Sample records for a1 abca1 mediates

  1. Puerarin promotes ABCA1-mediated cholesterol efflux and decreases cellular lipid accumulation in THP-1 macrophages.

    PubMed

    Li, Cong-Hui; Gong, Duo; Chen, Ling-Yan; Zhang, Min; Xia, Xiao-Dan; Cheng, Hai-Peng; Huang, Chong; Zhao, Zhen-Wang; Zheng, Xi-Long; Tang, Xiao-Er; Tang, Chao-Ke

    2017-09-15

    It was reported that puerarin decreases the total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and increases high-density lipoprotein cholesterol (HDL-C) level, but the underlying mechanism is unclear. This study was designed to determine whether puerarin decreased lipid accumulation via up-regulation of ABCA1-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. Our results showed that puerarin significantly promoted the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via the AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor-alpha (LXR-α) pathway and decreased cellular lipid accumulation in human THP-1 macrophage-derived foam cells. The miR-7 directly targeted 3' untranslated region of STK11 (Serine/Threonine Kinase 11), which activated the AMPK pathway. Transfection with miR-7 mimic significantly reduced STK11 expression in puerarin-treated macrophages, decreased the phosphorylation of AMPK, down-regulated the expression of the PPARγ-LXR-α-ABCA1 expression. Additionally, treatment with miR-7 decreased cholesterol efflux and increased cholesterol levels in THP-1 macrophage-derived foam cells. Our study demonstrates that puerarin promotes ABCA1-mediated cholesterol efflux and decreases intracellular cholesterol levels through the pathway involving miR-7, STK11, and the AMPK-PPARγ-LXR-α-ABCA1 cascade. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Purification and ATPase activity of human ABCA1.

    PubMed

    Takahashi, Kei; Kimura, Yasuhisa; Kioka, Noriyuki; Matsuo, Michinori; Ueda, Kazumitsu

    2006-04-21

    ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.

  3. Effects of miR-33a-5P on ABCA1/G1-Mediated Cholesterol Efflux under Inflammatory Stress in THP-1 Macrophages

    PubMed Central

    Mao, Min; Lei, Han; Liu, Qing; Chen, Yaxi; Zhao, Lei; Li, Qing; Luo, Suxin; Zuo, Zhong; He, Quan; Huang, Wei; Zhang, Nan; Zhou, Chao; Ruan, Xiong Z.

    2014-01-01

    The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages. PMID:25329888

  4. Effects of miR-33a-5P on ABCA1/G1-mediated cholesterol efflux under inflammatory stress in THP-1 macrophages.

    PubMed

    Mao, Min; Lei, Han; Liu, Qing; Chen, Yaxi; Zhao, Lei; Li, Qing; Luo, Suxin; Zuo, Zhong; He, Quan; Huang, Wei; Zhang, Nan; Zhou, Chao; Ruan, Xiong Z

    2014-01-01

    The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages.

  5. PPAR{gamma} activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mogilenko, Denis A., E-mail: denis@iem.sp.ru; Department of Embryology, St. Petersburg State University, 199034 St. Petersburg; Shavva, Vladimir S.

    Research highlights: {yields} PPAR{gamma} activates ABCA1 gene expression but decreases ABCA1 protein content in human hepatoma cell line HepG2. {yields} Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1-LXR{beta} complex. {yields} Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex. {yields} Activation of PPAR{gamma} leads to increasing of the level of LXR{beta} associated with LXRE within ABCA1 gene promoter. -- Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPAR{gamma} is known as activator of ABCA1 expression, but details of PPAR{gamma}-mediatedmore » regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPAR{gamma} activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXR{beta} binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1/LXR{beta} complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24 h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex, but does not block PPAR{gamma}-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPAR{gamma} may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPAR{gamma}, LXR{beta} and MEK1/2 in regulation of ABCA1 mRNA and protein expression.« less

  6. ABCA1 and biogenesis of HDL.

    PubMed

    Yokoyama, Shinji

    2006-02-01

    Mammalian somatic cells do not catabolize cholesterol and therefore export it for sterol homeostasis at cell and whole body levels. This mechanism may reduce intracellularly accumulated excess cholesterol, and thereby would contribute to the prevention or cure of the initial stage of atherosclerotic vascular lesion. High-density lipoprotein (HDL) plays a central role in this reaction by removing cholesterol from cells and transporting it to the liver, the major cholesterol catabolic site. Two independent mechanisms have been identified for cellular cholesterol release. The first is non-specific diffusion-mediated cholesterol "efflux" from the cell surface, in which cholesterol is trapped by various extracellular acceptors including lipoproteins. Extracellular cholesterol esterification of HDL provides a driving force for the net removal of cell cholesterol by this pathway, and some cellular factors may enhance this reaction. The other mechanism is an apolipoprotein-mediated process to generate new HDL particles by removing cellular phospholipid and cholesterol. This reaction is mediated by a membrane protein ATP-binding cassette transporter A1 (ABCA1), and lipid-free or lipid-poor helical apolipoproteins recruit cellular phospholipid and cholesterol to assemble HDL particles. The reaction is composed of two elements: the assembly of HDL particles with phospholipid by apolipoprotein, and cholesterol enrichment in HDL. ABCA1 is essential for the former step and the latter requires further intracellular events. ABCA1 is a rate-limiting factor of HDL assembly and is regulated by transcriptional and post-transcriptional factors. Post-transcriptional regulation of ABCA1 involves modulation of its calpain-mediated degradation.

  7. The E3 ubiquitin ligase, HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages.

    PubMed

    Aleidi, Shereen M; Yang, Alryel; Sharpe, Laura J; Rao, Geetha; Cochran, Blake J; Rye, Kerry-Anne; Kockx, Maaike; Brown, Andrew J; Gelissen, Ingrid C

    2018-04-01

    The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting β-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPAR{gamma}-LXR{alpha}-ABCA1 pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu, Yanni; Lai, Fangfang; Xu, Yang

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Using an ABCA1p-LUC HepG2 cell line, we found that MPA upregulated ABCA1 expression. Black-Right-Pointing-Pointer MPA induced ABCA1 and LXR{alpha} protein expression in HepG2 cells. Black-Right-Pointing-Pointer PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. Black-Right-Pointing-Pointer The effect of MPA upregulating ABCA1 was due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 pathway. -- Abstract: ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1.more » Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50 = 0.09 {mu}M). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPAR{gamma}; EC50 = 5.2-9.3 {mu}M). Liver X receptor {alpha} (LXR{alpha}) is a target gene of PPAR{gamma} and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXR{alpha} protein expression in HepG2 cells. Addition of PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPAR{gamma}. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism.« less

  9. Intestinal ABCA1 directly contributes to HDL biogenesis in vivo

    PubMed Central

    Brunham, Liam R.; Kruit, Janine K.; Iqbal, Jahangir; Fievet, Catherine; Timmins, Jenelle M.; Pape, Terry D.; Coburn, Bryan A.; Bissada, Nagat; Staels, Bart; Groen, Albert K.; Hussain, M. Mahmood; Parks, John S.; Kuipers, Folkert; Hayden, Michael R.

    2006-01-01

    Plasma HDL cholesterol levels are inversely related to risk for atherosclerosis. The ATP-binding cassette, subfamily A, member 1 (ABCA1) mediates the rate-controlling step in HDL particle formation, the assembly of free cholesterol and phospholipids with apoA-I. ABCA1 is expressed in many tissues; however, the physiological functions of ABCA1 in specific tissues and organs are still elusive. The liver is known to be the major source of plasma HDL, but it is likely that there are other important sites of HDL biogenesis. To assess the contribution of intestinal ABCA1 to plasma HDL levels in vivo, we generated mice that specifically lack ABCA1 in the intestine. Our results indicate that approximately 30% of the steady-state plasma HDL pool is contributed by intestinal ABCA1 in mice. In addition, our data suggest that HDL derived from intestinal ABCA1 is secreted directly into the circulation and that HDL in lymph is predominantly derived from the plasma compartment. These data establish a critical role for intestinal ABCA1 in plasma HDL biogenesis in vivo. PMID:16543947

  10. Cellular cholesterol regulates ubiquitination and degradation of the cholesterol export proteins ABCA1 and ABCG1.

    PubMed

    Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C; Brown, Andrew J; Sandoval, Cecilia; Hallab, Jeannette C; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

    2014-03-14

    The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes.

  11. Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

    PubMed Central

    Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

    2014-01-01

    The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

  12. Association of ABCA1 with syntaxin 13 and flotillin-1 and enhanced phagocytosis in tangier cells.

    PubMed

    Bared, Salim Maa; Buechler, Christa; Boettcher, Alfred; Dayoub, Rania; Sigruener, Alexander; Grandl, Margot; Rudolph, Christian; Dada, Ashraf; Schmitz, Gerd

    2004-12-01

    The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I-dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX-insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains.

  13. Association of ABCA1 with Syntaxin 13 and Flotillin-1 and Enhanced Phagocytosis in Tangier Cells

    PubMed Central

    Bared, Salim Maa; Buechler, Christa; Boettcher, Alfred; Dayoub, Rania; Sigruener, Alexander; Grandl, Margot; Rudolph, Christian; Dada, Ashraf; Schmitz, Gerd

    2004-01-01

    The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I–dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX–insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains. PMID:15469992

  14. Piperine inhibits ABCA1 degradation and promotes cholesterol efflux from THP‐1‐derived macrophages

    PubMed Central

    Wang, Limei; Palme, Veronika; Rotter, Susanne; Schilcher, Nicole; Cukaj, Malsor; Wang, Dongdong; Ladurner, Angela; Heiss, Elke H.; Stangl, Herbert; Dirsch, Verena M.

    2016-01-01

    1 Scope Increased macrophage cholesterol efflux (ChE) is considered to have anti‐atherosclerotic effect counteracting cardiovascular disease. The principle pungent ingredient of the fruits of Piper nigrum, piperine, is identified in this study as a ChE inducer in THP‐1‐derived macrophages, and mechanisms underlying this effect are explored. 2 Methods and results Without affecting cell viability, piperine concentration‐dependently enhances ChE in THP‐1‐derived macrophages from 25 to 100 μM. The expression level of the key cholesterol transporter protein ATP‐binding cassette transporter A1 (ABCA1) is significantly upregulated by piperine, as revealed by western blot analyses. However, two other ChE‐mediating transporter proteins, ATP‐binding cassette transporter G1 (ABCG1) and scavenger receptor class B member 1 (SR‐B1), remain unaffected. Piperine exerts no influence on ABCA1 mRNA levels, but significantly inhibits the degradation of ABCA1, as evident by an increased half‐life of the protein in the presence of cycloheximide. Furthermore, it is found that piperine likely interferes with the calpain‐mediated ABCA1 degradation pathway and exhibits significant inhibition of calpain activity. 3 Conclusion Our findings suggest that piperine promotes ChE in THP‐1‐derived macrophages by upregulation of ABCA1, which might be mediated by inhibition of calpain activity. This novel bioactivity makes the dietary constituent piperine a good candidate to be further explored for therapeutic or preventive applications in the context of atherosclerosis. PMID:27862930

  15. The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear

    PubMed Central

    Jacob, Daria; Hunegnaw, Ruth; Sabyrzyanova, Tatyana A.; Pushkarsky, Tatiana; Chekhov, Vladimir O.; Adzhubei, Alexei A.; Kalebina, Tatyana S.; Bukrinsky, Michael

    2014-01-01

    HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using coimmunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope. PMID:24406162

  16. Tangshen Formula Attenuates Diabetic Nephropathy by Promoting ABCA1-Mediated Renal Cholesterol Efflux in db/db Mice.

    PubMed

    Liu, Peng; Peng, Liang; Zhang, Haojun; Tang, Patrick Ming-Kuen; Zhao, Tingting; Yan, Meihua; Zhao, Hailing; Huang, Xiaoru; Lan, Huiyao; Li, Ping

    2018-01-01

    The commonly prescribed Tangshen Formula (TSF) is a traditional Chinese formulation that has been shown to reduce plasma lipid metabolism and proteinuria and improve the estimated glomerular filtration rate (eGFR) in patients with diabetic kidney disease. This study investigated the underlying mechanism whereby TSF regulates renal lipid accumulation and ameliorates diabetic renal injuries in spontaneous diabetic db/db mice and in vitro in sodium palmitate (PA)-stimulated and Abca1-SiRNA-transfected mouse tubular epithelial cells (mTECs). The results revealed that TSF treatment significantly ameliorated the renal injuries by lowering urinary albumin excretion and improving renal tissue injuries in diabetic (db/db) mice. Interestingly, the treatment with TSF also resulted in decreased cholesterol levels in the renal tissues of db/db mice, which was associated with increased expression of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), the Liver X receptors (LXR), and ATP-binding cassette subfamily A member 1 (ABCA1), suggesting that TSF might attenuate diabetic kidney injury via a mechanism associated with improving cholesterol efflux in the diabetic kidney. This was investigated in vitro in mTECs, and the results showed that TSF reduced the PA-stimulated cholesterol accumulation in mTECs. Mechanistically, the addition of TSF was capable of reversing PA-induced downregulation of PGC-1α, LXR, and ABCA1 expression and cholesterol accumulation in mTECs, suggesting that TSF might act the protection via the PGC-1α-LXR-ABCA1 pathway to improve the cholesterol efflux in the renal tissues of db/db mice. This was further confirmed by silencing ABCA1 to block the promotive effect of TSF on cholesterol efflux in vitro . In conclusion, TSF might ameliorate diabetic kidney injuries by promoting ABCA1-mediated renal cholesterol efflux.

  17. Pim-1L Protects Cell Surface-Resident ABCA1 From Lysosomal Degradation in Hepatocytes and Thereby Regulates Plasma High-Density Lipoprotein Level.

    PubMed

    Katsube, Akira; Hayashi, Hisamitsu; Kusuhara, Hiroyuki

    2016-12-01

    ATP-binding cassette transporter A1 (ABCA1) exerts an atheroprotective action through the biogenesis of high-density lipoprotein in hepatocytes and prevents the formation of foam cells from macrophages. Controlling ABCA1 is a rational approach to improving atherosclerotic cardiovascular disease. Although much is known about the regulatory mechanism of ABCA1 synthesis, the molecular mechanism underpinning its degradation remains to be clearly described. ABCA1 possesses potential sites of phosphorylation by serine/threonine-protein kinase Pim-1 (Pim-1). Pim-1 depletion decreased the expression of cell surface-resident ABCA1 (csABCA1) and apolipoprotein A-I-mediated [ 3 H]cholesterol efflux in the human hepatoma cell line HepG2, but not in peritoneal macrophages from mice. In vitro kinase assay, immunoprecipitation, and immunocytochemistry suggested phosphorylation of csABCA1 by the long form of Pim-1 (Pim-1L). Cell surface biotinylation indicated that Pim-1L inhibited lysosomal degradation of csABCA1 involving the liver X receptor β, which interacts with csABCA1 and thereby protects it from ubiquitination and subsequent lysosomal degradation. Cell surface coimmunoprecipitation with COS-1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that Pim-1L-mediated phosphorylation of csABCA1 facilitated the interaction between csABCA1 and liver X receptor β and thereby stabilized the csABCA1-Pim-1L complex. Mice deficient in Pim-1 kinase activity showed lower expression of ABCA1 in liver plasma membranes and lower plasma high-density lipoprotein levels than control mice. Pim-1L protects hepatic csABCA1 from lysosomal degradation by facilitating the physical interaction between csABCA1 and liver X receptor β and subsequent stabilization of the csABCA1-Pim-1L complex and thereby regulates the circulating level of high-density lipoprotein. Our findings may aid the development of high-density lipoprotein-targeted therapy. © 2016 American Heart Association

  18. BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein modulates ABCA1 trafficking and function

    PubMed Central

    Lin, Sisi; Zhou, Chun; Neufeld, Edward; Wang, Yu-Hua; Xu, Suo-Wen; Lu, Liang; Wang, Ying; Liu, Zhi-Ping; Li, Dong; Li, Cuixian; Chen, Shaorui; Le, Kang; Huang, Heqing; Liu, Peiqing; Moss, Joel; Vaughan, Martha; Shen, Xiaoyan

    2013-01-01

    Objective Cell surface localization and intracellular trafficking of ATP-binding cassette transporter A-1 (ABCA1) are essential for its function. However, regulation of these activities is still largely unknown. Brefeldin A (BFA), a uncompetitive inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1 trafficking and cholesterol efflux, and further to explore the potential mechanism. Methods and Results By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells and inhibited by 60% cholesterol release. In contrast, BIG1 over-expression increased surface ABCA1 and cholesterol secretion. With partial restoration of BIG1 through over-expression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione cleavage revealed that BIG1 siRNA dramatically decreased the internalization and recycling of ABCA1. This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation of ADP-ribosylation factor 1 (ARF1). Conclusions BIG1, through its ability to activate ARF1, regulates cell surface levels and function of ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action. PMID:23220274

  19. Assembly of high density lipoprotein by the ABCA1/apolipoprotein pathway.

    PubMed

    Yokoyama, Shinji

    2005-06-01

    Mammalian somatic cells do not catabolize cholesterol and therefore need to export it for sterol homeostasis at the levels of cells and whole bodies. This mechanism may reduce intracellularly accumulated cholesterol in excess, and thereby would contribute to the prevention or cure of the initial stage of atherosclerotic vascular lesions. HDL is thought to play a main role in this reaction on the basis of epidemiological evidence and in-vitro experimental data. Two independent mechanisms have been identified for this reaction. One is non-specific diffusion-mediated cholesterol 'efflux' from the cell surface, and cholesterol is trapped by various extracellular acceptors including lipoproteins. Extracellular cholesterol esterification on HDL provides a driving force for the net removal of cell cholesterol, and some cellular factors may enhance this reaction. The other mechanism is an apolipoprotein-mediated process to generate HDL by removing cellular phospholipid and cholesterol. This reaction is mediated by a membrane protein ABCA1, and lipid-free or lipid-poor helical apolipoproteins recruit cellular phospholipid and cholesterol to assemble HDL particles. The reaction is composed of two elements: the assembly of HDL particles with phospholipid by apolipoprotein, and cholesterol enrichment in HDL. ABCA1 is essential for the former step, and the latter step requires further intracellular events. ABCA1 is a rate-limiting factor of HDL assembly and is regulated by transcriptional factors and posttranscriptional factors. Posttranscriptional regulation of ABCA1 involves the modulation of its calpain-mediated degradation.

  20. Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1.

    PubMed

    Mizuno, Tadahaya; Hayashi, Hisamitsu; Kusuhara, Hiroyuki

    2015-06-01

    By excreting cellular cholesterol to apolipoprotein A-I, ATP-binding cassette transporter A1 (ABCA1) mediates the biogenesis of high-density lipoprotein in hepatocytes and prevents foam cell formation from macrophages. We recently showed that cell surface-resident ABCA1 (csABCA1) undergoes ubiquitination and later lysosomal degradation through the endosomal sorting complex required for transport system. Herein, we investigated the relevance of this degradation pathway to the turnover of csABCA1 in hypercholesterolemia. Immunoprecipitation and cell surface-biotinylation studies with HepG2 cells and mouse peritoneal macrophages showed that the ubiquitination level and degradation of csABCA1 were facilitated by treatment with a liver X receptor (LXR) agonist and acetylated low-density lipoprotein. The effects of an LXR agonist and acetylated low-density lipoprotein on the degradation of csABCA1 were repressed completely by treatment with bafilomycin, an inhibitor of lysosomal degradation, and by depletion of tumor susceptibility gene 101, a major component of endosomal sorting complex required for transport-I. RNAi analysis indicated that LXRβ inhibited the accelerated lysosomal degradation of csABCA1 by the LXR agonist, regardless of its transcriptional activity. Cell surface coimmunoprecipitation with COS1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that LXRβ interacted with csABCA1 and inhibited the ubiquitination of csABCA1. Immunoprecipitates with anti-ABCA1 antibodies from the liver plasma membranes showed less LXRβ and a higher ubiquitination level of ABCA1 in high-fat diet-fed mice than in normal chow-fed mice. Under conditions of high cellular cholesterol content, csABCA1 became susceptible to ubiquitination by dissociation of LXRβ from csABCA1, which facilitated the lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport system. © 2015 American Heart Association, Inc.

  1. A novel compound inhibits rHDL assembly and blocks nascent HDL biogenesis downstream of apoAI binding to ABCA1 expressing cells

    PubMed Central

    Lyssenko, Nicholas N.; Brubaker, Gregory; Smith, Bradley D.; Smith, Jonathan D.

    2011-01-01

    Objective Nascent high-density lipoprotein (HDL) particles form from cellular lipids and extracellular lipid-free apolipoprotein AI (apoAI) in a process mediated by ATP-binding cassette transporter A1 (ABCA1). We have sought out compounds that inhibit nascent HDL biogenesis without affecting ABCA1 activity. Methods and Results Reconstituted HDL (rHDL) formation and cellular cholesterol efflux assays were used to show that two compounds that bond via hydrogen with phospholipids inhibit rHDL and nascent HDL production. In rHDL formation assays, the inhibitory effect of compound 1 (methyl 3α-acetoxy-7α,12α-di[(phenylaminocarbonyl)amino]-5β-cholan-24-oate), the more active of the two, depended on its ability to associate with phospholipids. In cell assays, compound 1 suppressed ABCA1-mediated cholesterol efflux to apoAI, the 18A peptide, and taurocholate with high specificity, without affecting ABCA1-independent cellular cholesterol efflux to HDL and endocytosis of acetylated low-density lipoprotein (AcLDL) and transferrin. Furthermore, compound 1 did not affect ABCA1 activity adversely, as ABCA1-mediated shedding of microparticles proceeded unabated and apoAI binding to ABCA1-expressing cells increased in its presence. Conclusions The inhibitory effects of compound 1 support a three-step model of nascent HDL biogenesis: plasma membrane remodeling by ABCA1, apoAI binding to ABCA1, and lipoprotein particle assembly. The compound inhibits the final step, causing accumulation of apoAI in ABCA1-expressing cells. PMID:21836073

  2. Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains.

    PubMed

    Drobnik, Wolfgang; Borsukova, Hana; Böttcher, Alfred; Pfeiffer, Alexandra; Liebisch, Gerhard; Schütz, Gerhard J; Schindler, Hansgeorg; Schmitz, Gerd

    2002-04-01

    We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL3 reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL3-mediated lipid efflux.

  3. Lactobacillus acidophilus K301 Inhibits Atherogenesis via Induction of 24 (S), 25-Epoxycholesterol-Mediated ABCA1 and ABCG1 Production and Cholesterol Efflux in Macrophages

    PubMed Central

    Kim, Hye Sun; Park, Woo Jung; Kim, Joo-Yun; Chung, Dae Kyun

    2016-01-01

    Lactobacillus acidophilus species are well-known probiotics with the beneficial activity of regulating cholesterol levels. In this study, we showed that L. acidophilus K301 reduced the level of cholesterol through reverse transport in macrophages. L. acidophilus K301 upregulated the mRNA and protein levels of genes such as ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) under the control of liver X receptor (LXR), resulting in increased apoA-I-dependent cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. L. acidophilus K301 induced both ABCA1 and ABCG1 through the endogenous LXR agonist 24(S), 25-epoxcycholesterol, which is synthesized by intracellular cholesterol synthetic pathways. In vivo studies using L. acidophilus K301-treated ApoE-/- mice showed reduced accumulation of lipoproteins in the arterial lumen. The inhibitory effects of L. acidophilus K301 on accumulation of lipoprotein in atherosclerotic plaques were mediated by the induction of squalene reductase (SQLE) and oxidosqualene cyclase (OSC) and resulted in ABCA1-mediated cholesterol efflux. Taken together, our findings revealed that Lactobacillus acidophilus K301 regulates the expression of genes related to cholesterol reverse transport via the induction of endogenous LXR agonist, suggesting the therapeutic potential of Lactobacillus acidophilus K301 as an anti-atherosclerotic agent. PMID:27120199

  4. Lactobacillus acidophilus K301 Inhibits Atherogenesis via Induction of 24 (S), 25-Epoxycholesterol-Mediated ABCA1 and ABCG1 Production and Cholesterol Efflux in Macrophages.

    PubMed

    Hong, Yi-Fan; Kim, Hangeun; Kim, Hye Sun; Park, Woo Jung; Kim, Joo-Yun; Chung, Dae Kyun

    2016-01-01

    Lactobacillus acidophilus species are well-known probiotics with the beneficial activity of regulating cholesterol levels. In this study, we showed that L. acidophilus K301 reduced the level of cholesterol through reverse transport in macrophages. L. acidophilus K301 upregulated the mRNA and protein levels of genes such as ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) under the control of liver X receptor (LXR), resulting in increased apoA-I-dependent cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. L. acidophilus K301 induced both ABCA1 and ABCG1 through the endogenous LXR agonist 24(S), 25-epoxcycholesterol, which is synthesized by intracellular cholesterol synthetic pathways. In vivo studies using L. acidophilus K301-treated ApoE-/- mice showed reduced accumulation of lipoproteins in the arterial lumen. The inhibitory effects of L. acidophilus K301 on accumulation of lipoprotein in atherosclerotic plaques were mediated by the induction of squalene reductase (SQLE) and oxidosqualene cyclase (OSC) and resulted in ABCA1-mediated cholesterol efflux. Taken together, our findings revealed that Lactobacillus acidophilus K301 regulates the expression of genes related to cholesterol reverse transport via the induction of endogenous LXR agonist, suggesting the therapeutic potential of Lactobacillus acidophilus K301 as an anti-atherosclerotic agent.

