002-10-01

    Curcumin, traditionally used as a seasoning spice in Indian cuisine, has been reported to decrease the proliferation potential of prostate cancer cells, by a mechanism that is not fully understood. In the current study, we have evaluated the effects of curcumin in cell growth, activation of signal transduction, and transforming activities of both androgen-dependent and independent cell lines. Prostate cancer cell lines, LNCaP and PC-3, were treated with curcumin and its effects were further analyzed on signal transduction and expression of androgen receptor (AR) and AR-related cofactors using transient transfection assay and Western blotting. Our results show that curcumin down-regulates transactivation and expression of AR, activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), and CREB (cAMP response element-binding protein)-binding protein (CBP). Curcumin also inhibited the transforming activities of both cell lines as evidenced by the reduced colony forming ability in soft agar. The results obtained here demonstrate that curcumin has a potential therapeutic effect on prostate cancer cells through down-regulation of AR and AR-related cofactors (AP-1, NF-kappaB and CBP). PMID:12239622

  12. Down-Regulation of Brush Border Efflux Transporter Expression in the Kidneys of Pregnant Mice

    PubMed Central

    Yacovino, Lindsay L.; Gibson, Christopher J.

    2013-01-01

    Pregnancy increases the urinary excretion of chemicals in women and rodents. It is unknown whether the enhanced clearance of drugs during pregnancy involves changes in the expression of transporters that mediate chemical secretion and reabsorption. The purpose of this study was to quantify the mRNA and protein expression of efflux transporters in kidneys from virgin and pregnant mice on gestational days 7, 11, 14, and 17 and postnatal days 1, 15, and 30 with use of quantitative polymerase chain reaction, Western blot, and immunofluorescence. Multidrug resistance protein (Mdr) 1b mRNA, multidrug resistance-associated protein (Mrp) 4 mRNA, and protein levels decreased significantly by 25–75% throughout pregnancy and lactation. Similarly, Mrp2 and multidrug and toxin extrusion transporter (Mate) 1 mRNA expression were down-regulated 20–40% during mid to late gestation but returned to control levels by postnatal day 15. In contrast, Mrp3 mRNA and protein increased 225% and 31%, respectively, at gestational day 14. Coordinated down-regulation of brush border transporters Mate1, Mrp2, and Mrp4 and up-regulation of the basolateral Mrp3 transporter would reduce chemical secretion into urine. PMID:22896729

  13. Down-regulation of tissue N:P ratios in terrestrial plants by elevated CO2.

    PubMed

    Deng, Qi; Hui, Dafeng; Luo, Yiqi; Elser, James; Wang, Ying-ping; Loladze, Irakli; Zhang, Quanfa; Dennis, Sam

    2015-12-01

    Increasing atmospheric CO2 concentrations generally alter element stoichiometry in plants. However, a comprehensive evaluation of the elevated CO2 impact on plant nitrogen: phosphorus (N:P) ratios and the underlying mechanism has not been conducted. We synthesized the results from 112 previously published studies using meta-analysis to evaluate the effects of elevated CO2 on the N:P ratio of terrestrial plants and to explore the underlying mechanism based on plant growth and soil P dynamics. Our results show that terrestrial plants grown under elevated CO2 had lower N:P ratios in both above- and belowground biomass across different ecosystem types. The response ratio for plant N:P was negatively correlated with the response ratio for plant growth in croplands and grasslands, and showed a stronger relationship for P than for N. In addition, the CO2-induced down-regulation of plant N:P was accompanied by 19.3% and 4.2% increases in soil phosphatase activity and labile P, respectively, and a 10.1% decrease in total soil P. Our results show that down-regulation of plant N:P under elevated CO2 corresponds with accelerated soil P cycling. These findings should be useful for better understanding of terrestrial plant stoichiometry in response to elevated CO2 and of the underlying mechanisms affecting nutrient dynamics under climate change. PMID:26909440

  14. Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function

    SciTech Connect

    Yoshihara, Takashi; Collado, Denise; Hamaguchi, Masaaki . E-mail: hamaguchi@fordham.edu

    2007-07-13

    The expression of tumor suppressor gene DBC2 causes certain breast cancer cells to stop growing [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. Recently, DBC2 was found to participate in diverse cellular functions such as protein transport, cytoskeleton regulation, apoptosis, and cell cycle control [V. Siripurapu, J.L. Meth, N. Kobayashi, M. Hamaguchi, DBC2 significantly influences cell cycle, apoptosis, cytoskeleton, and membrane trafficking pathways. J. Mol. Biol. 346 (2005) 83-89]. Its tumor suppression mechanism, however, remains unclear. In this paper, we demonstrate that DBC2 suppresses breast cancer proliferation through down-regulation of Cyclin D1 (CCND1). Additionally, the constitutional overexpression of CCND1 prevented the negative impact of DBC2 expression on their growth. Under a CCND1 promoter, the expression of CCNE1 exhibited the same protective effect. Our results indicate that the down-regulation of CCND1 is an essential step for DBC2's growth suppression of cancer cells. We believe that this discovery contributes to a better understanding of DBC2's tumor suppressor function.

  15. TEL2 suppresses metastasis by down-regulating SERPINE1 in nasopharyngeal carcinoma

    PubMed Central

    Zhang, Ru-Hua; Wang, Li; Li, Mei; Luo, Rongzhen; Qian, Chao-Nan; Shao, Jian-Yong; Zeng, Yi-Xin; Kang, Tiebang

    2015-01-01

    Metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). However, the molecular mechanisms of NPC metastasis are poorly understood. Here, using our customized gene microarray containing all of the known human transcription factors and the current markers for epithelial-mesenchymal transition, we report that TEL2 was down-regulated in highly metastatic NPC cells and the metastatic tissues in lymph node. Mechanistically, TEL2 inhibits the cell migration and invasion in vitro and metastasis in vivo by releasing its direct suppression on the SERPINE1 promoter in NPC. Consistently, an inverse correlation was observed between the protein levels of TEL2 and SERPINE1 using clinical NPC samples. Collectively, we have provided the first evidence that TEL2 plays a key role in NPC metastasis by directly down-regulating SERPINE1, and that this novel axis of TEL2 / SERPINE1 may be valuable to develop new strategies for treating NPC patients with metastasis. PMID:26335051

  16. CyclinD1 Down-Regulation and Increased Apoptosis Are Common Features of Cohesinopathies.

    PubMed

    Fazio, Grazia; Gaston-Massuet, Carles; Bettini, Laura Rachele; Graziola, Federica; Scagliotti, Valeria; Cereda, Anna; Ferrari, Luca; Mazzola, Mara; Cazzaniga, Gianni; Giordano, Antonio; Cotelli, Franco; Bellipanni, Gianfranco; Biondi, Andrea; Selicorni, Angelo; Pistocchi, Anna; Massa, Valentina

    2016-03-01

    Genetic variants within components of the cohesin complex (NIPBL, SMC1A, SMC3, RAD21, PDS5, ESCO2, HDAC8) are believed to be responsible for a spectrum of human syndromes known as "cohesinopathies" that includes Cornelia de Lange Syndrome (CdLS). CdLS is a multiple malformation syndrome affecting almost any organ and causing severe developmental delay. Cohesinopathies seem to be caused by dysregulation of specific developmental pathways downstream of mutations in cohesin components. However, it is still unclear how mutations in different components of the cohesin complex affect the output of gene regulation. In this study, zebrafish embryos and SMC1A-mutated patient-derived fibroblasts were used to analyze abnormalities induced by SMC1A loss of function. We show that the knockdown of smc1a in zebrafish impairs neural development, increases apoptosis, and specifically down-regulates Ccnd1 levels. The same down-regulation of cohesin targets is observed in SMC1A-mutated patient fibroblasts. Previously, we have demonstrated that haploinsufficiency of NIPBL produces similar effects in zebrafish and in patients fibroblasts indicating a possible common feature for neurological defects and mental retardation in cohesinopathies. Interestingly, expression analysis of Smc1a and Nipbl in developing mouse embryos reveals a specific pattern in the hindbrain, suggesting a role for cohesins in neural development in vertebrates. PMID:26206533

  17. Down-Regulated CK8 Expression in Human Intervertebral Disc Degeneration

    PubMed Central

    Sun, Zhen; Wang, Hai-Qiang; Liu, Zhi-Heng; Chang, Le; Chen, Yu-Fei; Zhang, Yong-Zhao; Zhang, Wei-Lin; Gao, Yang; Wan, Zhong-Yuan; Che, Lu; Liu, Xu; Samartzis, Dino; Luo, Zhuo-Jing

    2013-01-01

    As an intermediate filament protein, cytokeratin 8 (CK8) exerts multiple cellular functions. Moreover, it has been identified as a marker of notochord cells, which play essential roles in human nucleus pulposus (NP). However, the distribution of CK8 positive cells in human NP and their relationship with intervertebral disc degeneration (IDD) have not been clarified until now. Here, we found the percentage of CK8 positive cells in IDD (25.7±4.14%) was significantly lower than that in normal and scoliosis NP (51.9±9.73% and 47.8±5.51%, respectively, p<0.05). Western blotting and qRT-PCR results confirmed the down-regulation of CK8 expression in IDD on both of protein and mRNA levels. Furthermore, approximately 37.4% of cell clusters were CK8 positive in IDD. Taken together, this is the first study to show a down-regulated CK8 expression and the percentage of CK8 positive cell clusters in IDD based upon multiple lines of evidence. Consequently, CK8 positive cells might be considered as a potential option in the development of cellular treatment strategies for NP repair. PMID:23801880

  18. Amino acid limitation induces down-regulation of WNT5a at transcriptional level

    SciTech Connect

    Wang Zuguang; Chen Hong

    2009-01-23

    An aberrant WNT signaling contributes to the development and progression of multiple cancers. WNT5a is one of the WNT signaling molecules. This study was designed to test the hypothesis that amino acid deprivation induces changes in the WNT signaling pathway in colon cancer cells. Results showed that targets of the amino acid response pathway, ATF3 and p21, were induced in the human colon cancer cell line SW480 during amino acid limitation. There was a significant decrease in the WNT5a mRNA level following amino acid deprivation. The down-regulation of WNT5a mRNA by amino acid deprivation is not due to mRNA destabilization. There is a reduction of nuclear {beta}-catenin protein level by amino acid limitation. Under amino acid limitation, phosphorylation of ERK1/2 was increased and the blockage of ERK1/2 by the inhibitor U0126 partially restored WNT5a mRNA level. In conclusion, amino acid limitation in colon cancer cells induces phosphorylation of ERK1/2, which then down-regulates WNT5a expression.

  19. Down-regulation of cyclooxygenase-2 (COX-2) by cannabidiolic acid in human breast cancer cells.

    PubMed

    Takeda, Shuso; Okazaki, Hiroyuki; Ikeda, Eriko; Abe, Satomi; Yoshioka, Yasushi; Watanabe, Kazuhito; Aramaki, Hironori

    2014-01-01

    Metastases are known to be responsible for approximately 90% of breast cancer-related deaths. Cyclooxygenase-2 (COX-2) is involved not only in inflammatory processes, but also in the metastasis of cancer cells; it is expressed in 40% of human invasive breast cancers. To comprehensively analyze the effects of cannabidiolic acid (CBDA), a selective COX-2 inhibitor found in the fiber-type cannabis plant (Takeda et al., 2008), on COX-2 expression and the genes involved in metastasis, we performed a DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are invasive breast cancer cells that express high levels of COX-2, treated with CBDA for 48 hr at 25 µM. The results obtained revealed that COX-2 and Id-1, a positive regulator of breast cancer metastasis, were down-regulated (0.19-fold and 0.52-fold, respectively), while SHARP1 (or BHLHE41), a suppressor of breast cancer metastasis, was up-regulated (1.72-fold) and CHIP (or STUB1) was unaffected (1.03-fold). These changes were confirmed by real-time RT-PCR analyses. Taken together, the results obtained here demonstrated that i) CBDA had dual inhibitory effects on COX-2 through down-regulation and enzyme inhibition, and ii) CBDA may possess the ability to suppress genes that are positively involved in the metastasis of cancer cells in vitro. PMID:25242400

  20. Phosphorylation-dependent down-regulation of apolipoprotein A5 by insulin

    SciTech Connect

    Nowak, Maxine; Helleboid-Chapman, Audrey; Jakel, Heidelinde; Rommens, Corinne; Martin, Genevieve; Duran-Sandoval, Daniel; Staels, Bart; Rubin, Edward M.; Pennacchio, Len A.; Taskinen, Marja-Riitta; Fruchart-Najib, Jamila; Fruchart, Jean-Charles

    2004-02-15

    The apolipoprotein A5 (APOA5) gene has been shown to be important in lowering plasma triglyceride levels. Since several studies have shown that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 gene is regulated by insulin. We show here that cell and mouse treatments with insulin down-regulated APOA5 expression in a dose-dependent manner. Furthermore, we determined that insulin decreases APOA5 promoter activity and subsequent deletion analyses revealed an E-box-containing fragment. We showed that Upstream Stimulatory Factors, USF1/USF2, bind to the identified E-box in the APOA5 promoter. Moreover, in cotransfection studies, USF1 stimulates APOA5 promoter activity. The treatment with insulin reduces the binding of USF1/USF2 to APOA5 promoter. The inhibition of PI3K pathway with wortmannin abolished the insulin s effect on APOA5 gene transcription. Using oligoprecipitation method of USF from nuclear extracts, we demonstrated that phosphorylated USF1 failed to bind to APOA5 promoter. This indicates that the APOA5 gene transrepression by insulin involves a phosphorylation of USF through PI3K, that modulate their binding to APOA5 promoter and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in healthy men shows a decrease of the plasma ApoAV level. These data suggest a potential mechanism involving APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.

  1. Effects of p21 Gene Down-Regulation through RNAi on Antler Stem Cells In Vitro

    PubMed Central

    Guo, Qianqian; Wang, Datao; Liu, Zhen; Li, Chunyi

    2015-01-01

    Cell cycle is an integral part of cell proliferation, and consists mainly of four phases, G1, S, G2 and M. The p21 protein, a cyclin dependent kinase inhibitor, plays a key role in regulating cell cyclevia G1 phase control. Cells capable of epimorphic regeneration have G2/M accumulation as their distinctive feature, whilst the majority of somatic cells rest at G1 phase. To investigate the role played byp21 in antler regeneration, we studied the cell cycle distribution of antler stem cells (ASCs), via down-regulation of p21 in vitro using RNAi. The results showed that ASCs had high levels of p21 mRNA expression and rested at G1 phase, which was comparable to the control somatic cells. Down-regulation of p21 did not result in ASC cell cycle re-distribution toward G2/M accumulation, but DNA damage and apoptosis of the ASCs significantly increased and the process of cell aging was slowed. These findings suggest that the ASCs may have evolved to use an alternative, p21-independent cell cycle regulation mechanism. Also a unique p21-dependent inhibitory effect may control DNA damage as a protective mechanism to ensure the fast proliferating ASCs do not become dysplastic/cancerous. Understanding of the mechanism underlying the role played by p21 in the ASCs could give insight into a mammalian system where epimorphic regeneration is initiated whilst the genome stability is effectively maintained. PMID:26308075

  2. Paeoniflorin ameliorates symptoms of experimental Sjogren's syndrome associated with down-regulating Cyr61 expression.

    PubMed

    Li, Huidan; Sun, Xiaoxuan; Zhang, Jie; Sun, Yue; Huo, Rongfen; Li, Haichuan; Zhai, Tianhang; Shen, Baihua; Zhang, Miaojia; Li, Ningli

    2016-01-01

    Paeoniflorin (PF), an active compound extracted from Paeony root, has been used in therapy of autoimmune diseases with effective clinical efficiency and higher safety. Sjogren's syndrome (SS) is a chronic, systemic, immune-mediated inflammatory disease. In this study, we demonstrated that novel pro-inflammatory factor Cyr61/CCN1 was up-regulated in epithelial cells of salivary glands of primary SS patients and submandibular gland autoantigen-induced experimental SS mice. Blocking Cyr61 expression with special monoclonal antibody improved saliva secretion by ameliorating inflammatory infiltration and cytokines production in vivo. Furthermore, we showed that PF could alleviate inflammation by down-regulating Cyr61 expression in experimental SS mice. In conclusion, our new findings revealed for the first time that Cyr61 involves the pathogenesis of primary SS and PF alleviates SS-like symptoms associated with inhibiting Cyr61 expression, providing new insights into the potential molecular mechanism of PF in primary SS treatment. PMID:26630293

  3. Natural polyphenols down-regulate universal stress protein in Mycobacterium tuberculosis: An in-silico approach.

    PubMed

    Aanandhi, M Vijey; Bhattacherjee, Debojit; George, P Samuel Gideon; Ray, Anirban

    2014-10-01

    Universal stress protein (USP) is a novel target to overcome the tuberculosis resistance. Our present study enlightens the possibilities of some natural polyphenols as an antioxidant for USP. The study has shown some molecular simulations of some selected natural antioxidants with USP. We have considered USP (Rv1636) strain for homology modeling and the selected template was taken for the docking study. Curcumin, catechin, reservetrol has shown ARG 136 (1.8Å) hydrogen bonding and two ionic bonding with carboxyl group of curcumin with LEU 130 (3.3Å) and ASN 144 (3.4Å) respectively. INH was taken for the standard molecule to perform molecular simulation. It showed poor binding interaction with the target, that is, -5.18 kcal, and two hydrogen bonding with SER 140 (1.887Å), ARG 147 (2.064Å) respectively. The study indicates possible new generation curcumin analogue for future therapy to down-regulate USP. PMID:25364695

  4. Lithium Down-regulates Histone Deacetylase 1 (HDAC1) and Induces Degradation of Mutant Huntingtin*

    PubMed Central

    Wu, Shuai; Zheng, Shui-Di; Huang, Hong-Ling; Yan, Li-Chong; Yin, Xiao-Fei; Xu, Hai-Neng; Zhang, Kang-Jian; Gui, Jing-Hua; Chu, Liang; Liu, Xin-Yuan

    2013-01-01

    Lithium is an effective mood stabilizer that has been clinically used to treat bipolar disorder for several decades. Recent studies have suggested that lithium possesses robust neuroprotective and anti-tumor properties. Thus far, a large number of lithium targets have been discovered. Here, we report for the first time that HDAC1 is a target of lithium. Lithium significantly down-regulated HDAC1 at the translational level by targeting HDAC1 mRNA. We also showed that depletion of HDAC1 is essential for the neuroprotective effects of lithium and for the lithium-mediated degradation of mutant huntingtin through the autophagic pathway. Our studies explain the multiple functions of lithium and reveal a novel mechanism for the function of lithium in neurodegeneration. PMID:24165128

  5. Hematopoietic lineage cell specific protein 1 associates with and down-regulates protein kinase CK2.

    PubMed

    Ruzzene, M; Brunati, A M; Sarno, S; Donella-Deana, A; Pinna, L A

    1999-11-12

    The catalytic (alpha) subunit of protein kinase CK2 and the hematopoietic specific protein 1 (HS1) display opposite effects on Ha-ras induced fibroblast transformation, by enhancing and counteracting it, respectively. Here we show the occurrence of physical association between HS1 and CK2alpha as judged from both far Western blot and plasmon resonance (BIAcore) analysis. Association of HS1 with CK2alpha is drastically reduced by the deletion of the HS1 C-terminal region (403-486) containing an SH3 domain. HS1, but not its deletion mutant HS1 Delta324-393, lacking a sequence similar to an acidic stretch of the regulatory beta-subunit of CK2, inhibits calmodulin phosphorylation by CK2alpha. These data indicate that HS1 physically interacts with CK2alpha and down-regulates its activity by a mechanism similar to the beta-subunit. PMID:10561491

  6. Down-regulation of telomerase activity in DLD-1 human colorectal adenocarcinoma cells by tocotrienol

    SciTech Connect

    Eitsuka, Takahiro; Nakagawa, Kiyotaka; Miyazawa, Teruo . E-mail: miyazawa@biochem.tohoku.ac.jp

    2006-09-15

    As high telomerase activity is detected in most cancer cells, inhibition of telomerase by drug or dietary food components is a new strategy for cancer prevention. Here, we investigated the inhibitory effect of vitamin E, with particular emphasis on tocotrienol (unsaturated vitamin E), on human telomerase in cell-culture study. As results, tocotrienol inhibited telomerase activity of DLD-1 human colorectal adenocarcinoma cells in time- and dose-dependent manner, interestingly, with {delta}-tocotrienol exhibiting the highest inhibitory activity. Tocotrienol inhibited protein kinase C activity, resulting in down-regulation of c-myc and human telomerase reverse transcriptase (hTERT) expression, thereby reducing telomerase activity. In contrast to tocotrienol, tocopherol showed very weak telomerase inhibition. These results provide novel evidence for First time indicating that tocotrienol acts as a potent candidate regulator of telomerase and supporting the anti-proliferative function of tocotrienol.

  7. Dioscin enhances methotrexate absorption by down-regulating MDR1 in vitro and in vivo.

    PubMed

    Wang, Lijuan; Wang, Changyuan; Peng, Jinyong; Liu, Qi; Meng, Qiang; Sun, Huijun; Huo, Xiaokui; Sun, Pengyuan; Yang, Xiaobo; Zhen, Yuhong; Liu, Kexin

    2014-06-01

    The purpose of this study was to investigate the enhancing effect of dioscin on the absorption of methotrexate (MTX) and clarify the molecular mechanism involved in vivo and in vitro. Dioscin increased MTX chemosensitivity and transepithelial flux in the absorptive direction, significantly inhibiting multidrug resistance 1 (MDR1) mRNA and protein expression and MDR1 promoter and nuclear factor κ-B (NF-κB) activities in Caco-2 cells. Moreover, inhibitor κB-α (IκB-α) degradation was inhibited by dioscin. Dioscin enhanced the intracellular concentration of MTX by down-regulating MDR1 expression through a mechanism that involves NF-κB signaling pathway inhibition in Caco-2 cells. Dioscin strengthened MTX absorption by inhibiting MDR1 expression in rat intestine. In addition, even though MTX is absorbed into the enterocytes, there was no increase in toxicity observed, and that, in fact, decreased toxicity was seen. PMID:24680847

  8. Mutations and Down-Regulation of CDX1 in Children with Anorectal Malformations

    PubMed Central

    Zhang, Tao; Tang, Xiao Bing; Wang, Li Li; Bai, Yu Zuo; Qiu, Guang Rong; Yuan, Zheng Wei; Wang, Wei Lin

    2013-01-01

    Background: Anorectal malformations (ARMs) represent a variety of congenital disorders that involve abnormal termination of the anorectum. This study was to reveal relation between CDX1 and human ARMs phenotypes. Methods: 108 Chinese patients and 120 Chinese controls were included in this study. We analyzed the relation between two by PCR, qRT-PCR, western blot and immunofluorescence. Results: Four heterozygous mutations in CDX1 gene were identified in ARMs patients (3.7%, 4/108), no found in controls. CDX1 protein expression was significantly decreased in the ARMs compared with the control anorectum. All samples analyzed in ARMs group exhibited down-regulated CDX1 mRNA expression in comparison to matched normal group, demonstrated significant differences statistically. Conclusion: The findings represented the relation between CDX1 mutations and CDX1 genotype. Furthermore, it was suggested that the downregulation of CDX1 might be related to the development of ARMs. PMID:23329892

  9. Matrix Rigidity Activates Wnt Signaling through Down-regulation of Dickkopf-1 Protein*

    PubMed Central

    Barbolina, Maria V.; Liu, Yiuying; Gurler, Hilal; Kim, Mijung; Kajdacsy-Balla, Andre A.; Rooper, Lisa; Shepard, Jaclyn; Weiss, Michael; Shea, Lonnie D.; Penzes, Peter; Ravosa, Matthew J.; Stack, M. Sharon

    2013-01-01

    Cells respond to changes in the physical properties of the extracellular matrix with altered behavior and gene expression, highlighting the important role of the microenvironment in the regulation of cell function. In the current study, culture of epithelial ovarian cancer cells on three-dimensional collagen I gels led to a dramatic down-regulation of the Wnt signaling inhibitor dickkopf-1 with a concomitant increase in nuclear β-catenin and enhanced β-catenin/Tcf/Lef transcriptional activity. Increased three-dimensional collagen gel invasion was accompanied by transcriptional up-regulation of the membrane-tethered collagenase membrane type 1 matrix metalloproteinase, and an inverse relationship between dickkopf-1 and membrane type 1 matrix metalloproteinase was observed in human epithelial ovarian cancer specimens. Similar results were obtained in other tissue-invasive cells such as vascular endothelial cells, suggesting a novel mechanism for functional coupling of matrix adhesion with Wnt signaling. PMID:23152495

  10. Genes down-regulated in spaceflight are involved in the control of longevity in Caenorhabditis elegans

    PubMed Central

    Honda, Yoko; Higashibata, Akira; Matsunaga, Yohei; Yonezawa, Yukiko; Kawano, Tsuyoshi; Higashitani, Atsushi; Kuriyama, Kana; Shimazu, Toru; Tanaka, Masashi; Szewczyk, Nathaniel J.; Ishioka, Noriaki; Honda, Shuji

    2012-01-01

    How microgravitational space environments affect aging is not well understood. We observed that, in Caenorhabditis elegans, spaceflight suppressed the formation of transgenically expressed polyglutamine aggregates, which normally accumulate with increasing age. Moreover, the inactivation of each of seven genes that were down-regulated in space extended lifespan on the ground. These genes encode proteins that are likely related to neuronal or endocrine signaling: acetylcholine receptor, acetylcholine transporter, choline acetyltransferase, rhodopsin-like receptor, glutamate-gated chloride channel, shaker family of potassium channel, and insulin-like peptide. Most of them mediated lifespan control through the key longevity-regulating transcription factors DAF-16 or SKN-1 or through dietary-restriction signaling, singly or in combination. These results suggest that aging in C. elegans is slowed through neuronal and endocrine response to space environmental cues. PMID:22768380

  11. RNF4 negatively regulates NF-κB signaling by down-regulating TAB2.

    PubMed

    Tan, Bo; Mu, Rui; Chang, Yan; Wang, Yu-Bo; Wu, Min; Tu, Hai-Qing; Zhang, Yu-Cheng; Guo, Sai-Sai; Qin, Xuan-He; Li, Tao; Li, Wei-Hua; Zhang, Xue-Min; Li, Ai-Ling; Li, Hui-Yan

    2015-09-14

    Most of NF-κB (nuclear factor kappa B) signaling molecules have various types of post-translational modifications. In this study, we focused on ubiquitination and designed a siRNA library including most ubiquitin-binding domains. With this library, we identified several candidate regulators of canonical NF-κB pathway, including RNF4. Overexpression of RNF4 impaired NF-κB activation in a dose-dependent manner, whereas RNF4 knockdown potentiated NF-κB activation. We showed that RNF4 interacts with the TAK1-TAB2-TAB3 complex, but not TAB1. Further, we found that RNF4 specifically down-regulated TAB2 through a lysosomal pathway, and knockdown of RNF4 impaired endogenous TAB2 degradation. Therefore, our findings will provide new insights into the negative regulation of NF-κB signaling. PMID:26299341

  12. Mechanism of endogenous myc gene down-regulation in E mu-N-myc tumors.

    PubMed

    Ma, A; Smith, R K; Tesfaye, A; Achacoso, P; Dildrop, R; Rosenberg, N; Alt, F W

    1991-01-01

    Transgenic mouse lines carrying the N-myc oncogene deregulated by the immunoglobulin heavy-chain enhancer spontaneously develop B-lymphoid tumors (R. Dildrop, A. Ma, K. Zimmerman, E. Hsu, A. Tesfaye, R. DePinho, and F. W. Alt, EMBO J. 8:1121-1128, 1989; H. Rosenbaum, E. Webb, J. M. Adams, S. Cory, and A. W. Harris, EMBO J. 8:749-755). Permanent cell lines derived from these tumors (E mu-N-myc cell lines) express extremely high levels of the N-myc transgene but little or no detectable endogenous N-myc or c-myc. We have employed nuclear run-on assays to show that down-regulation of endogenous N- and c-myc expression occurs at the transcriptional level. To determine whether the lack of endogenous myc gene transcription is a direct effect of high-level N-myc transgene expression, we have generated Abelson murine leukemia virus (A-MuLV)-transformed cell lines from prelymphomatous E mu-N-myc mice (A-MuLV/E mu-N-myc cell lines). Although these A-MuLV/E mu-N-myc lines express very high levels of the N-myc transgene, they continue to transcribe the endogenous c-myc gene. These findings demonstrate that high-level N-myc gene expression alone does not necessarily lead to down-regulation of endogenous myc gene expression and suggest that events associated with transformation by N-myc may be critical to this process. PMID:1986238

  13. In Vitro Ischemia Triggers a Transcriptional Response to Down-Regulate Synaptic Proteins in Hippocampal Neurons

    PubMed Central

    Fernandes, Joana; Vieira, Marta; Carreto, Laura; Santos, Manuel A. S.; Duarte, Carlos B.; Carvalho, Ana Luísa; Santos, Armanda E.

    2014-01-01

    Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells, which is partially associated with the induction or repression of genes that influence the ischemic response. However, the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults, we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD), an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD, total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes, whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD, confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins, such as those encoding for PICK1, GRIP1, TARPγ3, calsyntenin-2/3, SAPAP2 and SNAP-25, were down-regulated after OGD. Additionally, OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors, but increased the mRNA expression of the GluN3A subunit, thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together, our results present the expression profile elicited by in vitro ischemia in hippocampal neurons, and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins, suggesting that the synaptic proteome may change after ischemia. PMID:24960035

  14. Down-regulation of intestinal epithelial innate response by probiotic yeasts isolated from kefir.

    PubMed

    Romanin, David; Serradell, María; González Maciel, Dolores; Lausada, Natalia; Garrote, Graciela L; Rumbo, Martín

    2010-06-15

    Kefir is obtained by milk fermentation with a complex microbial population included in a matrix of polysaccharide and proteins. Several health-promoting activities has been attributed to kefir consumption. The aim of this study was to select microorganisms from kefir able to down-regulate intestinal epithelial innate response and further characterize this activity. Caco-2 cells stably transfected with a human CCL20 promoter luciferase reporter were used to screen a collection of 24 yeast and 23 bacterial strains isolated from kefir. The Toll-like receptor 5 agonist, flagellin was used to activate the reporter cells, while pre-incubation with the selected strains was tested to identify strains with the capacity to inhibit cell activation. In this system, 21 yeast strains from the genera Saccharomyces, Kluyveromyces and Issatchenkia inhibited almost 100% of the flagellin-dependent activation, whereas only some lactobacilli strains showed a partial effect. K. marxianus CIDCA 8154 was selected for further characterization. Inhibitory activity was confirmed at transcriptional level on Caco-2/TC-7 and HT-29 cells upon flagellin stimulation. A similar effect was observed using other pro-inflammatory stimulation such as IL-1beta and TNF-alpha. Pre-incubation with yeasts induced a down-regulation of NF-kappaB signalling in epithelial cells in vitro, as well as expression of other pro-inflammatory chemokines such as CXCL8 and CXCL2. Furthermore, modulation of CCL20 mRNA expression upon flagellin stimulation was evidenced in vivo, in a mouse ligated intestinal loop model. Results indicate kefir contains microorganisms able to abolish the intestinal epithelial inflammatory response that could explain some of the properties attributed to this fermented milk. PMID:20471126

  15. Additional Nitrogen Fertilization at Heading Time of Rice Down-Regulates Cellulose Synthesis in Seed Endosperm

    PubMed Central

    Midorikawa, Keiko; Kuroda, Masaharu; Terauchi, Kaede; Hoshi, Masako; Ikenaga, Sachiko; Ishimaru, Yoshiro; Abe, Keiko; Asakura, Tomiko

    2014-01-01

    The balance between carbon and nitrogen is a key determinant of seed storage components, and thus, is of great importance to rice and other seed-based food crops. To clarify the influence of the rhizosphere carbon/nitrogen balance during the maturation stage of several seed components, transcriptome analysis was performed on the seeds from rice plants that were provided additional nitrogen fertilization at heading time. As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed. Moreover, cellulose and sucrose synthases, which are involved in cellulose synthesis, were down-regulated. Therefore, we compared cellulose content of mature seeds that were treated with additional nitrogen fertilization with those from control plants using calcofluor staining. In these experiments, cellulose content in endosperm from plants receiving additional nitrogen fertilization was less than that in control endosperm. Other starch synthesis-related genes such as starch synthase 1, starch phosphorylase 2, and branching enzyme 3 were also down-regulated, whereas some α-amylase and β-amylase genes were up-regulated. On the other hand, mRNA expression of amino acid biosynthesis-related molecules was up-regulated. Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression. These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm. PMID:24905454

  16. Cortisol administration induces global down-regulation of the brain's reward circuitry.

    PubMed

    Montoya, Estrella R; Bos, Peter A; Terburg, David; Rosenberger, Lisa A; van Honk, Jack

    2014-09-01

    Research in rodents and humans has shown divergent effects of the glucocorticoids corticosterone and cortisol (CRT) on reward processing. In rodents, administration of CRT increases reward drive by facilitating dopamine release in the ventral striatum. In humans, correspondingly, risky decision-making increases when CRT levels are elevated. Human stress studies contrariwise show that elevated CRT is accompanied by a decrease in reward-related brain activity. There are however no direct insights into how CRT acts on the reward system in the human brain. Accordingly, we used pharmacological functional magnetic resonance imaging (pharmaco-fMRI) to investigate the effects of CRT on the brain's reward system. In a randomized within-subject design we administered a high dose of CRT (40 mg) and placebo to twenty healthy male volunteers on separate days, and used a monetary incentive delay task to assess the effects of the hormone on the striatum and the amygdala in anticipation of potential reward. In contrast to animal studies, we show that this high dose of CRT strongly decreases activity of the striatum in both reward and non-reward conditions. Furthermore, we observed reductions in activity in the basolateral amygdala, a key regulator of the brain's reward system. Crucially, the overall down-regulation of the brain's reward circuit was verified on the subjective level as subjects reported significantly reduced reward preference after CRT. In sum, we provide here direct evidence in humans that CRT acts on brain regions involved in reward-related behavior, that is, the basolateral amygdala and the striatum. Our findings suggest that CRT in the quantity and time course presently used globally down-regulates the reward system, and thereby decreases motivational processing in general. PMID:25001954

  17. Steatogenesis in adult-onset type II citrullinemia is associated with down-regulation of PPARα.

    PubMed

    Komatsu, Michiharu; Kimura, Takefumi; Yazaki, Masahide; Tanaka, Naoki; Yang, Yang; Nakajima, Takero; Horiuchi, Akira; Fang, Zhong-Ze; Joshita, Satoru; Matsumoto, Akihiro; Umemura, Takeji; Tanaka, Eiji; Gonzalez, Frank J; Ikeda, Shu-Ichi; Aoyama, Toshifumi

    2015-03-01

    SLC25A13 (citrin or aspartate-glutamate carrier 2) is located in the mitochondrial membrane in the liver and its genetic deficiency causes adult-onset type II citrullinemia (CTLN2). CTLN2 is one of the urea cycle disorders characterized by sudden-onset hyperammonemia due to reduced argininosuccinate synthase activity. This disorder is frequently accompanied with hepatosteatosis in the absence of obesity and ethanol consumption. However, the precise mechanism of steatogenesis remains unclear. The expression of genes associated with fatty acid (FA) and triglyceride (TG) metabolism was examined using liver samples obtained from 16 CTLN2 patients and compared with 7 healthy individuals. Although expression of hepatic genes associated with lipogenesis and TG hydrolysis was not changed, the mRNAs encoding enzymes/proteins involved in FA oxidation (carnitine palmitoyl-CoA transferase 1α, medium- and very-long-chain acyl-CoA dehydrogenases, and acyl-CoA oxidase 1), very-low-density lipoprotein secretion (microsomal TG transfer protein), and FA transport (CD36 and FA-binding protein 1), were markedly suppressed in CTLN2 patients. Serum concentrations of ketone bodies were also decreased in these patients, suggesting reduced mitochondrial β-oxidation activity. Consistent with these findings, the expression of peroxisome proliferator-activated receptor α (PPARα), a master regulator of hepatic lipid metabolism, was significantly down-regulated. Hepatic PPARα expression was inversely correlated with severity of steatosis and circulating ammonia and citrulline levels. Additionally, phosphorylation of c-Jun-N-terminal kinase was enhanced in CTLN2 livers, which was likely associated with lower hepatic PPARα. Collectively, down-regulation of PPARα is associated with steatogenesis in CTLN2 patients. These findings provide a novel link between urea cycle disorder, lipid metabolism, and PPARα. PMID:25533124

  18. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways.