  5. 7-ketocholesteryl-9-carboxynonanoate enhances ATP binding cassette transporter A1 expression mediated by PPARγ in THP-1 macrophages.

    PubMed

    Chi, Yan; Wang, Le; Liu, Yuanyuan; Ma, Yanhua; Wang, Renjun; Han, Xiaofei; Qiao, Hui; Lin, Jiabin; Matsuura, Eiji; Liu, Shuqian; Liu, Qingping

    2014-06-01

    ATP binding cassette transporter A1 (ABCA1) is a member of the ATP-binding cassette transporter family. It plays an essential role in mediating the efflux of excess cholesterol. It is known that peroxisome proliferator-activated receptor gamma (PPARγ) promoted ABCA1 expression. We previously found 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) upregulated ABCA1 partially through CD36 mediated signals. In the present study, we intended to test if PPARγ signally is involved in the upregulation mediated by oxLig-1. First, we docked oxLig-1 and the ligand-binding domain (LBD) of PPARγ by using AutoDock 3.05 and subsequently confirmed the binding by ELISA assay. Western blotting analyses showed that oxLig-1 induces liver X receptor alpha (LXRα), PPARγ and consequently ABCA1 expression. Furthermore, oxLig-1 significantly enhanced ApoA-I-mediated cholesterol efflux. Pretreatment with an inhibitor for PPARγ (GW9662) or/and LXRα (GGPP) attenuated oxLig-1-induced ABCA1 expression. Under PPARγ knockdown by using PPARγ-shRNA, oxLig-1-induced ABCA1 expression and cholesterol efflux in THP-1 macrophages was blocked by 62% and 25% respectively. These observations suggest that oxLig-1 is a novel PPARγ agonist, promoting ApoA-I-mediated cholesterol efflux from THP-1 macrophages by increasing ABCA1 expression via induction of PPARγ. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Activation of Liver X Receptor Decreases Atherosclerosis in Ldlr−/− mice in the Absence of ABCA1 and ABCG1 in Myeloid Cells

    PubMed Central

    Kappus, Mojdeh S.; Murphy, Andrew J.; Abramowicz, Sandra; Ntonga, Vusisizwe; Welch, Carrie L.; Tall, Alan R.; Westerterp, Marit

    2014-01-01

    Objective Liver X Receptor (LXR) activators decrease atherosclerosis in mice. LXR activators (1) directly up-regulate genes involved in reverse cholesterol transport (RCT) and (2) exert anti-inflammatory effects mediated by transrepression of NFκB target genes. We investigated whether myeloid cell deficiency of ATP-binding cassette transporters A1 and G1 (ABCA1/G1), principal targets of LXR that promote macrophage cholesterol efflux and initiate RCT, would abolish the beneficial effects of LXR activation on atherosclerosis. Approach and Results LXR activator T0901317 (T0) substantially reduced inflammatory gene expression in macrophages lacking ABCA1/G1. Ldlr−/− mice were transplanted with Abca1−/−Abcg1−/− or wild-type bone marrow (BM) and fed a Western-type diet (WTD) for 6 weeks with or without T0 supplementation. Abca1/g1 BM deficiency increased atherosclerotic lesion complexity and inflammatory cell infiltration into the adventitia and myocardium. T0 markedly decreased lesion area, complexity and inflammatory cell infiltration in the Abca1−/−Abcg1−/− BM transplanted mice. To investigate whether this was due to macrophage Abca1/g1 deficiency, Ldlr−/− mice were transplanted with LysmCreAbca1fl/flAbcg1fl/fl or Abca1fl/flAbcg1fl/fl BM and fed WTD with or without the more specific LXR agonist GW3965 for 12 weeks. GW3965 decreased lesion size in both groups and the decrease was more prominent in the LysmCreAbca1fl/flAbcg1fl/fl group. Conclusions The results suggest that anti-inflammatory effects of LXR activators are of key importance to their anti-atherosclerotic effects in vivo independent of cholesterol efflux pathways mediated by macrophage ABCA1/G1. This has implications for the development of LXR activators that lack adverse effects on lipogenic genes while maintaining the ability to trans-repress inflammatory genes. PMID:24311381

  7. Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum*♦

    PubMed Central

    Yamauchi, Yoshio; Iwamoto, Noriyuki; Rogers, Maximillian A.; Abe-Dohmae, Sumiko; Fujimoto, Toyoshi; Chang, Catherine C. Y.; Ishigami, Masato; Kishimoto, Takuma; Kobayashi, Toshihide; Ueda, Kazumitsu; Furukawa, Koichi; Chang, Ta-Yuan; Yokoyama, Shinji

    2015-01-01

    Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process. PMID:26198636

  8. IL-8 negatively regulates ABCA1 expression and cholesterol efflux via upregulating miR-183 in THP-1 macrophage-derived foam cells.

    PubMed

    Tang, Xiao-Er; Li, Heng; Chen, Ling-Yan; Xia, Xiao-Dan; Zhao, Zhen-Wang; Zheng, Xi-Long; Zhao, Guo-Jun; Tang, Chao-Ke

    2018-04-24

    Previous studies suggest that IL-8 has an important role in the regulation of cholesterol efflux, but whether miRNAs are involved in this process is still unknown. The purpose of this study is to explore whether IL-8 promotes cholesterol accumulation by enhancing miR-183 expression in macrophages and its underlying mechanism. Treatment of THP-1 macrophage-derived foam cells with IL-8 decreased ABCA1 expression and cholesterol efflux. Using bioinformatics analyses and dual-luciferase reporter assays, we found that miR-183 was highly conserved during evolution and directly inhibited ABCA1 protein and mRNA expression by targeting ABCA1 3'UTR. MiR-183 directly regulated endogenous ABCA1 expression levels. Furthermore, IL-8 enhanced the expression of miR-183 and decrease ABCA1 expression. Cholesterol transport assays confirmed that IL-8 dramatically inhibited apolipoprotein AI-mediated ABCA1-dependent cholesterol efflux by increasing miR-183 expression. In contrast, treatment with anti-IL-8 antibody reversed these effects. IL-8 enhances the expression of miR-183, which then inhibits ABCA1 expression and cholesterol efflux. Our studies suggest that the IL-8-miR-183-ABCA1 axis may play an intermediary role in the development of atherosclerosis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Silymarin Constituents Enhance ABCA1 Expression in THP-1 Macrophages

    PubMed Central

    Wang, Limei; Rotter, Susanne; Ladurner, Angela; Heiss, Elke H.; Oberlies, Nicholas H.; Dirsch, Verena M.; Atanasov, Atanas G.

    2016-01-01

    Silymarin is a hepatoprotective mixture of flavonolignans and flavonoids extracted from the seeds of milk thistle (Silybum marianum L. Gaertn). This study investigates the effect of major bioactive constituents from silymarin, silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, isosilychristin, and taxifolin, on the expression of ABCA1, an important cholesterol efflux transporter, in THP-1-derived macrophages. Four of the studied compounds, isosilybin A, silybin B, silychristin and isosilychristin, were found to significantly induce ABCA1 protein expression without affecting cell viability. Moreover, isosilybin A, a partial PPARγ agonist, was found to promote cholesterol efflux from THP-1 macrophages in a concentration-dependent manner. These findings first show ABCA1 protein up-regulating activity of active constituents of silymarin and provide new avenues for their further study in the context of cardiovascular disease. PMID:26729088

  10. Silymarin Constituents Enhance ABCA1 Expression in THP-1 Macrophages.

    PubMed

    Wang, Limei; Rotter, Susanne; Ladurner, Angela; Heiss, Elke H; Oberlies, Nicholas H; Dirsch, Verena M; Atanasov, Atanas G

    2015-12-31

    Silymarin is a hepatoprotective mixture of flavonolignans and flavonoids extracted from the seeds of milk thistle (Silybum marianum L. Gaertn). This study investigates the effect of major bioactive constituents from silymarin, silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, isosilychristin, and taxifolin, on the expression of ABCA1, an important cholesterol efflux transporter, in THP-1-derived macrophages. Four of the studied compounds, isosilybin A, silybin B, silychristin and isosilychristin, were found to significantly induce ABCA1 protein expression without affecting cell viability. Moreover, isosilybin A, a partial PPARγ agonist, was found to promote cholesterol efflux from THP-1 macrophages in a concentration-dependent manner. These findings first show ABCA1 protein up-regulating activity of active constituents of silymarin and provide new avenues for their further study in the context of cardiovascular disease.

  11. ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

    PubMed

    Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

    2014-01-01

    ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.

  12. ABCA1, ABCG1, and ABCG4 Are Distributed to Distinct Membrane Meso-Domains and Disturb Detergent-Resistant Domains on the Plasma Membrane

    PubMed Central

    Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

    2014-01-01

    ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters. PMID:25302608

  13. ABCA1 agonist peptides for the treatment of disease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bielicki, John K.

    Purpose of review The review summarizes information pertaining to the preclinical development of new apolipoprotein (apo) E mimetic peptides that stimulate cellular cholesterol efflux. Recent findings Small α-helical peptides based on the C-terminal domain of apoE have been developed for therapeutic applications. These peptides stimulate cellular cholesterol efflux via the ATP-binding cassette transporter A1 (ABCA1) with high potency, like native apolipoproteins on a molar basis. This potent activity has been related to the unique ability of these peptides to maintain α-helix structure upon dilution. Recent structure-activity studies improving the safety features of these mimetic peptides have greatly improved their potentialmore » for clinical use. Structural features of the class A α-helix motif that induce muscle toxicity and hypertriglyceridemia have been identified. These may have implications for the design of other HDL mimetic peptides. Summary ABCA1 is an integral membrane protein that plays a central role in biology. Its principal function is to mediate the efflux of cholesterol and phospholipid from cells to extracellular apo, preventing a build-up of excess cholesterol in membranes. This process generates HDL particles that perform a variety of functions to protect against disease. A number of these functions can be viewed as directly or indirectly supporting ABCA1 activity, thus constituting a positive feedback system to optimize cellular lipid efflux responses and disease prevention. Consequently, therapeutic approaches that mimic the activities of apos may prove highly effective to combat disease. One such approach involves the use of peptides. The broad biological relevance of ABCA1 suggests these apo mimetic peptides may be useful for the treatment of a number of diseases, such as atherosclerosis, diabetes, and Alzheimer's disease.« less

  14. ABCA1 agonist peptides for the treatment of disease

    DOE PAGES

    Bielicki, John K.

    2016-02-01

    Purpose of review The review summarizes information pertaining to the preclinical development of new apolipoprotein (apo) E mimetic peptides that stimulate cellular cholesterol efflux. Recent findings Small α-helical peptides based on the C-terminal domain of apoE have been developed for therapeutic applications. These peptides stimulate cellular cholesterol efflux via the ATP-binding cassette transporter A1 (ABCA1) with high potency, like native apolipoproteins on a molar basis. This potent activity has been related to the unique ability of these peptides to maintain α-helix structure upon dilution. Recent structure-activity studies improving the safety features of these mimetic peptides have greatly improved their potentialmore » for clinical use. Structural features of the class A α-helix motif that induce muscle toxicity and hypertriglyceridemia have been identified. These may have implications for the design of other HDL mimetic peptides. Summary ABCA1 is an integral membrane protein that plays a central role in biology. Its principal function is to mediate the efflux of cholesterol and phospholipid from cells to extracellular apo, preventing a build-up of excess cholesterol in membranes. This process generates HDL particles that perform a variety of functions to protect against disease. A number of these functions can be viewed as directly or indirectly supporting ABCA1 activity, thus constituting a positive feedback system to optimize cellular lipid efflux responses and disease prevention. Consequently, therapeutic approaches that mimic the activities of apos may prove highly effective to combat disease. One such approach involves the use of peptides. The broad biological relevance of ABCA1 suggests these apo mimetic peptides may be useful for the treatment of a number of diseases, such as atherosclerosis, diabetes, and Alzheimer's disease.« less

  15. Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

    PubMed

    Sorrenson, Brie; Suetani, Rachel J; Williams, Michael J A; Bickley, Vivienne M; George, Peter M; Jones, Gregory T; McCormick, Sally P A

    2013-01-01

    Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

  16. A survey of ABCA1 sequence variation confirms association with dementia

    PubMed Central

    Reynolds, Chandra A.; Hong, Mun-Gwan; Eriksson, Ulrika K.; Blennow, Kaj; Bennet, Anna M.; Johansson, Boo; Malmberg, Bo; Berg, Stig; Wiklund, Fredrik; Gatz, Margaret; Pedersen, Nancy L.; Prince, Jonathan A.

    2009-01-01

    We and others have conducted targeted genetic association analyses of ABCA1 in relation to Alzheimer disease risk with a resultant mixture of both support and refutation, but all previous studies have been based upon only a few markers. Here, a detailed survey of genetic variation in the ABCA1 region has been performed in a total of 1567 Swedish dementia cases (including 1275 with Alzheimer disease) and 2203 controls, providing evidence of association with maximum significance at marker rs2230805 (OR = 1.39; 95% CI 1.23–1.57, P = 7.7 × 10−8). Haplotype-based tests confirmed association of this genomic region after excluding rs2230805, and imputation did not reveal additional markers with greater support. Significantly associating markers reside in two distinct linkage disequilibrium blocks with maxima near the promoter and in the terminal exon of a truncated ABCA1 splice-form. The putative risk allele of rs2230805 was also found to be associated with reduced cerebrospinal fluid levels of β-amyloid. The strongest evidence of association was obtained when all forms of dementia were considered together, but effect sizes were similar when only confirmed Alzheimer disease cases were assessed. Results further implicate ABCA1 in dementia, reinforcing the putative involvement of lipid transport in neurodegenerative disease. PMID:19606474

  17. Glucagon-like peptide-1 contributes to increases ABCA1 expression by downregulating miR-758 to regulate cholesterol homeostasis.

    PubMed

    Yao, Yue; Li, Qiang; Gao, Ping; Wang, Wei; Chen, Lili; Zhang, Jinchao; Xu, Yi

    2018-03-04

    Abnormal regulation of lipid metabolism is associated with type 2 diabetes mellitus (T2DM). GLP-1 as a new treatment for T2DM, has unique effects in modulating cholesterol homeostasis. However, the mechanism of this effect is largely missing. The aim of this study was to determine the effects of GLP-1 on cholesterol-induced lipotoxicity in hepatocytes and examine the underlying mechanisms. The cell viability was determined, and caspase-3 was used to detect the effects of GLP-1 on cholesterol-induced apoptosis. The alterations of miR-758 and ATP-binding cassette transporter A1 (ABCA1) resulting from cholesterol incubation or GLP-1 were detected by qRT-PCR and Western blot assays. Overexpression of miR-758 abrogated the GLP-1-mediated ABCA1 expression, and conversely, down-regulation of miR-758 aggravated GLP-1's action and revealed significant promotion effects. BODIPY-Cholesterol efflux assay revealed that treatment with miR-758 inhibitor significantly enhanced ABCA1-dependent cholesterol efflux, resulting in reduced total cholesterol. Furthermore, Oil red O staining and cholesterol measurement were used to detect lipid accumulation. As a result, cholesterol significantly attenuated cell viability, promoted cell apoptosis, and facilitated lipid accumulation, and these effects were reversed by GLP-1. This study provides evidence that, in HepG2 cells, GLP-1 may affect cholesterol homeostasis by regulating the expression of miR-758 and ABCA1. These data can inform the development of biomarkers for miR-758, and potentially other drugs, on the key pathways of lipid metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. On the mechanism for PPAR agonists to enhance ABCA1 gene expression

    PubMed Central

    Ogata, Masaki; Tsujita, Maki; Hossain, Mohammad Anwar; Akita, Nobukatsu; Gonzalez, Frank J.; Staels, Bart; Suzuki, Shogo; Fukutomi, Tatsuya; Kimura, Genjiro; Yokoyama, Shinji

    2009-01-01

    Expression of ATP binding cassette transporter A1 (ABCA1), a major regulator of high density lipoprotein (HDL) biogenesis, is known to be up-regulated by the transcription factor liver X receptor (LXR) α, and expression is further enhanced by activation of the peroxisome proliferator activated receptors (PPARs). We investigated this complex regulatory network using specific PPAR agonists: four fibrates (fenofibrate, bezafibrate, gemfibrozil and LY518674), a PPAR δ agonist (GW501516) and a PPAR γ agonist (pioglitazone). All of these compounds increased the expression of LXRs, PPARs and ABCA1 mRNAs, and associated apoA-I-mediated lipid release in THP-1 macrophage, WI38 fibroblast and mouse fibroblast. When mouse fibroblasts lacking expression of PPAR α were examined, the effects of fenofibrate and LY518674 were markedly diminished while induction by other ligands were retained. The PPAR α promoter was activated by all of these compounds in an LXR α-dependent manner, and partially in a PPAR α-dependent manner, in mouse fibroblast. The LXR responsive element (LXRE)-luciferase activity was enhanced by all the compounds in an LXR α-dependent manner in mouse fibroblast. This activation was exclusively PPAR α-dependent by fenofibrate and LY518674, but nonexclusively by the others. We conclude that PPARs and LXRs are involved in the regulation of ABCA1 expression and HDL biogenesis in a cooperative signal transduction pathway. PMID:19201410

  19. Modulation of ABCA1 by an LXR Agonist Reduces Beta-Amyloid Levels and Improves Outcome after Traumatic Brain Injury

    PubMed Central

    Loane, David J.; Washington, Patricia M.; Vardanian, Lilit; Pocivavsek, Ana; Hoe, Hyang-Sook; Duff, Karen E.; Cernak, Ibolja; Rebeck, G. William; Faden, Alan I.

    2011-01-01

    Abstract Traumatic brain injury (TBI) increases brain beta-amyloid (Aβ) in humans and animals. Although the role of Aβ in the injury cascade is unknown, multiple preclinical studies have demonstrated a correlation between reduced Aβ and improved outcome. Therefore, therapeutic strategies that enhance Aβ clearance may be beneficial after TBI. Increased levels of ATP-binding cassette A1 (ABCA1) transporters can enhance Aβ clearance through an apolipoprotein E (apoE)-mediated pathway. By measuring Aβ and ABCA1 after experimental TBI in C57BL/6J mice, we found that Aβ peaked early after injury (1–3 days), whereas ABCA1 had a delayed response (beginning at 3 days). As ABCA1 levels increased, Aβ levels returned to baseline levels—consistent with the known role of ABCA1 in Aβ clearance. To test if enhancing ABCA1 levels could block TBI-induced Aβ, we treated TBI mice with the liver X-receptor (LXR) agonist T0901317. Pre- and post-injury treatment increased ABCA1 levels at 24 h post-injury, and reduced the TBI-induced increase in Aβ. This reduction in Aβ was not due to decreased amyloid precursor protein processing, or a shift in the solubility of Aβ, indicating enhanced clearance. T0901317 also limited motor coordination deficits in injured mice and reduced brain lesion volume. These data indicate that activation of LXR can reduce Aβ accumulation after TBI, and is accompanied by improved functional recovery. PMID:21175399

  20. ABCA1 in adipocytes regulates adipose tissue lipid content, glucose tolerance, and insulin sensitivity.

    PubMed

    de Haan, Willeke; Bhattacharjee, Alpana; Ruddle, Piers; Kang, Martin H; Hayden, Michael R

    2014-03-01

    Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1(-ad/-ad)). When fed a high-fat, high-cholesterol diet, ABCA1(-ad/-ad) mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1(-ad/-ad) mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis.

  1. Local TNF causes NFATc1-dependent cholesterol-mediated podocyte injury

    PubMed Central

    Pedigo, Christopher E.; Ducasa, Gloria Michelle; Leclercq, Farah; Sloan, Alexis; Hashmi, Tahreem; Molina-David, Judith; Ge, Mengyuan; Lassenius, Mariann I.; Groop, Per-Henrik; Kretzler, Matthias; Martini, Sebastian; Reich, Heather; Wahl, Patricia; Ghiggeri, GianMarco; Burke, George W.; Kretz, Oliver; Huber, Tobias B.; Mendez, Armando J.; Merscher, Sandra

    2016-01-01

    High levels of circulating TNF and its receptors, TNFR1 and TNFR2, predict the progression of diabetic kidney disease (DKD), but their contribution to organ damage in DKD remains largely unknown. Here, we investigated the function of local and systemic TNF in podocyte injury. We cultured human podocytes with sera collected from DKD patients, who displayed elevated TNF levels, and focal segmental glomerulosclerosis (FSGS) patients, whose TNF levels resembled those of healthy patients. Exogenous TNF administration or local TNF expression was equally sufficient to cause free cholesterol–dependent apoptosis in podocytes by acting through a dual mechanism that required a reduction in ATP-binding cassette transporter A1mediated (ABCA1-mediated) cholesterol efflux and reduced cholesterol esterification by sterol-O-acyltransferase 1 (SOAT1). TNF-induced albuminuria was aggravated in mice with podocyte-specific ABCA1 deficiency and was partially prevented by cholesterol depletion with cyclodextrin. TNF-stimulated free cholesterol–dependent apoptosis in podocytes was mediated by nuclear factor of activated T cells 1 (NFATc1). ABCA1 overexpression or cholesterol depletion was sufficient to reduce albuminuria in mice with podocyte-specific NFATc1 activation. Our data implicate an NFATc1/ABCA1-dependent mechanism in which local TNF is sufficient to cause free cholesterol–dependent podocyte injury irrespective of TNF, TNFR1, or TNFR2 serum levels. PMID:27482889

  2. Myeloid-specific genetic ablation of ATP-binding cassette transporter ABCA1 is protective against cancer

    PubMed Central

    Zamanian-Daryoush, Maryam; Lindner, Daniel J.; DiDonato, Joseph A.; Wagner, Matthew; Buffa, Jennifer; Rayman, Patricia; Parks, John S.; Westerterp, Marit; Tall, Alan R.; Hazen, Stanley L.

    2017-01-01

    Increased circulating levels of apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), by genetic manipulation or infusion, protects against melanoma growth and metastasis. Herein, we explored potential roles in melanoma tumorigenesis for host scavenger receptor class B, type 1 (SR-B1), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), all mediators of apoA-I and HDL sterol and lipid transport function. In a syngeneic murine melanoma tumor model, B16F10, mice with global deletion of SR-B1 expression exhibited increased plasma HDL cholesterol (HDLc) levels and decreased tumor volume, indicating host SR-B1 does not directly contribute to HDL-associated anti-tumor activity. In mice with myeloid-specific loss of ABCA1 (Abca1−M/−M; A1−M/−M), tumor growth was inhibited by ∼4.8-fold relative to wild type (WT) animals. Abcg1−M/−M (G1−M/−M) animals were also protected by 2.5-fold relative to WT, with no further inhibition of tumor growth in Abca1/Abcg1 myeloid-specific double knockout animals (DKO). Analyses of tumor-infiltrating immune cells revealed a correlation between tumor protection and decreased presence of the immune suppressive myeloid-derived suppressor cell (MDSC) subsets, Ly-6G+Ly-6CLo and Ly-6GnegLy-6CHi cells. The growth of the syngeneic MB49 murine bladder cancer cells was also inhibited in A1−M/−M mice. Collectively, our studies provide further evidence for an immune modulatory role for cholesterol homeostasis pathways in cancer. PMID:29069761

  3. ABCA1 in adipocytes regulates adipose tissue lipid content, glucose tolerance, and insulin sensitivity[S

    PubMed Central

    de Haan, Willeke; Bhattacharjee, Alpana; Ruddle, Piers; Kang, Martin H.; Hayden, Michael R.

    2014-01-01

    Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1−ad/−ad). When fed a high-fat, high-cholesterol diet, ABCA1−ad/−ad mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1−ad/−ad mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis. PMID:24443560

  4. Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages.

    PubMed

    Fournier, Natalie; Tardivel, Sylviane; Benoist, Jean-François; Vedie, Benoît; Rousseau-Ralliard, Delphine; Nowak, Maxime; Allaoui, Fatima; Paul, Jean-Louis

    2016-04-01

    A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70μM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro

    PubMed Central

    Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.; Genest, Jacques

    2015-01-01

    Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are

  6. Novel apo E-derived ABCA1 agonist peptide (CS-6253) promotes reverse cholesterol transport and induces formation of preβ-1 HDL in vitro

    DOE PAGES

    Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.; ...