    PubMed

    Zhang, Jian; Lazarenko, Oxana P; Blackburn, Michael L; Badger, Thomas M; Ronis, Martin J J; Chen, Jin-Ran

    2014-07-01

    It has been suggested that the beneficial effects of soy protein isolate (SPI) on bone quality are due to either stimulation of estrogenic signaling via isoflavones or through a novel and as yet uncharacterized nonestrogenic pathway. In our study, SPI-fed rat serum inhibited the osteoblastic cell senescence pathway. This effect was accompanied by stimulation of cell differentiation, proliferation, and significant restoration of replicative senescent bone marrow mesenchymal ST2 cells (passaged 30 times). These effects were reproduced in bone from 5-wk-old intact and 10-wk-old ovariectomized female rats fed SPI diets. Caveolin-1 and p53 expression was decreased in bone in SPI-fed, but not in 17β-estradiol (E2)-treated rats. In cell culture studies, membranous caveolin-1 and nuclear p53 expression was greater in replicative senescent ST2 cell cultures than in earlier passaged cells. SPI-fed rat serum significantly down-regulated both caveolin-1 and p53 in senescent and nonsenescent cells. Replicative senescent ST2 cells exhibited a strong association among caveolin-1, p53, and mouse double minute 2 homologue (mdm2), which was inhibited by SPI-fed rat serum. Overexpression of caveolin-1 in ST2 cells resulted in increased expression of p53 and p21, whereas, knockdown of caveolin-1 using shRNA led to increases in mdm2 and eliminated SPI-fed rat serum's effects on p53 and p21 expression. In contrast, manipulation of caveolin-1 expression did not affect the actions of E2 or isoflavones on p53 expression in either ST2 or OB6 cells. These results suggest that caveolin-1 is a mediator of nonestrogenic SPI effects on bone cells.-Zhang, J., Lazarenko, O. P., Blackburn, M. L., Badger, T. M., Ronis, M. J. J., Chen, J.-R. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways. PMID:24719353

  19. BMP4 and LGL1 are Down Regulated in an Ovine Model of Congenital Diaphragmatic Hernia

    PubMed Central

    Emmerton-Coughlin, Heather M. A.; Martin, K. Kathryn; Chiu, Jacky S. S.; Zhao, Lin; Scott, Leslie A.; Regnault, Timothy R. H.; Bütter, Andreana

    2014-01-01

    Background/Purpose: The molecular pathophysiology of lung hypoplasia in congenital diaphragmatic hernia (CDH) remains poorly understood. The Wnt signaling pathway and downstream targets, such as bone morphogenetic proteins (BMP) 4 and other factors such as late gestation lung protein 1 (LGL1), are essential to normal lung development. Nitrofen-induced hypoplastic CDH rodent lungs demonstrate down regulation of the Wnt pathway including BMP4 and reduced LGL1 expression. The aim of the current study was to examine the molecular pathophysiology associated with a surgically induced CDH in an ovine model. Methods: Left thoracotomy was performed at 80 days in 14 fetal sheep; CDH was created in seven experimental animals. Lungs were harvested at 136 days (term = 145 days). Lung weight (LW) and mean terminal bronchiole density (MTBD) were measured to determine the degree of pulmonary hypoplasia. Quantitative real time PCR was undertaken to analyze Wnt2, Wnt7b, BMP4, and LGL1 mRNA expression. Results: Total LW was decreased while MTBD was increased in the CDH group (p < 0.05), confirming pulmonary hypoplasia. BMP4 and LGL1 mRNA was significantly reduced in CDH lungs (p < 0.05). Wnt2 mRNA was decreased, although not significantly (p < 0.06). Conclusion: For the first time, down regulation of BMP4 and LGL1 are reported in an ovine CDH model. In contrast to other animal models, these changes are persistent to near term. These findings suggest that mechanical compression from herniated viscera may play a more important role in causing pulmonary hypoplasia in CDH, rather than a primary defect in lung organogenesis. PMID:25593968

  20. Acylpeptide Hydrolase Inhibition as Targeted Strategy to Induce Proteasomal Down-Regulation

    PubMed Central

    Luini, Alberto; Ruvo, Menotti; Gogliettino, Marta; Langella, Emma; Saviano, Michele; Hegde, Ramanath N.; Sandomenico, Annamaria; Rossi, Mose

    2011-01-01

    Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy. Here, we illustrate a new pathway modulating UPS and proteasome activity through inhibition of APEH. To find novel molecules able to down-regulate APEH activity, we screened a set of synthetic peptides, reproducing the reactive-site loop of a known archaeal inhibitor of APEH (SsCEI), and the conjugated linoleic acid (CLA) isomers. A 12-mer SsCEI peptide and the trans10-cis12 isomer of CLA, were identified as specific APEH inhibitors and their effects on cell-based assays were paralleled by a dose-dependent reduction of proteasome activity and the activation of the pro-apoptotic caspase cascade. Moreover, cell treatment with the individual compounds increased the cytoplasm levels of several classic hallmarks of proteasome inhibition, such as NFkappaB, p21, and misfolded or polyubiquitinylated proteins, and additive effects were observed in cells exposed to a combination of both inhibitors without any cytotoxicity. Remarkably, transfection of human bronchial epithelial cells with APEH siRNA, promoted a marked accumulation of a mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), herein used as a model of misfolded protein typically degraded by UPS. Finally, molecular modeling studies, to gain insights into the APEH inhibition by the trans10-cis12 CLA isomer, were performed. Our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of strategies aimed at modulation of cancer progression. PMID:22016782

  1. Hyperinsulinemia Down-Regulates TLR4 Expression in the Mammalian Heart

    PubMed Central

    de Laat, Melody A.; Gruntmeir, Kaylynn J.; Pollitt, Christopher C.; McGowan, Catherine M.; Sillence, Martin N.; Lacombe, Véronique A.

    2014-01-01

    Toll-like receptors (TLR) are key regulators of innate immune and inflammatory responses and their activation is linked to impaired glucose metabolism during metabolic disease. Determination of whether TLR4 signaling can be activated in the heart by insulin may shed light on the pathogenesis of diabetic cardiomyopathy, a process that is often complicated by obesity and insulin resistance. The aim of the current study was to determine if supraphysiological insulin concentrations alter the expression of TLR4, markers of TLR4 signaling and glucose transporters (GLUTs) in the heart. Firstly, the effect of insulin on TLR4 protein expression was investigated in vitro in isolated rat cardiac myocytes. Secondly, protein expression of TLR4, the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) suppressor of cytokine signaling 3 (SOCS3) and GLUTs (1, 4, 8, 12) were examined in the equine ventricular myocardium following a prolonged, euglycemic, hyperinsulinemic clamp. Down-regulation of TLR4 protein content in rat cardiac myocytes was observed after incubation with a supraphysiologic concentration of insulin as well as in the equine myocardium after prolonged insulin infusion. Further, cardiac TLR4 expression was negatively correlated with serum insulin concentration. Markers of cardiac TLR4 signaling and GLUT expression were not affected by hyperinsulinemia and concomitant TLR4 down-regulation. Since TLRs are major determinants of the inflammatory response, our findings suggest that insulin infusion exerts an anti-inflammatory effect in the hearts of non-obese individuals. Understanding the regulation of cardiac TLR4 signaling during metabolic dysfunction will facilitate improved management of cardiac sequela to metabolic syndrome and diabetes. PMID:25101057

  2. RASSF6 expression in adipocytes is down-regulated by interaction with macrophages.

    PubMed

    Sanada, Yohei; Kumoto, Takahiro; Suehiro, Haruna; Nishimura, Fusanori; Kato, Norihisa; Hata, Yutaka; Sorisky, Alexander; Yanaka, Noriyuki

    2013-01-01

    Macrophage infiltration into adipose tissue is associated with obesity and the crosstalk between adipocytes and infiltrated macrophages has been investigated as an important pathological phenomenon during adipose tissue inflammation. Here, we sought to identify adipocyte mRNAs that are regulated by interaction with infiltrated macrophages in vivo. An anti-inflammatory vitamin, vitamin B6, suppressed macrophage infiltration into white adipose tissue and altered mRNA expression. We identified >3500 genes whose expression is significantly altered during the development of obesity in db/db mice, and compared them to the adipose tissue mRNA expression profile of mice supplemented with vitamin B6. We identified PTX3 and MMP3 as candidate genes regulated by macrophage infiltration. PTX3 and MMP3 mRNA expression in 3T3-L1 adipocytes was up-regulated by activated RAW264.7 cells and these mRNA levels were positively correlated with macrophage number in adipose tissue in vivo. Next, we screened adipose genes down-regulated by the interaction with macrophages, and isolated RASSF6 (Ras association domain family 6). RASSF6 mRNA in adipocytes was decreased by culture medium conditioned by activated RAW264.7 cells, and RASSF6 mRNA level was negatively correlated with macrophage number in adipose tissue, suggesting that adipocyte RASSF6 mRNA expression is down-regulated by infiltrated macrophages in vivo. Finally, this study also showed that decreased RASSF6 expression up-regulates mRNA expression of several genes, such as CD44 and high mobility group protein HMGA2. These data provide novel insights into the biological significance of interactions between adipocytes and macrophages in adipose tissue during the development of obesity. PMID:23626755

  3. Mediobasal hypothalamic and adenohypophyseal TRH-degrading enzyme (PPII) is down-regulated by zinc deficiency.

    PubMed

    Alvarez-Salas, Elena; Alcántara-Alonso, Viridiana; Matamoros-Trejo, Gilberto; Vargas, Miguel Angel; Morales-Mulia, Marcela; de Gortari, Patricia

    2015-11-01

    Thyrotropin-releasing hormone (TRH) synthesized in hypothalamic paraventricular nucleus directs hypothalamus-pituitary-thyroid (HPT) axis function, regulating thyrotropin release from adenohypophysis and thyroid hormones serum concentration. Pyroglutamyl aminopeptidase II (PPII), a Zn-dependent metallopeptidase located in adenohypophysis and medial-basal-hypothalamus degrades TRH released from the median eminence and participates in HPT axis function by regulating TRH-induced thyrotropin release from adenohypophysis. It is unknown whether dietary Zn deficiency down-regulates PPII. Our aim was to compare adenohypohyseal and medial-basal-hypothalamic PPII activity and expression of adult rats fed a Zn-deficient diet (2ppm) throughout their lifespan (DD), prenatally (DC) or after weaning (CD) vs. that of animals fed a control diet (20ppm:CC). Female rats consumed a Zn-deficient or control diet from two weeks before gestation and up to the end of lactation. We analyzed adenohypophyseal and medial-basal-hypothalamic PPII activity of dams and male offspring when adults; its relation to median eminence TRH, serum thyrotropin, leptin and thyroid hormones concentration. Offspring ate the same diet as their dams (CC, DD) or were switched from dietary regime after weaning (CD, DC) and until 2.5 months of age. DD males showed decreased adenohypophyseal and medial-basal-hypothalamic PPII activity, along with high thyrotropin serum concentration. Post-weaning Zn-deficiency (CD) decreased PPII activity only in adenohypophysis and increased thyrotropin circulating levels. Zn-replenishment (DC) normalized PPII activity in both regions and serum thyrotropin concentration. Adenohypophyseal PPII activity decreased and prolactin levels increased in Zn-deficient dams. We concluded that long-term changes in dietary Zn down-regulate PPII activity independently of T3, increasing thyrotropin serum concentration, overall resembling sub-clinical hypothyroidism. PMID:26315400

  4. Down regulation of hepatic PPARalpha function by AhR ligand.

    PubMed

    Shaban, Zein; El-Shazly, Samir; Abdelhady, Shawky; Fattouh, Ibrahim; Muzandu, Kaampwe; Ishizuka, Mayumi; Kimura, Kazuhiro; Kazusaka, Akio; Fujita, Shoichi

    2004-11-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachloro dibenzo-p-dioxin (TCDD) and related compounds. Peroxisome proliferator activated receptor alpha (PPARalpha) is a nuclear receptor involved in the maintenance of lipid and glucose homeostasis. In this study we hypothesized that one of the possible mechanisms for the effect of TCDD and its related chemicals on fat metabolism could be through down regulation of PPARalpha functions. We treated Wistar rats with an AhR ligand, Sudan III (S.III), and/or PPARalpha ligand, Clofibric Acid (CA), for 3 days. We analysed the expression of one of the PPARalpha-target gene products, CYP4A protein and its mRNA. We also tested HepG2 cells with the afore-mentioned treatments and evaluated their effects on PPARalpha and RXRalpha protein. Treatment of Wistar rats with S.III was found to down regulates CYP4A protein expression and reduced its induction with CA. It also decreased mRNA expressions of CYP4A1, CYP4A2, CYP4A3 and PPARalpha. In HepG2 cells, PPARalpha and RXRalpha protein expression was decreased by S.III treatment in a dose dependent manner. Our results suggest that AhR has an inhibitory effect on PPARalpha function and a new pathway by which AhR ligands could disturb lipid metabolism. PMID:15585952

  5. Down-Regulation of Nogo-B Expression as a Newly Identified Feature of Intrahepatic Cholangiocarcinoma.

    PubMed

    Nanashima, Atsushi; Hatachi, Go; Tominaga, Tetsurou; Murakami, Goushi; Takagi, Katsunori; Arai, Junichi; Wada, Hideo; Nagayasu, Takeshi; Sumida, Yorihisa

    2016-01-01

    Nogo-B, located in the endoplasmic reticulum, is an isoform belonging to the reticulon protein family, which is expressed specifically in cholangiocytes and non-parenchymal cells in the liver. Nogo-B expression is down-regulated with the progression of liver fibrosis, but its distinct function in liver malignancies has not been fully clarified. We have hypothesized that Nogo-B expression may be altered in intrahepatic cholangiocarcinoma (ICC), a relatively rare type of primary liver cancer with highly malignant behavior. The present study aimed to investigate the relationship between Nogo-B expression, assessed by immunohistochemical staining, and clinicopathological factors and prognosis in 34 ICC patients. Positive expression was observed in 19 (56%) of 34 ICC specimens: 6 patients (18%) with positivity levels of 1+ (positive cells in 10-50% of cancer cells) and 13 patients (38%) with 2+ (positive cells over 50%). Importantly, the remaining 15 patients (44%) were categorized as negative expression (Nogo-B-positive cells, less than 10%). Conversely, the mass-forming type of ICC tended to express Nogo-B with the degree of 2+ positivity, compared to the periductal infiltration type (p = 0.064), and the mass-forming type showed a better 5-year survival rate (66% vs. 5%) after hepatectomy (p < 0.05). However, the degree of positivity was not associated with tumor relapse rate, disease-free and overall survival, although each of the periductal infiltration type, intrahepatic metastasis, larger tumor size, and lower microvessel counts was associated with lower survival rates. We propose that Nogo-B expression is down-regulated in ICC, the implication of which, however, remains to be investigated. PMID:26656426

  6. Let-7c down-regulation in Helicobacter pylori-related gastric carcinogenesis.

    PubMed

    Fassan, Matteo; Saraggi, Deborah; Balsamo, Laura; Cascione, Luciano; Castoro, Carlo; Coati, Irene; De Bernard, Marina; Farinati, Fabio; Guzzardo, Vincenza; Valeri, Nicola; Zambon, Carlo Federico; Rugge, Massimo

    2016-01-26

    Aberrant let-7c microRNA (miRNA) expression has been observed in Helicobacter pylori-related gastric cancer (GC) but fragmentary information is available on the let-7c dysregulation occurring with each phenotypic change involved in gastric carcinogenesis. Let-7c expression was assessed (qRT-PCR) in a series of 175 gastric biopsy samples representative of the whole spectrum of phenotypic changes involved in H. pylori-related gastric oncogenesis including: i) normal gastric mucosa, as obtained from dyspeptic controls (40 biopsy samples); ii) non-atrophic gastritis (40 samples); iii) atrophic-metaplastic gastritis (35 samples); iv) intra-epithelial neoplasia (30 samples); v) GC (30 samples). Let-7c expression was also tested in 20 biopsy samples obtained from 10 patients before and after H. pylori eradication therapy (median follow-up: 10 weeks; range: 7-14). The results obtained were further validated by in situ hybridization on multiple tissue specimens obtained from 5 surgically treated H. pylori-related GCs. The study also included 40 oxyntic biopsy samples obtained from serologically/histologically confirmed autoimmune gastritis (AIG: 20 corpus-restricted, non-atrophic; 20 corpus-restricted, atrophic-metaplastic). Let-7c expression dropped from non-atrophic gastritis to atrophic-metaplastic gastritis, intra-epithelial neoplasia, and invasive GC (p<0.001). It rose again significantly following H. pylori eradication (p=0.009). As in the H. pylori model, AIG also featured a significant let-7c down-regulation (p<0.001). The earliest phases of the two pathways to gastric oncogenesis (H. pylori-environmental and autoimmune host-related) are characterized by similar let-7c dysregulations. In H. pylori infection, let-7c down-regulation regresses after the bacterium's eradication, while it progresses significantly with the increasing severity of the histological lesions. PMID:26701848