    2015-07-24

    Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from themore » carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are

  7. ABCA1 gene variants regulate postprandial lipid metabolism in healthy men

    PubMed Central

    Delgado-Lista, Javier; Perez-Martinez, Pablo; Perez-Jimenez, Francisco; Garcia-Rios, Antonio; Fuentes, Francisco; Marin, Carmen; Gómez-Luna, Purificación; Camargo, Antonio; Parnell, Laurence D; Ordovas, Jose Maria; Lopez-Miranda, Jose

    2010-01-01

    Objective Genetic variants of ABCA1, an ATP-binding cassette (ABC) transporter, have been linked to altered atherosclerosis progression and fasting lipid concentration, mainly high density lipoproteins (HDL) and Apolipoprotein A1 (APOA1), but results from different studies have been inconsistent. Methods and results In order to further characterize the effects of ABCA1 variants in human postprandial lipid metabolism, we studied the influence of three single nucleotide polymorphisms (SNPs) [i27943 (rs2575875); i48168 (rs4149272); R219K (rs2230806)] in the postprandial lipemia of 88 normolipidemic young men, who were given a fatty meal. For i27943 and i48168 SNPs, fasting and postprandial values of APOA1 were higher, and postprandial lipemia was much lower in homozygotes for the major alleles, for total triglycerides in plasma, and large-triglyceride rich lipoproteins (TRL) triglycerides. These persons also showed higher APOA1/APOB ratio. Major allele homozygotes for i48168 and i27943 showed additionally higher HDL and lower postprandial Apolipoprotein B (ApoB). Conclusions Our work shows that major allele homozygotes for ABCA1 SNPs i27943 and i48168 have a lower postprandial response as compared to minor allele carriers. This finding may further characterize the role of ABCA1 in lipid metabolism. PMID:20185793

  8. Hsp27 promotes ABCA1 expression and cholesterol efflux through the PI3K/PKCζ/Sp1 pathway in THP-1 macrophages.

    PubMed

    Kuang, Hai-Jun; Zhao, Guo-Jun; Chen, Wu-Jun; Zhang, Min; Zeng, Gao-Feng; Zheng, Xi-Long; Tang, Chao-Ke

    2017-09-05

    Heat shock protein 27 (Hsp27) is a putative biomarker and therapeutic target in atherosclerosis. This study was to explore the potential mechanisms underlying Hsp27 effects on ATP-binding cassette transporter A1 (ABCA1) expression and cellular cholesterol efflux. THP-1 macrophage-derived foam cells were infected with adenovirus to express wild-type Hsp27, hyper-phosphorylated Hsp27 mimic (3D Hsp27), antisense Hsp27 or hypo-phosphorylated Hsp27 mimic (3A Hsp27). Wild-type and 3D Hsp27 were found to up-regulate ABCA1 mRNA and protein expression and increase cholesterol efflux from cells. Expression of antisense or 3A Hsp27 suppressed the expression of ABCA1 and cholesterol efflux. Furthermore, over-expression of wild-type and 3D Hsp27 significantly increased the levels of phosphorylated specificity protein 1 (Sp1), protein kinase C ζ (PKCζ) and phosphatidylinositol 3-kinase (PI3K). In addition, the up-regulation of ABCA1 expression and cholesterol efflux induced by 3D Hsp27 was suppressed by inhibition of Sp1, PKCζ and PI3K with specific kinase inhibitors. Taken together, our results revealed that Hsp27 may up-regulate the expression of ABCA1 and promotes cholesterol efflux through activation of the PI3K/PKCζ/Sp1 signal pathway in THP-1 macrophage-derived foam cells. Our findings may partly explain the mechanisms underlying the anti-atherogenic effect of Hsp27. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Large-scale deletions of the ABCA1 gene in patients with hypoalphalipoproteinemia.

    PubMed

    Dron, Jacqueline S; Wang, Jian; Berberich, Amanda J; Iacocca, Michael A; Cao, Henian; Yang, Ping; Knoll, Joan; Tremblay, Karine; Brisson, Diane; Netzer, Christian; Gouni-Berthold, Ioanna; Gaudet, Daniel; Hegele, Robert A

    2018-06-04

    Copy-number variations (CNVs) have been studied in the context of familial hypercholesterolemia but have not yet been evaluated in patients with extremes of high-density lipoprotein (HDL) cholesterol levels. We evaluated targeted next-generation sequencing data from patients with very low HDL cholesterol (i.e. hypoalphalipoproteinemia) using the VarSeq-CNV caller algorithm to screen for CNVs disrupting the ABCA1, LCAT or APOA1 genes. In four individuals, we found three unique deletions in ABCA1: a heterozygous deletion of exon 4, a heterozygous deletion spanning exons 8 to 31, and a heterozygous deletion of the entire ABCA1 gene. Breakpoints were identified using Sanger sequencing, and the full-gene deletion was also confirmed using exome sequencing and the Affymetrix CytoScanTM HD Array. Before now, large-scale deletions in candidate HDL genes have not been associated with hypoalphalipoproteinemia; our findings indicate that CNVs in ABCA1 may be a previously unappreciated genetic determinant of low HDL cholesterol levels. By coupling bioinformatic analyses with next-generation sequencing data, we can successfully assess the spectrum of genetic determinants of many dyslipidemias, now including hypoalphalipoproteinemia. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Neopterin negatively regulates expression of ABCA1 and ABCG1 by the LXRα signaling pathway in THP-1 macrophage-derived foam cells.

    PubMed

    Yan, Jin-quan; Tan, Chun-zhi; Wu, Jin-hua; Zhang, Dong-cui; Chen, Ji-ling; Zeng, Bin-yuan; Jiang, Yu-ping; Nie, Jin; Liu, Wei; Liu, Qin; Dai, Hao

    2013-07-01

    To investigate the effects of neopterin on ABCA1 expression and cholesterol efflux in human THP-1 macrophage-derived foam cells, and to explore the role of the liver X receptor alpha (LXRα) involved. In the present study, THP-1 cells were pre-incubated with ox-LDL to become foam cells. The protein and mRNA expression were examined by Western blot assays and real-time quantitative PCR, respectively. Liquid scintillation counting and high performance liquid chromatography assays were used to test cellular cholesterol efflux and cholesterol content. Neopterin decreased ABCA1 expression and cholesterol efflux in a time- and concentration-dependent manner in THP-1 macrophage-derived foam cells, and the LXRα siRNA can reverse the inhibitory effects induced by neopterin. Neoterin has a negative regulation on ABCA1 expression via the LXRα signaling pathway, which suggests the aggravated effects of neopterin on atherosclerosis.

  11. 13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages

    PubMed Central

    2011-01-01

    Background Synthetic activators of peroxisome proliferator-activated receptors (PPARs) stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα) and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR-BI). The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA), 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages. Methods RAW264.7 macrophages were treated without (control) or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux. Results Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P < 0.05). In addition, 13-HODE enhanced cholesterol concentration in the medium but decreased cellular cholesterol concentration during incubation of cells with the extracellular lipid acceptor apolipoprotein A-I (P < 0.05). Pre-treatment of cells with a selective PPARα or PPARγ antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated. In contrast to 13-HODE, LA had no effect on either of these parameters compared to control cells. Conclusion 13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway. PMID:22129452

  12. Differences in ABCA1 R219K Polymorphisms and Serum Indexes in Alzheimer and Parkinson Diseases in Northern China

    PubMed Central

    Ya, Lagai; Lu, Zuneng

    2017-01-01

    Background ABCA1 R219K single-nucleotide polymorphisms (SNPs) was related to Alzheimer disease (AD) but not Parkinson disease (PD). Here, we analyzed the associations among ABCA1 R219K distribution, serum biomarkers, AD, and PD in a population in northern China. Material/Methods We used the Mini-Mental State Examination (MMSE) and the Hoehn and Yahr scale (H-Y) to evaluate AD and PD progression, separately. ABCA1 R219K was analyzed by matrix-assisted laser desorption ionization time of flight time mass spectrometry (MALDI-TOF-MS). Serum indexes were determined by enzyme-linked immunosorbent assay (ELISA). Results ABCA1 R219K RR+RK genotype frequency in AD and PD patients was lower than that in normal controls (NC), while ABCA1 R219K KK genotype frequency was significantly higher. ABCA1 R219K RR genotype frequency in AD patients and NC was lower than that in PD patients, while ABCA1 R219K RK+KK genotype frequency was significantly higher. ABCA1 R219K RR genotype was positively correlated to MMSE value in AD patients, while ABCA1 R219K KK genotype was negatively correlated to H-Y value in PD patients. Serum factors were significantly different among AD and PD patients and NC. Serum ABCA1, ApoA1, ApoA2, ApoB, HDL, TC, IL-1β, IL-6, and TNF-α were significantly different between AD and PD patients. Conclusions ABCA1 R219K R allele was the risk factor inducing abnormal serum levels of ApoA2, LDL, and TG in AD patients, and abnormal levels of serum ABCA1, HDL, IL-1β, IL-6, and TNF-α in PD patients, while ABCA1 R219K K allele was the risk factor inducing lower ABCA1 in AD patients. IL-1β, IL-6, and TNF-α were negatively correlated to MMSE in AD patients but positively correlated to H-Y in PD patients, while HDL was positively related to H-Y in PD patients. PMID:28943632

  13. Differences in ABCA1 R219K Polymorphisms and Serum Indexes in Alzheimer and Parkinson Diseases in Northern China.

    PubMed

    Ya, Lagai; Lu, Zuneng

    2017-09-25

    BACKGROUND ABCA1 R219K single-nucleotide polymorphisms (SNPs) was related to Alzheimer disease (AD) but not Parkinson disease (PD). Here, we analyzed the associations among ABCA1 R219K distribution, serum biomarkers, AD, and PD in a population in northern China. MATERIAL AND METHODS We used the Mini-Mental State Examination (MMSE) and the Hoehn and Yahr scale (H-Y) to evaluate AD and PD progression, separately. ABCA1 R219K was analyzed by matrix-assisted laser desorption ionization time of flight time mass spectrometry (MALDI-TOF-MS). Serum indexes were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS ABCA1 R219K RR+RK genotype frequency in AD and PD patients was lower than that in normal controls (NC), while ABCA1 R219K KK genotype frequency was significantly higher. ABCA1 R219K RR genotype frequency in AD patients and NC was lower than that in PD patients, while ABCA1 R219K RK+KK genotype frequency was significantly higher. ABCA1 R219K RR genotype was positively correlated to MMSE value in AD patients, while ABCA1 R219K KK genotype was negatively correlated to H-Y value in PD patients. Serum factors were significantly different among AD and PD patients and NC. Serum ABCA1, ApoA1, ApoA2, ApoB, HDL, TC, IL-1β, IL-6, and TNF-α were significantly different between AD and PD patients. CONCLUSIONS ABCA1 R219K R allele was the risk factor inducing abnormal serum levels of ApoA2, LDL, and TG in AD patients, and abnormal levels of serum ABCA1, HDL, IL-1b, IL-6, and TNF-α in PD patients, while ABCA1 R219K K allele was the risk factor inducing lower ABCA1 in AD patients. IL-1β, IL-6, and TNF-α were negatively correlated to MMSE in AD patients but positively correlated to H-Y in PD patients, while HDL was positively related to H-Y in PD patients.

  14. Insulin-like Growth Factor 1 Regulates the Expression of ATP-Binding Cassette Transporter A1 in Pancreatic Beta Cells.

    PubMed

    Lyu, J; Imachi, H; Iwama, H; Zhang, H; Murao, K

    2016-05-01

    ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and cholesterol homeostasis. The present study investigates whether insulin-like growth factor 1 (IGF-1), which mediates stimulation of ABCA1 gene expression, could also interfere with the phosphatidylinositol 3-kinase (PI3-K) cascade.ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and a reporter gene assay in rat insulin-secreting INS-1 cells incubated with IGF-1. The binding of forkhead box O1 (FoxO1) protein to the ABCA1 promoter was assessed by a chromatin immunoprecipitation (ChIP) assay. ABCA1 protein levels increased in response to rising concentrations of IGF-1. Real-time PCR analysis showed a significant increase in ABCA1 mRNA expression. However, both effects were suppressed after silencing the IGF-1 receptor. In parallel with its effect on endogenous ABCA1 mRNA levels, IGF-1 induced the activity of a reporter construct containing the ABCA1 promoter, while it was abrogated by LY294002, a specific inhibitor of PI3-K. Constitutively active Akt stimulated activity of the ABCA1 promoter, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element in the ABCA1 promoter abolished the ability of IGF-1 to stimulate promoter activity. A ChIP assay showed that FoxO1 mediated its transcriptional activity by directly binding to the ABCA1 promoter region. The knockdown of FoxO1 disrupted the effect of IGF-1 on ABCA1 expression. Furthermore, IGF-1 promoted cholesterol efflux and reduced the pancreatic lipotoxicity. These results demonstrate that the PI3-K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF-1 stimulation. © Georg Thieme Verlag KG Stuttgart · New York.

  15. The cholesterol transporter ABCA1 is expressed in stallion spermatozoa and reproductive tract tissues.

    PubMed

    Merkl, M; Ertl, R; Handschuh, S; Aurich, C; Schäfer-Somi, S

    2016-04-01

    In the present study, we assessed the presence of the ATP-binding-cassette (ABC) transporter molecules ABCA1 in spermatozoa of adult stallions and in testicular and epididymal tissue of prepubertal and adult stallions. For this purpose, semen samples from six fertile Shetland pony stallions aged 4 to 19 years were collected. Semen was collected from each stallion on three consecutive days. Ejaculates were analyzed immediately after collection, and only ejaculates meeting minimal requirements for fertile stallions were further evaluated. ABCA1 immunosignal was localized after staining of semen smears with different antibodies and counterstaining with Fluorescein isothiocyanate (FITC)-peanut agglutinin (PNA) and 4',6-Diamidin-2-phenylindol (DAPI). In a total of three samples, capacitation and acrosome reaction were induced by means of capacitation medium and progesterone substitution, respectively. Testicular and epididymal tissues were obtained from five prepubertal stallions aged 8 to 12 months and five adult stallions aged 4 to 9 years. For quantitative RT-PCR (qPCR), testicular and epididymal tissue of another seven adult (aged 1.5-14.5 years) and five prepupertal stallions (6-8 months) was used. For immunohistochemistry, sections from the caput, corpus, and cauda of the testes and epididymes were stained with the same specific antibodies as for immunocytochemistry. In stallion spermatozoa, strong immunosignal for ABCA1 was detected in the acrosomal area, the equatorial zone, and the principle piece of the flagellum but not in the caudal part of the head and the midpiece. In damaged or acrosome-reacted spermatozoa the FITC-PNA signal vanished together with the ABCA1 signal in most spermatozoa. In testicular tissue, strong immunostaining for ABCA1 was mainly visible in the heads and flagella of round spermatids and weaker signals in late spermatids and released spermatozoa. No staining was assessed in the Sertoli cells and spermatogonia of adult stallions, whereas

  16. Amphipathic Polyproline Peptides Stimulate Cholesterol Efflux by the ABCA1 Transporter

    PubMed Central

    Sviridov, D.O.; Drake, S.K.; Freeman, L.A.; Remaley, A.T.

    2016-01-01

    ApoA-I mimetics are short synthetic peptides that contain an amphipathic αα-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenol group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop - Prog - Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-αhelical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20-35% (p<0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides. PMID:26879139

  17. HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE-/- Mice: Evidence of ABCA1 Down-Regulation

    PubMed Central

    Tardy, Claudine; Goffinet, Marine; Boubekeur, Nadia; Cholez, Guy; Ackermann, Rose; Sy, Gavin; Keyserling, Constance; Lalwani, Narendra; Paolini, John F.; Dasseux, Jean-Louis; Barbaras, Ronald; Baron, Rudi

    2015-01-01

    high doses of HDL and CER-001 induce a rapid and strong down-regulation of ABCA1 both in vitro and in vivo. In conclusion, maximally efficient HDL- or CER-001-mediated cholesterol removal from atherosclerotic plaque is achieved by maximizing macrophage-mediated efflux from the plaque while minimizing dose-dependent down-regulation of ABCA1 expression. These observations may help define the optimal dose of HDL mimetics for testing in clinical trials of atherosclerotic burden regression. PMID:26335690

  18. HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE-/- Mice: Evidence of ABCA1 Down-Regulation.

    PubMed

    Tardy, Claudine; Goffinet, Marine; Boubekeur, Nadia; Cholez, Guy; Ackermann, Rose; Sy, Gavin; Keyserling, Constance; Lalwani, Narendra; Paolini, John F; Dasseux, Jean-Louis; Barbaras, Ronald; Baron, Rudi

    2015-01-01

    CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and charged phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the dose-dependent regulation of ABCA1 expression in vitro and in vivo in the presence of CER-001 and native HDL (HDL3). CER-001 induced cholesterol efflux from J774 macrophages in a dose-dependent manner similar to natural HDL. A strong down-regulation of the ATP-binding cassette A1 (ABCA1) transporter mRNA (- 50%) as well as the ABCA1 membrane protein expression (- 50%) was observed at higher doses of CER-001 and HDL3 compared to non-lipidated apoA-I. In vivo, in an apoE-/- mouse "flow cessation model," in which the left carotid artery was ligatured to induce local inflammation, the inhibition of atherosclerotic plaque burden progression in response to a dose-range of every-other-day CER-001 or HDL in the presence of a high-fat diet for two weeks was assessed. We observed a U-shaped dose-response curve: inhibition of the plaque total cholesterol content increased with increasing doses of CER-001 or HDL3 up to a maximum inhibition (- 51%) at 5 mg/kg; however, as the dose was increased above this threshold, a progressively less pronounced inhibition of progression was observed, reaching a complete absence of inhibition of progression at doses of 20 mg/kg and over. ABCA1 protein expression in the same atherosclerotic plaque was decreased by-45% and-68% at 50 mg/kg for CER-001 and HDL respectively. Conversely, a-12% and 0% decrease in ABCA1 protein expression was observed at the 5 mg/kg dose for CER-001 and HDL respectively. These data demonstrate that high doses of HDL and CER-001 are less effective at slowing progression of atherosclerotic plaque in apoE-/- mice compared to lower doses, following a U-shaped dose-response curve. A potential mechanism for this phenomenon is supported by the observation that high doses of HDL and CER-001 induce a rapid and

  19. Investigation of ABCA1 C69T polymorphism in patients with type 2 diabetes mellitus.

    PubMed

    Ergen, H Arzu; Zeybek, Umit; Gök, Ozlem; Karaali, Z Ermis

    2012-01-01

    Non insulin dependent diabetes mellitus is the most common type of diabetes. Genetic factors, lipid profiles, hypertension are potential risk factors for diabetes mellitus. Adenosine binding cassette transporter proteins 1 (ABCA1) plays a role in cholesterol metabolism, especially high density lipoprotein (HDL-cholesterol). There are multiple mechanisms by which HDL-cholesterol can be atheroprotective, it is clear that the relative activity of ABCA1 plays a major role. We aimed to investigate association of ABCA1 C69T gene polymorphism with lipid levels in Turkish type 2 diabetic patients. After isolation of DNA by ethanol precipitation we determined ABCA1 gene polymorphism by using polimerase chain reaction--restriction fragment lenght polymorphism (PCR-RFLP) method in 107 type 2 diabetic patients and 50 healthy controls. We have observed that the frequency of TT genotype is significantly higher in healthy controls compared to patients (14% vs. 3%; P = 0.008). Also frequency of T allele was higher in controls than in patients (34% vs. 21%; P = 0.020; OR (95% CI) = 0.52 (0.30-0.88)). There was no association of lipid levels and ABCA1 C69T polymorphism subgroups. We have found significantly higher frequency of both T allele and genotype in control group when compared to patients that made us think that T allele may be a protective factor against diabetes mellitus. But, we could not find a relationship between genotypes and lipid concentrations in our two groups. Larger studies will help us to understand the relationship between ABCA1 C69T genotype and lipid parameters in diabetes mellitus.

  20. Allicin induces the upregulation of ABCA1 expression via PPARγ/LXRα signaling in THP-1 macrophage-derived foam cells

    PubMed Central

    Lin, Xiao-Long; Hu, Hui-Jun; Liu, Yuan-Bo; Hu, Xue-Mei; Fan, Xiao-Juan; Zou, Wei-Wen; Pan, Yong-Quan; Zhou, Wen-Quan; Peng, Min-Wen; Gu, Cai-Hong

    2017-01-01

    Allicin is considered anti-atherosclerotic due to its antioxidant and anti-inflammatory effects, which makes it an important drug for the prevention and treatment of atherosclerosis. However, the effects of allicin on foam cells are unclear. Thus, in this study, we examined the effects of allicin on lipid accumulation via peroxisome proliferator-activated receptor γ (PPARγ)/liver X receptor α (LXRα) in THP-1 macrophage-derived foam cells. THP-1 cells were exposed to 100 nM phorbol myristate acetate (PMA) for 24 h, and then to oxydized low-density lipoprotein (ox-LDL; 50 mg/ml) to induce foam cell formation. The results of Oil Red O staining and high-performance liquid chromatography (HPLC) revealed showed that pre-treatment of the foam cells with allicin decreased total cholesterol, free cholesterol (FC) and cholesterol ester levels in cells, and also decreased lipid accumulation. Moreover, allicin upregulated ATP binding cassette transporter A1 (ABCA1) expression and promoted cholesterol efflux. However, these effects were significantly abolished by transfection with siRNA targeting ABCA1. Furthermore, PPARγ/LXRα signaling was activated by allicin treatment. The allicin-induced upregulation of ABCA1 expression was also abolished by PPARγ inhibitor (GW9662) and siRNA or LXRα siRNA co-treatment. Overall, our data demonstrate that the allicin-induced upregulation of ABCA1 promotes cholesterol efflux and reduces lipid accumulation via PPARγ/LXRα signaling in THP-1 macrophage-derived foam cells. PMID:28440421

  1. High glucose upregulates BACE1-mediated Aβ production through ROS-dependent HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization in SK-N-MC cells

    PubMed Central

    Lee, Hyun Jik; Ryu, Jung Min; Jung, Young Hyun; Lee, Sei-Jung; Kim, Jeong Yeon; Lee, Sang Hun; Hwang, In Koo; Seong, Je Kyung; Han, Ho Jae

    2016-01-01

    There is an accumulation of evidence indicating that the risk of Alzheimer’s disease is associated with diabetes mellitus, an indicator of high glucose concentrations in blood plasma. This study investigated the effect of high glucose on BACE1 expression and amyloidogenesis in vivo, and we present details of the mechanism associated with those effects. Our results, using ZLC and ZDF rat models, showed that ZDF rats have high levels of amyloid-beta (Aβ), phosphorylated tau, BACE1, and APP-C99. In vitro result with mouse hippocampal neuron and SK-N-MC, high glucose stimulated Aβ secretion and apoptosis in a dose-dependent manner. In addition, high glucose increased BACE1 and APP-C99 expressions, which were reversed by a reactive oxygen species (ROS) scavenger. Indeed, high glucose increased intracellular ROS levels and HIF-1α expression, associated with regulation of BACE1 and Liver X Receptor α (LXRα). In addition, high glucose induced ATP-binding cassette transporter A1 (ABCA1) down-regulation, was associated with LXR-induced lipid raft reorganization and BACE1 localization on the lipid raft. Furthermore, silencing of BACE1 expression was shown to regulate Aβ secretion and apoptosis of SK-N-MC. In conclusion, high glucose upregulates BACE1 expression and activity through HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization, leading to Aβ production and apoptosis of SK-N-MC. PMID:27829662

  2. Quercetin-3-O-glucuronide induces ABCA1 expression by LXRα activation in murine macrophages

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohara, Kazuaki, E-mail: Kazuaki_Ohara@kirin.co.jp; Wakabayashi, Hideyuki; Taniguchi, Yoshimasa

    Highlights: •The major circulating quercetin metabolite (Q3GA) activated LXRα. •Q3GA induced ABCA1 via LXRα activation in macrophages. •Nelumbo nucifera leaf extracts contained quercetin glycosides. •N. nucifera leaf extract feeding elevated HDLC in mice. -- Abstract: Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found thatmore » quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.« less

  3. Deficiency of ABCA1 and ABCG1 in Macrophages Increases Inflammation and Accelerates Atherosclerosis in Mice

    PubMed Central

    Westerterp, Marit; Murphy, Andrew J.; Wang, Mi; Pagler, Tamara A.; Vengrenyuk, Yuliya; Kappus, Mojdeh S.; Gorman, Darren J.; Nagareddy, Prabhakara R.; Zhu, Xuewei; Abramowicz, Sandra; Parks, John S.; Welch, Carrie; Fisher, Edward A.; Wang, Nan; Yvan-Charvet, Laurent; Tall, Alan R.

    2013-01-01

    Rationale Plasma HDL levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is due to the ability of HDL to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. Objective To assess the role of macrophage cholesterol efflux pathways in atherogenesis. Methods and Results We developed MAC-ABCDKO mice with efficient deletion of the ATP Binding Cassette Transporters A1 and G1 (ABCA1 and ABCG1) in macrophages but not in hematopoietic stem or progenitor populations. MAC-ABCDKO bone marrow (BM) was transplanted into Ldlr-/- recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared to controls. On the Western type diet (WTD), MAC-ABCDKO BM transplanted Ldlr-/- mice had disproportionate atherosclerosis, considering they also had lower VLDL/LDL cholesterol levels than controls. ABCA1/G1 deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, WTD-fed MAC-ABCDKO BM transplanted Ldlr-/- mice displayed monocytosis and neutrophilia in the absence of HSPC proliferation. Mechanistic studies revealed increased expression of M-CSF and G-CSF in splenic macrophage foam cells, driving BM monocyte and neutrophil production. Conclusion These studies 1) show that macrophage deficiency of ABCA1/G1 is pro-atherogenic likely by promoting plaque inflammation and 2) uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways. PMID:23572498

  4. Gender-specific association of ATP-binding cassette transporter 1 (ABCA1) polymorphisms with the risk of late-onset Alzheimer's disease.

    PubMed

    Sundar, Purnima Desai; Feingold, Eleanor; Minster, Ryan L; DeKosky, Steven T; Kamboh, M Ilyas

    2007-06-01

    Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder caused by a complex interaction of genetic and environmental factors. Increasing evidence highlights a potential role for cholesterol in the pathophysiology of AD. The ABCA1 gene, located in close vicinity to the 9q linkage peaks identified by genome-wide AD linkage studies, plays an important role in cellular cholesterol efflux, and is likely a good candidate gene. However, results from published genetic association studies between ABCA1 and AD are ambiguous. In the present study, we examined the role of two ABCA1 polymorphisms, R219K (rs2230806) and G-17C (rs2740483) in modifying the risk of late-onset AD (LOAD) in a large American white cohort of 992 AD cases and 699 controls. We observed significant gender x R219K interaction (p=0.00008). Female carriers of the 219K allele showed a 1.75-fold increased risk of developing AD compared to non-219K carrier females (95% CI 1.34-2.29; p=0.00004). The overall two-site haplotype distribution was also significant between female AD cases and controls (p=0.017). The risk associated with the R219K polymorphism was independent of the recently reported significant association in the ubiquilin (UBQLN1) gene in this region on chromosome 9q. Our data suggest a gender-specific and APOE and UBQLN1 independent association between the ABCA1/R219K polymorphism and LOAD.

  5. Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling.

    PubMed

    Chowdhury, Saiful M; Zhu, Xuewei; Aloor, Jim J; Azzam, Kathleen M; Gabor, Kristin A; Ge, William; Addo, Kezia A; Tomer, Kenneth B; Parks, John S; Fessler, Michael B

    2015-07-01

    Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. © 2015 by The American Society for Biochemistry and

  6. Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling*

    PubMed Central

    Chowdhury, Saiful M.; Zhu, Xuewei; Aloor, Jim J.; Azzam, Kathleen M.; Gabor, Kristin A.; Ge, William; Addo, Kezia A.; Tomer, Kenneth B.; Parks, John S.; Fessler, Michael B.

    2015-01-01

    Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. PMID:25910759

  7. Common variants near ABCA1, AFAP1 and GMDS confer risk of primary open-angle glaucoma

    PubMed Central

    Fogarty, Rhys; Sharma, Shiwani; Hewitt, Alex W.; Martin, Sarah; Law, Matthew H.; Cremin, Katie; Bailey, Jessica N. Cooke; Loomis, Stephanie J.; Pasquale, Louis R.; Haines, Jonathan L.; Hauser, Michael A.; Viswanathan, Ananth C.; McGuffin, Peter; Topouzis, Fotis; Foster, Paul J.; Graham, Stuart L; Casson, Robert J; Chehade, Mark; White, Andrew J; Zhou, Tiger; Souzeau, Emmanuelle; Landers, John; Fitzgerald, Jude T; Klebe, Sonja; Ruddle, Jonathan B; Goldberg, Ivan; Healey, Paul R; Mills, Richard A.; Wang, Jie Jin; Montgomery, Grant W.; Martin, Nicholas G.; Radford-Smith, Graham; Whiteman, David C.; Brown, Matthew A.; Wiggs, Janey L.; Mackey, David A; Mitchell, Paul; MacGregor, Stuart; Craig, Jamie E.

    2014-01-01

    Primary open-angle glaucoma (POAG) is a major cause of irreversible blindness worldwide. We performed a genome-wide association study in an Australian discovery cohort comprising 1,155 advanced POAG cases and 1,992 controls. Association of the top SNPs from the discovery stage was investigated in two Australian replication cohorts (total 932 cases, 6,862 controls) and two US replication cohorts (total 2,616 cases, 2,634 controls). Meta-analysis of all cohorts revealed three novel loci associated with development of POAG. These loci are located upstream of ABCA1 (rs2472493 [G] OR=1.31, P= 2.1 × 10−19), within AFAP1 (rs4619890 [G] OR=1.20, P= 7.0 × 10−10) and within GMDS (rs11969985 [G] OR=1.31, and P= 7.7 × 10−10). Using RT-PCR and immunolabelling, we also showed that these genes are expressed within human retina, optic nerve and trabecular meshwork and that ABCA1 and AFAP1 are also expressed in retinal ganglion cells. PMID:25173105

  8. ABCA transporter gene expression and poor outcome in epithelial ovarian cancer.

    PubMed

    Hedditch, Ellen L; Gao, Bo; Russell, Amanda J; Lu, Yi; Emmanuel, Catherine; Beesley, Jonathan; Johnatty, Sharon E; Chen, Xiaoqing; Harnett, Paul; George, Joshy; Williams, Rebekka T; Flemming, Claudia; Lambrechts, Diether; Despierre, Evelyn; Lambrechts, Sandrina; Vergote, Ignace; Karlan, Beth; Lester, Jenny; Orsulic, Sandra; Walsh, Christine; Fasching, Peter; Beckmann, Matthias W; Ekici, Arif B; Hein, Alexander; Matsuo, Keitaro; Hosono, Satoyo; Nakanishi, Toru; Yatabe, Yasushi; Pejovic, Tanja; Bean, Yukie; Heitz, Florian; Harter, Philipp; du Bois, Andreas; Schwaab, Ira; Hogdall, Estrid; Kjaer, Susan K; Jensen, Allan; Hogdall, Claus; Lundvall, Lene; Engelholm, Svend Aage; Brown, Bob; Flanagan, James; Metcalf, Michelle D; Siddiqui, Nadeem; Sellers, Thomas; Fridley, Brooke; Cunningham, Julie; Schildkraut, Joellen; Iversen, Ed; Weber, Rachel P; Berchuck, Andrew; Goode, Ellen; Bowtell, David D; Chenevix-Trench, Georgia; deFazio, Anna; Norris, Murray D; MacGregor, Stuart; Haber, Michelle; Henderson, Michelle J

    2014-07-01

    ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA-mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan-Meier analysis and log-rank tests. All statistical tests were two-sided. Associations with outcome were observed with ABC transporters of the "A" subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e-6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Expression of ABCA transporters was associated with poor outcome in serous ovarian cancer, implicating lipid

  9. ABCA Transporter Gene Expression and Poor Outcome in Epithelial Ovarian Cancer

    PubMed Central

    Hedditch, Ellen L.; Gao, Bo; Russell, Amanda J.; Lu, Yi; Emmanuel, Catherine; Beesley, Jonathan; Johnatty, Sharon E.; Chen, Xiaoqing; Harnett, Paul; George, Joshy; Williams, Rebekka T.; Flemming, Claudia; Lambrechts, Diether; Despierre, Evelyn; Lambrechts, Sandrina; Vergote, Ignace; Karlan, Beth; Lester, Jenny; Orsulic, Sandra; Walsh, Christine; Fasching, Peter; Beckmann, Matthias W.; Ekici, Arif B.; Hein, Alexander; Matsuo, Keitaro; Hosono, Satoyo; Nakanishi, Toru; Yatabe, Yasushi; Pejovic, Tanja; Bean, Yukie; Heitz, Florian; Harter, Philipp; du Bois, Andreas; Schwaab, Ira; Hogdall, Estrid; Kjaer, Susan K.; Jensen, Allan; Hogdall, Claus; Lundvall, Lene; Engelholm, Svend Aage; Brown, Bob; Flanagan, James; Metcalf, Michelle D; Siddiqui, Nadeem; Sellers, Thomas; Fridley, Brooke; Cunningham, Julie; Schildkraut, Joellen; Iversen, Ed; Weber, Rachel P.; Berchuck, Andrew; Goode, Ellen; Bowtell, David D.; Chenevix-Trench, Georgia; deFazio, Anna; Norris, Murray D.; MacGregor, Stuart; Haber, Michelle; Henderson, Michelle J.

    2014-01-01

    Background ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. Methods The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA–mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan–Meier analysis and log-rank tests. All statistical tests were two-sided. Results Associations with outcome were observed with ABC transporters of the “A” subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e−6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Conclusions Expression of ABCA transporters was associated with poor

  10. ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines

    PubMed Central

    Sporstøl, Marita; Mousavi, Seyed Ali; Eskild, Winnie; Roos, Norbert; Berg, Trond

    2007-01-01

    Background Scavenger receptor type B class I (SR-BI), ABC transporter A1 (ABCA1) -and G1 (ABCG1) all play important roles in the reverse cholesterol transport. Reverse cholesterol transport is a mechanism whereby the body can eliminate excess cholesterol. Here, the regulation of SR-BI, ABCA1, and ABCG1 by dexamethasone (a synthetic glucocorticoid) and insulin were studied in order to gain more insight into the role of these two hormones in the cholesterol metabolism. Results By use of real time RT-PCR and Western blotting we examined the expression of our target genes. The results show that SR-BI, ABCA1 and ABCG1 mRNA expression increased in response to dexamethasone while insulin treatment reduced the expression in primary rat hepatocytes. The stimulatory effect of dexamethasone was reduced by the addition of the anti-glucocorticoid mifepristone. In HepG2 cells and THP-1 macrophages, however, the effect of dexamethasone was absent or inhibitory with no significant change in the presence of mifepristone. The latter observation may be a result of the low protein expression of glucocorticoid receptor (GR) in these cell lines. Conclusion Our results illustrates that insulin and glucocorticoids, two hormones crucial in the carbohydrate metabolism, also play an important role in the regulation of genes central in reverse cholesterol transport. We found a marked difference in mRNA expression between the primary cells and the two established cell lines when studying the effect of dexamethasone which may result from the varying expression levels of GR. PMID:17241464

  11. ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines.

    PubMed

    Sporstøl, Marita; Mousavi, Seyed Ali; Eskild, Winnie; Roos, Norbert; Berg, Trond

    2007-01-22

    Scavenger receptor type B class I (SR-BI), ABC transporter A1 (ABCA1) -and G1 (ABCG1) all play important roles in the reverse cholesterol transport. Reverse cholesterol transport is a mechanism whereby the body can eliminate excess cholesterol. Here, the regulation of SR-BI, ABCA1, and ABCG1 by dexamethasone (a synthetic glucocorticoid) and insulin were studied in order to gain more insight into the role of these two hormones in the cholesterol metabolism. By use of real time RT-PCR and Western blotting we examined the expression of our target genes. The results show that SR-BI, ABCA1 and ABCG1 mRNA expression increased in response to dexamethasone while insulin treatment reduced the expression in primary rat hepatocytes. The stimulatory effect of dexamethasone was reduced by the addition of the anti-glucocorticoid mifepristone. In HepG2 cells and THP-1 macrophages, however, the effect of dexamethasone was absent or inhibitory with no significant change in the presence of mifepristone. The latter observation may be a result of the low protein expression of glucocorticoid receptor (GR) in these cell lines. Our results illustrates that insulin and glucocorticoids, two hormones crucial in the carbohydrate metabolism, also play an important role in the regulation of genes central in reverse cholesterol transport. We found a marked difference in mRNA expression between the primary cells and the two established cell lines when studying the effect of dexamethasone which may result from the varying expression levels of GR.

  12. ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis

    PubMed Central

    Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan

    2016-01-01

    Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway. PMID:26497474

  13. ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis.

    PubMed

    Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan

    2016-01-01

    Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

  14. Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol[S

    PubMed Central

    Zhu, Xuewei; Owen, John S.; Wilson, Martha D.; Li, Haitao; Griffiths, Gary L.; Thomas, Michael J.; Hiltbold, Elizabeth M.; Fessler, Michael B.; Parks, John S.

    2010-01-01

    We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1-M/-M) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1-M/-M macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1-M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1-M/-M and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1-M/-M macrophages. Abca1-M/-M macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts. PMID:20650929

  15. Differential regulation of ATP binding cassette protein A1 expression and ApoA-I lipidation by Niemann-Pick type C1 in murine hepatocytes and macrophages.

    PubMed

    Wang, Ming-Dong; Franklin, Vivian; Sundaram, Meenakshi; Kiss, Robert S; Ho, Kenneth; Gallant, Michel; Marcel, Yves L

    2007-08-03

    Niemann-Pick type C1 (Npc1) protein inactivation results in lipid accumulation in late endosomes and lysosomes, leading to a defect of ATP binding cassette protein A1 (Abca1)-mediated lipid efflux to apolipoprotein A-I (apoA-I) in macrophages and fibroblasts. However, the role of Npc1 in Abca1-mediated lipid efflux to apoA-I in hepatocytes, the major cells contributing to HDL formation, is still unknown. Here we show that, whereas lipid efflux to apoA-I in Npc1-null macrophages is impaired, the lipidation of endogenously synthesized apoA-I by low density lipoprotein-derived cholesterol or de novo synthesized cholesterol or phospholipids in Npc1-null hepatocytes is significantly increased by about 1-, 3-, and 8-fold, respectively. The increased cholesterol efflux reflects a major increase of Abca1 protein in Npc1-null hepatocytes, which contrasts with the decrease observed in Npc1-null macrophages. The increased Abca1 expression is largely post-transcriptional, because Abca1 mRNA is only slightly increased and Lxr alpha mRNA is not changed, and Lxr alpha target genes are reduced. This differs from the regulation of Abcg1 expression, which is up-regulated at both mRNA and protein levels in Npc1-null cells. Abca1 protein translation rate is higher in Npc1-null hepatocytes, compared with wild type hepatocytes as measured by [(35)S]methionine incorporation, whereas there is no difference for the degradation of newly synthesized Abca1 in these two types of hepatocytes. Cathepsin D, which we recently identified as a positive modulator of Abca1, is markedly increased at both mRNA and protein levels by Npc1 inactivation in hepatocytes but not in macrophages. Consistent with this, inhibition of cathepsin D with pepstatin A reduced the Abca1 protein level in both Npc1-inactivated and WT hepatocytes. Therefore, Abca1 expression is specifically regulated in hepatocytes, where Npc1 activity modulates cathepsin D expression and Abca1 protein translation rate.

  16. Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4*

    PubMed Central

    Biswas-Fiss, Esther E.; Affet, Stephanie; Ha, Malissa; Biswas, Subhasis B.

    2012-01-01

    The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport. PMID:23144455

  17. Gene-Gene Combination Effect and Interactions among ABCA1, APOA1, SR-B1, and CETP Polymorphisms for Serum High-Density Lipoprotein-Cholesterol in the Japanese Population

    PubMed Central

    Nakamura, Akihiko; Niimura, Hideshi; Kuwabara, Kazuyo; Takezaki, Toshiro; Morita, Emi; Wakai, Kenji; Hamajima, Nobuyuki; Nishida, Yuichiro; Turin, Tanvir Chowdhury; Suzuki, Sadao; Ohnaka, Keizo; Uemura, Hirokazu; Ozaki, Etsuko; Hosono, Satoyo; Mikami, Haruo; Kubo, Michiaki; Tanaka, Hideo

    2013-01-01

    Background/Objective Gene-gene interactions in the reverse cholesterol transport system for high-density lipoprotein-cholesterol (HDL-C) are poorly understood. The present study observed gene-gene combination effect and interactions between single nucleotide polymorphisms (SNPs) in ABCA1, APOA1, SR-B1, and CETP in serum HDL-C from a cross-sectional study in the Japanese population. Methods The study population comprised 1,535 men and 1,515 women aged 35–69 years who were enrolled in the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study. We selected 13 SNPs in the ABCA1, APOA1, CETP, and SR-B1 genes in the reverse cholesterol transport system. The effects of genetic and environmental factors were assessed using general linear and logistic regression models after adjusting for age, sex, and region. Principal Findings Alcohol consumption and daily activity were positively associated with HDL-C levels, whereas smoking had a negative relationship. The T allele of CETP, rs3764261, was correlated with higher HDL-C levels and had the highest coefficient (2.93 mg/dL/allele) among the 13 SNPs, which was statistically significant after applying the Bonferroni correction (p<0.001). Gene-gene combination analysis revealed that CETP rs3764261 was associated with high HDL-C levels with any combination of SNPs from ABCA1, APOA1, and SR-B1, although no gene-gene interaction was apparent. An increasing trend for serum HDL-C was also observed with an increasing number of alleles (p<0.001). Conclusions The present study identified a multiplier effect from a polymorphism in CETP with ABCA1, APOA1, and SR-B1, as well as a dose-dependence according to the number of alleles present. PMID:24376512

  18. Multi-Layer Identification of Highly-Potent ABCA1 Up-Regulators Targeting LXRβ Using Multiple QSAR Modeling, Structural Similarity Analysis, and Molecular Docking.

    PubMed

    Chen, Meimei; Yang, Fafu; Kang, Jie; Yang, Xuemei; Lai, Xinmei; Gao, Yuxing

    2016-11-29

    In this study, in silico approaches, including multiple QSAR modeling, structural similarity analysis, and molecular docking, were applied to develop QSAR classification models as a fast screening tool for identifying highly-potent ABCA1 up-regulators targeting LXRβ based on a series of new flavonoids. Initially, four modeling approaches, including linear discriminant analysis, support vector machine, radial basis function neural network, and classification and regression trees, were applied to construct different QSAR classification models. The statistics results indicated that these four kinds of QSAR models were powerful tools for screening highly potent ABCA1 up-regulators. Then, a consensus QSAR model was developed by combining the predictions from these four models. To discover new ABCA1 up-regulators at maximum accuracy, the compounds in the ZINC database that fulfilled the requirement of structural similarity of 0.7 compared to known potent ABCA1 up-regulator were subjected to the consensus QSAR model, which led to the discovery of 50 compounds. Finally, they were docked into the LXRβ binding site to understand their role in up-regulating ABCA1 expression. The excellent binding modes and docking scores of 10 hit compounds suggested they were highly-potent ABCA1 up-regulators targeting LXRβ. Overall, this study provided an effective strategy to discover highly potent ABCA1 up-regulators.

  19. Activation of liver X receptor decreases atherosclerosis in Ldlr⁻/⁻ mice in the absence of ATP-binding cassette transporters A1 and G1 in myeloid cells.

    PubMed

    Kappus, Mojdeh S; Murphy, Andrew J; Abramowicz, Sandra; Ntonga, Vusisizwe; Welch, Carrie L; Tall, Alan R; Westerterp, Marit

    2014-02-01

    Liver X receptor (LXR) activators decrease atherosclerosis in mice. LXR activators (1) directly upregulate genes involved in reverse cholesterol transport and (2) exert anti-inflammatory effects mediated by transrepression of nuclear factor-κB target genes. We investigated whether myeloid cell deficiency of ATP-binding cassette transporters A1 and G1 (ABCA1/G1), principal targets of LXR that promote macrophage cholesterol efflux and initiate reverse cholesterol transport, would abolish the beneficial effects of LXR activation on atherosclerosis. LXR activator T0901317 substantially reduced inflammatory gene expression in macrophages lacking ABCA1/G1. Ldlr(-/-) mice were transplanted with Abca1(-/-)Abcg1(-/-) or wild-type bone marrow (BM) and fed a Western-type diet for 6 weeks with or without T0901317 supplementation. Abca1/g1 BM deficiency increased atherosclerotic lesion complexity and inflammatory cell infiltration into the adventitia and myocardium. T0901317 markedly decreased lesion area, complexity, and inflammatory cell infiltration in the Abca1(-/-)Abcg1(-/-) BM-transplanted mice. To investigate whether this was because of macrophage Abca1/g1 deficiency, Ldlr(-/-) mice were transplanted with LysmCreAbca1(fl/fl)Abcg1(fl/fl) or Abca1(fl/fl)Abcg1(fl/fl) BM and fed Western-type diet with or without the more specific LXR agonist GW3965 for 12 weeks. GW3965 decreased lesion size in both groups, and the decrease was more prominent in the LysmCreAbca1(fl/fl)Abcg1(fl/fl) group. The results suggest that anti-inflammatory effects of LXR activators are of key importance to their antiatherosclerotic effects in vivo independent of cholesterol efflux pathways mediated by macrophage ABCA1/G1. This has implications for the development of LXR activators that lack adverse effects on lipogenic genes while maintaining the ability to transrepress inflammatory genes.

  20. Recurrent amplification of RTEL1 and ABCA13 and its synergistic effect associated with clinicopathological data of gastric adenocarcinoma.

    PubMed

    Araújo, T M; Seabra, A D; Lima, E M; Assumpção, P P; Montenegro, R C; Demachki, S; Burbano, R M; Khayat, A S

    2016-01-01

    Despite progression in treatment of gastric cancer, prognosis of patients remains poor, in part due to the low rate of diagnosis during its early stages. This paradigm implies the necessity to identify molecular biomarkers for early gastric cancer diagnosis, as well as for disease monitoring, thus contributing to the development of new therapeutic approaches. In a previous study, performed by array-Comparative Genomic Hybridization, we described for the first time in literature recurrent amplification of RTEL1 and ABCA13 genes in gastric cancer. Thus, the aim of the present study was to validate recurrent amplification of RTEL1 and ABCA13 genes and associate CNV status with clinicopathological data. Results showed RTEL1 and ABCA13 amplification in 38 % of samples. Statistical analysis demonstrated that RTEL amplification is more common in older patients and more associated with intestinal type and ABCA13 amplification increases the risk of lymph node metastasis and is more common in men. Co-amplification of these genes showed a significant association with advanced staging. aCGH is a very useful tool for investigating novel genes associated with carcinogenesis and RTEL1 amplification may be important for the development of gastric cancer in older patients, besides being a probable event contributing for chromosomal instability in intestinal gastric carcinogenesis. ABCA13 amplification may have age-specific function and could be considered a useful marker for predicting lymph node metastasis in resected gastric cancer patients in early stage. Lastly, RTEL1 and ABCA13 synergistic effect may be considered as a putative marker for advanced staging in gastric cancer patients.

  1. Evidence That Chromium Modulates Cellular Cholesterol Homeostasis and ABCA1 Functionality Impaired By Hyperinsulinemia

    PubMed Central

    Sealls, Whitney; Penque, Brent A.; Elmendorf, Jeffrey S.

    2011-01-01

    Objective Trivalent chromium (Cr3+) is an essential micronutrient. Findings since the 1950s suggest that Cr3+ might benefit cholesterol homeostasis. Here we present mechanistic evidence in support of this role of Cr3+. Method and Results High-density lipoprotein cholesterol generation in 3T3-L1 adipocytes, rendered ineffective by hyperinsulinemia, known to accompany disorders of lipid metabolism was corrected by Cr3+. Mechanistically, Cr3+ reversed hyperinsulinemia-induced cellular cholesterol accrual and associated defects in cholesterol transporter ABCA1 trafficking and apolipoprotein A1-mediated cholesterol efflux. Moreover, direct activation of AMP-activated protein kinase (AMPK), known to be activated by Cr3+, and/or inhibition of hexosamine biosynthesis pathway (HBP) activity, known to be elevated by hyperinsulinemia, mimics Cr3+ action. Conclusion These findings suggest a mechanism of Cr3+ action that fits with long-standing claims of its role in cholesterol homeostasis. Furthermore, these data implicate a mechanistic basis for the coexistence of dyslipidemia with hyperinsulinemia. PMID:21311039

  2. Interaction between FTO rs9939609 and the Native American-origin ABCA1 rs9282541 affects BMI in the admixed Mexican population.

    PubMed

    Villalobos-Comparán, Marisela; Antuna-Puente, Bárbara; Villarreal-Molina, María Teresa; Canizales-Quinteros, Samuel; Velázquez-Cruz, Rafael; León-Mimila, Paola; Villamil-Ramírez, Hugo; González-Barrios, Juan Antonio; Merino-García, José Luis; Thompson-Bonilla, María Rocío; Jarquin, Diego; Sánchez-Hernández, Osvaldo Erik; Rodríguez-Arellano, Martha Eunice; Posadas-Romero, Carlos; Vargas-Alarcón, Gilberto; Campos-Pérez, Francisco; Quiterio, Manuel; Salmerón-Castro, Jorge; Carnevale, Alessandra; Romero-Hidalgo, Sandra

    2017-05-02

    The aim of this study was to explore whether interactions between FTO rs9939609 and ABCA1 rs9282541 affect BMI and waist circumference (WC), and could explain previously reported population differences in FTO-obesity and FTO-BMI associations in the Mexican and European populations. A total of 3938 adults and 636 school-aged children from Central Mexico were genotyped for both polymorphisms. Subcutaneous and visceral adipose tissue biopsies from 22 class III obesity patients were analyzed for FTO and ABCA1 mRNA expression. Generalized linear models were used to test for associations and gene-gene interactions affecting BMI, WC and FTO expression. FTO and ABCA1 risk alleles were not individually associated with higher BMI or WC. However, in the absence of the ABCA1 risk allele, the FTO risk variant was significantly associated with higher BMI (P = 0.043) and marginally associated with higher WC (P = 0.067), as reported in Europeans. The gene-gene interaction affecting BMI and WC was statistically significant only in adults. FTO mRNA expression in subcutaneous abdominal adipose tissue according to ABCA1 genotype was consistent with these findings. This is the first report showing evidence of FTO and ABCA1 gene variant interactions affecting BMI, which may explain previously reported population differences. Further studies are needed to confirm this interaction.

  3. Seminal Plasma Characteristics and Expression of ATP-binding Cassette Transporter A1 (ABCA1) in Canine Spermatozoa from Ejaculates with Good and Bad Freezability.

    PubMed

    Schäfer-Somi, S; Palme, N

    2016-04-01

    The composition of seminal plasma and the localization of the ATP-binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen-thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm-rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC-PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post-thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as < 50% progressive motility and/or > 40% morphologically abnormal sperm and/or < 50% viability. Bad freezers were older than good freezers (5.3 vs 3.4 years, p < 0.05). In bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p < 0.01) and the percentage of sperm with complete loss of ABCA1 signal higher (46.7% vs 30%, p < 0.01); the percentage of dead spermatozoa was higher (36.1% vs 25.5%, p < 0.05), and the concentration of cholesterol and sodium in seminal plasma was lower than in good freezers (p < 0.05). We conclude that in thawed bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow-up studies revealed whether a relation exists between these findings. © 2016 Blackwell Verlag GmbH.