  7. Capsaicin inhibits the Wnt/β-catenin signaling pathway by down-regulating PP2A.

    PubMed

    Park, Dong-Seok; Yoon, Gang-Ho; Lee, Hyun-Shik; Choi, Sun-Cheol

    2016-09-01

    Xenopus embryo serves as an ideal model for teratogenesis assays to examine the effects of any substances on the cellular processes critical for early development and adult tissue homeostasis. In our chemical library screening with frog embryo, capsaicin was found to repress the Wnt/β-catenin signaling. Depending on the stages at which embryos became exposed to capsaicin, it could disrupt formation of dorsal or posterior body axis of embryo, which is associated with inhibition of maternal or zygotic Wnt signal in early development. In agreement with these phenotypes, capsaicin suppressed the expression of Wnt target genes such as Siamois and Chordin in the organizer region of embryo and in Wnt signals-stimulated tissue explants. In addition, the cellular level of β-catenin, a key component of Wnt pathway, was down-regulated in capsaicin-treated embryonic cells. Unlike wild-type β-catenin, its non-phosphorylatable mutant in which serine and threonine residues phosphorylated by GSK3 are substituted with alanine was not destabilized by capsaicin, indicative of the effect of this chemical on the phosphorylation status of β-catenin. In support of this, capsaicin up-regulated the level of GSK3- or CK1-phosphorylated β-catenin, concomitantly lowering that of its de-phosphorylated version. Notably, capsaicin augmented the phosphorylation of a phosphatase, PP2A at tyrosine 307, suggesting its repression of the enzymatic activity of the phosphatase. Furthermore, capsaicin still enhanced β-catenin phosphorylation in cells treated with a GSK3 inhibitor, LiCl but not in those treated with a phosphatase inhibitor, okadaic acid. Together, these results indicate that capsaicin inhibits the patterning of the dorso-ventral and anterior-posterior body axes of embryo by repressing PP2A and thereby down-regulating the Wnt/β-catenin signaling. PMID:27318088

  8. Rapamycin Attenuates Mouse Liver Ischemia and Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress.

    PubMed

    Zhu, J; Hua, X; Li, D; Zhang, J; Xia, Q

    2015-01-01

    The roles of endoplasmic reticulum (ER) stress in liver ischemia and reperfusion injury (IRI) have been well recognized. However, the impact of rapamycin (Rapa), a broadly used immunosuppressive agent in human liver transplantation, on ER stress during IRI remains unclear. This study was designed to investigate the roles of Rapa in the regulation of ER stress in vivo and in vitro. In a mouse liver partial warm ischemia and reperfusion mode, we demonstrated that Rapa markedly protected livers from IRI, as evidenced by serum alanine aminotransferase (sALT) levels and liver histology. Then we also confirmed the protection of Rapa from thapsigargin (Tg)-induced cell death in primary hepatocytes. Both in vivo and in vitro experiments showed that the ER stress markers were markedly up-regulated by IRI and Tg treatment, whereas they were down-regulated by Rapa pretreatment, as monitored by Western blot at the protein levels and by quantitative reverse transcription polymerase chain reaction (RT-PCR) at the messenger RNA (mRNA) levels. In addition, it was also revealed that Rapa was able to remarkably inhibit the mammalian target of rapamycin (mTOR) pathway and enhance autophagy both in IR-stressed livers and Tg-treated primary hepatocytes. Thus, these results suggest that Rapa protects livers from IRI through inhibiting the ER stress pathway. PMID:26293028

  9. Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells.

    PubMed

    Akhade, Vijay Suresh; Dighe, Shrinivas Nivrutti; Kataruka, Shubhangini; Rao, Manchanahalli R Satyanarayana

    2016-01-01

    Long non coding RNAs (lncRNAs) have emerged as important regulators of various biological processes. LncRNAs also behave as response elements or targets of signaling pathway(s) mediating cellular function. Wnt signaling is important in regulating mammalian spermatogenesis. Mrhl RNA negatively regulates canonical Wnt pathway and gets down regulated upon Wnt signaling activation in mouse spermatogonial cells. Also, mrhl RNA regulates expression of genes pertaining to Wnt pathway and spermatogenesis by binding to chromatin. In the present study, we delineate the detailed molecular mechanism of Wnt signaling induced mrhl RNA down regulation in mouse spermatogonial cells. Mrhl RNA has an independent transcription unit and our various experiments like Chromatin Immunoprecipitation (in cell line as well as mouse testis) and shRNA mediated down regulation convincingly show that β-catenin and TCF4, which are the key effector proteins of the Wnt signaling pathway are required for down regulation of mrhl RNA. We have identified Ctbp1 as the co-repressor and its occupancy on mrhl RNA promoter depends on both β-catenin and TCF4. Upon Wnt signaling activation, Ctbp1 mediated histone repression marks increase at the mrhl RNA promoter. We also demonstrate that Wnt signaling induced mrhl RNA down regulation results in an up regulation of various meiotic differentiation marker genes. PMID:26446991

  10. Down-regulation of Bcl-2-interacting protein BAG-1 confers resistance to anti-cancer drugs.

    PubMed

    Takahashi, Noriko; Yanagihara, Miyako; Ogawa, Yuzi; Yamanoha, Banri; Andoh, Toshiwo

    2003-02-14

    BAG-1 was originally identified as a binding partner of anti-apoptotic factor Bcl-2 [Takayama et al., Cell 80 (1995) 279-284]. Exogenous expression of BAG-1 was reported to confer cells resistance to several stresses [Chen et al., Oncogene 21 (2002) 7050]. We have obtained human cervical cancer HeLa cells with down-regulated BAG-1 levels by using a highly specific and efficient RNA interference approach. Surprisingly, cells with down-regulated BAG-1 exhibited significantly lower sensitivity against several anti-cancer drugs than parental cells expressing normal levels of the protein. Furthermore, growth rate of the cells was reduced when BAG-1 was down-regulated. Activity of ERK pathway appeared to be decreased in BAG-1 down-regulated cells, as shown by the reduced phosphorylation of ERK1/2 proteins. Taken together resistance against anti-cancer drugs acquired by BAG-1 down-regulated cells may well be accounted for by the retardation of cell cycle progression, implicating the importance of BAG-1 in cell growth regulation. PMID:12565851

  11. Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells

    PubMed Central

    Akhade, Vijay Suresh; Dighe, Shrinivas Nivrutti; Kataruka, Shubhangini; Rao, Manchanahalli R. Satyanarayana

    2016-01-01

    Long non coding RNAs (lncRNAs) have emerged as important regulators of various biological processes. LncRNAs also behave as response elements or targets of signaling pathway(s) mediating cellular function. Wnt signaling is important in regulating mammalian spermatogenesis. Mrhl RNA negatively regulates canonical Wnt pathway and gets down regulated upon Wnt signaling activation in mouse spermatogonial cells. Also, mrhl RNA regulates expression of genes pertaining to Wnt pathway and spermatogenesis by binding to chromatin. In the present study, we delineate the detailed molecular mechanism of Wnt signaling induced mrhl RNA down regulation in mouse spermatogonial cells. Mrhl RNA has an independent transcription unit and our various experiments like Chromatin Immunoprecipitation (in cell line as well as mouse testis) and shRNA mediated down regulation convincingly show that β-catenin and TCF4, which are the key effector proteins of the Wnt signaling pathway are required for down regulation of mrhl RNA. We have identified Ctbp1 as the co-repressor and its occupancy on mrhl RNA promoter depends on both β-catenin and TCF4. Upon Wnt signaling activation, Ctbp1 mediated histone repression marks increase at the mrhl RNA promoter. We also demonstrate that Wnt signaling induced mrhl RNA down regulation results in an up regulation of various meiotic differentiation marker genes. PMID:26446991

  12. The target of rapamycin (TOR) proteins

    PubMed Central

    Raught, Brian; Gingras, Anne-Claude; Sonenberg, Nahum

    2001-01-01

    Rapamycin potently inhibits downstream signaling from the target of rapamycin (TOR) proteins. These evolutionarily conserved protein kinases coordinate the balance between protein synthesis and protein degradation in response to nutrient quality and quantity. The TOR proteins regulate (i) the initiation and elongation phases of translation, (ii) ribosome biosynthesis, (iii) amino acid import, (iv) the transcription of numerous enzymes involved in multiple metabolic pathways, and (v) autophagy. Intriguingly, recent studies have also suggested that TOR signaling plays a critical role in brain development, learning, and memory formation. PMID:11416184

  13. Down-regulation of SOSTDC1 promotes thyroid cancer cell proliferation via regulating cyclin A2 and cyclin E2

    PubMed Central

    He, Xiaoying; Ke, Weijian; Xu, Lijuan; Liu, Liehua; Xiao, Haipeng; Li, Yanbing

    2015-01-01

    Sclerostin domain containing protein 1 (SOSTDC1) is down-regulated and acts as a tumor suppressor in some kinds of cancers. However, the expression pattern and biological significance of SOSTDC1 in thyroid cancer are largely unknown. We demonstrated that SOSTDC1 was significantly down-regulated in thyroid cancer. Ectopic over-expression of SOSTDC1 inhibited proliferation and induced G1/S arrest in thyroid cancer cells. Moreover, SOSTDC1 over-expression suppressed the growth of tumor xenografts in nude mice. We also found that elevated SOSTDC1 led to inhibition of cyclin A2 and cyclin E2. Together, our results demonstrate that SOSTDC1 is down-regulated in thyroid cancer and might be a potential therapeutic target in the treatment of thyroid cancer. PMID:26378658

  14. Down-regulation of gibberellic acid in poplar has negligible effects on host-plant suitability and insect pest response

    SciTech Connect

    Buhl, Christine; Strauss, Steven H.; Lindroth, Richard L.

    2015-01-06

    Abstract Endogenous levels and signaling of gibberellin plant hormones such as gibberellic acid (GA) have been genetically down-regulated to create semi-dwarf varieties of poplar. The potential benefits of semi-dwarf stature include reduced risk of wind damage, improved stress tolerance, and improved wood quality. Despite these benefits, modification of growth traits may have consequences for non-target traits that confer defense against insect herbivores. According to the growth-differentiation balance hypothesis, reductions in growth may shift allocation of carbon from growth to chemical resistance traits, thereby altering plant defense. To date, host-plant suitability and pest response have not been comprehensively evaluated in GA down-regulated plants. We quantified chemical resistance and nitrogen (an index of protein) in GA down-regulated and wild-type poplar (Populus alba × P. tremula) genotypes. We also evaluated performance of both generalist (Lymantria dispar) and specialist (Chrysomela scripta) insect pests reared on these genotypes. Our evaluation of resistance traits in four GA down-regulated genotypes revealed increased phenolic glycosides in one modified genotype and reduced lignin in two modified genotypes relative to the non-transgenic wild type. Nitrogen levels did not vary significantly among the experimental genotypes. Generalists reared on the four GA down-regulated genotypes exhibited reduced performance on only one modified genotype relative to the wild type. Specialists, however, performed similarly across all genotypes. Results from this study indicate that although some non-target traits varied among GA down-regulated genotypes, the differences in poplar pest susceptibility were modest and highly genotype-specific.

  15. Down-regulation of gibberellic acid in poplar has negligible effects on host-plant suitability and insect pest response

    DOE PAGESBeta

    Buhl, Christine; Strauss, Steven H.; Lindroth, Richard L.

    2015-01-06

    Abstract Endogenous levels and signaling of gibberellin plant hormones such as gibberellic acid (GA) have been genetically down-regulated to create semi-dwarf varieties of poplar. The potential benefits of semi-dwarf stature include reduced risk of wind damage, improved stress tolerance, and improved wood quality. Despite these benefits, modification of growth traits may have consequences for non-target traits that confer defense against insect herbivores. According to the growth-differentiation balance hypothesis, reductions in growth may shift allocation of carbon from growth to chemical resistance traits, thereby altering plant defense. To date, host-plant suitability and pest response have not been comprehensively evaluated in GAmore » down-regulated plants. We quantified chemical resistance and nitrogen (an index of protein) in GA down-regulated and wild-type poplar (Populus alba × P. tremula) genotypes. We also evaluated performance of both generalist (Lymantria dispar) and specialist (Chrysomela scripta) insect pests reared on these genotypes. Our evaluation of resistance traits in four GA down-regulated genotypes revealed increased phenolic glycosides in one modified genotype and reduced lignin in two modified genotypes relative to the non-transgenic wild type. Nitrogen levels did not vary significantly among the experimental genotypes. Generalists reared on the four GA down-regulated genotypes exhibited reduced performance on only one modified genotype relative to the wild type. Specialists, however, performed similarly across all genotypes. Results from this study indicate that although some non-target traits varied among GA down-regulated genotypes, the differences in poplar pest susceptibility were modest and highly genotype-specific.« less

  16. Involvement of the MEK/ERK pathway in EGF-induced E-cadherin down-regulation.

    PubMed

    Tashiro, Etsu; Henmi, Shizuka; Odake, Hiroyuki; Ino, Seitaro; Imoto, Masaya

    2016-09-01

    E-cadherin is a major component of the epithelial adherens junction. However, the regulatory mechanism of E-cadherin expression is still poorly understood. In this study, we found that EGF decreased E-cadherin expression at both mRNA and protein levels in colorectal carcinoma LoVo cells. Since E-cadherin down-regulation is a well-known hallmark of the EMT (Epithelial-Mesenchymal Transition), we investigated whether EGF induced E-cadherin down-regulation during the EMT. EGF was unable to affect the expression of mesenchymal markers (such as N-cadherin, vimentin or fibronectin) or EMT-regulating transcription factors (such as SNAIL, SLUG, ZEB1, ZEB2 or TWIST), suggesting that EGF induced E-cadherin down-regulation via an EMT-independent mechanism. On the other hand, the MEK inhibitor U0126 was found to suppress EGF-induced E-cadherin down-regulation at the transcriptional level, suggesting that the MEK/ERK pathway is involved in EGF-induced E-cadherin down-regulation. Moreover, we also found that EGF disrupted cell-cell contact, stimulated cells to form an elongated shape with filamentous protrusions, and induced cell migration in LoVo cells. These effects were suppressed by U0126. Therefore, EGF is suggested to induce E-cadherin down-regulation at the transcriptional level through the MEK/ERK pathway, which might result in, at least in part, the induction of cellular morphological changes and cell migration in LoVo cells. PMID:27369075

  17. Luteinizing Hormone Receptor mRNA Down-Regulation Is Mediated through ERK-Dependent Induction of RNA Binding Protein

    PubMed Central

    Menon, Bindu; Franzo-Romain, Megan; Damanpour, Shadi

    2011-01-01

    The ligand-induced down-regulation of LH receptor (LHR) expression in the ovaries, at least in part, is regulated by a posttranscriptional process mediated by a specific LH receptor mRNA binding protein (LRBP). The LH-mediated signaling pathways involved in this process were examined in primary cultures of human granulosa cells. Treatment with 10 IU human chorionic gonadotropin (hCG) for 12 h resulted in the down-regulation of LHR mRNA expression while producing an increase in LHR mRNA binding to LRBP as well as a 2-fold increase in LRBP levels. The activation of ERK½ pathway in LH-mediated LHR mRNA down-regulation was also established by demonstrating the translocation of ERK½ from the cytosol to the nucleus using confocal microcopy. Inhibition of protein kinase A using H-89 or ERK½ by U0126 abolished the LH-induced LHR mRNA down-regulation. These treatments also abrogated both the increases in LRBP levels as well as the LHR mRNA binding activity. The abolishment of the hCG-induced increase in LRBP levels and LHR mRNA binding activity was further confirmed by transfecting granulosa cells with ERK½ specific small interfering RNA. This treatment also reversed the hCG-induced down-regulation of LHR mRNA. These data show that LH-regulated ERK½ signaling is required for the LRBP-mediated down-regulation of LHR mRNA. PMID:21147848

  18. LPS Down-Regulates Specificity Protein 1 Activity by Activating NF-κB Pathway in Endotoxemic Mice

    PubMed Central

    Ye, Xiaobing; Liu, Hong; Gong, Yong-Sheng; Liu, Shu Fang

    2015-01-01

    Background Specificity protein (Sp) 1 mediates the transcription of a large number of constitutive genes encoding physiological mediators. NF-κB mediates the expression of hundreds of inducible genes encoding pathological mediators. Crosstalk between Sp1 and NF-κB pathways could be pathophysiologically significant, but has not been studied. This study examined the crosstalk between the two pathways and defined the role of NF-κB signaling in LPS-induced down-regulation of Sp1 activity. Methods and Main Findings Challenge of wild type mice with samonelia enteritidis LPS (10 mg/kg, i.p.) down-regulated Sp1 binding activity in lungs in a time-dependent manner, which was concomitantly associated with an increased NF-κB activity. LPS down-regulates Sp1 activity by inducing an LPS inducible Sp1-degrading enzyme (LISPDE) activity, which selectively degrades Sp1 protein, resulting in Sp1 down-regulation. Blockade of NF-κB activation in mice deficient in NF-κB p50 gene (NF-κB-KO) suppressed LISPDE activity, prevented Sp1 protein degradation, and reversed the down-regulation of Sp1 DNA binding activity and eNOS expression (an indicator of Sp1 transactivation activity). Inhibition of LISPDE activity using a selective LISPDE inhibitor mimicked the effects of NF-κB blockade. Pretreatment of LPS-challenged WT mice with a selective LISPDE inhibitor increased nuclear Sp1 protein content, restored Sp1 DNA binding activity and reversed eNOS protein down-regulation in lungs. Enhancing tissue level of Sp1 activity by inhibiting NF-κB-mediated Sp1 down-regulation increased tissue level of IL-10 and decreased tissue level of TNF- αin the lungs. Conclusions NF-κB signaling mediates LPS-induced down-regulation of Sp1 activity. Activation of NF-κB pathway suppresses Sp1 activity and Sp1-mediated anti-inflammatory signals. Conversely, Sp1 signaling counter-regulates NF-κB-mediated inflammatory response. Crosstalk between NF-κB and Sp1 pathways regulates the balance between pro

  19. Down-Regulation of NDUFB9 Promotes Breast Cancer Cell Proliferation, Metastasis by Mediating Mitochondrial Metabolism.