  4. Reduced Plasma HDL Cholesterol in Hyperthyroid Mice Coincides with Decreased Hepatic ABCA1 Expression

    PubMed Central

    TANCEVSKI, IVAN; WEHINGER, ANDREAS; DEMETZ, EGON; ELLER, PHILIPP; DUWENSEE, KRISTINA; HUBER, JULIA; HOCHEGGER, KATHRIN; SCHGOER, WILFRIED; FIEVET, CATHERINE; STELLAARD, FRANS; RUDLING, MATS; PATSCH, JOSEF R.; RITSCH, ANDREAS

    2010-01-01

    The aim of the study was to investigate the influence of severe hyperthyroidism on plasma high-density lipoprotein cholesterol (HDL-C). Recently, it was shown in mice that increasing doses of triiodothyronine (T3) upregulate hepatic expression of scavenger receptor-BI (SR-BI), resulting in increased clearance of plasma HDL-C. Here we show that severe hyperthyroidism in mice did not affect hepatic expression of SR-BI, but reduced hepatic expression of ATP-binding cassette transporter 1 (ABCA1), accompanied by a 40%-reduction of HDL-C. Sterol content of bile, liver and feces was markedly increased, accompanied by upregulation of hepatic CYP7A1, and ATP-binding cassette half-transporter ABCG5, which is known to promote biliary sterol secretion upon dimerization with ABCG8. Both control and hyperthyroid mice exerted identical plasma clearance of intravenously injected [3H] HDL-C, supporting the view that severe hyperthyroidism does not affect HDL-C clearance, but rather its formation via hepatic ABCA1. PMID:18388200

  5. Genetic variants of ABCA1 modify Alzheimer disease risk and quantitative traits related to beta-amyloid metabolism.

    PubMed

    Katzov, Hagit; Chalmers, Katy; Palmgren, Juni; Andreasen, Niels; Johansson, Boo; Cairns, Nigel J; Gatz, Margaret; Wilcock, Gordon K; Love, Seth; Pedersen, Nancy L; Brookes, Anthony J; Blennow, Kaj; Kehoe, Patrick G; Prince, Jonathan A

    2004-04-01

    Linkage studies have provided evidence that one or more loci on chromosome 9q influence Alzheimer disease (AD). The gene encoding the ATP-binding cassette A1 transporter (ABCA1) resides within proximity of previously identified linkage peaks and represents a plausible biological candidate for AD due to its central role in cellular lipid homeostasis. Several single nucleotide polymorphisms (SNPs) spanning ABCA1 have been genotyped and haplotype-based association analyses performed in four independent case-control samples, consisting of over 1,750 individuals from three European populations representing both early and late-onset AD. Prominent effects were observed for a common (H2) and rarer haplotype (H5) that were enriched in AD cases across studied populations (odds ratio [OR] 1.59, 95% confidence interval [CI] 1.36-1.82; P<0.00001 and OR 2.90; 95% CI 2.54-3.27; P<0.00001, respectively). Two other common haplotypes in the studied region (H1 and H3) were significantly under-represented in AD cases, suggesting that they may harbor alleles that decrease disease risk (OR 0.79, 95% CI 0.64-0.94; P=0.0065 and OR 0.70, 95% CI 0.46-0.93; P=0.011, respectively). While findings were significant in both early and late-onset samples, haplotype effects were more distinct in early-onset materials. For late-onset samples, ancillary evidence was obtained that both single marker alleles and haplotypes of ABCA1 contribute to variable cerebrospinal fluid tau and beta amyloid (Abeta42) protein levels, and brain Abeta load. Results indicate that variants of ABCA1 may affect the risk of AD, providing further support for a genetic link between AD and cholesterol metabolism. Copyright 2004 Wiley-Liss, Inc.

  6. Ligand, receptor, and cell type-dependent regulation of ABCA1 and ABCG1 mRNA in prostate cancer epithelial cells

    USDA-ARS?s Scientific Manuscript database

    Recent evidence suggests that the liver X receptor (LXR) is a potential anti-cancer target in prostate carcinoma. There is little characterization, however, of how the two major isoforms LXRa or LXRß regulate the LXR-responsive genes ATP-binding cassette sub-family A 1 (ABCA1) and sub-family member ...

  7. Genetic and Epigenetic Factors at COL2A1 and ABCA4 Influence Clinical Outcome in Congenital Toxoplasmosis

    PubMed Central

    Jamieson, Sarra E.; de Roubaix, Lee-Anne; Cortina-Borja, Mario; Tan, Hooi Kuan; Mui, Ernest J.; Cordell, Heather J.; Kirisits, Michael J.; Miller, E. Nancy; Peacock, Christopher S.; Hargrave, Aubrey C.; Coyne, Jessica J.; Boyer, Kenneth; Bessieres, Marie-Hélène; Buffolano, Wilma; Ferret, Nicole; Franck, Jacqueline; Kieffer, François; Meier, Paul; Nowakowska, Dorota E.; Paul, Malgorzata; Peyron, François; Stray-Pedersen, Babill; Prusa, Andrea-Romana; Thulliez, Philippe; Wallon, Martine; Petersen, Eskild; McLeod, Rima; Gilbert, Ruth E.; Blackwell, Jenefer M.

    2008-01-01

    Background Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute. Methods and Findings In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting. Conclusions These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite. PMID:18523590

  8. The impact of APOA5, APOB, APOC3 and ABCA1 gene polymorphisms on ischemic stroke: Evidence from a meta-analysis.

    PubMed

    Au, Anthony; Griffiths, Lyn R; Irene, Looi; Kooi, Cheah Wee; Wei, Loo Keat

    2017-10-01

    Genetic studies have been reported on the association between APOA5, APOB, APOC3 and ABCA1 gene polymorphisms and ischemic stroke, but results remain controversial. Hence, this meta-analysis aimed to infer the causal relationships of APOA5 (rs662799, rs3135506), APOB (rs693, rs1042031, rs1801701), APOC3 (rs4520, rs5128, rs2854116, rs2854117) and ABCA1 rs2230806 with ischemic stroke risk. A systematic review was performed for all the articles retrieved from multiple databases, up until March 2017. Data were extracted from all eligible studies, and meta-analysis was carried out using RevMan 5.3 and R package 3.2.1. The strength of association between each studied polymorphism and ischemic stroke risk was measured as odds ratios (ORs) and 95% confidence intervals (CIs), under fixed- and random-effect models. A total of 79 studies reporting on the association between the studied polymorphisms and ischemic stroke risk were identified. The pooled data indicated that all genetic models of APOA5 rs662799 (ORs = 1.23-1.43), allelic and over-dominant models of APOA5 rs3135506 (ORs = 1.77-1.97), APOB rs1801701 (ORs = 1.72-2.13) and APOB rs1042031 (ORs = 1.66-1.88) as well as dominant model of ABCA1 rs2230806 (OR = 1.31) were significantly associated with higher risk of ischemic stroke. However, no significant associations were observed between ischemic stroke and the other five polymorphisms, namely ApoB (rs693) and APOC3 (rs4520, rs5128, rs2854116 and rs2854117), under any genetic model. The present meta-analysis confirmed a significant association of APOA5 rs662799 CC, APOA5 rs3135506 CG, APOB rs1801701 GA, APOB rs1042031 GA and ABCA1 rs2230806 GG with increased risk of ischemic stroke. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Evaluation of the Association Between Common Genetic Variants Near the ABCA1 Gene and Primary Angle Closure Glaucoma in a Han Chinese Population.

    PubMed

    Luo, Huaichao; Chen, Yuhong; Ye, Zimeng; Sun, Xinghuai; Shi, Yi; Luo, Qian; Gong, Bo; Shuai, Ping; Yang, Jiyun; Zhou, Yu; Liu, Xiaoqi; Zhang, Kaijiong; Tan, Chang; Li, Yuanfeng; Lin, Ying; Yang, Zhenglin

    2015-10-01

    Recently, three large genome-wide association studies have identified multiple variants associated with primary open angle glaucoma (POAG) near the ABCA1 gene. Considering that POAG and primary angle closure glaucoma (PACG) share many similar clinical manifestations, the present study was conducted to investigate whether these genetic variants were also associated with PACG in a Han Chinese population. A case-control association study of 1122 cases (PACG/PAC) and 1311 normal, matched controls was undertaken. Seven single-nucleotide polymorphisms (SNPs) near the ABCA1 gene, including rs2422493, rs2487042, rs2472496, rs2472493, rs2487032, rs2472459, and rs2472519, were genotyped. Genotype and allele frequencies were assessed using χ² tests. Linkage disequilibrium (LD) structure was analyzed by computer software. Among the SNPs genotyped, no association was observed between these SNPs and PACG. However, we discovered that two haplotypes, CATTTAC (corrected P = 0.048) and CGCCCGC (corrected P = 0.048), remained significantly associated with PACG/PAC after Bonferroni correction. Subjects with the CATTTAC haplotype have a 1.71-fold increased possibility of having PACG/PAC, whereas subjects with the CGCCCGC haplotype have 0.47-fold decreased possibility of developing PACG. Our findings suggest that the genetic backgrounds of PACG and POAG might be different. However, whether or not ABCA1 plays a role in the development of PACG is still not made certain by this study. Thus, further research is needed to find the role of ABCA1 in the progress of PACG.

  10. A Mouse Model of Harlequin Ichthyosis Delineates a Key Role for Abca12 in Lipid Homeostasis

    PubMed Central

    Smyth, Ian; Mukhamedova, Nigora; Meikle, Peter J.; Ellis, Sarah; Slattery, Keith; Collinge, Janelle E.; de Graaf, Carolyn A.; Bahlo, Melanie; Sviridov, Dmitri

    2008-01-01

    Harlequin Ichthyosis (HI) is a severe and often lethal hyperkeratotic skin disease caused by mutations in the ABCA12 transport protein. In keratinocytes, ABCA12 is thought to regulate the transfer of lipids into small intracellular trafficking vesicles known as lamellar bodies. However, the nature and scope of this regulation remains unclear. As part of an original recessive mouse ENU mutagenesis screen, we have identified and characterised an animal model of HI and showed that it displays many of the hallmarks of the disease including hyperkeratosis, loss of barrier function, and defects in lipid homeostasis. We have used this model to follow disease progression in utero and present evidence that loss of Abca12 function leads to premature differentiation of basal keratinocytes. A comprehensive analysis of lipid levels in mutant epidermis demonstrated profound defects in lipid homeostasis, illustrating for the first time the extent to which Abca12 plays a pivotal role in maintaining lipid balance in the skin. To further investigate the scope of Abca12's activity, we have utilised cells from the mutant mouse to ascribe direct transport functions to the protein and, in doing so, we demonstrate activities independent of its role in lamellar body function. These cells have severely impaired lipid efflux leading to intracellular accumulation of neutral lipids. Furthermore, we identify Abca12 as a mediator of Abca1-regulated cellular cholesterol efflux, a finding that may have significant implications for other diseases of lipid metabolism and homeostasis, including atherosclerosis. PMID:18802465

  11. Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol efflux capacity via the ABCA1 pathway[S

    PubMed Central

    Pamir, Nathalie; Hutchins, Patrick; Ronsein, Graziella; Vaisar, Tomas; Reardon, Catherine A.; Getz, Godfrey S.; Lusis, Aldons J.; Heinecke, Jay W.

    2016-01-01

    Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL’s size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL’s content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL’s content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL’s ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL’s APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages. PMID:26673204

  12. ABCG1-mediated generation of extracellular cholesterol microdomains[S

    PubMed Central

    Freeman, Sebastian R.; Jin, Xueting; Anzinger, Joshua J.; Xu, Qing; Purushothaman, Sonya; Fessler, Michael B.; Addadi, Lia; Kruth, Howard S.

    2014-01-01

    Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 μg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space. PMID:24212237

  13. ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells.

    PubMed

    Reddy, Srinivasa T; Hama, Susan; Ng, Carey; Grijalva, Victor; Navab, Mohamad; Fogelman, Alan M

    2002-11-01

    We have previously reported that products of the lipoxygenase pathway, hydroperoxyoctadecadienoic acid and hydroperoxyeicosatetraenoic acid, as well as cholesterol linoleate hydroperoxides, collectively termed seeding molecules, are removed by apolipoprotein A-I (apoA-I) from the artery wall cells and render low density lipoprotein (LDL) resistant to oxidation by human artery wall cells. The mechanisms by which oxidized lipids are transported and/or transferred to lipoproteins and the pathways by which apoA-I facilitates their removal remain unclear. ATP-binding cassette transporter 1 (ABCA1) is known to facilitate the release of cellular phospholipids and cholesterol from the plasma membrane to apoA-I and high density lipoprotein. Therefore, we evaluated whether ABCA1 participates in LDL oxidation. In this report, we show that (1) chemical inhibitors of ABCA1 function, glyburide and DIDS, block artery wall cell-mediated oxidative modification of LDL, (2) inhibition of ABCA1 with the use of antisense (but not sense) oligonucleotides prevents LDL-induced lipid hydroperoxide formation and LDL-induced monocyte chemotactic activity by the artery wall cells, and (3) oxysterols that induce ABCA1 expression, such as 22(R)hydroxycholesterol, enhance cell-mediated LDL oxidation. Furthermore, we also show that 22(R)hydroxycholesterol induces the production of reactive oxygen species in the artery wall cells, which can be removed by incubating the artery wall cells with apoA-I. Our data suggest that ABCA1 plays an important role in artery wall cell-mediated modification/oxidation of LDL by modulating the release of reactive oxygen species from artery wall cells that are necessary for LDL oxidation.

  14. Increase in HDL-C concentration by a dietary portfolio with soy protein and soluble fiber is associated with the presence of the ABCA1R230C variant in hyperlipidemic Mexican subjects.

    PubMed

    Guevara-Cruz, Martha; Tovar, Armando R; Larrieta, Elena; Canizales-Quinteros, Samuel; Torres, Nimbe

    2010-01-01

    A dietary portfolio has been used to reduce blood lipids in hyperlipidemic subjects. To increase the effectiveness of these dietary treatments in specific populations, it is important to study the genetic variability associated with the development of certain types of hyperlipidemias. Low plasma high-density lipoprotein cholesterol (HDL-C) levels are the most common dyslipidemia in Mexican adults and are coupled with the presence of the ABCA1 R230C genotype. Therefore, the aim of this study was to assess the response of HDL-C concentration to a dietary portfolio in a group of Mexican hyperlipidemic subjects with ABCA1R230C (rs9282541) and R219K (rs2230806) polymorphisms. Forty-three hyperlipidemic subjects (20 men and 23 women) were given a low saturated fat (LSF) diet for one month, followed by a LSF diet that included 25g of soy protein and 15g of soluble fiber daily for 2months. We analyzed two ABCA1 polymorphisms and studied their association with serum lipids before and after treatment. Hyperlipidemic subjects with the ABCA1 R230C genotype showed lower HDL-C concentrations at the beginning of the study and were better responders to the dietary treatment than subjects with the ABCA1 R230R genotype (+4.6% vs. +14.6%) (p=.05). According to gender and the presence of the R230C genotype, women responded more significantly to the dietary treatment, reflected by an increase of 21.9% in HDL concentration (p=.022), than women with R230R genotype who only experienced an increase of 2.7% in HDL-C concentration. There was no association between the presence of the ABCA1 R219K variant (p=.544) and HDL concentration. Hyperlipidemic Mexican subjects with the ABCA1 R230C genotype showed lower HDL-concentrations and were better responders to dietary portfolio treatments for increasing HDL-C concentrations than subjects with the R230R genotype. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Mutations in GPR143/OA1 and ABCA4 Inform Interpretations of Short-Wavelength and Near-Infrared Fundus Autofluorescence

    PubMed Central

    Paavo, Maarjaliis; Zhao, Jin; Kim, Hye Jin; Lee, Winston; Zernant, Jana; Cai, Carolyn; Allikmets, Rando; Tsang, Stephen H.; Sparrow, Janet R.

    2018-01-01

    Purpose We sought to advance interpretations and quantification of short-wavelength fundus autofluorescence (SW-AF) emitted from bisretinoid lipofuscin and near-infrared autofluoresence (NIR-AF) originating from melanin. Methods Carriers of mutations in X-linked GPR143/OA1, a common form of ocular albinism; patients with confirmed mutations in ABCA4 conferring increased SW-AF; and subjects with healthy eyes were studied. SW-AF (488 nm excitation, 500–680 nm emission) and NIR-AF (excitation 787 nm, emission >830 nm) images were acquired with a confocal scanning laser ophthalmoscope. SW-AF images were analyzed for quantitative autofluoresence (qAF). Analogous methods of image acquisition and analysis were performed in albino and pigmented Abca4−/− mice and wild-type mice. Results Quantitation of SW-AF (qAF), construction of qAF color-coded maps, and examination of NIR-AF images from GPR143/OA1 carriers revealed mosaics in which patches of fundus exhibiting NIR-AF signal had qAF levels within normal limits whereas the hypopigmented areas in the NIR-AF image corresponded to foci of elevated qAF. qAF also was increased in albino versus pigmented mice. Although melanin contributes to fundus infrared reflectance, the latter appeared to be uniform in en face reflectance images of GPR143/OA1-carriers. In patients diagnosed with ABCA4-associated disease, NIR-AF increased in tandem with increased qAF originating in bisretinoid lipofuscin. Similarly in Abca4−/− mice having increased SW-AF, NIR-AF was more pronounced than in wild-type mice. Conclusions These studies corroborate RPE melanin as the major source of NIR-AF but also indicate that bisretinoid lipofuscin, when present at sufficient concentrations, contributes to the NIR-AF signal. Ocular melanin attenuates the SW-AF signal.

  16. Effect Of G2706A and G1051A polymorphisms of the ABCA1 gene on the lipid, oxidative stress and homocystein levels in Turkish patients with polycystıc ovary syndrome

    PubMed Central

    2011-01-01

    Background Obesity, insulin resistance and hyperandrogenism, crucial parameters of Polycystic ovary syndrome (PCOS) play significant pathophysiological roles in lipidemic aberrations associated within the syndrome. Parts of the metabolic syndrome (low HDL and insulin resistance) appeared to facilitate the association between PCOS and coronary artery disease, independently of obesity. ABCA1 gene polymorphism may be altered this components in PCOS patients. In this study, we studied 98 PCOS patients and 93 healthy controls. All subjects underwent venous blood drawing for complete hormonal assays, lipid profile, glucose, insulin, malondialdehyde, nitric oxide, disulfide levels and ABCA genetic study. Results In PCOS group fasting glucose, DHEAS, 17-OHP, free testosterone, total-cholesterol, triglyceride, LDL-cholesterol and fibrinogen were significantly different compare to controls. The genotype ABCA G2706A distribution differed between the control group (GG 60.7%, GA 32.1%, AA 7.1%) and the PCOS patients (GG 8.7%, GA 8.7%, AA 76.8%). The frequency of the A allele (ABCAG2706A) was higher in PCOS patients than control group with 13,0% and 23,2%, respectively. In this study, the homocystein and insulin levels were significantly higher in PCOS patients with ABCA G1051A mutant genotype than those with heterozygote and wild genotypes. Conclusions We found higher percentage of AA genotype and A allele of ABCA G2706A in PCOS patients compare to controls. The fasting insulin and homocystein levels were significantly higher in PCOS patients with ABCA G1051A mutant genotype than those with heterozygote and wild genotypes. PMID:22035022

  17. miR-27b inhibits LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels in mice

    PubMed Central

    Goedeke, Leigh; Rotllan, Noemi; Ramírez, Cristina M.; Aranda, Juan F.; Canfrán-Duque, Alberto; Araldi, Elisa; Fernández-Hernando, Ana; Langhi, Cedric; de Cabo, Rafael; Baldán, Ángel; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-01-01

    Rationale Recently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. Methods and Results Our results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50–fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10–20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. Conclusions The results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels. PMID:26520906

  18. Association studies of several cholesterol-related genes (ABCA1, CETP and LIPC) with serum lipids and risk of Alzheimer’s disease

    PubMed Central

    2012-01-01

    Objectives Accumulating evidence suggested that dysregulation of cholesterol homeostasis might be a major etiologic factor in initiating and promoting neurodegeneration in Alzheimer’s disease (AD). ATP-binding cassette transporter A1 (ABCA1), hepatic lipase (HL, coding genes named LIPC) and cholesteryl ester transfer protein (CETP) are important components of high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT) implicated in atherosclerosis and neurodegenerative diseases. In the present study, we will investigate the possible association of several common polymorphisms (ABCA1R219K, CETPTaqIB and LIPC-250 G/A) with susceptibility to AD and plasma lipid levels. Methods Case–control study of 208 Han Chinese (104 AD patients and 104 non-demented controls) from Changsha area in Hunan Province was performed using the PCR-RFLP analysis. Cognitive decline was assessed using Mini Mental State Examination (MMSE) as a standardized method. Additionally, fasting lipid profile and the cognitive testing scores including Wechsler Memory Scale (WMS) and Wisconsin Card Sorting Test (WCST) were recorded. Results and conclusions We found significant differences among the genotype distributions of these three genes in AD patients when compared with controls. But after adjusting other factors, multivariate logistic regression analysis showed only ABCA1R219K (B = −0.903, P = 0.005, OR = 0.405, 95%CI:0.217-0.758) and LIPC-250 G/A variants(B = −0.905, P = 0.018, OR = 0.405, 95%CI:0.191-0.858) were associated with decreased AD risk. There were significantly higher levels of high-density lipoprotein cholesterol (HDL-C) and apolipoproteinA-I in the carriers of KK genotype and K allele (P < 0.05), and B2B2 genotype of CETP Taq1B showed significant association with higher HDL-C levels than other genotypes (F = 5.598, P = 0.004), while -250 G/A polymorphisms had no significant effect on HDL-C. In total population, subjects

  19. Association of Brain DNA methylation in SORL1, ABCA7, HLA-DRB5, SLC24A4, and BIN1 with pathological diagnosis of Alzheimer disease.

    PubMed

    Yu, Lei; Chibnik, Lori B; Srivastava, Gyan P; Pochet, Nathalie; Yang, Jingyun; Xu, Jishu; Kozubek, James; Obholzer, Nikolaus; Leurgans, Sue E; Schneider, Julie A; Meissner, Alexander; De Jager, Philip L; Bennett, David A

    2015-01-01

    Recent large-scale genome-wide association studies have discovered several genetic variants associated with Alzheimer disease (AD); however, the extent to which DNA methylation in these AD loci contributes to the disease susceptibility remains unknown. To examine the association of brain DNA methylation in 28 reported AD loci with AD pathologies. Ongoing community-based clinical pathological cohort studies of aging and dementia (the Religious Orders Study and the Rush Memory and Aging Project) among 740 autopsied participants 66.0 to 108.3 years old. DNA methylation levels at individual CpG sites generated from dorsolateral prefrontal cortex tissue using a bead assay. Pathological diagnosis of AD by National Institute on Aging-Reagan criteria following a standard postmortem examination. Overall, 447 participants (60.4%) met the criteria for pathological diagnosis of AD. Brain DNA methylation in SORL1, ABCA7, HLA-DRB5, SLC24A4, and BIN1 was associated with pathological AD. The association was robustly retained after replacing the binary trait of pathological AD with 2 quantitative and molecular specific hallmarks of AD, namely, Aβ load and paired helical filament tau tangle density. Furthermore, RNA expression of transcripts of SORL1 and ABCA7 was associated with paired helical filament tau tangle density, and the expression of BIN1 was associated with Aβ load. Brain DNA methylation in multiple AD loci is associated with AD pathologies. The results provide further evidence that disruption of DNA methylation is involved in the pathological process of AD.

  20. Novel mutations in CRB1 and ABCA4 genes cause Leber congenital amaurosis and Stargardt disease in a Swedish family

    PubMed Central

    Jonsson, Frida; Burstedt, Marie S; Sandgren, Ola; Norberg, Anna; Golovleva, Irina

    2013-01-01

    This study aimed to identify genetic mechanisms underlying severe retinal degeneration in one large family from northern Sweden, members of which presented with early-onset autosomal recessive retinitis pigmentosa and juvenile macular dystrophy. The clinical records of affected family members were analysed retrospectively and ophthalmological and electrophysiological examinations were performed in selected cases. Mutation screening was initially performed with microarrays, interrogating known mutations in the genes associated with recessive retinitis pigmentosa, Leber congenital amaurosis and Stargardt disease. Searching for homozygous regions with putative causative disease genes was done by high-density SNP-array genotyping, followed by segregation analysis of the family members. Two distinct phenotypes of retinal dystrophy, Leber congenital amaurosis and Stargardt disease were present in the family. In the family, four patients with Leber congenital amaurosis were homozygous for a novel c.2557C>T (p.Q853X) mutation in the CRB1 gene, while of two cases with Stargardt disease, one was homozygous for c.5461-10T>C in the ABCA4 gene and another was carrier of the same mutation and a novel ABCA4 mutation c.4773+3A>G. Sequence analysis of the entire ABCA4 gene in patients with Stargardt disease revealed complex alleles with additional sequence variants, which were evaluated by bioinformatics tools. In conclusion, presence of different genetic mechanisms resulting in variable phenotype within the family is not rare and can challenge molecular geneticists, ophthalmologists and genetic counsellors. PMID:23443024

  1. Gene therapy for Stargardt disease associated with ABCA4 gene.

    PubMed

    Han, Zongchao; Conley, Shannon M; Naash, Muna I

    2014-01-01

    Mutations in the photoreceptor-specific flippase ABCA4 lead to accumulation of the toxic bisretinoid A2E, resulting in atrophy of the retinal pigment epithelium (RPE) and death of the photoreceptor cells. Many blinding diseases are associated with these mutations including Stargardt's disease (STGD1), cone-rod dystrophy, retinitis pigmentosa (RP), and increased susceptibility to age-related macular degeneration. There are no curative treatments for any of these dsystrophies. While the monogenic nature of many of these conditions makes them amenable to treatment with gene therapy, the ABCA4 cDNA is 6.8 kb and is thus too large for the AAV vectors which have been most successful for other ocular genes. Here we review approaches to ABCA4 gene therapy including treatment with novel AAV vectors, lentiviral vectors, and non-viral compacted DNA nanoparticles. Lentiviral and compacted DNA nanoparticles in particular have a large capacity and have been successful in improving disease phenotypes in the Abca4 (-/-) murine model. Excitingly, two Phase I/IIa clinical trials are underway to treat patients with ABCA4-associated Startgardt's disease (STGD1). As a result of the development of these novel technologies, effective therapies for ABCA4-associated diseases may finally be within reach.