    PubMed

    Li, Liang-Dong; Sun, He-Fen; Liu, Xue-Xiao; Gao, Shui-Ping; Jiang, Hong-Lin; Hu, Xin; Jin, Wei

    2015-01-01

    Despite advances in basic and clinical research, metastasis remains the leading cause of death in breast cancer patients. Genetic abnormalities in mitochondria, including mutations affecting complex I and oxidative phosphorylation, are found in breast cancers and might facilitate metastasis. Genes encoding complex I components have significant breast cancer prognostic value. In this study, we used quantitative proteomic analyses to compare a highly metastatic cancer cell line and a parental breast cancer cell line; and observed that NDUFB9, an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I), was down-regulated in highly metastatic breast cancer cells. Furthermore, we demonstrated that loss of NDUFB9 promotes MDA-MB-231 cells proliferation, migration, and invasion because of elevated levels of mtROS, disturbance of the NAD+/NADH balance, and depletion of mtDNA. We also showed that, the Akt/mTOR/p70S6K signaling pathway and EMT might be involved in this mechanism. Thus, our findings contribute novel data to support the hypothesis that misregulation of mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, suggesting that complex I deficiency is a potential and important biomarker for further basic research or clinical application. PMID:26641458

  20. Physicians down-regulate their pain empathy response: an event-related brain potential study.

    PubMed

    Decety, Jean; Yang, Chia-Yan; Cheng, Yawei

    2010-05-01

    Watching or imagining other people experiencing pain activates the central nervous system's pain matrix in the observer. Without emotion regulation skills, repeated exposure to the suffering of others in healthcare professionals may be associated with the adverse consequences of personal distress, burnout and compassion fatigue, which are detrimental to their wellbeing. Here, we recorded event-related potentials (ERP) from physicians and matched controls as they were presented with visual stimuli depicting body parts pricked by a needle (pain) or touched by a Q-tip (no-pain). The results showed early N110 differentiation between pain and no-pain over the frontal area as well as late P3 over the centro-parietal regions were observed in the control participants. In contrast, no such early and late ERP responses were detected in the physicians. Our results indicate that emotion regulation in physicians has very early effects, inhibiting the bottom-up processing of the perception of pain in others. It is suggested that physicians' down-regulation of the pain response dampens their negative arousal in response to the pain of others and thus may have many beneficial consequences including freeing up cognitive resources necessary for being of assistance. PMID:20080194

  1. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    NASA Astrophysics Data System (ADS)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  2. Silkworm Hemolymph Down-Regulates the Expression of Endoplasmic Reticulum Chaperones under Radiation-Irradiation

    PubMed Central

    Lee, Kyeong Ryong; Kim, Seung-Whan; Kim, Young Kook; Kwon, Kisang; Choi, Jong-Soon; Yu, Kweon; Kwon, O-Yu

    2011-01-01

    We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, “ischemia-responsive protein 94 kDa” (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation. PMID:21845089

  3. The Mycobacterium tuberculosis H37Ra gene MRA_1916 causes growth defects upon down-regulation

    PubMed Central

    Singh, Kumar Sachin; Singh, Sudheer Kumar

    2015-01-01

    D-amino acid oxidases play an important role in converting D-amino acids to their corresponding α-keto acids. MRA_1916 of Mycobacterium tuberculosis H37Ra (Mtb-Ra) is annotated to be a D-amino acid oxidase (DAO). However, not much information is available about its physiological role during Mtb-Ra growth and survival. The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth. Recombinant Mtb-Ra strains with DAO and GlcB (malate synthase: MRA_1848) gene knockdown were developed and their growth was studied using Microtiter Alamar Blue Assay (MABA) with glycerol, acetate and glycine as a carbon source. Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB. MABA study showed inhibition of wild-type (WT) and knockdowns in the presence of BrP (2.5mM). However, growth inhibition of WT was less noticeable at lower concentrations of BrP. Mtb-Ra with DAO knockdown showed poor utilization of glycine in the presence of BrP. The DAO localization study showed its prominent distribution in cytosolic fraction and to some extent in cell wall and membrane fractions. Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104). PMID:26531045

  4. Down-Regulation of NDUFB9 Promotes Breast Cancer Cell Proliferation, Metastasis by Mediating Mitochondrial Metabolism

    PubMed Central

    Gao, Shui-Ping; Jiang, Hong-Lin; Hu, Xin; Jin, Wei

    2015-01-01

    Despite advances in basic and clinical research, metastasis remains the leading cause of death in breast cancer patients. Genetic abnormalities in mitochondria, including mutations affecting complex I and oxidative phosphorylation, are found in breast cancers and might facilitate metastasis. Genes encoding complex I components have significant breast cancer prognostic value. In this study, we used quantitative proteomic analyses to compare a highly metastatic cancer cell line and a parental breast cancer cell line; and observed that NDUFB9, an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I), was down-regulated in highly metastatic breast cancer cells. Furthermore, we demonstrated that loss of NDUFB9 promotes MDA-MB-231 cells proliferation, migration, and invasion because of elevated levels of mtROS, disturbance of the NAD+/NADH balance, and depletion of mtDNA. We also showed that, the Akt/mTOR/p70S6K signaling pathway and EMT might be involved in this mechanism. Thus, our findings contribute novel data to support the hypothesis that misregulation of mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, suggesting that complex I deficiency is a potential and important biomarker for further basic research or clinical application. PMID:26641458

  5. Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy

    PubMed Central

    Pawar, Kamlesh; Hanisch, Carlos; Palma Vera, Sergio Eliseo; Einspanier, Ralf; Sharbati, Soroush

    2016-01-01

    Small non-coding RNA play a major part in host response to bacterial agents. However, the role of long non-coding RNA (lncRNA) in this context remains unknown. LncRNA regulate gene expression by acting e.g. as transcriptional coactivators, RNA decoys or microRNA sponges. They control development, differentiation and cellular processes such as autophagy in disease conditions. Here, we provide an insight into the role of lncRNA in mycobacterial infections. Human macrophages were infected with Mycobacterium bovis BCG and lncRNA expression was studied early post infection. For this purpose, lncRNA with known immune related functions were preselected and a lncRNA specific RT-qPCR protocol was established. In addition to expression-based prediction of lncRNA function, we assessed strategies for thorough normalisation of lncRNA. Arrayed quantification showed infection-dependent repression of several lncRNA including MEG3. Pathway analysis linked MEG3 to mTOR and PI3K-AKT signalling pointing to regulation of autophagy. Accordingly, IFN-γ induced autophagy in infected macrophages resulted in sustained MEG3 down regulation and lack of IFN-γ allowed for counter regulation of MEG3 by viable M. bovis BCG. Knockdown of MEG3 in macrophages resulted in induction of autophagy and enhanced eradication of intracellular M. bovis BCG. PMID:26757825

  6. Down-Regulation of CD9 Expression and its Correlation to Tumor Progression in B Lymphomas

    PubMed Central

    Yoon, Sun-Ok; Zhang, Xin; Freedman, Arnold S.; Zahrieh, David; Lossos, Izidore S.; Li, Li; Choi, Yong Sung

    2010-01-01

    Histological transformation, a pivotal event in the natural history of cancers including lymphomas, is typically associated with more aggressive clinical behavior. L3055, a B lymphoma cell line of germinal center (GC) origin, is dependent on follicular dendritic cells (FDCs) for survival and proliferation, similar to GC-B cells. However, L3055 cells become less FDC-dependent after prolonged culture, which is analogous to transformation in vivo. Comparison of two L3055 subclones (i.e., the FDC-dependent indolent clone 12 and the FDC-independent aggressive clone 33) by DNA microarray revealed that CD9 was the most differentially expressed gene (P = 0.05). L3055-12 expresses high levels of CD9 while L3055-33 does not. Reduced levels or loss of CD9 expression is also observed in other CD9-positive B lymphoma cell lines. The resultant CD9-negative cells grow faster than CD9-positive cells due to their greater resistance to apoptosis. Furthermore, CD9-negative cells are less dependent on FDCs for their survival and growth compared with CD9-positive cells. CD9 down-regulation in B lymphomas appears to be controlled epigenetically, mainly through histone modifications. These findings imply that CD9 is inversely correlated with B lymphoma progression, and CD9 inactivation may play an important role in B lymphoma transformation. PMID:20566742

  7. TGFβ-pathway is down-regulated in a uterine carcinosarcoma: a case study.

    PubMed

    Semczuk, Andrzej; Zakrzewski, Piotr K; Forma, Ewa; Cygankiewicz, Adam I; Semczuk-Sikora, Anna; Bryś, Magdalena; Rechberger, Tomasz; Krajewska, Wanda M

    2013-11-01

    Data assessing the role of various genetic alterations in uterine carcinosarcoma (CS), particularly the transforming growth factors-β (TGFβ) that play a crucial role in many cellular processes, including proliferation, differentiation, adhesion and migration, are scarce. TGFβ exert their effects through specific receptors and associated auxiliary receptors. In the current study, we investigated the expression of TGFβ isoforms and their receptors, as well as selected genes in a case of CS. We applied the real-time fluorescence detection PCR method with FAM dye-labeled TaqMan specific probes. In a comparison to the normal counterpart, TGFB1, TGFB2, TGFBRII, TGFBR3, ENG and CD109 were all down-regulated in uterine CS samples at different extents. BIRC5 and hTERT, markers of tumor survival, were up-regulated in CS as compared with normal counterparts. A concomitant increase of the hypoxia marker HIF1A expression pattern was noted, whereas the expression of GPR120, responsible for free fatty acids sensing, was not different in both counterparts evaluated. In conclusion, deregulation of various cellular mechanisms in uterine CS is associated with alterations at many levels - cell growth and proliferation, apoptosis, and impaired response to stimuli from extracellular environment. PMID:23932095

  8. Down-Regulation of Negative Emotional Processing by Transcranial Direct Current Stimulation: Effects of Personality Characteristics

    PubMed Central

    Peña-Gómez, Cleofé; Vidal-Piñeiro, Dídac; Clemente, Immaculada C.; Pascual-Leone, Álvaro; Bartrés-Faz, David

    2011-01-01

    Evidence from neuroimaging and electrophysiological studies indicates that the left dorsolateral prefrontal cortex (DLPFC) is a core region in emotional processing, particularly during down-regulation of negative emotional conditions. However, emotional regulation is a process subject to major inter-individual differences, some of which may be explained by personality traits. In the present study we used transcranial direct current stimulation (tDCS) over the left DLPFC to investigate whether transiently increasing the activity of this region resulted in changes in the ratings of positive, neutral and negative emotional pictures. Results revealed that anodal, but not cathodal, tDCS reduced the perceived degree of emotional valence for negative stimuli, possibly due to an enhancement of cognitive control of emotional expression. We also aimed to determine whether personality traits (extraversion and neuroticism) might condition the impact of tDCS. We found that individuals with higher scores on the introversion personality dimension were more permeable than extraverts to the modulatory effects of the stimulation. The present study underlines the role of the left DLPFC in emotional regulation, and stresses the importance of considering individual personality characteristics as a relevant variable, although replication is needed given the limited sample size of our study. PMID:21829522

  9. Transient down-regulation and restoration of glycogen synthase levels in axotomized rat facial motoneurons.

    PubMed

    Takezawa, Yosuke; Kohsaka, Shinichi; Nakajima, Kazuyuki

    2014-10-24

    In adult rats, transection of the facial nerve causes a functional down-regulation of motoneurons and glial activation/proliferation. It has not been clear how energy-supplying systems are regulated in an axotomized facial nucleus. Here we investigated the regulation of molecules involved in glycogen degradation/synthesis in axotomized facial nuclei in rats. Immunoblotting revealed that the amounts of glycogen phosphorylase in the contralateral and ipsilateral nuclei were unchanged for the first 14 days, whereas the amount of glycogen synthase in the axotomized facial nuclei was significantly decreased from days 7-14 post-insult. A quantitative analysis estimated that the glycogen synthase levels in the transected nucleus were reduced to approx. 50% at 14 days post-injury. An immunohistochemical study showed that the injured motoneurons had decreased expressions of glycogen synthase proteins. The glycogen synthase levels in the axotomized facial nucleus had returned to control levels by 5 weeks post-insult, as had the cholinergic markers. The immunohistochemical study also revealed the recovery of glycogen synthase levels at the later stage. The glycogen phosphorylase levels in the injured nucleus were not significantly changed during weeks 3-5 post-insult. Taken together, these results demonstrated that the injured facial motoneurons transiently reduced glycogen synthase levels at around 1-2 weeks post-insult, but restored the levels at 4-5 weeks post-insult. PMID:25152465

  10. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    SciTech Connect

    Walsh, Erica M.; Niu, MengMeng; Bergholz, Johann; Jim Xiao, Zhi-Xiong

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  11. Autophagic down-regulation in motor neurons remarkably prolongs the survival of ALS mice.

    PubMed

    Hsueh, Kuo-Wei; Chiou, Tzyy-Wen; Chiang, Shu-Fen; Yamashita, Toru; Abe, Koji; Borlongan, Cesar V; Sanberg, Paul R; Huang, Angela Yu Hsuan; Lin, Shinn-Zong; Harn, Horng-Jyh

    2016-09-01

    Amyotrophic lateral sclerosis (ALS) is a lethal degenerating disease, characterized by progressive muscular atrophy without any effective treatment. Here, we demonstrated the efficacy of abrograting autophagy in motor neurons (MN) by treatment with n-butylidenephthalide (n-BP) in ALS transgenic mice (SOD1(G93A)). Pre-symptomatic oral administration of 250 mg/kg/bid n-BP significantly prolonged the survival period (203.9 ± 18.3 days), improved motor function, and attenuated MN loss compared to vehicle control (126.4 ± 7.2 days). This prolonged survival of ALS mice is much more robust than that reported with riluzole (140 days), which is an approved clinical therapy for ALS. The therapeutic mechanism targeted by n-BP involved the autophagic pathway as evidenced by decreased LC3-II expression (a biomarker of autophagy), enhanced mTOR levels, and attenuated autophagic activity, altogether increasing MN survival in a dose-dependent manner. This result was also confirmed by double transgenic mice (SOD1(G93A):LC3-GFP) which showed that oral administration of n-BP reduced GFP density and decreased caspase-3 expression. In addition, electron microscopy revealed that n-BP administration not only decreased autophagosome number but also reduced morphological dysfunction of mitochondria. In summary, these results indicate that down-regulation of autophagy activation via n-BP may pose as a therapeutic regimen for ALS and relevant neurodegenerative diseases. PMID:27059126

  12. Influenza peptide-induced self-lysis and down-regulation of cloned cytotoxic T cells.

    PubMed Central

    Pemberton, R M; Wraith, D C; Askonas, B A

    1990-01-01

    Virus-specific cytotoxic T-cell (Tc) clones can lyse target cells in vitro in the presence of their specific peptide epitopes. The lytic potency of murine influenza nucleoprotein (NP)-specific Tc clones was investigated after observing that target cell killing was reduced in the presence of high (greater than 0.2 microM) concentrations of specific NP peptide antigen. Following incubation of Tc for 16 hr in the presence of a range of peptide concentrations, two effects were observed; (i) a peptide dose-dependent mortality of Tc, which has been attributed to self-lysis by clonal Tc in the presence of specific peptide; (ii) and a reduced ability to specifically lyse NP-expressing target cells whilst retaining lectin-dependent lytic activity in the surviving Tc. This functional down-regulation was reversible after 24 hr incubation in the absence of peptide. Toxic effects were excluded, since inhibition of specific target lysis by Tc was mediated only be pretreatment with specifically recognized peptide. PMID:2373520

  13. Ginsenoside Rb1 inhibits matrix metalloproteinase 13 through down-regulating Notch signaling pathway in osteoarthritis.

    PubMed

    Wang, Wei; Zeng, Li; Wang, Ze-ming; Zhang, Sihan; Rong, Xiao-Feng; Li, Rong-Heng

    2015-12-01

    Mounting evidence suggests that an excess of matrix metalloproteinase-13 (MMP-13) plays an important role in the breakdown of extracellular matrix in osteoarthritis (OA). Here, the effects of ginsenoside Rb1 (GRb1) on the expression of MMP-13 in IL-1β-induced SW 1353 chondrosarcoma cells and an experimental rat model of OA induced by anterior cruciate ligament transection (ACLT) were investigated. SW1353 chondrosarcoma cells were pretreated with or without GRb1 and Notch signaling pathway inhibitor, DAPT, then were stimulated with IL-1β. In rats, experimental OA was induced by ACLT. These rats then received intra-articular injections of vehicle, an inhibitor of γ-secretase, DAPT, and/or GRb1. Expression of MMP-13, collagen type II (CII), Notch1, and jagged 1 (JAG1) were verified by western blotting and immunohistochemistry. In addition, levels of MMP-13 mRNA were detected using quantitative real-time PCR. In histological analyses, treatment with DAPT reduced the number of cartilage lesions present and the expressions of MMP-13, CII, Notch1, and JAG1. In addition, treatment with GRb1 was associated with lower levels of Notch1 and JAG1 in both IL-1β-induced SW1353 chondrosarcoma cells and in the rat OA model. Furthermore, the suppressive effect of GRb1 on MMP-13 was greater than that exhibited by the signaling pathway inhibitor. In conclusion, GRb1 inhibits MMP-13 through down-regulating Notch signaling pathway in OA. PMID:26062798

  14. Down-regulation of lipoprotein lipase increases ABCA1-mediated cholesterol efflux in THP-1 macrophages.

    PubMed

    Kawashima, Ryoko L; Medh, Jheem D

    2014-08-01

    The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of excess cholesterol from foam cells to lipid-poor apolipoprotein A-I, in a process called reverse cholesterol transport. Lipoprotein lipase (LPL) is a lipolytic enzyme expressed by macrophages within atherosclerotic lesions. Lentivirus-mediated RNA interference was used to genetically knock-down (KD) the expression of LPL in THP-1 macrophages. Silencing of the LPL gene was confirmed by end-point PCR, real time PCR, and protein analysis. Suppression of LPL expression correlated with a 1.6-fold up-regulation of ABCA1 mRNA levels, and resulted in a 4.5-fold increase in ABCA1-dependent cholesterol efflux. Replenishing LPL by addition of purified bovine LPL to the cell culture media resulted in down-regulation of ABCA1-mediated cholesterol efflux in both wild-type and LPL knockdown cells. These findings suggest an inverse correlation between macrophage LPL levels and ABCA1 cholesterol transport activity. PMID:25017912

  15. Down-regulation of osteoprotegerin expression as a novel biomarker for colorectal carcinoma

    PubMed Central

    Kim, Hyun-Soo; Yoon, Gun; Do, Sung-Im; Kim, Sung-Joo; Kim, Youn-Wha

    2016-01-01

    A better understanding of tumor biology is important in the identification of molecules that are down-regulated in malignancy and in determining their role in tumor suppression. The aim of this study was to analyze osteoprotegerin (OPG) expression in colorectal carcinoma (CRC) and to investigate the underlying mechanism for changes in the expression of OPG. OPG expression was assessed in CRC tissue samples and cell lines. The methylation status of the OPG promoter region was determined, and the effects of demethylation on OPG expression were analyzed. The effects of recombinant OPG (rOPG) administration on cellular functions were also investigated. Clinical and prognostic implications of OPG protein expression in CRC patients were analyzed. The CRC tissues and cells showed significantly lower OPG expression. Pyrosequencing of OPG-silenced CRC cells revealed that the OPG gene promoter was highly methylated. Treatment with demethylating agent significantly elevated OPG mRNA and protein expression. rOPG significantly decreased cell viability and MMP-2 and VEGF-A production in CRC cells. Reduced OPG immunoreactivity was associated with aggressive oncogenic behavior in CRC. Also, OPG expression was found to be an independent predictor of recurrent hepatic metastasis and independent prognostic factor for worse survival rates. We demonstrated that OPG silencing in CRC occurs through epigenetic repression, and is involved in the development and progression of CRC. Our data suggest that OPG is a novel prognostic biomarker and a new therapeutic target for the treatment of patients with CRC. PMID:26942563

  16. The down regulation of target genes by photo activated DNA nanoscissors.

    PubMed

    Tsai, Tsung-Lin; Shieh, Dar-Bin; Yeh, Chen-Sheng; Tzeng, Yonhua; Htet, Khant; Chuang, Kao-Shu; Hwu, Jih Ru; Su, Wu-Chou

    2010-09-01

    An artificial, targeted, light-activated nanoscissor (ATLANS) was developed for precision photonic cleavage of DNA at selectable target sequences. The ATLANS is comprised of nanoparticle core and a monolayer of hydrazone-modified triplex-forming oligonucleotides (TFOs), which recognize and capture the targeted DNA duplex. Upon photo-illumination (lambda = 460 nm), the attached hydrazone scissor specifically cleaves the targeted DNA at a pre-designed nucleotide pair. Electrophoretic mobility shift and co-precipitation assays revealed sequence-specific binding with the short-fragment and long-form plasmid DNA of both TFO and TFO-nanoparticle probes. Upon photo-illumination, ATLANS introduced a precise double-stranded break 12bp downstream the TFO binding sequence and down-regulated the target gene in HeLa cell system. Gold nanoparticles multiplexed the cutting efficiency and potential for simultaneous manipulation of multiple targets, as well as protected DNA from non-specific photo-damage. This photon-mediated DNA manipulation technology will facilitate high spatial and temporal precision in simultaneous silencing at the genome level, and advanced simultaneous manipulation of multiple targeted genes. PMID:20605206

  17. Down-regulated Lotus japonicus GCR1 plants exhibit nodulation signalling pathways alteration.

    PubMed

    Rogato, Alessandra; Valkov, Vladimir Totev; Alves, Ludovico Martins; Apone, Fabio; Colucci, Gabriella; Chiurazzi, Maurizio

    2016-06-01

    G Protein Coupled Receptor (GPCRs) are integral membrane proteins involved in various signalling pathways by perceiving many extracellular signals and transducing them to heterotrimeric G proteins, which further transduce these signals to intracellular downstream effectors. GCR1 is the only reliable plant candidate as a member of the GPCRs superfamily. In the legume/rhizobia symbiotic interaction, G proteins are involved in signalling pathways controlling different steps of the nodulation program. In order to investigate the putative hierarchic role played by GCR1 in these symbiotic pathways we identified and characterized the Lotus japonicus gene encoding the seven transmembrane GCR1 protein. The detailed molecular and topological analyses of LjGCR1 expression patterns that are presented suggest a possible involvement in the early steps of nodule organogenesis. Furthermore, phenotypic analyses of independent transgenic RNAi lines, showing a significant LjGCR1 expression down regulation, suggest an epistatic action in the control of molecular markers of nodulation pathways, although no macroscopic symbiotic phenotypes could be revealed. PMID:27095401

  18. Hemopexin down-regulates LPS-induced proinflammatory cytokines from macrophages

    PubMed Central

    Liang, Xueya; Lin, Tian; Sun, Guangjie; Beasley-Topliffe, Laura; Cavaillon, Jean-Marc; Warren, H. Shaw

    2009-01-01

    Detection of LPS in tissues is an integral component of innate immunity that acts to protect against invasion by Gram-negative bacteria. Plasma down-regulates LPS-induced cytokine production from macrophages, thereby limiting systemic inflammation in blood and distant tissues. To identify the protein(s) involved in this process, we used classical biochemical chromatographic techniques to identify fractions of mouse sera that suppress LPS-induced TNF from bone marrow-derived macrophages (BMDMs). Fractionation yielded microgram quantities of a protein that was identified by MS to be hemopexin (Hx). Mouse Hx purified on hemin-agarose beads and rhHx decreased the production of cytokines from BMDMs and peritoneal macrophages induced by LPS. Preincubation of LPS with Hx did not affect the activity of LPS on LAL, whereas preincubation of Hx with macrophages followed by washing resulted in decreased activity of these cells in response to LPS, suggesting that Hx acts on macrophages rather than LPS. Heme-free Hx did not stimulate HO-1 in the macrophages. Purified Hx also decreased TNF and IL-6 from macrophages induced by the synthetic TLR2 agonist Pam3Cys. Our data suggest that Hx, which is an acute-phase protein that increases during inflammation, limits TLR4 and TLR2 agonist-induced macrophage cytokine production directly through a mechanism distinct from HO-1. PMID:19395472

  19. Carnosine reverses the aging-induced down regulation of brain regional serotonergic system.

    PubMed

    Banerjee, Soumyabrata; Ghosh, Tushar K; Poddar, Mrinal K

    2015-12-01

    The purpose of the present investigation was to study the role of carnosine, an endogenous dipeptide biomolecule, on brain regional (cerebral cortex, hippocampus, hypothalamus and pons-medulla) serotonergic system during aging. Results showed an aging-induced brain region specific significant (a) increase in Trp (except cerebral cortex) and their 5-HIAA steady state level with an increase in their 5-HIAA accumulation and declination, (b) decrease in their both 5-HT steady state level and 5-HT accumulation (except cerebral cortex). A significant decrease in brain regional 5-HT/Trp ratio (except cerebral cortex) and increase in 5-HIAA/5-HT ratio were also observed during aging. Carnosine at lower dosages (0.5-1.0μg/Kg/day, i.t. for 21 consecutive days) didn't produce any significant response in any of the brain regions, but higher dosages (2.0-2.5μg/Kg/day, i.t. for 21 consecutive days) showed a significant response on those aging-induced brain regional serotonergic parameters. The treatment with carnosine (2.0μg/Kg/day, i.t. for 21 consecutive days), attenuated these brain regional aging-induced serotonergic parameters and restored towards their basal levels that observed in 4 months young control rats. These results suggest that carnosine attenuates and restores the aging-induced brain regional down regulation of serotonergic system towards that observed in young rats' brain regions. PMID:26364584

  20. Top-down regulation of default mode activity in spatial visual attention.

    PubMed

    Wen, Xiaotong; Liu, Yijun; Yao, Li; Ding, Mingzhou

    2013-04-10

    Dorsal anterior cingulate and bilateral anterior insula form a task control network (TCN) whose primary function includes initiating and maintaining task-level cognitive set and exerting top-down regulation of sensorimotor processing. The default mode network (DMN), comprising an anatomically distinct set of cortical areas, mediates introspection and self-referential processes. Resting-state data show that TCN and DMN interact. The functional ramifications of their interaction remain elusive. Recording fMRI data from human subjects performing a visual spatial attention task and correlating Granger causal influences with behavioral performance and blood oxygen level-dependent (BOLD) activity we report three main findings. First, causal influences from TCN to DMN, i.e., TCN → DMN, are positively correlated with behavioral performance. Second, causal influences from DMN to TCN, i.e., DMN → TCN, are negatively correlated with behavioral performance. Third, stronger DMN → TCN are associated with less elevated BOLD activity in TCN, whereas the relationship between TCN → DMN and DMN BOLD activity is unsystematic. These results suggest that, during visual spatial attention, top-down signals from TCN to DMN regulate the activity in DMN to enhance behavioral performance, whereas signals from DMN to TCN, acting possibly as internal noise, interfere with task control, leading to degraded behavioral performance. PMID:23575842

  1. Down-regulation of the small conductance calcium-activated potassium channels in diabetic mouse atria.

    PubMed

    Yi, Fu; Ling, Tian-You; Lu, Tong; Wang, Xiao-Li; Li, Jingchao; Claycomb, William C; Shen, Win-Kuang; Lee, Hon-Chi

    2015-03-13

    The small conductance Ca(2+)-activated K(+) (SK) channels have recently been found to be expressed in the heart, and genome-wide association studies have shown that they are implicated in atrial fibrillation. Diabetes mellitus is an independent risk factor of atrial fibrillation, but the ionic mechanism underlying this relationship remains unclear. We hypothesized that SK channel function is abnormal in diabetes mellitus, leading to altered cardiac electrophysiology. We found that in streptozotocin-induced diabetic mice, the expression of SK2 and SK3 isoforms was down-regulated by 85 and 92%, respectively, whereas that of SK1 was not changed. SK currents from isolated diabetic mouse atrial myocytes were significantly reduced compared with controls. The resting potentials of isolated atrial preparations were similar between control and diabetic mice, but action potential durations were significantly prolonged in the diabetic atria. Exposure to apamin significantly prolonged action potential durations in control but not in diabetic atria. Production of reactive oxygen species was significantly increased in diabetic atria and in high glucose-cultured HL-1 cells, whereas exposure of HL-1 cells in normal glucose culture to H2O2 reduced the expression of SK2 and SK3. Tyrosine nitration in SK2 and SK3 was significantly increased by high glucose culture, leading to accelerated channel turnover. Treatment with Tiron prevented these changes. Our results suggest that increased oxidative stress in diabetes results in SK channel-associated electrical remodeling in diabetic atria and may promote arrhythmogenesis. PMID:25605734

  2. Dioscin enhances methotrexate absorption by down-regulating MDR1 in vitro and in vivo

    SciTech Connect

    Wang, Lijuan; Wang, Changyuan; Peng, Jinyong; Liu, Qi; Meng, Qiang; Sun, Huijun; Huo, Xiaokui; and others

    2014-06-01

    The purpose of this study was to investigate the enhancing effect of dioscin on the absorption of methotrexate (MTX) and clarify the molecular mechanism involved in vivo and in vitro. Dioscin increased MTX chemosensitivity and transepithelial flux in the absorptive direction, significantly inhibiting multidrug resistance 1 (MDR1) mRNA and protein expression and MDR1 promoter and nuclear factor κ-B (NF-κB) activities in Caco-2 cells. Moreover, inhibitor κB-α (IκB-α) degradation was inhibited by dioscin. Dioscin enhanced the intracellular concentration of MTX by down-regulating MDR1 expression through a mechanism that involves NF-κB signaling pathway inhibition in Caco-2 cells. Dioscin strengthened MTX absorption by inhibiting MDR1 expression in rat intestine. In addition, even though MTX is absorbed into the enterocytes, there was no increase in toxicity observed, and that, in fact, decreased toxicity was seen. - Highlights: • Dioscin raised MTX concentration by inhibiting MDR1 in Caco-2 cells. • Dioscin suppresses MDR1 by inhibiting NF-κB signaling pathway in Caco-2 cells. • Dioscin can enhance MTX absorption via inhibiting MDR1 in vivo and in vitro. • Dioscin did not increase MTX-induced gastrointestinal mucosal toxicity.

  3. The Mycobacterium tuberculosis H37Ra gene MRA_1916 causes growth defects upon down-regulation.

    PubMed

    Singh, Kumar Sachin; Singh, Sudheer Kumar

    2015-01-01

    D-amino acid oxidases play an important role in converting D-amino acids to their corresponding α-keto acids. MRA_1916 of Mycobacterium tuberculosis H37Ra (Mtb-Ra) is annotated to be a D-amino acid oxidase (DAO). However, not much information is available about its physiological role during Mtb-Ra growth and survival. The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth. Recombinant Mtb-Ra strains with DAO and GlcB (malate synthase: MRA_1848) gene knockdown were developed and their growth was studied using Microtiter Alamar Blue Assay (MABA) with glycerol, acetate and glycine as a carbon source. Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB. MABA study showed inhibition of wild-type (WT) and knockdowns in the presence of BrP (2.5mM). However, growth inhibition of WT was less noticeable at lower concentrations of BrP. Mtb-Ra with DAO knockdown showed poor utilization of glycine in the presence of BrP. The DAO localization study showed its prominent distribution in cytosolic fraction and to some extent in cell wall and membrane fractions. Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104). PMID:26531045

  4. Silkworm hemolymph down-regulates the expression of endoplasmic reticulum chaperones under radiation-irradiation.

    PubMed

    Lee, Kyeong Ryong; Kim, Seung-Whan; Kim, Young Kook; Kwon, Kisang; Choi, Jong-Soon; Yu, Kweon; Kwon, O-Yu

    2011-01-01

    We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, "ischemia-responsive protein 94 kDa" (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation. PMID:21845089

  5. Down-regulation of osteopontin attenuates breast tumour progression in vivo.

    PubMed

    Chakraborty, Goutam; Jain, Shalini; Patil, Tushar V; Kundu, Gopal C

    2008-12-01

    Development of breast tumour malignancies results in enhanced expression of various oncogenic molecules. Elevated expression of osteopontin (OPN) in higher grades of breast carcinoma correlates with enhanced expressions of several oncogenic molecules (urokinase-type plasminogen activator [uPA], matrix metalloproteinase-2/-9 [MMP-2 and -9]) and increased angiogenic potential of breast carcinoma. In this study, using in vitro and multiple in vivo models, we have demonstrated that silencing of OPN by its specific small interfering RNA (siRNA) down-regulates the expressions of oncogenic molecules such as uPA, MMP-2 and -9 resulting in inhibition of in vitro cell motility and in vivo tumourigenicity in mice. Moreover our results demonstrated that OPN-/- mice showed slower progression of tumour growth in breast cancer model as compared to wild-type mice. Furthermore, the data showed that injection of carcinogenic compound, pristane (2, 6,10,14-tetramethylpen-tadecane) induces breast tumour progression leading to enhanced expression of OPN and other oncogenic molecules in mammary fat pad of nude- and wild-type mice but not in OPN-/- mice. However, intratumoural injection of OPN siRNA to pristane-induced tumour significantly suppressed these effects. Our data revealed that knocking down of OPN effectively curb breast cancer progression and further suggested that developing of OPN-based therapeutics might be an emerging approach for the next generation of breast cancer management. PMID:18266970

  6. Minocycline Down-Regulates Topical Mucosal Inflammation during the Application of Microbicide Candidates

    PubMed Central

    Li, Liangzhu; Ben, Yinyin; Zhu, Zhaoqin; Li, Weihua; Xu, Jianqing; Zhang, Xiaoyan

    2012-01-01

    An effective anti-human immunodeficiency virus-1 (HIV-1) microbicide should exert its action in the absence of causing aberrant activation of topical immunity that will increase the risk of HIV acquisition. In the present study, we demonstrated that the vaginal application of cellulose sulfate (CS) gel induced topical mucosal inflammatory responses; the addition of minocycline to CS gel could significantly attenuate the inflammation in a mice model. The combined gel of CS plus minocycline not only reduced the production of inflammatory cytokines in cervicovaginal lavages (CVLs), also down-regulated the activation of CD4+ T cells and the recruitment of other immune cells including HIV target cells into vaginal tissues. Furthermore, an In vitro HIV-1 pseudovirus infection inhibition assay showed that the combined gel decreased the infection efficacy of different subtypes of HIV-1 pseudoviruses compared with that of CS gel alone. These results implicate that minocycline could be integrated into microbicide formulation to suppress the aberrant activation of topical mucosal immunity and enhance the safety profile during the application of microbicides. PMID:22905236

  7. Top-down regulation of default mode activity in spatial visual attention

    PubMed Central

    Wen, Xiaotong; Liu, Yijun; Yao, Li; Ding, Mingzhou

    2013-01-01

    Dorsal anterior cingulate and bilateral anterior insula form a task control network (TCN) whose primary function includes initiating and maintaining task-level cognitive set and exerting top-down regulation of sensorimotor processing. The default mode network (DMN), comprising an anatomically distinct set of cortical areas, mediates introspection and self-referential processes. Resting-state data show that TCN and DMN interact. The functional ramifications of their interaction remain elusive. Recording fMRI data from human subjects performing a visual spatial attention task and correlating Granger causal influences with behavioral performance and blood-oxygen-level-dependent (BOLD) activity we report three main findings. First, causal influences from TCN to DMN, i.e., TCN→DMN, are positively correlated with behavioral performance. Second, causal influences from DMN to TCN, i.e., DMN→TCN, are negatively correlated with behavioral performance. Third, stronger DMN→TCN are associated with less elevated BOLD activity in TCN, whereas the relationship between TCN→DMN and DMN BOLD activity is unsystematic. These results suggest that during visual spatial attention, top-down signals from TCN to DMN regulate the activity in DMN to enhance behavioral performance, whereas signals from DMN to TCN, acting possibly as internal noise, interfere with task control, leading to degraded behavioral performance. PMID:23575842

  8. Galectin-3 down-regulates IL-5 gene expression on different cell types.

    PubMed

    Cortegano, I; del Pozo, V; Cárdaba, B; de Andrés, B; Gallardo, S; del Amo, A; Arrieta, I; Jurado, A; Palomino, P; Liu, F T; Lahoz, C

    1998-07-01

    Galectin-3 is an animal lectin, formerly named epsilon-binding protein or Mac-2, which has been described to play an important role in some inflammatory processes by the implication of different cells and the increase in cell adhesion functions through laminin binding activity. In this work we analyzed the role of galectin-3 in the modulation of Th2 cytokines that have an important role in the development of the inflammatory response. We have found that the addition of galectin-3 to human eosinophils, the eosinophilic cell line EoL-3, PBMC, and an Ag-specific T cell line (CD4+) produced a selective inhibition of IL-5 transcription. No inhibitory effect was found on the IL-4 mRNA transcription rate. The inhibitory effect on IL-5 transcription was reversed by incubation with lactose and using specific Ab against galectin-3. Galectin-3 is able to induce inhibition of the IL-5 released in the supernatants from PBMC stimulated with phorbol 12,13-dibutyrate and anti-CD3. Similar results were obtained when a T-specific cell line was stimulated with Ag. Also, EoL-3 stimulated with anti-CD32 produced IL-5 protein, the synthesis of which was partially inhibited by galectin-3. The present results demonstrate that galectin-3 induces a selective down-regulation of IL-5 expression in different cell types, opening important new possibilities in the regulation of the allergic reactions. PMID:9647247

  9. Down Regulation of Gene Expression Between the Diapause Initiation and Maintenance Phases of the Colorado Potato Beetle, Leptinotarsa Decemlineata

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The diapause initiation and maintenance phases of the Colorado potato beetle, Leptinotarsa decemlineata were screened. Eight transcripts were found to be down regulated as the beetles enter the diapause maintenance phase of diapause development after day 15 postemergence. These transcripts were also...