  2. Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

    PubMed

    Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

    2013-01-01

    pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

  3. Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux.

    PubMed

    Daniil, Georgios; Zannis, Vassilis I; Chroni, Angeliki

    2013-01-01

    ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185-243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69-99% of control by double deletion mutants apoA-I[Δ(1-41)Δ(185-243)] and apoA-I[Δ(1-59)Δ(185-243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185-243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I.

  4. Regulation of Expression of abcA and Its Response to Environmental Conditions

    PubMed Central

    Villet, Regis A.; Truong-Bolduc, Que Chi; Wang, Yin; Estabrooks, Zoe; Medeiros, Heidi

    2014-01-01

    The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic β-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2ΔabcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA. PMID:24509312

  5. Static pressure accelerates ox-LDL-induced cholesterol accumulation via SREBP-1-mediated caveolin-1 downregulation in cultured vascular smooth muscle cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Luo, Di-xian, E-mail: luodixian_2@163.com; Institute of Pharmacy and Pharmacology, College of Science and Technology, University of South China, Hengyang 421001, Hunan; First People's Hospital of Chenzhou City, Chenzhou 423000, Hunan

    Research highlights: {yields} Vertical static pressure accelerates ox-LDL-induced cholesterol accumulation in cultured vascular smooth muscle cells. {yields} Static pressure induces SREBP-1 activation. {yields} Static pressure downregulates the expressions of caveolin-1 by activating SREBP-1. {yields} Static pressure also downregulates the transcription of ABCA1 by activating SREBP-1. {yields} Static pressure increases ox-LDL-induced cholesterol accumulation by SREBP-1-mediated caveolin-1 downregulation in vascular smooth muscle cells cultured in vitro. -- Abstract: Objective: To investigate the effect of static pressure on cholesterol accumulation in vascular smooth muscle cells (VSMCs) and its mechanism. Methods: Rat-derived VSMC cell line A10 treated with 50 mg/L ox-LDL and different staticmore » pressures (0, 60, 90, 120, 150, 180 mm Hg) in a custom-made pressure incubator for 48 h. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC; The mRNA levels of caveolin-1 and ABCA1, the protein levels of caveolin-1 SREBP-1 and mature SREBP-1 were respectively detected by RT-PCR or western blot. ALLN, an inhibitor of SREBP metabolism, was used to elevate SREBP-1 protein level in VSMCs treated with static pressure. Results: Static pressures significantly not only increase intracellular lipid droplets in VSMCs, but also elevate cellular lipid content in a pressure-dependent manner. Intracellular free cholesterol (FC), cholesterol ester (CE), total cholesterol (TC) were respectively increased from 60.5 {+-} 2.8 mg/g, 31.8 {+-} 0.7 mg/g, 92.3 {+-} 2.1 mg/g at atmosphere pressure (ATM, 0 mm Hg) to 150.8 {+-} 9.4 mg/g, 235.9 {+-} 3.0 mg/g, 386.7 {+-} 6.4 mg/g at 180 mm Hg. At the same time, static pressures decrease the mRNA and protein levels of caveolin-1, and induce the activation and nuclear translocation of SREBP-1. ALLN increases the protein level of mature SREBP-1 and decreases caveolin-1 expression, so that cellular lipid levels

  6. MicroRNA-20a/b regulates cholesterol efflux through post-transcriptional repression of ATP-binding cassette transporter A1.

    PubMed

    Liang, Bin; Wang, Xin; Song, Xiaosu; Bai, Rui; Yang, Huiyu; Yang, Zhiming; Xiao, Chuanshi; Bian, Yunfei

    2017-09-01

    ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in reverse cholesterol transport and exhibits anti-atherosclerosis effects. Some microRNAs (miRs) regulate ABCA1 expression, and recent studies have shown that miR-20a/b might play a critical role in atherosclerotic diseases. Here, we attempted to clarify the potential contribution of miR-20a/b in post-transcriptional regulation of ABCA1, cholesterol efflux, and atherosclerosis. We performed bioinformatics analysis and found that miR-20a/b was highly conserved and directly bound to ABCA1 mRNA with low binding free energy. Luciferase-reporter assay also confirmed that miR-20a/b significantly reduced luciferase activity associated with the ABCA1 3' untranslated region reporter construct. Additionally, miR-20a/b decreased ABCA1 expression, which, in turn, decreased cholesterol efflux and increased cholesterol content in THP-1 and RAW 264.7 macrophage-derived foam cells. In contrast, miR-20a/b inhibitors increased ABCA1 expression and cholesterol efflux, decreased cholesterol content, and inhibited foam-cell formation. Consistent with our in vitro results, miR-20a/b-treated ApoE -/- mice showed decreased ABCA1expression in the liver and reductions of reverse cholesterol transport in vivo. Furthermore, miR-20a/b regulated the formation of nascent high-density lipoprotein and promoted atherosclerotic development, whereas miR-20a/b knockdown attenuated atherosclerotic formation. miR-20 is a new miRNA capable of targeting ABCA1 and regulating ABCA1 expression. Therefore, miR-20 inhibition constitutes a new strategy for ABCA1-based treatment of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Extremely hypomorphic and severe deep intronic variants in the ABCA4 locus result in varying Stargardt disease phenotypes.

    PubMed

    Zernant, Jana; Lee, Winston; Nagasaki, Takayuki; Collison, Frederick T; Fishman, Gerald A; Bertelsen, Mette; Rosenberg, Thomas; Gouras, Peter; Tsang, Stephen H; Allikmets, Rando

    2018-05-30

    Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of the ABCA4 locus in STGD1 patients identifies two expected disease-causing alleles in ~75% of patients and only one mutation in ~15% of patients. Recently, many possibly pathogenic variants in deep intronic sequences of ABCA4 have been identified in the latter group. We extended our analyses of deep intronic ABCA4 variants and determined that one of these, c.4253+43G>A (rs61754045), is present in 29/1155 (2.6%) of STGD1 patients. The variant is found at statistically significantly higher frequency in patients with only one pathogenic ABCA4 allele, 23/160 (14.38%), MAF=0.072, compared to MAF=0.013 in all STGD1 cases and MAF=0.006 in the matching general population (P<1x10-7). The variant, which is not predicted to have any effect on splicing, is the first reported intronic "extremely hypomorphic allele" in the ABCA4 locus; i.e., it is pathogenic only when in trans with a loss-of-function ABCA4 allele. It results in a distinct clinical phenotype characterized by late-onset of symptoms and foveal sparing. In ~70% of cases the variant was allelic with the c.6006-609T>A (rs575968112) variant, which was deemed non-pathogenic. Another rare deep intronic variant, c.5196+1056A>G (rs886044749), found in 5/834 (0.6%) of STGD1 cases is, conversely, a severe allele. This study determines pathogenicity for three non-coding variants in STGD1 patients of European descent accounting for ~3% of the disease. Defining disease-associated alleles in the non-coding sequences of the ABCA4 locus can be accomplished by integrated clinical and genetic analyses. Cold Spring Harbor Laboratory Press.

  8. Hepatic Overexpression of Endothelial Lipase Lowers HDL (High-Density Lipoprotein) but Maintains Reverse Cholesterol Transport in Mice: Role of SR-BI (Scavenger Receptor Class B Type I)/ABCA1 (ATP-Binding Cassette Transporter A1)-Dependent Pathways.

    PubMed

    Takiguchi, Shunichi; Ayaori, Makoto; Yakushiji, Emi; Nishida, Takafumi; Nakaya, Kazuhiro; Sasaki, Makoto; Iizuka, Maki; Uto-Kondo, Harumi; Terao, Yoshio; Yogo, Makiko; Komatsu, Tomohiro; Ogura, Masatsune; Ikewaki, Katsunori

    2018-05-10

    Reverse cholesterol transport (RCT) is a major mechanism by which HDL (high-density lipoprotein) protects against atherosclerosis. Endothelial lipase (EL) reportedly reduces HDL levels, which, in theory, would increase atherosclerosis. However, it remains unclear whether EL affects RCT in vivo. Adenoviral vectors expressing EL or luciferase were intravenously injected into mice, and a macrophage RCT assay was performed. As expected, hepatic EL overexpression markedly reduced HDL levels. In parallel, plasma 3 H-cholesterol counts from the EL-expressing mice decreased by 85% compared with control. Surprisingly, there was no difference in fecal 3 H-cholesterol excretion between the groups. Kinetic studies revealed increased catabolism/hepatic uptake of 3 HDL-cholesteryl ether, resulting in no change in fecal HDL-cholesteryl ester excretion in the mice. To explore underlying mechanisms for the preservation of RCT despite low HDL levels in the EL-expressing mice, we investigated the effects of hepatic SR-BI (scavenger receptor class B type I) knockdown. RCT assay revealed that knockdown of SR-BI alone reduced fecal excretion of macrophage-derived 3 H-cholesterol. Interestingly, hepatic EL overexpression under SR-BI inhibition further attenuated fecal tracer counts as compared with control. Finally, we observed that EL overexpression enhanced in vivo RCT under pharmacological inhibition of hepatic ABCA1 (ATP-binding cassette transporter A1) by probucol. Hepatic EL expression compensates for reduced macrophage-derived cholesterol efflux to plasma because of low HDL levels by promoting cholesterol excretion to bile/feces via an SR-BI pathway, maintaining overall RCT in vivo. In contrast, EL-modified HDL might negatively regulate RCT via hepatic ABCA1. Despite extreme hypoalphalipoproteinemia, RCT is maintained in EL-expressing mice via SR-BI/ABCA1-dependent pathways. © 2018 American Heart Association, Inc.

  9. Single nucleotide polymorphisms in CETP, SLC46A1, SLC19A1, CD36, BCMO1, APOA5, and ABCA1 are significant predictors of plasma HDL in healthy adults

    PubMed Central

    2013-01-01

    Background In a marker-trait association study we estimated the statistical significance of 65 single nucleotide polymorphisms (SNP) in 23 candidate genes on HDL levels of two independent Caucasian populations. Each population consisted of men and women and their HDL levels were adjusted for gender and body weight. We used a linear regression model. Selected genes corresponded to folate metabolism, vitamins B-12, A, and E, and cholesterol pathways or lipid metabolism. Methods Extracted DNA from both the Sacramento and Beltsville populations was analyzed using an allele discrimination assay with a MALDI-TOF mass spectrometry platform. The adjusted phenotype, y, was HDL levels adjusted for gender and body weight only statistical analyses were performed using the genotype association and regression modules from the SNP Variation Suite v7. Results Statistically significant SNP (where P values were adjusted for false discovery rate) included: CETP (rs7499892 and rs5882); SLC46A1 (rs37514694; rs739439); SLC19A1 (rs3788199); CD36 (rs3211956); BCMO1 (rs6564851), APOA5 (rs662799), and ABCA1 (rs4149267). Many prior association trends of the SNP with HDL were replicated in our cross-validation study. Significantly, the association of SNP in folate transporters (SLC46A1 rs37514694 and rs739439; SLC19A1 rs3788199) with HDL was identified in our study. Conclusions Given recent literature on the role of niacin in the biogenesis of HDL, focus on status and metabolism of B-vitamins and metabolites of eccentric cleavage of β-carotene with lipid metabolism is exciting for future study. PMID:23656756

  10. Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy.

    PubMed

    Maugeri, A; Klevering, B J; Rohrschneider, K; Blankenagel, A; Brunner, H G; Deutman, A F; Hoyng, C B; Cremers, F P

    2000-10-01

    The photoreceptor cell-specific ATP-binding cassette transporter gene (ABCA4; previously denoted "ABCR") is mutated, in most patients, with autosomal recessive (AR) Stargardt disease (STGD1) or fundus flavimaculatus (FFM). In addition, a few cases with AR retinitis pigmentosa (RP) and AR cone-rod dystrophy (CRD) have been found to have ABCA4 mutations. To evaluate the importance of the ABCA4 gene as a cause of AR CRD, we selected 5 patients with AR CRD and 15 patients from Germany and The Netherlands with isolated CRD. Single-strand conformation-polymorphism analysis and sequencing revealed 19 ABCA4 mutations in 13 (65%) of 20 patients. In six patients, mutations were identified in both ABCA4 alleles; in seven patients, mutations were detected in one allele. One complex ABCA4 allele (L541P;A1038V) was found exclusively in German patients with CRD; one patient carried this complex allele homozygously, and five others were compound heterozygous. These findings suggest that mutations in the ABCA4 gene are the major cause of AR CRD. A primary role of the ABCA4 gene in STGD1/FFM and AR CRD, together with the gene's involvement in an as-yet-unknown proportion of cases with AR RP, strengthens the idea that mutations in the ABCA4 gene could be the most frequent cause of inherited retinal dystrophy in humans.

  11. MicroRNA-19b promotes macrophage cholesterol accumulation and aortic atherosclerosis by targeting ATP-binding cassette transporter A1.

    PubMed

    Lv, Yun-Cheng; Tang, Yan-Yan; Peng, Juan; Zhao, Guo-Jun; Yang, Jing; Yao, Feng; Ouyang, Xin-Ping; He, Ping-Ping; Xie, Wei; Tan, Yu-Lin; Zhang, Min; Liu, Dan; Tang, Deng-Pei; Cayabyab, Francisco S; Zheng, Xi-Long; Zhang, Da-Wei; Tian, Guo-Ping; Tang, Chao-Ke

    2014-09-01

    Macrophage accumulation of cholesterol leads to foam cell formation which is a major pathological event of atherosclerosis. Recent studies have shown that microRNA (miR)-19b might play an important role in cholesterol metabolism and atherosclerotic diseases. Here, we have identified miR-19b binding to the 3'UTR of ATP-binding cassette transporter A1 (ABCA1) transporters, and further determined the potential roles of this novel interaction in atherogenesis. To investigate the molecular mechanisms involved in a miR-19b promotion of macrophage cholesterol accumulation and the development of aortic atherosclerosis. We performed bioinformatics analysis using online websites, and found that miR-19b was highly conserved during evolution and directly bound to ABCA1 mRNA with very low binding free energy. Luciferase reporter assay confirmed that miR-19b bound to 3110-3116 sites within ABCA1 3'UTR. MiR-19b directly regulated the expression levels of endogenous ABCA1 in foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by qRT-PCR and western blot. Cholesterol transport assays revealed that miR-19b dramatically suppressed apolipoprotein AI-mediated ABCA1-dependent cholesterol efflux, resulting in the increased levels of total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) as revealed by HPLC. The excretion of (3)H-cholesterol originating from cholesterol-laden MPMs into feces was decreased in mice overexpressing miR-19b. Finally, we evaluated the proatherosclerotic role of miR-19b in apolipoprotein E deficient (apoE(-/-)) mice. Treatment with miR-19b precursor reduced plasma high-density lipoprotein (HDL) levels, but increased plasma low-density lipoprotein (LDL) levels. Consistently, miR-19b precursor treatment increased aortic plaque size and lipid content, but reduced collagen content and ABCA1 expression. In contrast, treatment with the inhibitory miR-19b antisense oligonucleotides (ASO) prevented or

  12. The R230C variant of the ATP binding cassette protein A1 (ABCA1) gene is associated with a decreased response to glyburide therapy in patients with type 2 diabetes mellitus.

    PubMed

    Aguilar-Salinas, Carlos A; Muñoz-Hernandez, Linda Liliana; Cobos-Bonilla, Monica; Ramírez-Márquez, Marcos Rafael; Ordoñez-Sanchez, Maria Luisa; Mehta, Roopa; Medina-Santillan, Roberto; Tusie-Luna, Maria Teresa

    2013-05-01

    To test the hypothesis that persons with the R230C allele of ABCA1 show a decreased glycemic response to glyburide. This polymorphism is exclusively found in Ameri-indian populations and is associated with type 2 diabetes. This is a single blind controlled study including participants with type 2 diabetes (fasting glucose levels 126-250 mg/dl and HbA1c 7%-10%) managed with metformin and a lifestyle program. Each person with the risk allele (R230C) was matched by age, gender and BMI to three others with the wild type variant (R230R). Following a four week stabilization period, only participants with a greater than 70% adherence to metformin and a stable body weight were prescribed glyburide therapy for a further 16 weeks. The main outcome variable was the glyburide dose required to achieve treatment goals. No significant difference was observed in the glucose lowering effect of glyburide between subjects with the R230C and R230R alleles. However, the dose of sulfonylurea was significantly higher in the R230C participants compared with the R230R subjects (3.3±2.1 vs 6.3±5 mg/day, p<0.001). A higher percentage of R230C participants required at least 5mg of glyburide per day to achieve treatment goals. The glyburide dose was determined by the presence of the risk allele, among other factors. Patients with type 2 diabetes who have the R230C allele of ABCA1 needed a higher dose of glyburide in order to achieve the same glucose lowering effect as that in persons with the wild type variant. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. ABCA7 frameshift deletion associated with Alzheimer disease in African Americans

    PubMed Central

    Cukier, Holly N.; Kunkle, Brian W.; Vardarajan, Badri N.; Rolati, Sophie; Hamilton-Nelson, Kara L.; Kohli, Martin A.; Whitehead, Patrice L.; Dombroski, Beth A.; Van Booven, Derek; Lang, Rosalyn; Dykxhoorn, Derek M.; Farrer, Lindsay A.; Cuccaro, Michael L.; Vance, Jeffery M.; Gilbert, John R.; Beecham, Gary W.; Martin, Eden R.; Carney, Regina M.; Mayeux, Richard; Schellenberg, Gerard D.; Byrd, Goldie S.; Haines, Jonathan L.

    2016-01-01

    Objective: To identify a causative variant(s) that may contribute to Alzheimer disease (AD) in African Americans (AA) in the ATP-binding cassette, subfamily A (ABC1), member 7 (ABCA7) gene, a known risk factor for late-onset AD. Methods: Custom capture sequencing was performed on ∼150 kb encompassing ABCA7 in 40 AA cases and 37 AA controls carrying the AA risk allele (rs115550680). Association testing was performed for an ABCA7 deletion identified in large AA data sets (discovery n = 1,068; replication n = 1,749) and whole exome sequencing of Caribbean Hispanic (CH) AD families. Results: A 44-base pair deletion (rs142076058) was identified in all 77 risk genotype carriers, which shows that the deletion is in high linkage disequilibrium with the risk allele. The deletion was assessed in a large data set (531 cases and 527 controls) and, after adjustments for age, sex, and APOE status, was significantly associated with disease (p = 0.0002, odds ratio [OR] = 2.13 [95% confidence interval (CI): 1.42–3.20]). An independent data set replicated the association (447 cases and 880 controls, p = 0.0117, OR = 1.65 [95% CI: 1.12–2.44]), and joint analysis increased the significance (p = 1.414 × 10−5, OR = 1.81 [95% CI: 1.38–2.37]). The deletion is common in AA cases (15.2%) and AA controls (9.74%), but in only 0.12% of our non-Hispanic white cohort. Whole exome sequencing of multiplex, CH families identified the deletion cosegregating with disease in a large sibship. The deleted allele produces a stable, detectable RNA strand and is predicted to result in a frameshift mutation (p.Arg578Alafs) that could interfere with protein function. Conclusions: This common ABCA7 deletion could represent an ethnic-specific pathogenic alteration in AD. PMID:27231719

  14. IL-17A, MCP-1, CCR-2, and ABCA1 polymorphisms in children with non-alcoholic fatty liver disease.

    PubMed

    Akbulut, Ulas Emre; Emeksiz, Hamdi Cihan; Citli, Senol; Cebi, Alper Han; Korkmaz, Hatice Ayca Ata; Baki, Gaye

    2018-05-05

    The prevalence of non-alcoholic fatty liver disease in children has risen significantly, owing to the worldwide childhood obesity epidemic in the last two decades. Non-alcoholic fatty liver disease is closely linked to sedentary lifestyle, increased body mass index, and visceral adiposity. In addition, individual genetic variations also have a role in the development and progression of non-alcoholic fatty liver disease. The aim of this study was to investigate the gene polymorphisms of MCP-1 (-2518 A/G) (rs1024611), CCR-2 (190 G/A) (rs1799864), ABCA1 (883 G/A) (rs4149313), and IL-17A (-197 G/A) (rs2275913) in obese Turkish children with non-alcoholic fatty liver disease. The study recruited 186 obese children aged 10-17 years, including 101 children with non-alcoholic fatty liver disease and 85 children without non-alcoholic fatty liver disease. Anthropometric measurements, insulin resistance, a liver panel, a lipid profile, liver ultrasound examination, and genotyping of the four variants were performed. No difference was found between the groups in respect to age and gender, body mass index, waist/hip ratio, or body fat ratio. In addition to the elevated ALT levels, AST and GGT levels were found significantly higher in the non-alcoholic fatty liver disease group compared to the non non-alcoholic fatty liver disease group (p<0.05). The A-allele of IL-17A (-197 G/A) (rs2275913) was associated with non-alcoholic fatty liver disease (odds ratio 2.05, 95% confidence interval: 1.12-3.77, p=0.02). The findings of this study suggest that there may be an association between IL-17A (-197 G/A) (rs2275913) polymorphism and non-alcoholic fatty liver disease development in obese Turkish children. Copyright © 2018 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  15. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    PubMed Central

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K.; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D.; de Ángeles Granados-Silvestre, Ma; Montufar-Robles, Isela; Tito-Alvarez, Ana M.; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P.; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L.; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A.; Lisker, Ruben; Moises, Regina S.; Menjivar, Marta; Salzano, Francisco M.; Knowler, William C.; Bortolini, M. Cátira; Hayden, Michael R.; Baier, Leslie J.; Canizales-Quinteros, Samuel

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was associated with low high-density lipoprotein cholesterol (HDL-C) levels, obesity and type 2 diabetes in Mexican Mestizos. We performed a more extensive analysis of this variant in 4405 Native Americans and 863 individuals from other ethnic groups to investigate genetic evidence of positive selection, to assess its functional effect in vitro and to explore associations with HDL-C levels and other metabolic traits. The C230 allele was found in 29 of 36 Native American groups, but not in European, Asian or African individuals. C230 was observed on a single haplotype, and C230-bearing chromosomes showed longer relative haplotype extension compared with other haplotypes in the Americas. Additionally, single-nucleotide polymorphism data from the Human Genome Diversity Panel Native American populations were enriched in significant integrated haplotype score values in the region upstream of the ABCA1 gene. Cells expressing the C230 allele showed a 27% cholesterol efflux reduction (P< 0.001), confirming this variant has a functional effect in vitro. Moreover, the C230 allele was associated with lower HDL-C levels (P = 1.77 × 10−11) and with higher body mass index (P = 0.0001) in the combined analysis of Native American populations. This is the first report of a common functional variant exclusive to Native American and descent populations, which is a major determinant of HDL-C levels and may have contributed to the adaptive evolution of Native American populations. PMID:20418488

  16. Mutations in the ABCA4 (ABCR) Gene Are the Major Cause of Autosomal Recessive Cone-Rod Dystrophy

    PubMed Central

    Maugeri, Alessandra; Klevering, B. Jeroen; Rohrschneider, Klaus; Blankenagel, Anita; Brunner, Han G.; Deutman, August F.; Hoyng, Carel B.; Cremers, Frans P. M.