  10. DNA Methylation mediated down-regulating of MicroRNA-33b and its role in gastric cancer

    PubMed Central

    Yin, Haixin; Song, Peng; Su, Rui; Yang, Guihua; Dong, Lei; Luo, Min; Wang, Bin; Gong, Bei; Liu, Changzheng; Song, Wei; Wang, Fang; Ma, Yanni; Zhang, Junwu; Wang, Weibin; Yu, Jia

    2016-01-01

    The discovery of microRNAs (miRNAs) provides a new and powerful tool for studying the mechanism, diagnosis and treatment of human cancers. Currently, down-regulation of tumor suppressive miRNAs by CpG island hypermethylation is emerging as a common hallmark of cancer. Here, we reported that the down-regulation of miR-33b was associated with pM stage of gastric cancer (GC) patients. Ectopic expression of miR-33b in HGC-27 and MGC-803 cells inhibited cell proliferation, migration and invasion, which might be due to miR-33b targeting oncogene c-Myc. Moreover, enhanced methylation level of the CpG island upstream of miR-33b in GC patients with down-regulated miR-33b was confirmed by methylation-specific PCR (MSP) amplification. Furthermore, re-introduction of miR-33b significantly suppressed tumorigenesis of GC cells in the nude mice. In conclusion, miR-33b acts as a tumor suppressor and hypermethylation of the CpG island upstream of miR-33b is responsible for its down-regulation in gastric cancer. PMID:26729612

  11. Transcriptional down regulation of the nov proto-oncogene in fibroblasts transformed by p60v-src.

    PubMed Central

    Scholz, G; Martinerie, C; Perbal, B; Hanafusa, H

    1996-01-01

    We have sought to identify genes whose expression is altered as a consequence of transformation by p60v-src. Using the mRNA differential display method, we have identified the nov proto-oncogene as one gene that is down regulated in chicken embryo fibroblasts (CEFs) transformed by p60v-src. nov transcripts were also found to be present at only very low levels in proliferating CEFs in comparison with quiescent CEFs. Serum stimulation of quiescent CEFs also resulted in a decline in the steady-state level of nov transcripts. Taken together, these findings suggest that the nov gene is expressed only in quiescent fibroblasts and that its down regulation may contribute to cellular transformation by the v-src oncogene. Down regulation of the nov gene appears to occur at both the transcriptional and posttranscriptional levels. Results obtained from experiments with a protein kinase inhibitor suggest that protein kinase C may be a key downstream effector in mediating the down regulation of nov transcripts in response to activation of p60src or serum stimulation. In addition, we found that transcription of an unknown gene is required for the decline in the steady-state level of nov transcripts in response to serum stimulation. PMID:8552074

  12. Prostaglandin E2 mediates growth arrest in NFS-60 cells by down-regulating interleukin-6 receptor expression.

    PubMed Central

    de Silva, Kumudika I; Daud, Asif N; Deng, JiangPing; Jones, Stephen B; Gamelli, Richard L; Shankar, Ravi

    2003-01-01

    Interleukin-6 (IL-6), a potent myeloid mitogen, and the immunosuppressive prostanoid prostaglandin E2 (PGE2) are elevated following thermal injury and sepsis. We have previously demonstrated that bone marrow myeloid commitment shifts toward monocytopoiesis and away from granulocytopoiesis during thermal injury and sepsis and that PGE2 plays a central role in this alteration. Here we investigated whether PGE2 can modulate IL-6-stimulated growth in the promyelocytic cell line, NFS-60, by down-regulating IL-6 receptor (IL-6r) expression. Exposure of NFS-60 cells to PGE2 suppressed IL-6-stimulated proliferation as well as IL-6r expression. Receptor down-regulation is functionally significant since IL-6-induced signal transduction through activators of transcription (STAT)-3 is also decreased. Down-regulation of IL-6r correlated with the ability of PGE2 to arrest cells in the G0/G1 phase of the cell cycle. PGE2 appears to signal through EP2 receptors. Butaprost (EP2 agonist) but not sulprostone (EP3 agonist) inhibited IL-6-stimulated proliferation. In addition, an EP2 antagonist (AH6809) alleviated the anti-proliferative effects of PGE2. NFS-60 cells express predominantly EP2 and EP4 receptors. While PGE2 down-regulated both the IL-6r protein and mRNA expression, it had no influence on EP2 or EP4 mRNA expression. The present study demonstrates that PGE2 is a potent down-regulator of IL-6r expression and thus may provide a mechanistic explanation for the granulocytopenia seen in thermal injury and sepsis. PMID:12429018

  13. Down-regulation of TRPS1 stimulates epithelial-mesenchymal transition and metastasis through repression of FOXA1.

    PubMed

    Huang, Jin-Zhou; Chen, Min; Zeng, Ming; Xu, Song-Hui; Zou, Fei-Yan; Chen, De; Yan, Guang-Rong

    2016-06-01

    The tricho-rhino-phalangeal syndrome 1 gene (TRPS1), which was initially found to be associated with tricho-rhino-phalangeal syndrome, is critical for the development and differentiation of bone, hair follicles and kidney. However, its role in cancer progression is largely unknown. In this study, we demonstrated that down-regulation of TRPS1 correlated with distant metastasis, tumour recurrence and poor survival rate in cancer patients. TRPS1 was frequently down-regulated in high-metastatic cancer cell lines from the breast, colon and nasopharynx. Silencing of TRPS1 stimulated epithelial-mesenchymal transition (EMT), migration and invasion in vitro and metastasis in vivo, while TRPS1 over-expression exhibited the opposite effects. Using quantitative proteomics, FOXA1, a negative regulator of epithelial-mesenchymal transition (EMT), was shown to be down-regulated by TRPS1 knockdown. Ectopic expression of FOXA1 blocked the enhancement of EMT, migration and invasion induced by TRPS1 silencing. Mechanistically, TRPS1, acting as a transcription activator, directly induced FOXA1 transcription by binding to the FOXA1 promoter. We further showed that down-regulation of TRPS1 was induced by miR-373 binding to the 3' UTR of TRPS1. Over-expression of TRPS1, but not TRPS1 3' UTR, blocked the enhancement of migration and invasion induced by miR-373. Taken together, we consider that down-regulation of TRPS1 by miR-373, acting as a transcriptional activator, promotes EMT and metastasis by repressing FOXA1 transcription, expanding upon its previously reported role as a transcription repressor. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:26969828

  14. Irradiation of protoporphyric mice induces down-regulation of epidermal eicosanoid metabolism

    SciTech Connect

    He, D.; Lim, H.W. )

    1991-09-01

    This study investigated the effect of radiation on clinical and histologic changes, and on cutaneous eicosanoid metabolism, in Skh:HR-1 hairless albino mice rendered protoporphyric by the administration of collidine. At 0.1-18 h after exposure to 12 kJ/m2 of 396-406 nm irradiation, thicknesses of back skin and ears were measured, and histologic changes were evaluated by using hematoxylin and eosin (H-E) and Giemsa's stains. Activities of eicosanoid-metabolizing enzymes in epidermal and dermal homogenates were assessed by incubating the tissue homogenates with 3H-AA, followed by quantitation of the eicosanoids generated by radio-TLC. In irradiated protoporphyric mice, an increase of back-skin thickness was noted at 0.1 h, reaching a peak at 18 h, whereas maximal increase in ear thickness was observed at 12 h. Histologic changes included dermal edema, increased mast cell degranulation, and mononuclear cells in the dermis. In these irradiated protoporphyric animals, generations of 6 keto-PGF1a, PGF2a, PGE2, PGD2, and HETE by epidermal eicosanoid-metabolizing enzymes were markedly suppressed at all the timepoints studied. Dermal eicosanoid-metabolizing enzymes of irradiated protoporphyric mice generated increased amounts of PGE2 and HETE at 18 h, probably reflecting the presence of dermal cellular infiltrates. The suppression of the activities of epidermal eicosanoid-metabolizing enzymes was prevented by intraperitoneal injection of WR-2721, a sulfhydryl group generator, prior to irradiation, suggesting that the suppression was secondary to photo-oxidative damage of the enzymes during the in vivo phototoxic response. These results suggest that the effect of protoporphyrin and radiation on cutaneous eicosanoid metabolism in this animal model in vivo is that of a down regulation of the activities of epidermal eicosanoid-metabolizing enzymes.

  15. MicroRNA-21 Down-regulates Rb1 Expression by Targeting PDCD4 in Retinoblastoma

    PubMed Central

    Shen, Fengmei; Mo, Meng-Hsuan; Chen, Liang; An, Shejuan; Tan, Xiaohui; Fu, Yebo; Rezaei, Katayoon; Wang, Zuoren; Zhang, Lin; Fu, Sidney W.

    2014-01-01

    Retinoblastoma (RB) is a children's ocular cancer caused by mutated retinoblastoma 1 (Rb1) gene on both alleles. Rb1 and other related genes could be regulated by microRNAs (miRNA) via complementarily pairing with their target sites. MicroRNA-21 (miR-21) possesses the oncogenic potential to target several tumor suppressor genes, including PDCD4, and regulates tumor progression and metastasis. However, the mechanism of how miR-21 regulates PDCD4 is poorly understood in RB. We investigated the expression of miRNAs in RB cell lines and identified that miR-21 is one of the most deregulated miRNAs in RB. Using qRT-PCR, we verified the expression level of several miRNAs identified by independent microarray assays, and analyzed miRNA expression patterns in three RB cell lines, including Weri-Rb1, Y79 and RB355. We found that miR-19b, -21, -26a, -195 and -222 were highly expressed in all three cell lines, suggesting their potential role in RB tumorigenesis. Using the TargetScan program, we identified a list of potential target genes of these miRNAs, of which PDCD4 is one the targets of miR-21. In this study, we focused on the regulatory mechanism of miR-21 on PDCD4 in RB. We demonstrated an inverse correlation between miR-21 and PDCD4 expression in Weri-Rb1 and Y79 cells. These data suggest that miR-21 down-regulates Rb1 by targeting PDCD4 tumor suppressor. Therefore, miR-21 could serve as a therapeutic target for retinoblastoma. PMID:25520758

  16. Impacts on the metabolome of down-regulating polyphenol oxidase in potato tubers.

    PubMed

    Shepherd, Louise Vida Traill; Alexander, Colin James; Hackett, Christine Anne; McRae, Diane; Sungurtas, Julia Anne; Verrall, Susan Ramsay; Morris, Jennifer Anne; Hedley, Peter Edward; Rockhold, David; Belknap, William; Davies, Howard Vivian

    2015-06-01

    Tubers of potato (Solanum tuberosum L. cv. Estima) genetically modified to reduce polyphenol oxidase (PPO) activity and enzymatic discolouration were assessed for changes in the metabolome using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography (GC)-MS. Metabolome changes induced over a 48 hour (h) period by tuber wounding (sliced transverse sections) were also assessed using two PPO antisense lines (asPPO) and a wild-type (WT) control. Data were analysed using Principal Components Analysis and Analysis of Variance to assess differences between genotypes and temporal changes post-tuber wounding (by slicing). The levels of 15 metabolites (out of a total of 134 that were detected) differed between the WT and asPPO lines in mature tubers at harvest. A considerably higher number (63) of these metabolites changed significantly over a 48 h period following tuber wounding. For individual metabolites the magnitude of the differences between the WT and asPPO lines at harvest were small compared with the impacts of tuber wounding on metabolite levels. Some of the observed metabolite changes are explicable in terms of pathways known to be affected by wound responses. Whilst some statistically significant interactions (11 metabolites) were observed between line and time after wounding, very few profiles were consistent when comparing the WT with both asPPO lines, and the underlying metabolites appeared to be random in terms of the pathways they occupy. Overall, mechanical damage to tubers has a considerably greater impact on the metabolite profile than any potential unintended effects resulting from the down-regulation of PPO gene expression. PMID:25417184

  17. Delta-Like Ligand 4 Modulates Liver Damage by Down-Regulating Chemokine Expression.

    PubMed

    Shen, Zhe; Liu, Yan; Dewidar, Bedair; Hu, Junhao; Park, Ogyi; Feng, Teng; Xu, Chengfu; Yu, Chaohui; Li, Qi; Meyer, Christoph; Ilkavets, Iryna; Müller, Alexandra; Stump-Guthier, Carolin; Munker, Stefan; Liebe, Roman; Zimmer, Vincent; Lammert, Frank; Mertens, Peter R; Li, Hai; Ten Dijke, Peter; Augustin, Hellmut G; Li, Jun; Gao, Bin; Ebert, Matthias P; Dooley, Steven; Li, Youming; Weng, Hong-Lei

    2016-07-01

    Disrupting Notch signaling ameliorates experimental liver fibrosis. However, the role of individual Notch ligands in liver damage is unknown. We investigated the effects of Delta-like ligand 4 (Dll4) in liver disease. DLL4 expression was measured in 31 human liver tissues by immunohistochemistry. Dll4 function was examined in carbon tetrachloride- and bile duct ligation-challenged mouse models in vivo and evaluated in hepatic stellate cells, hepatocytes, and Kupffer cells in vitro. DLL4 was expressed in patients' Kupffer and liver sinusoidal endothelial cells. Recombinant Dll4 protein (rDll4) ameliorated hepatocyte apoptosis, inflammation, and fibrosis in mice after carbon tetrachloride challenge. In vitro, rDll4 significantly decreased lipopolysaccharide-dependent chemokine expression in both Kupffer and hepatic stellate cells. In bile duct ligation mice, rDll4 induced massive hepatic necrosis, resulting in the death of all animals within 1 week. Inflammatory cell infiltration and chemokine ligand 2 (Ccl2) expression were significantly reduced in rDll4-receiving bile duct ligation mice. Recombinant Ccl2 rescued bile duct ligation mice from rDll4-mediated death. In patients with acute-on-chronic liver failure, DLL4 expression was inversely associated with CCL2 abundance. Mechanistically, Dll4 regulated Ccl2 expression via NF-κB. Taken together, Dll4 modulates liver inflammatory response by down-regulating chemokine expression. rDll4 application results in opposing outcomes in two models of liver damage. Loss of DLL4 may be associated with CCL2-mediated cytokine storm in patients with acute-on-chronic liver failure. PMID:27171900

  18. Dexamethasone acutely down-regulates genes involved in steroidogenesis in stallion testes.

    PubMed

    Ing, Nancy H; Forrest, David W; Riggs, Penny K; Loux, Shavahn; Love, Charlie C; Brinsko, Steven P; Varner, Dickson D; Welsh, Thomas H

    2014-09-01

    In rodents, livestock and primate species, a single dose of the synthetic glucocorticoid dexamethasone acutely lowers testosterone biosynthesis. To determine the mechanism of decreased testosterone biosynthesis, stallions were treated with 0.1mg/kg dexamethasone 12h prior to castration. Dexamethasone decreased serum concentrations of testosterone by 60% compared to saline-treated control stallions. Transcriptome analyses (microarrays, northern blots and quantitative PCR) of testes discovered that dexamethasone treatment decreased concentrations of glucocorticoid receptor alpha (NR3C1), alpha actinin 4 (ACTN4), luteinizing hormone receptor (LHCGR), squalene epoxidase (SQLE), 24-dehydrocholesterol reductase (DHCR24), glutathione S-transferase A3 (GSTA3) and aromatase (CYP19A1) mRNAs. Dexamethasone increased concentrations of NFkB inhibitor A (NFKBIA) mRNA in testes. SQLE, DHCR24 and GSTA3 mRNAs were predominantly expressed by Leydig cells. In man and livestock, the GSTA3 protein provides a major 3-ketosteroid isomerase activity: conversion of Δ(5)-androstenedione to Δ(4)-androstenedione, the immediate precursor of testosterone. Consistent with the decrease in GSTA3 mRNA, dexamethasone decreased the 3-ketosteroid isomerase activity in testicular extracts. In conclusion, dexamethasone acutely decreased the expression of genes involved in hormone signaling (NR3C1, ACTN4 and LHCGR), cholesterol synthesis (SQLE and DHCR24) and steroidogenesis (GSTA3 and CYP19A1) along with testosterone production. This is the first report of dexamethasone down-regulating expression of the GSTA3 gene and a very late step in testosterone biosynthesis. Elucidation of the molecular mechanisms involved may lead to new approaches to modulate androgen regulation of the physiology of humans and livestock in health and disease. PMID:25010478

  19. Down-regulation of voltage-dependent sodium channels initiated by sodium influx in developing neurons.

    PubMed Central

    Dargent, B; Couraud, F

    1990-01-01

    To address the issue of whether regulatory feedback exists between the electrical activity of a neuron and ion-channel density, we investigated the effect of Na(+)-channel activators (scorpion alpha toxin, batrachotoxin, and veratridine) on the density of Na+ channels in fetal rat brain neurons in vitro. A partial but rapid (t1/2, 15 min) disappearance of surface Na+ channels was observed as measured by a decrease in the specific binding of [3H]saxitoxin and 125I-labeled scorpion beta toxin and a decrease in specific 22Na+ uptake. Moreover, the increase in the number of Na+ channels that normally occurs during neuronal maturation in vitro was inhibited by chronic channel activator treatment. The induced disappearance of Na+ channels was abolished by tetrodotoxin, was found to be dependent on the external Na+ concentration, and was prevented when either choline (a nonpermeant ion) or Li+ (a permeant ion) was substituted for Na+. Amphotericin B, a Na+ ionophore, and monensin were able to mimick the effect of Na(+)-channel activators, while a KCl depolarization failed to do this. This feedback regulation seems to be a neuronal property since Na(+)-channel density in cultured astrocytes was not affected by channel activator treatment or by amphotericin B. The present evidence suggests that an increase in intracellular Na+ concentration, whether elicited by Na(+)-channel activators or mediated by a Na+ ionophore, can induce a decrease in surface Na+ channels and therefore is involved in down-regulation of Na(+)-channel density in fetal rat brain neurons in vitro. PMID:2165609

  20. Down-regulating alpha-galactosidase enhances freezing tolerance in transgenic petunia.

    PubMed

    Pennycooke, Joyce C; Jones, Michelle L; Stushnoff, Cecil

    2003-10-01

    Alpha-galactosidase (alpha-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the alpha-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of alpha-Gal was modified in transgenic petunia (Petunia x hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that alpha-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of alpha-Gal mRNAs. alpha-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from -4 degrees C for cold-acclimated wild-type plants to -8 degrees C for the most tolerant antisense line. Down-regulating alpha-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the alpha-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with alpha-Gal provides an additional method for improving the freezing tolerance of plants. PMID:14500789

  1. Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia1

    PubMed Central

    Pennycooke, Joyce C.; Jones, Michelle L.; Stushnoff, Cecil

    2003-01-01

    α-Galactosidase (α-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the α-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia (Petunia × hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that α-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of α-Gal mRNAs. α-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating α-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the α-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with α-Gal provides an additional method for improving the freezing tolerance of plants. PMID:14500789

  2. Growth differentiation factor 8 down-regulates pentraxin 3 in human granulosa cells.

    PubMed

    Chang, Hsun-Ming; Fang, Lanlan; Cheng, Jung-Chien; Klausen, Christian; Sun, Ying-Pu; Leung, Peter C K

    2015-03-15

    Growth differentiation factor 8 (GDF8), also known as myostatin, is highly expressed in the mammalian musculoskeletal system and plays critical roles in the regulation of skeletal muscle growth. Though not exclusively expressed in the musculoskeletal system, the expression and biological function of GDF8 has never been examined in the human ovary. Pentraxin 3 (PTX3) plays a key role in the assembly of extracellular matrix, which is essential for cumulus expansion, ovulation and in vivo fertilization. The aim of this study was to investigate GDF8 expression and function in human granulosa cells and to examine its underlying molecular determinants. An established immortalized human granulosa cell line (SVOG), granulosa cell tumor cell line (KGN) and primary granulosa-lutein cells were used as study models. We now demonstrate for the first time that GDF8 is expressed in human granulosa cells and follicular fluid. All 16 follicular fluid samples tested contained GDF8 protein at an average concentration of 3 ng/ml. In addition, GDF8 treatment significantly decreased PTX3 mRNA and protein levels. These suppressive effects, along with the induction of SMAD2/3 phosphorylation, were abolished by co-treatment with the ALK4/5/7 inhibitor SB431542. Knockdown of ALK5, ACVR2A/ACVR2B or SMAD4 reversed the effects of GDF8-induced PTX3 suppression. These results indicate that GDF8 down-regulates PTX3 expression via ACVR2A/ACVR2B-ALK5-mediated SMAD-dependent signaling in human granulosa cells. These novel findings support a potential role for GDF8 in the regulation of follicular function, likely via autocrine effects on human granulosa cells. PMID:25641196

  3. FOXL2 down-regulates vitellogenin expression at mature stage in Eriocheir sinensis

    PubMed Central

    Li, Qing; Xie, Jing; He, Lin; Wang, Yuanli; Yang, Hongdan; Duan, Zelin; Wang, Qun

    2015-01-01

    Ovarian development in crustaceans is characterized by rapid production of egg yolk protein in a process called vitellogenesis. In the present study, we investigated the involvement of a DEAD (Asp-Glu-Ala-Asp) box RNA helicase 20 (DDX20), forkhead transcription factor (FOXL)2 and fushi tarazu factor (FTZ-F)1 in the regulation of vitellogenesis. Based on ESTs from the testis and accessory gland of Eriocheir sinensis, we cloned the full-length cDNAs of foxl2 and fushitarazu factor 1 (ftz-f1), which include the conserved structural features of the forkhead family and nuclear receptor 5A (NR5A) family respectively. The expression of foxl2 mRNA surged at the mature stage of the ovary, when vtg mRNA swooped, suggesting that foxl2 negatively affects the vitellogenin (VTG) synthesis at this developmental stage. Etoposide (inducing germ cell apoptosis) treatment up-regulated FOXL2 and DDX20 at both the mRNA and the protein levels, primarily in the follicular cells as shown by immunofluorescence analysis. Furthermore, foxl2, ddx20 and ftz-f1 mRNA levels increased significantly with right-eyestalk ablation. Interactions between FOXL2 and DDX20 or FTZ-F1 were confirmed by co-immunoprecipitation and the forkhead domain of FOXL2 was identified as the specific structure interacting with FTZ-F1. In conclusion, FOXL2 down-regulates VTG expression by binding with DDX20 in regulation of follicular cell apoptosis and with FTZ-F1 to repress the synthesis of VTG at the mature stage. This report is the first to describe the molecular mechanism of VTG synthesis in E. sinensis and may shed new light on the regulation of cytochrome P450 enzyme by FOXL2 and FTZ-F1 in vitellogenesis. PMID:26430246

  4. FOXL2 down-regulates vitellogenin expression at mature stage in Eriocheir sinensis.

    PubMed

    Li, Qing; Xie, Jing; He, Lin; Wang, Yuanli; Yang, Hongdan; Duan, Zelin; Wang, Qun

    2015-01-01

    Ovarian development in crustaceans is characterized by rapid production of egg yolk protein in a process called vitellogenesis. In the present study, we investigated the involvement of a DEAD (Asp-Glu-Ala-Asp) box RNA helicase 20 (DDX20), forkhead transcription factor (FOXL)2 and fushi tarazu factor (FTZ-F)1 in the regulation of vitellogenesis. Based on ESTs from the testis and accessory gland of Eriocheir sinensis, we cloned the full-length cDNAs of foxl2 and fushitarazu factor 1 (ftz-f1), which include the conserved structural features of the forkhead family and nuclear receptor 5A (NR5A) family respectively. The expression of foxl2 mRNA surged at the mature stage of the ovary, when vtg mRNA swooped, suggesting that foxl2 negatively affects the vitellogenin (VTG) synthesis at this developmental stage. Etoposide (inducing germ cell apoptosis) treatment up-regulated FOXL2 and DDX20 at both the mRNA and the protein levels, primarily in the follicular cells as shown by immunofluorescence analysis. Furthermore, foxl2, ddx20 and ftz-f1 mRNA levels increased significantly with right-eyestalk ablation. Interactions between FOXL2 and DDX20 or FTZ-F1 were confirmed by co-immunoprecipitation and the forkhead domain of FOXL2 was identified as the specific structure interacting with FTZ-F1. In conclusion, FOXL2 down-regulates VTG expression by binding with DDX20 in regulation of follicular cell apoptosis and with FTZ-F1 to repress the synthesis of VTG at the mature stage. This report is the first to describe the molecular mechanism of VTG synthesis in E. sinensis and may shed new light on the regulation of cytochrome P450 enzyme by FOXL2 and FTZ-F1 in vitellogenesis. PMID:26430246

  5. ATP synthase subunit-β down-regulation aggravates diabetic nephropathy

    PubMed Central

    Guan, Siao-Syun; Sheu, Meei-Ling; Wu, Cheng-Tien; Chiang, Chih-Kang; Liu, Shing-Hwa

    2015-01-01

    In this study, we investigated the role of ATP synthase subunit-β (ATP5b) in diabetic nephropathy. Histopathological changes, fibrosis, and protein expressions of α-smooth muscle actin (α-SMA), advanced glycation end-products (AGEs), and ATP5b were obviously observed in the kidneys of db/db diabetic mice as compared with the control db/m+ mice. The increased ATP5b expression was majorly observed in diabetic renal tubules and was notably observed to locate in cytoplasm of tubule cells, but no significant increase of ATP5b in diabetic glomeruli. AGEs significantly increased protein expression of ATP5b and fibrotic factors and decreased ATP content in cultured renal tubular cells via an AGEs-receptor for AGEs (RAGE) axis pathway. Oxidative stress was also induced in diabetic kidneys and AGEs-treated renal tubular cells. The increase of ATP5b and CTGF protein expression in AGEs-treated renal tubular cells was reversed by antioxidant N-acetylcysteine. ATP5b-siRNA transfection augmented the increased protein expression of α-SMA and CTGF and CTGF promoter activity in AGEs-treated renal tubular cells. The in vivo ATP5b-siRNA delivery significantly enhanced renal fibrosis and serum creatinine in db/db mice with ATP5b down-regulation. These findings suggest that increased ATP5b plays an important adaptive or protective role in decreasing the rate of AGEs-induced renal fibrosis during diabetic condition. PMID:26449648

  6. Quantitative cell signalling analysis reveals down-regulation of MAPK pathway activation in colorectal cancer.

    PubMed

    Gulmann, Christian; Sheehan, Katherine M; Conroy, Ronán M; Wulfkuhle, Julia D; Espina, Virginia; Mullarkey, Michelle J; Kay, Elaine W; Liotta, Lance A; Petricoin, Emanuel F

    2009-08-01

    Mitogen-activated protein kinases (MAPK) are considered to play significant roles in colonic carcinogenesis and kinase inhibitor therapy has been proposed as a potential tool in the treatment of this disease. Reverse-phase microarray assays using phospho-specific antibodies can directly measure levels of phosphorylated protein isoforms. In the current study, samples from 35 cases of untreated colorectal cancer colectomies were laser capture-microdissected to isolate epithelium and stroma from cancer as well as normal (i.e. uninvolved) mucosa. Lysates generated from these four tissue types were spotted onto reverse-phase protein microarrays and probed with a panel of antibodies to ERK, p-ERK, p38, p-p38, p-JNK, MEK and p-MEK. Whereas total protein levels were unchanged, or slightly elevated (p38, p = 0.0025) in cancers, activated isoforms, including p-ERK, p-p38 and p-JNK, were decreased two- to four-fold in cancers compared with uninvolved mucosa (p < 0.0023 in all cases except for p-JNK in epithelium, where decrement was non-significant). This was backed up by western blotting. Dukes' stage B and C cancers displayed lower p-ERK and p-p38 expression than Dukes' stage A cancers, although this was not statistically significant. It is concluded that MAPK activity may be down-regulated in colorectal cancer and that further exploration of inhibitory therapy in this system should be carefully evaluated if this finding is confirmed in larger series. PMID:19396842

  7. Down-regulation of voltage-dependent sodium channels initiated by sodium influx in developing neurons

    SciTech Connect

    Dargent, B.; Couraud, F. )

    1990-08-01

    To address the issue of whether regulatory feedback exists between the electrical activity of a neuron and ion-channel density, the authors investigated the effect of Na{sup +}-channel activators (scorpion {alpha} toxin, batrachotoxin, and veratridine) on the density of Na{sup +} channels in fetal rat brain neurons in vitro. A partial but rapid (t{sub 1/2}, 15 min) disappearance of surface Na{sup +} channels was observed as measured by a decrease in the specific binding of ({sup 3}H)saxitoxin and {sup 125}I-labeled scorpion {beta} toxin and a decrease in specific {sup 22}Na{sup +} uptake. Moreover, the increase in the number of Na{sup +} channels that normally occurs during neuronal maturation in vitro was inhibited by chronic channel activator treatment. The induced disappearance of Na{sup +} channels was abolished by tetrodotoxin, was found to be dependent on the external Na{sup +} concentration, and was prevented when either choline (a nonpermeant ion) or Li{sup +} (a permeant ion) was substituted for Na{sup +}. Amphotericin B, a Na{sup +} ionophore, and monensin were able to mimick the effect of Na{sup +}-channel activators, while a KCl depolarization failed to do this. This feedback regulation seems to be a neuronal property since Na{sup +}-channel density in cultured astrocytes was not affected by channel activator treatment or by amphotericin B. The present evidence suggests that an increase in intracellular Na{sup +} concentration, whether elicited by Na{sup +}-channel activators or mediated by a Na{sup +} ionophore, can induce a decrease in surface Na{sup +} channels and therefore is involved in down-regulation of Na{sup +}-channel density in fetal rat brain neurons in vitro.

  8. Physiological concentrations of bile acids down-regulate agonist induced secretion in colonic epithelial cells.

    PubMed

    Keating, Niamh; Mroz, Magdalena S; Scharl, Michael M; Marsh, Christine; Ferguson, Gail; Hofmann, Alan F; Keely, Stephen J

    2009-08-01

    In patients with bile acid malabsorption, high concentrations of bile acids enter the colon and stimulate Cl(-) and fluid secretion, thereby causing diarrhoea. However, deoxycholic acid (DCA), the predominant colonic bile acid, is normally present at lower concentrations where its role in regulating transport is unclear. Thus, the current study set out to investigate the effects of physiologically relevant DCA concentrations on colonic epithelial secretory function. Cl(-) secretion was measured as changes in short-circuit current across voltage-clamped T(84) cell monolayers. At high concentrations (0.5-1 mM), DCA acutely stimulated Cl(-) secretion but this effect was associated with cell injury, as evidenced by decreased transepithelial resistance (TER) and increased lactate dehydrogenase (LDH) release. In contrast, chronic (24 hrs) exposure to lower DCA concentrations (10-200 microM) inhibited responses to Ca(2+) and cAMP-dependent secretagogues without altering TER, LDH release, or secretagogue-induced increases in intracellular second messengers. Other bile acids - taurodeoxycholic acid, chenodeoxycholic acid and cholic acid - had similar antisecretory effects. DCA (50 microM) rapidly stimulated phosphorylation of the epidermal growth factor receptor (EGFr) and both ERK and p38 MAPKs (mitogen-activated protein kinases). The EGFr inhibitor, AG1478, and the protein synthesis inhibitor, cycloheximide, reversed the antisecretory effects of DCA, while the MAPK inhibitors, PD98059 and SB203580, did not. In summary, our studies suggest that, in contrast to its acute prosecretory effects at pathophysiological concentrations, lower, physiologically relevant, levels of DCA chronically down-regulate colonic epithelial secretory function. On the basis of these data, we propose a novel role for bile acids as physiological regulators of colonic secretory capacity. PMID:19583809

  9. Slug down-regulation by RNA interference inhibits invasion growth in human esophageal squamous cell carcinoma

    PubMed Central

    2011-01-01

    Background Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas of the gastrointestinal tract. We assessed the relevance of Slug in measuring the invasive potential of ESCC cells in vitro and in vivo in immunodeficient mice. Methods We utilized RNA interference to knockdown Slug gene expression, and effects on survival and invasive carcinoma were evaluated using a Boyden chamber transwell assay in vitro. We evaluated the effect of Slug siRNA-transfection and Slug cDNA-transfection on E-cadherin and Bcl-2 expression in ESCC cells. A pseudometastatic model of ESCC in immunodeficient mice was used to assess the effects of Slug siRNA transfection on tumor metastasis development. Results The EC109 cell line was transfected with Slug-siRNA to knockdown Slug expression. The TE13 cell line was transfected with Slug-cDNA to increase Slug expression. EC109 and TE13 cell lines were tested for the expression of apoptosis-related genes bcl-2 and metastasis-related gene E-cadherin identified previously as Slug targets. Bcl-2 expression was increased and E-cadherin was decreased in Slug siRNA-transfected EC109 cells. Bcl-2 expression was increased and E-cadherin was decreased in Slug cDNA-transfected TE13 cells. Invasion of Slug siRNA-transfected EC109 cells was reduced and apoptosis was increased whereas invasion was greater in Slug cDNA-transfected cells. Animals injected with Slug siRNA-transfected EC109 cells exhihited fewer seeded nodes and demonstrated more apoptosis. Conclusions Slug down-regulation promotes cell apoptosis and decreases invasion capability in vitro and in vivo. Slug inhibition may represent a novel strategy for treatment of metastatic ESCC. PMID:21599940

  10. CCoAOMT Down-Regulation Activates Anthocyanin Biosynthesis in Petunia.

    PubMed

    Shaipulah, Nur Fariza M; Muhlemann, Joëlle K; Woodworth, Benjamin D; Van Moerkercke, Alex; Verdonk, Julian C; Ramirez, Aldana A; Haring, Michel A; Dudareva, Natalia; Schuurink, Robert C

    2016-02-01

    Anthocyanins and volatile phenylpropenes (isoeugenol and eugenol) in petunia (Petunia hybrida) flowers have the precursor 4-coumaryl coenzyme A (CoA) in common. These phenolics are produced at different stages during flower development. Anthocyanins are synthesized during early stages of flower development and sequestered in vacuoles during the lifespan of the flowers. The production of isoeugenol and eugenol starts when flowers open and peaks after anthesis. To elucidate additional biochemical steps toward (iso)eugenol production, we cloned and characterized a caffeoyl-coenzyme A O-methyltransferase (PhCCoAOMT1) from the petals of the fragrant petunia 'Mitchell'. Recombinant PhCCoAOMT1 indeed catalyzed the methylation of caffeoyl-CoA to produce feruloyl CoA. Silencing of PhCCoAOMT1 resulted in a reduction of eugenol production but not of isoeugenol. Unexpectedly, the transgenic plants had purple-colored leaves and pink flowers, despite the fact that cv Mitchell lacks the functional R2R3-MYB master regulator ANTHOCYANIN2 and has normally white flowers. Our results indicate that down-regulation of PhCCoAOMT1 activated the anthocyanin pathway through the R2R3-MYBs PURPLE HAZE (PHZ) and DEEP PURPLE, with predominantly petunidin accumulating. Feeding cv Mitchell flowers with caffeic acid induced PHZ expression, suggesting that the metabolic perturbation of the phenylpropanoid pathway underlies the activation of the anthocyanin pathway. Our results demonstrate a role for PhCCoAOMT1 in phenylpropene production and reveal a link between PhCCoAOMT1 and anthocyanin production. PMID:26620524