    2000-01-01

    The photoreceptor cell–specific ATP-binding cassette transporter gene (ABCA4; previously denoted “ABCR”) is mutated in most patients with autosomal recessive (AR) Stargardt disease (STGD1) or fundus flavimaculatus (FFM). In addition, a few cases with AR retinitis pigmentosa (RP) and AR cone-rod dystrophy (CRD) have been found to have ABCA4 mutations. To evaluate the importance of the ABCA4 gene as a cause of AR CRD, we selected 5 patients with AR CRD and 15 patients with isolated CRD, all from Germany and The Netherlands . Single-strand conformation–polymorphism analysis and sequencing revealed 19 ABCA4 mutations in 13 (65%) of 20 patients. In six patients, mutations were identified in both ABCA4 alleles; in seven patients, mutations were detected in one allele. One complex ABCA4 allele (L541P;A1038V) was found exclusively in German patients with CRD; one patient carried this complex allele homozygously, and five others were compound heterozygous. These findings suggest that mutations in the ABCA4 gene are the major cause of AR CRD. A primary role of the ABCA4 gene in STGD1/FFM and AR CRD, together with the gene's involvement in an as-yet-unknown proportion of cases with AR RP, strengthens the idea that mutations in the ABCA4 gene could be the most frequent cause of inherited retinal dystrophy in humans. PMID:10958761

  17. Presenting Papers at ABCA Conferences: Opinions and Recommendations.

    ERIC Educational Resources Information Center

    Mier, Denise; Myers, Robert

    1981-01-01

    Offers guidelines for preparing and presenting papers at American Business Communication Association (ABCA) conferences. Topics covered include: (1) types of delivery, (2) extemporaneous speaking, and (3) manuscript speaking. (FL)

  18. Increased maternal and fetal cholesterol efflux capacity and placental CYP27A1 expression in preeclampsia.

    PubMed

    Mistry, Hiten D; Kurlak, Lesia O; Mansour, Yosef T; Zurkinden, Line; Mohaupt, Markus G; Escher, Geneviève

    2017-06-01

    Preeclampsia is a pregnancy-specific condition that leads to increased cardiovascular risk in later life. A decrease in cholesterol efflux capacity is linked to CVD. We hypothesized that in preeclampsia there would be a disruption of maternal/fetal plasma to efflux cholesterol, as well as differences in the concentrations of both placental sterol 27-hydroxylase (CYP27A1) and apoA1 binding protein (AIBP). Total, HDL-, and ABCA1-mediated cholesterol effluxes were performed with maternal and fetal plasma from women with preeclampsia and normotensive controls (both n = 17). apoA1 and apoE were quantified by chemiluminescence, and 27-hydroxycholesterol (27-OHC) by GC-MS. Immunohistochemistry was used to determine placental expression/localization of CYP27A1, AIBP, apoA1, apoE, and SRB1. Maternal and fetal total and HDL-mediated cholesterol efflux capacities were increased in preeclampsia (by 10-20%), but ABCA1-mediated efflux was decreased (by 20-35%; P < 0.05). Maternal and fetal apoE concentrations were higher in preeclampsia. Fetal plasma 27-OHC levels were decreased in preeclamptic samples ( P < 0.05). Placental protein expression of both CYP27A1 and AIBP were localized around fetal vessels and significantly increased in preeclampsia ( P = 0.04). Placental 27-OHC concentrations were also raised in preeclampsia ( P < 0.05). Increased HDL-mediated cholesterol efflux capacity and placental CYP27A1/27-OHC could be a rescue mechanism in preeclampsia, to remove cholesterol from cells to limit lipid peroxidation and increase placental angiogenesis. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  19. Deleterious ABCA7 mutations and transcript rescue mechanisms in early onset Alzheimer's disease.

    PubMed

    De Roeck, Arne; Van den Bossche, Tobi; van der Zee, Julie; Verheijen, Jan; De Coster, Wouter; Van Dongen, Jasper; Dillen, Lubina; Baradaran-Heravi, Yalda; Heeman, Bavo; Sanchez-Valle, Raquel; Lladó, Albert; Nacmias, Benedetta; Sorbi, Sandro; Gelpi, Ellen; Grau-Rivera, Oriol; Gómez-Tortosa, Estrella; Pastor, Pau; Ortega-Cubero, Sara; Pastor, Maria A; Graff, Caroline; Thonberg, Håkan; Benussi, Luisa; Ghidoni, Roberta; Binetti, Giuliano; de Mendonça, Alexandre; Martins, Madalena; Borroni, Barbara; Padovani, Alessandro; Almeida, Maria Rosário; Santana, Isabel; Diehl-Schmid, Janine; Alexopoulos, Panagiotis; Clarimon, Jordi; Lleó, Alberto; Fortea, Juan; Tsolaki, Magda; Koutroumani, Maria; Matěj, Radoslav; Rohan, Zdenek; De Deyn, Peter; Engelborghs, Sebastiaan; Cras, Patrick; Van Broeckhoven, Christine; Sleegers, Kristel

    2017-09-01

    Premature termination codon (PTC) mutations in the ATP-Binding Cassette, Sub-Family A, Member 7 gene (ABCA7) have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer's disease (LOAD). High variability, however, is observed in downstream ABCA7 mRNA and protein expression, disease penetrance, and onset age, indicative of unknown modifying factors. Here, we investigated the prevalence and disease penetrance of ABCA7 PTC mutations in a large early onset AD (EOAD)-control cohort, and examined the effect on transcript level with comprehensive third-generation long-read sequencing. We characterized the ABCA7 coding sequence with next-generation sequencing in 928 EOAD patients and 980 matched control individuals. With MetaSKAT rare variant association analysis, we observed a fivefold enrichment (p = 0.0004) of PTC mutations in EOAD patients (3%) versus controls (0.6%). Ten novel PTC mutations were only observed in patients, and PTC mutation carriers in general had an increased familial AD load. In addition, we observed nominal risk reducing trends for three common coding variants. Seven PTC mutations were further analyzed using targeted long-read cDNA sequencing on an Oxford Nanopore MinION platform. PTC-containing transcripts for each investigated PTC mutation were observed at varying proportion (5-41% of the total read count), implying incomplete nonsense-mediated mRNA decay (NMD). Furthermore, we distinguished and phased several previously unknown alternative splicing events (up to 30% of transcripts). In conjunction with PTC mutations, several of these novel ABCA7 isoforms have the potential to rescue deleterious PTC effects. In conclusion, ABCA7 PTC mutations play a substantial role in EOAD, warranting genetic screening of ABCA7 in genetically unexplained patients. Long-read cDNA sequencing revealed both varying degrees of NMD and transcript-modifying events, which may influence ABCA7 dosage, disease severity, and may

  20. Lipid Absorption Defects in Intestine-specific Microsomal Triglyceride Transfer Protein and ATP-binding Cassette Transporter A1-deficient Mice*

    PubMed Central

    Iqbal, Jahangir; Parks, John S.; Hussain, M. Mahmood

    2013-01-01

    We have previously described apolipoprotein B (apoB)-dependent and -independent cholesterol absorption pathways and the role of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter A1 (ABCA1) in these pathways. To assess the contribution of these pathways to cholesterol absorption and to determine whether there are other pathways, we generated mice that lack MTP and ABCA1, individually and in combination, in the intestine. Intestinal deletions of Mttp and Abca1 decreased plasma cholesterol concentrations by 45 and 24%, respectively, whereas their combined deletion reduced it by 59%. Acute cholesterol absorption was reduced by 28% in the absence of ABCA1, and it was reduced by 92–95% when MTP was deleted in the intestine alone or together with ABCA1. MTP deficiency significantly reduced triglyceride absorption, although ABCA1 deficiency had no effect. ABCA1 deficiency did not affect cellular lipids, but Mttp deficiency significantly increased intestinal levels of triglycerides and free fatty acids. Accumulation of intestinal free fatty acids, but not triglycerides, in Mttp-deficient intestines was prevented when mice were also deficient in intestinal ABCA1. Combined deficiency of these genes increased intestinal fatty acid oxidation as a consequence of increased expression of peroxisome proliferator-activated receptor-γ (PPARγ) and carnitine palmitoyltransferase 1α (CPT1α). These studies show that intestinal MTP and ABCA1 are critical for lipid absorption and are the main determinants of plasma and intestinal lipid levels. Reducing their activities might lower plasma lipid concentrations. PMID:24019513

  1. Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway.

    PubMed

    Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

    2013-11-15

    Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

    PubMed

    Wang, Dongdong; Tosevska, Anela; Heiß, Elke H; Ladurner, Angela; Mölzer, Christine; Wallner, Marlies; Bulmer, Andrew; Wagner, Karl-Heinz; Dirsch, Verena M; Atanasov, Atanas G

    2017-04-28

    Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 μmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease. © 2017 The Authors. Published on

  3. Hydrogen Sulfide Up-Regulates the Expression of ATP-Binding Cassette Transporter A1 via Promoting Nuclear Translocation of PPARα.

    PubMed

    Li, Dong; Xiong, Qinghui; Peng, Jin; Hu, Bin; Li, Wanzhen; Zhu, Yizhun; Shen, Xiaoyan

    2016-04-29

    ATP binding cassette transporter A1 (ABCA1) plays a key role in atherogenesis. Hydrogen sulfide (H₂S), a gasotransmitter, has been reported to play an anti-atherosclerotic role. However, the underlying mechanisms are largely unknown. In this study we examined whether and how H₂S regulates ABCA1 expression. The effect of H₂S on ABCA1 expression and lipid metabolism were assessed in vitro by cultured human hepatoma cell line HepG2, and in vivo by ApoE(-/-) mice with a high-cholesterol diet. NaHS (an exogenous H₂S donor) treatment significantly increased the expression of ABCA1, ApoA1, and ApoA2 and ameliorated intracellular lipid accumulation in HepG2 cells. Depletion of the endogenous H₂S generator cystathionine γ-lyase (CSE) by small RNA interference (siRNA) significantly decreased the expression of ABCA1 and resulted in the accumulation of lipids in HepG2 cells. In vivo NaHS treatment significantly reduced the serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoproteins (LDL), diminished atherosclerotic plaque size, and increased hepatic ABCA1 expression in fat-fed ApoE(-/-) mice. Further study revealed that NaHS upregulated ABCA1 expression by promoting peroxisome proliferator-activated receptor α (PPARα) nuclear translocation. H₂S up-regulates the expression of ABCA1 by promoting the nuclear translocation of PPARα, providing a fundamental mechanism for the anti-atherogenic activity of H₂S. H₂S may be a promising potential drug candidate for the treatment of atherosclerosis.

  4. Advanced glycation end products affect cholesterol homeostasis by impairing ABCA1 expression on macrophages.

    PubMed

    Kamtchueng Simo, Olivier; Ikhlef, Souade; Berrougui, Hicham; Khalil, Abdelouahed

    2017-08-01

    Reverse cholesterol transport (RCT), which is intimately linked to high-density lipoproteins (HDLs), plays a key role in cholesterol homeostasis and the prevention of atherosclerosis. The goal of the present study was to investigate the effect of aging and advanced glycation end products (AGEs) on RCT as well as on other factors that may affect the antiatherogenic property of HDLs. The transfer of macrophage-derived cholesterol to the plasma and liver and then to the feces for elimination was significantly lower in aged mice than in young mice. Chronic injection of d -galactose (D-gal) or AGEs also significantly reduced RCT (65.3% reduction in [ 3 H]cholesterol levels in the plasma of D-gal-treated mice after 48 h compared with control mice, P < 0.01). The injection of both D-gal and aminoguanidine hydrochloride increased [ 3 H]cholesterol levels in the plasma, although the levels were lower than those of control mice. The in vitro incubation of HDLs with dicarbonyl compounds increased the carbonyl and conjugated diene content of HDLs and significantly reduced PON1 paraoxonase activity (87.4% lower than control HDLs, P < 0.0001). Treating J774A.1 macrophages with glycated fetal bovine serum increased carbonyl formation (39.5% increase, P < 0.003) and reduced ABCA1 protein expression and the capacity of macrophages to liberate cholesterol (69.1% decrease, P < 0.0001). Our results showed, for the first time, that RCT is altered with aging and that AGEs contribute significantly to this alteration.

  5. Melanocortin 1 Receptor Signaling Regulates Cholesterol Transport in Macrophages.

    PubMed

    Rinne, Petteri; Rami, Martina; Nuutinen, Salla; Santovito, Donato; van der Vorst, Emiel P C; Guillamat-Prats, Raquel; Lyytikäinen, Leo-Pekka; Raitoharju, Emma; Oksala, Niku; Ring, Larisa; Cai, Minying; Hruby, Victor J; Lehtimäki, Terho; Weber, Christian; Steffens, Sabine

    2017-07-04

    The melanocortin 1 receptor (MC1-R) is expressed by monocytes and macrophages, where it exerts anti-inflammatory actions on stimulation with its natural ligand α-melanocyte-stimulating hormone. The present study was designed to investigate the specific role of MC1-R in the context of atherosclerosis and possible regulatory pathways of MC1-R beyond anti-inflammation. Human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was assessed in apolipoprotein E-deficient mice. Characterization of human and mouse atherosclerotic plaques revealed that MC1-R expression localizes in lesional macrophages and is significantly associated with the ATP-binding cassette transporters ABCA1 and ABCG1, which are responsible for initiating reverse cholesterol transport. Using bone marrow-derived macrophages, we observed that α-melanocyte-stimulating hormone and selective MC1-R agonists similarly promoted cholesterol efflux, which is a counterregulatory mechanism against foam cell formation. Mechanistically, MC1-R activation upregulated the levels of ABCA1 and ABCG1. These effects were accompanied by a reduction in cell surface CD36 expression and in cholesterol uptake, further protecting macrophages from excessive lipid accumulation. Conversely, macrophages deficient in functional MC1-R displayed a phenotype with impaired efflux and enhanced uptake of cholesterol. Pharmacological targeting of MC1-R in atherosclerotic apolipoprotein E-deficient mice reduced plasma cholesterol levels and aortic CD36 expression and increased plaque ABCG1 expression and signs of plaque stability. Our findings identify a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R confers protection against macrophage foam cell formation through a dual mechanism: It prevents cholesterol uptake while concomitantly promoting ABCA1- and ABCG1-mediated reverse

  6. Mutations in the Cholesterol Transporter Gene ABCA5 Are Associated with Excessive Hair Overgrowth

    PubMed Central

    DeStefano, Gina M.; Kurban, Mazen; Anyane-Yeboa, Kwame; Dall'Armi, Claudia; Di Paolo, Gilbert; Feenstra, Heather; Silverberg, Nanette; Rohena, Luis; López-Cepeda, Larissa D.; Jobanputra, Vaidehi; Fantauzzo, Katherine A.; Kiuru, Maija; Tadin-Strapps, Marija; Sobrino, Antonio; Vitebsky, Anna; Warburton, Dorothy; Levy, Brynn; Salas-Alanis, Julio C.; Christiano, Angela M.

    2014-01-01

    Inherited hypertrichoses are rare syndromes characterized by excessive hair growth that does not result from androgen stimulation, and are often associated with additional congenital abnormalities. In this study, we investigated the genetic defect in a case of autosomal recessive congenital generalized hypertrichosis terminalis (CGHT) (OMIM135400) using whole-exome sequencing. We identified a single base pair substitution in the 5′ donor splice site of intron 32 in the ABC lipid transporter gene ABCA5 that leads to aberrant splicing of the transcript and a decrease in protein levels throughout patient hair follicles. The homozygous recessive disruption of ABCA5 leads to reduced lysosome function, which results in an accumulation of autophagosomes, autophagosomal cargos as well as increased endolysosomal cholesterol in CGHT keratinocytes. In an unrelated sporadic case of CGHT, we identified a 1.3 Mb cryptic deletion of chr17q24.2-q24.3 encompassing ABCA5 and found that ABCA5 levels are dramatically reduced throughout patient hair follicles. Collectively, our findings support ABCA5 as a gene underlying the CGHT phenotype and suggest a novel, previously unrecognized role for this gene in regulating hair growth. PMID:24831815

  7. Hydrogen Sulfide Up-Regulates the Expression of ATP-Binding Cassette Transporter A1 via Promoting Nuclear Translocation of PPARα

    PubMed Central

    Li, Dong; Xiong, Qinghui; Peng, Jin; Hu, Bin; Li, Wanzhen; Zhu, Yizhun; Shen, Xiaoyan

    2016-01-01

    ATP binding cassette transporter A1 (ABCA1) plays a key role in atherogenesis. Hydrogen sulfide (H2S), a gasotransmitter, has been reported to play an anti-atherosclerotic role. However, the underlying mechanisms are largely unknown. In this study we examined whether and how H2S regulates ABCA1 expression. The effect of H2S on ABCA1 expression and lipid metabolism were assessed in vitro by cultured human hepatoma cell line HepG2, and in vivo by ApoE−/− mice with a high-cholesterol diet. NaHS (an exogenous H2S donor) treatment significantly increased the expression of ABCA1, ApoA1, and ApoA2 and ameliorated intracellular lipid accumulation in HepG2 cells. Depletion of the endogenous H2S generator cystathionine γ-lyase (CSE) by small RNA interference (siRNA) significantly decreased the expression of ABCA1 and resulted in the accumulation of lipids in HepG2 cells. In vivo NaHS treatment significantly reduced the serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoproteins (LDL), diminished atherosclerotic plaque size, and increased hepatic ABCA1 expression in fat-fed ApoE−/− mice. Further study revealed that NaHS upregulated ABCA1 expression by promoting peroxisome proliferator-activated receptor α (PPARα) nuclear translocation. H2S up-regulates the expression of ABCA1 by promoting the nuclear translocation of PPARα, providing a fundamental mechanism for the anti-atherogenic activity of H2S. H2S may be a promising potential drug candidate for the treatment of atherosclerosis. PMID:27136542

  8. Functional Validation of ABCA3 as a Miltefosine Transporter in Human Macrophages: IMPACT ON INTRACELLULAR SURVIVAL OF LEISHMANIA (VIANNIA) PANAMENSIS.

    PubMed

    Dohmen, Luuk C T; Navas, Adriana; Vargas, Deninson Alejandro; Gregory, David J; Kip, Anke; Dorlo, Thomas P C; Gomez, Maria Adelaida

    2016-04-29

    Within its mammalian host, Leishmania resides and replicates as an intracellular parasite. The direct activity of antileishmanials must therefore depend on intracellular drug transport, metabolism, and accumulation within the host cell. In this study, we explored the role of human macrophage transporters in the intracellular accumulation and antileishmanial activity of miltefosine (MLF), the only oral drug available for the treatment of visceral and cutaneous leishmaniasis (CL). Membrane transporter gene expression in primary human macrophages infected in vitro with Leishmania Viannia panamensis and exposed to MLF showed modulation of ABC and solute liquid carrier transporters gene transcripts. Among these, ABCA3, a lipid transporter, was significantly induced after exposure to MLF, and this induction was confirmed in primary macrophages from CL patients. Functional validation of MLF as a substrate for ABCA3 was performed by shRNA gene knockdown (KD) in THP-1 monocytes. Intracellular accumulation of radiolabeled MLF was significantly higher in ABCA3(KD) macrophages. ABCA3(KD) resulted in increased cytotoxicity induced by MLF exposure. ABCA3 gene expression inversely correlated with intracellular MLF content in primary macrophages from CL patients. ABCA3(KD) reduced parasite survival during macrophage infection with an L. V. panamensis strain exhibiting low in vitro susceptibility to MLF. Confocal microscopy showed ABCA3 to be located in the cell membrane of resting macrophages and in intracellular compartments in L. V. panamensis-infected cells. These results provide evidence of ABCA3 as an MLF efflux transporter in human macrophages and support its role in the direct antileishmanial effect of this alkylphosphocholine drug. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Allelic and Phenotypic Heterogeneity in ABCA4 mutations

    PubMed Central

    Burke, Tomas R; Tsang, Stephen H

    2011-01-01

    Since the discovery of the ABCA4 gene as the cause of autosomal recessive Stargardt disease/fundus flavimaculatus much has been written of the phenotypic variability in ABCA4 retinopathy. In this review the authors discuss the findings seen on examination and the disease features detected using various clinical tests. Important differential diagnoses are presented and unusual presentations of ABCA4 disease highlighted. PMID:21510770

  10. CCAR1/CoCoA pair-mediated recruitment of the Mediator defines a novel pathway for GATA1 function.

    PubMed

    Mizuta, Shumpei; Minami, Tomoya; Fujita, Haruka; Kaminaga, Chihiro; Matsui, Keiji; Ishino, Ruri; Fujita, Azusa; Oda, Kasumi; Kawai, Asami; Hasegawa, Natsumi; Urahama, Norinaga; Roeder, Robert G; Ito, Mitsuhiro

    2014-01-01

    The MED1 subunit of the Mediator transcriptional coregulator complex coactivates GATA1 and induces erythropoiesis. Here, we show the dual mechanism of GATA1- and MED1-mediated transcription. MED1 expression levels in K562 erythroleukemia cells paralleled the levels of GATA1-targeted gene transcription and erythroid differentiation. An N-terminal fragment of MED1, MED1(1-602), which is incapable of interacting with GATA1, enhanced GATA1-targeted gene transcription and erythroid differentiation, and introduction of MED1(1-602) into Med1(-/-) mouse embryonic fibroblasts (MEFs) partially rescued GATA1-mediated transcription. The C-terminal zinc-finger domain of GATA1 interacts with the MED1(1-602)-interacting coactivator CCAR1, CoCoA and MED1(681-715). CCAR1 and CoCoA synergistically enhanced GATA1-mediated transcription from the γ-globin promoter in MEFs. Recombinant GATA1, CCAR1, CoCoA and MED1(1-602) formed a complex in vitro, and GATA1, CCAR1, CoCoA and MED1 were recruited to the γ-globin promoter in K562 cells during erythroid differentiation. Therefore, in addition to the direct interaction between GATA1 and MED1, CoCoA and CCAR1 appear to relay the GATA1 signal to MED1, and multiple modes of the GATA1-MED1 axis may help to fine-tune GATA1 function during GATA1-mediated homeostasis events. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  11. Heart ABCA1 and PPAR- α Genes Expression Responses in Male rats: Effects of High Intensity Treadmill Running Training and Aqueous Extraction of Black Crataegus-Pentaegyna.

    PubMed

    Ghanbari-Niaki, Abbass; Ghanbari-Abarghooi, Safieyh; Rahbarizadeh, Fatemeh; Zare-Kookandeh, Navabeh; Gholizadeh, Monireh; Roudbari, Fatemeh; Zare-Kookandeh, Asghar

    2013-11-01

    Heart as a high metabolic and aerobic tissue is consuming lipid as a fuel for its energy provision at rest during light and moderate exercise, except when lactate level is higher in blood circulation. It has been shown that any type of regular exercise and crataegus species would improve cardiovascular function and minimizes several risk factors via stimulating lipid metabolism by acting on enzymes and genes expression such as ABCA1 and PPAR α which are involving in this process. Twenty Wistar male rats (4-6 weeks old, 140-173 g weight) were used. Animals were randomly classified into training (n = 10) and control (n = 10) groups and then divided into saline-control (SC), saline-training (ST), Crataegus-Pentaegyna -control (CPC), and Crataegus-Pentaegyna -training (CPT) groups. Training groups have performed a high-intensity running program (at 34 m/min (0% grade), 60 min/day, 5 days/week) on a motor-driven treadmill for eight weeks. Animals were orally fed with Crataegus-Pentaegyna extraction (500mg/kg) and saline solution for six weeks. Seventy- two hours after the last training session, rats were sacrificed, hearts were excised, cleaned and immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Plasma also was collected for plasma variable measurements. Statistical analysis was performed using a two way analysis of variance, and significance was accepted at P < 0.05. A non-significant (P < 0.4, P < 0.79, respectively) increase in ABCA1 and PPAR α genes expression was accompanied by a significant (P < 0.01, P < 0.04, P < 0.04, respectively) reduction in TC, TG, and VLDL-C levels in Crataegus-Pentaegyna groups. Our findings show that a high intensity treadmill running was able to express ABCA1 and PPAR α in rat heart. Data also possibly indicate that the Crataeguse-Pentaegyna supplementation solely could mimic training effect on the mentioned genes and lipid profiles via different mechanism(s).

  12. Heart ABCA1 and PPAR- α Genes Expression Responses in Male rats: Effects of High Intensity Treadmill Running Training and Aqueous Extraction of Black Crataegus-Pentaegyna

    PubMed Central

    Ghanbari-Niaki, Abbass; Ghanbari-Abarghooi, Safieyh; Rahbarizadeh, Fatemeh; Zare-Kookandeh, Navabeh; Gholizadeh, Monireh; Roudbari, Fatemeh; Zare-Kookandeh, Asghar

    2013-01-01

    Introduction: Heart as a high metabolic and aerobic tissue is consuming lipid as a fuel for its energy provision at rest during light and moderate exercise, except when lactate level is higher in blood circulation. It has been shown that any type of regular exercise and crataegus species would improve cardiovascular function and minimizes several risk factors via stimulating lipid metabolism by acting on enzymes and genes expression such as ABCA1 and PPAR α which are involving in this process. Materials and Methods: Twenty Wistar male rats (4-6 weeks old, 140-173 g weight) were used. Animals were randomly classified into training (n = 10) and control (n = 10) groups and then divided into saline-control (SC), saline-training (ST), Crataegus-Pentaegyna -control (CPC), and Crataegus-Pentaegyna -training (CPT) groups. Training groups have performed a high-intensity running program (at 34 m/min (0% grade), 60 min/day, 5 days/week) on a motor-driven treadmill for eight weeks. Animals were orally fed with Crataegus-Pentaegyna extraction (500mg/kg) and saline solution for six weeks. Seventy- two hours after the last training session, rats were sacrificed, hearts were excised, cleaned and immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Plasma also was collected for plasma variable measurements. Statistical analysis was performed using a two way analysis of variance, and significance was accepted at P < 0.05. Results: A non-significant (P < 0.4, P < 0.79, respectively) increase in ABCA1 and PPAR α genes expression was accompanied by a significant (P < 0.01, P < 0.04, P < 0.04, respectively) reduction in TC, TG, and VLDL-C levels in Crataegus-Pentaegyna groups. Conclusions: Our findings show that a high intensity treadmill running was able to express ABCA1 and PPAR α in rat heart. Data also possibly indicate that the Crataeguse-Pentaegyna supplementation solely could mimic training effect on the mentioned genes and lipid profiles via

  13. The ATP-binding cassette transporter A1 regulates phosphoantigen release and Vγ9Vδ2 T cell activation by dendritic cells

    PubMed Central

    Castella, Barbara; Kopecka, Joanna; Sciancalepore, Patrizia; Mandili, Giorgia; Foglietta, Myriam; Mitro, Nico; Caruso, Donatella; Novelli, Francesco; Riganti, Chiara; Massaia, Massimo

    2017-01-01

    Vγ9Vδ2 T cells are activated by phosphoantigens, such as isopentenyl pyrophosphate (IPP), which is generated in the mevalonate pathway of antigen-presenting cells. IPP is released in the extracellular microenvironment via unknown mechanisms. Here we show that the ATP-binding cassette transporter A1 (ABCA1) mediates extracellular IPP release from dendritic cells (DC) in cooperation with apolipoprotein A-I (apoA-I) and butyrophilin-3A1. IPP concentrations in the supernatants are sufficient to induce Vγ9Vδ2 T cell proliferation after DC mevalonate pathway inhibition with zoledronic acid (ZA). ZA treatment increases ABCA1 and apoA-I expression via IPP-dependent LXRα nuclear translocation and PI3K/Akt/mTOR pathway inhibition. These results close the mechanistic gap in our understanding of extracellular IPP release from DC and provide a framework to fine-tune Vγ9Vδ2 T cell activation via mevalonate and PI3K/Akt/mTOR pathway modulation. PMID:28580927

  14. The detection of autoantibodies to ATP-binding cassette transporter A1 and its role in the pathogenesis of atherosclerosis in patients with systemic lupus erythematosus.

    PubMed

    Zeng, Ting; Li, Shu-Jie; Ao, Wen; Zheng, Hui; Wu, Feng-Xia; Chen, Yi; Yang, Cheng-De

    2012-11-01

    To investigate the prevalence of autoantibodies against ATP-binding cassette transporter A1 (ABCA1) in SLE patients, and evaluate the association between anti-ABCA1 autoantibodies and atherosclerosis in SLE. The sera of 75 SLE patients and 75 healthy controls were tested by immunoblotting. Then, we examined the effect of anti-ABCA1 autoantibodies on cholesterol efflux in vitro. The prevalence of anti-ABCA1 antibodies in SLE patients was significantly higher than the controls (p<0.05). The prevalence in the SLE-plaque group was higher than that in the SLE-non-plaque group (p<0.05). The IgG purified from anti-ABCA1-antibody positive sera can inhibit cellular cholesterol efflux from THP-1 cells in vitro with a significantly higher inhibition ratio than that of the healthy controls. Our observations suggest that anti-ABCA1 autoantibodies are involved in the pathogenesis of lupus atherosclerosis and that autoantibodies against ABCA1 may act as biomarkers for atherosclerosis in SLE. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  15. Double hyperautofluorescent ring on fundus autofluorescence in ABCA4.

    PubMed

    Abalem, Maria Fernanda; Qian, Cynthia X; Branham, Kari; Schlegel, Dana; Fahim, Abigail T; Khan, Naheed W; Heckenlively, John R; Jayasundera, K Thiran

    2018-01-01

    We report an unusual phenotype in a child with a clinical diagnosis of recessive Stargardt disease (STGD1) and two pathogenic variants in the ABCA4 gene. Typically, the diagnosis of early-onset STGD1 is challenging because children may present with a variety of fundus changes and a variable rate of progression. At the time of his initial visit, the 6-year-old boy presented with 20/200 OD (right eye) and 20/150 OS (left eye), symmetrical mild foveal atrophy without flecks on fundus exam, and foveal hypoautofluorescence surrounded by a homogeneous hyperautofluorescent background on wide-field fundus autofluorescence. Over 4 years of follow-up, the retinal atrophy continued to progress, resulting in two well-defined and concentric hyperautofluorescent rings: one ring located at the posterior pole and the other located around the peripapillary region. Visual acuity also deteriorated to counting fingers at 4ft OD and 20/500 OS. To the best of our knowledge, this phenotype has not been previously described with the ABCA4 gene.

  16. 29 CFR 1202.1 - Mediation.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 4 2010-07-01 2010-07-01 false Mediation. 1202.1 Section 1202.1 Labor Regulations Relating to Labor (Continued) NATIONAL MEDIATION BOARD RULES OF PROCEDURE § 1202.1 Mediation. The mediation..., or where conferences are refused. The National Mediation Board may proffer its services in case any...

  17. Inhibition of cholesterol absorption associated with a PPAR alpha-dependent increase in ABC binding cassette transporter A1 in mice.

    PubMed

    Knight, Brian L; Patel, Dilip D; Humphreys, Sandy M; Wiggins, David; Gibbons, Geoffrey F

    2003-11-01

    Dietary supplementation with the peroxisome proliferator-activated receptor alpha (PPAR alpha) ligand WY 14,643 gave rise to a 4- to 5-fold increase in the expression of mRNA for the ATP binding cassette transporter A1 (ABCA1) in the intestine of normal mice. There was no effect in the intestine of PPAR alpha-null mice. Consumption of a high-cholesterol diet also increased intestinal ABCA1 expression. The effects of WY 14,643 and the high-cholesterol diet were not additive. WY 14,643 feeding reduced intestinal absorption of cholesterol in the normal mice, irrespective of the dietary cholesterol concentration, and this resulted in lower diet-derived cholesterol and cholesteryl ester concentrations in plasma and liver. At each concentration of dietary cholesterol, there was a similar significant inverse correlation between intestinal ABCA1 mRNA content and the amount of cholesterol absorbed. The fibrate-induced changes in the intestines of the normal mice were accompanied by an increased concentration of the mRNA encoding the sterol-regulatory element binding protein-1c gene (SREBP-1c), a known target gene for the oxysterol receptor liver X receptor alpha (LXR alpha). There was a correlation between intestinal ABCA1 mRNA and SREBP-1c mRNA contents, but not between SREBP-1c mRNA content and cholesterol absorption. These results suggest that PPAR alpha influences cholesterol absorption through modulating ABCA1 activity in the intestine by a mechanism involving LXR alpha.

  18. Mutation Spectrum of the ABCA4 Gene in a Greek Cohort with Stargardt Disease: Identification of Novel Mutations and Evidence of Three Prevalent Mutated Alleles

    PubMed Central

    Vassiliki, Kokkinou; George, Koutsodontis; Polixeni, Stamatiou; Christoforos, Giatzakis; Minas, Aslanides Ioannis; Stavrenia, Koukoula; Ioannis, Datseris

    2018-01-01

    Aim To evaluate the frequency and pattern of disease-associated mutations of ABCA4 gene among Greek patients with presumed Stargardt disease (STGD1). Materials and Methods A total of 59 patients were analyzed for ABCA4 mutations using the ABCR400 microarray and PCR-based sequencing of all coding exons and flanking intronic regions. MLPA analysis as well as sequencing of two regions in introns 30 and 36 reported earlier to harbor deep intronic disease-associated variants was used in 4 selected cases. Results An overall detection rate of at least one mutant allele was achieved in 52 of the 59 patients (88.1%). Direct sequencing improved significantly the complete characterization rate, that is, identification of two mutations compared to the microarray analysis (93.1% versus 50%). In total, 40 distinct potentially disease-causing variants of the ABCA4 gene were detected, including six previously unreported potentially pathogenic variants. Among the disease-causing variants, in this cohort, the most frequent was c.5714+5G>A representing 16.1%, while p.Gly1961Glu and p.Leu541Pro represented 15.2% and 8.5%, respectively. Conclusions By using a combination of methods, we completely molecularly diagnosed 48 of the 59 patients studied. In addition, we identified six previously unreported, potentially pathogenic ABCA4 mutations. PMID:29854428

  19. Prenatal Ethanol Exposure Up-Regulates the Cholesterol Transporters ATP-Binding Cassette A1 and G1 and Reduces Cholesterol Levels in the Developing Rat Brain.

    PubMed

    Zhou, Chunyan; Chen, Jing; Zhang, Xiaolu; Costa, Lucio G; Guizzetti, Marina

    2014-11-01

    Cholesterol plays a pivotal role in many aspects of brain development; reduced cholesterol levels during brain development, as a consequence of genetic defects in cholesterol biosynthesis, leads to severe brain damage, including microcephaly and mental retardation, both of which are also hallmarks of the fetal alcohol syndrome. We had previously shown that ethanol up-regulates the levels of two cholesterol transporters, ABCA1 (ATP binding cassette-A1) and ABCG1, leading to increased cholesterol efflux and decreased cholesterol content in astrocytes in vitro. In the present study we investigated whether similar effects could be seen in vivo. Pregnant Sprague-Dawley rats were fed liquid diets containing 36% of the calories from ethanol from gestational day (GD) 6 to GD 21. A pair-fed control groups and an ad libitum control group were included in the study. ABCA1 and ABCG1 protein expression and cholesterol and phospholipid levels were measured in the neocortex of female and male fetuses at GD 21. Body weights were decreased in female fetuses as a consequence of ethanol treatments. ABCA1 and ABCG1 protein levels were increased, and cholesterol levels were decreased, in the neocortex of ethanol-exposed female, but not male, fetuses. Levels of phospholipids were unchanged. Control female fetuses fed ad libitum displayed an up-regulation of ABCA1 and a decrease in cholesterol content compared with pair-fed controls, suggesting that a compensatory up-regulation of cholesterol levels may occur during food restriction. Maternal ethanol consumption may affect fetal brain development by increasing cholesterol transporters' expression and reducing brain cholesterol levels. © The Author 2014. Medical Council on Alcohol and Oxford University Press. All rights reserved.

  20. Microarray-based mutation analysis of the ABCA4 (ABCR) gene in autosomal recessive cone-rod dystrophy and retinitis pigmentosa.

    PubMed

    Klevering, B Jeroen; Yzer, Suzanne; Rohrschneider, Klaus; Zonneveld, Marijke; Allikmets, Rando; van den Born, L Ingeborgh; Maugeri, Alessandra; Hoyng, Carel B; Cremers, Frans P M

    2004-12-01

    Mutations in the ABCA4 gene have been associated with autosomal recessive Stargardt disease (STGD1), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). We employed a recently developed genotyping microarray, the ABCR400-chip, to search for known ABCA4 mutations in patients with isolated or autosomal recessive CRD (54 cases) or RP (90 cases). We performed detailed ophthalmologic examinations and identified at least one ABCA4 mutation in 18 patients (33%) with CRD and in five patients (5.6%) with RP. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequencing revealed four novel missense mutations (R24C, E161K, P597S, G618E) and a novel 1-bp deletion (5888delG). Ophthalmoscopic abnormalities in CRD patients ranged from minor granular pigmentary changes in the posterior pole to widespread atrophy. In 12 patients with recordable electroretinogram (ERG) tracings, a cone-rod pattern was detected. Three patients demonstrated progression from a retinal dystrophy resembling STGD1 to a more widespread degeneration, and were subsequently diagnosed as CRD. In addition to a variable degree of atrophy, all RP patients displayed ophthalmologic characteristics of classic RP. When detectable, ERG recordings in these patients demonstrated rod-cone patterns of photoreceptor degeneration. In conclusion, in this study, we show that the ABCA4 mutation chip is an efficient first screening tool for arCRD.

  1. Possible protective role of the ABCA4 gene c.1268A>G missense variant in Stargardt disease and syndromic retinitis pigmentosa in a Sicilian family: Preliminary data.

    PubMed

    D'Angelo, Rosalia; Donato, Luigi; Venza, Isabella; Scimone, Concetta; Aragona, Pasquale; Sidoti, Antonina

    2017-04-01

    In the wide horizon of ophthalmologically rare diseases among retinitis pigmentosa forms, Stargardt disease has gradually assumed a significant role due to its heterogeneity. In the present study, we aimed to support one of two opposite hypotheses concerning the causative or protective role of heterozygous c.1268A>G missense variant of the ABCA4 gene in Stargardt disease and in syndromic retinitis pigmentosa. This study was based on a family consisting of three members: proband, age 54, with high myopia, myopic chorioretinitis and retinal dystrophy; wife, age 65, with mild symptoms; daughter, age 29, asymptomatic. After genetic counseling, ABCA4 and RP1 gene analysis was performed. The results highlighted an important genetic picture. The proband was found to carry two variant RP1 SNPs, rs2293869 (c.2953A>T) and rs61739567 (c.6098G>A), and, a wild-type condition for four RP1 polymorphisms, rs444772 (c.2623G>A) and three SNPs in the 'hot-spot' region, exon 4. The proband's wife, instead, showed an opposite condition compared to her husband: a homozygous mutated condition for the first four SNPs analyzed, while the last two were wild-type. Regarding the ABCA4 gene, the proband evidenced a wild-type condition. Furthermore, the wife showed a heterozygous condition of ABCA4 rs3112831 (c.1268A>G). As expected, the daughter presented heterozygosity for all variants of both genes. In conclusion, even though the c.1268A>G missense variant of the ABCA4 gene has often been reported as causative of disease, and in other cases protective of disease, in our family case, the variant appears to reduce or delay the risk of onset of Stargardt disease.

  2. 29 CFR 1203.1 - Mediation services.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 4 2010-07-01 2010-07-01 false Mediation services. 1203.1 Section 1203.1 Labor Regulations Relating to Labor (Continued) NATIONAL MEDIATION BOARD APPLICATIONS FOR SERVICE § 1203.1 Mediation services. Applications for the mediation services of the National Mediation Board under section 5, First, of the Railway...

  3. [A novel compound heterozygous mutation in ABCA3 gene in a child with diffuse parenchymal lung disease].

    PubMed

    Bao, Y M; Liu, X L; Liu, X L; Chen, J H; Zheng, Y J

    2017-11-02

    Objective: To summarize the clinical characteristics of the diffuse parenchymal lung diseases in a child caused by a novel compound heterozygous ABCA3 mutation and explore the association between the phenotype and ABCA3 mutation. Method: The clinical material of a patient diagnosed with diffuse parenchymal lung disease with ABCA3 mutation in December 2016 in Shenzhen Children's Hospital was analyzed. The information about ABCA3 gene mutation updated before April, 2017 was searched and collected from the gene databases (including 1000Genomes, HGMD, EXAC) and the literatures (including Wanfang Chinese database and Pubmed). Result: The girl was one year and nine months old. She presented with chronic cough, tachypnea, cyanosis and failure to thrive since she was one year and three months old. Her condition gradually deteriorated after she was empirically treated. Physical examination showed malnutrition, tachypnea and clubbed-fingers. Her high resolution computed tomography (HRCT) revealed diffused ground-glass opacities, thickened interlobular septum, and multiple subpleural small air-filled lung cysts. The second generation sequencing study identified a novel compound heterozygous mutation (c.1755delC+c.2890G>A) in her ABCA3 gene, which derived respectively from her parents and has not been reported in the database and the literatures mentioned above. Conclusion: c.1755delC+c.2890G>A is a new kind of compound heterozygous mutation in ABCA3, which can cause children's diffuse parenchymal lung disease. Its phenotype is related to its genotype.

  4. GLP-1 mediates antiapoptotic effect by phosphorylating Bad through a beta-arrestin 1-mediated ERK1/2 activation in pancreatic beta-cells.

    PubMed

    Quoyer, Julie; Longuet, Christine; Broca, Christophe; Linck, Nathalie; Costes, Safia; Varin, Elodie; Bockaert, Joël; Bertrand, Gyslaine; Dalle, Stéphane

    2010-01-15

    Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the beta-cells is of major importance. In beta-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Galpha(s)/cAMP/cAMP-dependent protein kinase (PKA) or beta-arrestin 1, a scaffold protein. Using pharmacological inhibitors, beta-arrestin 1 small interfering RNA, and islets isolated from beta-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the beta-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the beta-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the beta-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. beta-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in beta-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a beta-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of beta-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.

  5. Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflammation and accelerates atherosclerosis in mice.

    PubMed

    Westerterp, Marit; Murphy, Andrew J; Wang, Mi; Pagler, Tamara A; Vengrenyuk, Yuliya; Kappus, Mojdeh S; Gorman, Darren J; Nagareddy, Prabhakara R; Zhu, Xuewei; Abramowicz, Sandra; Parks, John S; Welch, Carrie; Fisher, Edward A; Wang, Nan; Yvan-Charvet, Laurent; Tall, Alan R

    2013-05-24

    Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. To assess the role of macrophage cholesterol efflux pathways in atherogenesis. We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABC(DKO) mice) but not in hematopoietic stem or progenitor populations. MAC-ABC(DKO) bone marrow (BM) was transplanted into Ldlr(-/-) recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet-fed MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production. These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.

  6. Novel association of the R230C variant of the ABCA1 gene with high triglyceride levels and low high-density lipoprotein cholesterol levels in Mexican school-age children with high prevalence of obesity.

    PubMed

    Gamboa-Meléndez, Marco Alberto; Galindo-Gómez, Carlos; Juárez-Martínez, Liliana; Gómez, F Enrique; Diaz-Diaz, Eulises; Ávila-Arcos, Marco Antonio; Ávila-Curiel, Abelardo

    2015-08-01

    Metabolic syndrome (MetS) is a disorder that includes a cluster of several risk factors for the development of type 2 diabetes and cardiovascular disease. The R230C variant of the ABCA1 gene has been associated with low HDL-cholesterol in several studies, but its association with MetS in children remains to be determined. The aim of this study was to analyze the association of the R230C variant with MetS and other metabolic traits in school-aged Mexican children. The study was performed in seven urban primary schools in the State of Mexico. Four hundred thirty-two Mexican school-age children 6-13 years old were recruited. MetS was identified using the International Diabetes Federation definition. The R230C variant of the ABCA1 gene was genotyped to seek associations with MetS and other metabolic traits. The prevalence of MetS was 29% in children aged 10-13 years. The R230C variant was not associated with MetS (OR = 1.65; p = 0.139). Furthermore, in the whole population, the R230C variant was associated with low HDL-cholesterol levels (β coefficient = -3.28, p <0.001). Interestingly, in the total population we found a novel association of this variant with high triglyceride levels (β coefficient = 14.34; p = 0.027). We found a new association of the R230C variant of the ABCA1 gene with high triglyceride levels. Our findings also replicate the association of this variant with low HDL-cholesterol levels in Mexican school-age children. Copyright © 2015 IMSS. Published by Elsevier Inc. All rights reserved.

  7. A Computational Approach From Gene to Structure Analysis of the Human ABCA4 Transporter Involved in Genetic Retinal Diseases.

    PubMed

    Trezza, Alfonso; Bernini, Andrea; Langella, Andrea; Ascher, David B; Pires, Douglas E V; Sodi, Andrea; Passerini, Ilaria; Pelo, Elisabetta; Rizzo, Stanislao; Niccolai, Neri; Spiga, Ottavia

    2017-10-01

    The aim of this article is to report the investigation of the structural features of ABCA4, a protein associated with a genetic retinal disease. A new database collecting knowledge of ABCA4 structure may facilitate predictions about the possible functional consequences of gene mutations observed in clinical practice. In order to correlate structural and functional effects of the observed mutations, the structure of mouse P-glycoprotein was used as a template for homology modeling. The obtained structural information and genetic data are the basis of our relational database (ABCA4Database). Sequence variability among all ABCA4-deposited entries was calculated and reported as Shannon entropy score at the residue level. The three-dimensional model of ABCA4 structure was used to locate the spatial distribution of the observed variable regions. Our predictions from structural in silico tools were able to accurately link the functional effects of mutations to phenotype. The development of the ABCA4Database gathers all the available genetic and structural information, yielding a global view of the molecular basis of some retinal diseases. ABCA4 modeled structure provides a molecular basis on which to analyze protein sequence mutations related to genetic retinal disease in order to predict the risk of retinal disease across all possible ABCA4 mutations. Additionally, our ABCA4 predicted structure is a good starting point for the creation of a new data analysis model, appropriate for precision medicine, in order to develop a deeper knowledge network of the disease and to improve the management of patients.

  8. Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex vivo.

    PubMed

    De Silva, Samantha R; Charbel Issa, Peter; Singh, Mandeep S; Lipinski, Daniel M; Barnea-Cramer, Alona O; Walker, Nathan J; Barnard, Alun R; Hankins, Mark W; MacLaren, Robert E

    2016-11-01

    Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4 -/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4 -/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.

  9. Retinoic Acid Isomers Up-Regulate ATP Binding Cassette A1 and G1 and Cholesterol Efflux in Rat Astrocytes: Implications for Their Therapeutic and Teratogenic Effects

    PubMed Central

    Chen, Jing; Costa, Lucio G.

    2011-01-01

    Recent studies suggest that retinoids may be effective in the treatment of Alzheimer's disease, although exposure to an excess of retinoids during gestation causes teratogenesis. Cholesterol is essential for brain development, but high levels of cholesterol have been associated with Alzheimer's disease. We hypothesized that retinoic acid may affect cholesterol homeostasis in rat astrocytes, which regulate cholesterol distribution in the brain, through the up-regulation of cholesterol transporters ATP binding cassette (Abc)a1 and Abcg1. Tretinoin, 13-cis retinoic acid (13-cis-RA), 9-cis-RA, and the selective retinoid X receptor (RXR) agonist methoprene significantly increased cholesterol efflux induced by cholesterol acceptors and protein levels of Abca1 by 2.3- (±0.25), 3.6- (±0.42), 4.1- (±0.5), and 1.75- (±0.43) fold, respectively, and Abcg1 by 2.1- (±0.26), 2.2- (±0.33), 2.5- (±0.23), and 2.2- (±0.21) fold, respectively. 13-cis-RA and 9-cis-RA also significantly increased mRNA levels of Abca1 (maximal induction 7.3 ± 0.42 and 2.7 ± 0.17, respectively) and Abcg1 (maximal induction 2.0 ± 0.18 and 1.8 ± 0.09, respectively), and the levels of membrane-bound Abca1 (2.5 ± 0.3 and 2.5 ± 0.40-fold increase, respectively), whereas they significantly decreased intracellular cholesterol content without affecting cholesterol synthesis. The effect of 9-cis-RA on cholesterol homeostasis in astrocytes can be ascribed to the activation of RXR, whereas the effects of 13-cis-RA and tretinoin were independent of either RXRs or retinoic acid receptors. These findings suggest that retinoids affect cholesterol homeostasis in astrocytes and that this effect may be involved in both their therapeutic and teratogenic actions. PMID:21628419

  10. Analysis of ABCA4 in mixed Spanish families segregating different retinal dystrophies.

    PubMed

    Paloma, Eva; Coco, Rosa; Martínez-Mir, Amalia; Vilageliu, Lluïsa; Balcells, Susana; Gonzàlez-Duarte, Roser

    2002-12-01

    Genotype-phenotype correlations highlighted the function of ABCA4 in retinitis pigmentosa (RP),cone-rod dystrophy (CRD) and Stargardt/Fundus Flavimaculatus disease (STGD/FFM). Initial screening of ABCA4 variants showed a correlation between the type of mutation and the severity of the disease. In the present study we have undertaken mutational and haplotype analysis of ABCA4 in three mixed pedigrees segregating different retinal dystrophies. In family I, we have shown cosegregation of different ABCA4 alleles with CRD (homozygosity for L1940P) and three subtypes of STGD/FFM. The first, a mild form, consisting on fundus flavimaculatus-like distribution of flecks, but good visual acuity and absence of dark choroid, was found to cosegregate with alleles R1097C and F553L; the second, a conventional Stargardt phenotype was associated to alleles L1940P/R1097C and the third, displaying severely reduced visual acuity and dark choroid (named FFM), was associated to L1940P/F553L. In family II, segregating STGD and RP phenotypes, while the involvement of ABCA4 in STGD seems clear this is not the case for RP. Finally, in family III, also segregating STGD and RP, ABCA4 fails to explain either phenotype. Our data highlight the wide allelic heterogeneity involving this gene and support the genetic variability (beyond ABCA4) of mixed STGD/RP pedigrees. Copyright 2002 Wiley-Liss, Inc.

  11. The Role of the Photoreceptor ABC Transporter ABCA4 in Lipid Transport and Stargardt Macular Degeneration

    PubMed Central

    Molday, Robert S.; Zhong, Ming; Quazi, Faraz

    2009-01-01

    ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone-rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of abca4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidene-phosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders. PMID:19230850

  12. ATP binding cassette G1-dependent cholesterol efflux during inflammation.

    PubMed

    de Beer, Maria C; Ji, Ailing; Jahangiri, Anisa; Vaughan, Ashley M; de Beer, Frederick C; van der Westhuyzen, Deneys R; Webb, Nancy R

    2011-02-01

    ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

  13. PECAM1 regulates flow-mediated Gab1 tyrosine phosphorylation and signaling

    PubMed Central

    Xu, Suowen; Ha, Chang Hoon; Wang, Weiye; Xu, Xiangbin; Yin, Meimei; Jin, Felix Q.; Mastrangelo, Michael; Koroleva, Marina; Fujiwara, Keigi; Jin, Zheng Gen

    2016-01-01

    Endothelial dysfunction, characterized by impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued decrease of NO production, is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Laminar blood flow-mediated specific signaling cascades modulate vascular endothelial cells (ECs) structure and functions. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation in ECs, which in part confers laminar flow atheroprotective action. However, the molecular mechanisms whereby flow regulates Gab1 tyrosine phosphorylation and its downstream signaling events remain unclear. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in flow signaling and HGF signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K decreased flow-, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs. PMID:26706435

  14. MACC1 regulates Fas mediated apoptosis through STAT1/3 - Mcl-1 signaling in solid cancers.

    PubMed

    Radhakrishnan, Harikrishnan; Ilm, Katharina; Walther, Wolfgang; Shirasawa, Senji; Sasazuki, Takehiko; Daniel, Peter T; Gillissen, Bernhard; Stein, Ulrike

    2017-09-10

    MACC1 was identified as a novel player in cancer progression and metastasis, but its role in death receptor-mediated apoptosis is still unexplored. We show that MACC1 knockdown sensitizes cancer cells to death receptor-mediated apoptosis. For the first time, we provide evidence for STAT signaling as a MACC1 target. MACC1 knockdown drastically reduced STAT1/3 activating phosphorylation, thereby regulating the expression of its apoptosis targets Mcl-1 and Fas. STAT signaling inhibition by the JAK1/2 inhibitor ruxolitinib mimicked MACC1 knockdown-mediated molecular signatures and apoptosis sensitization to Fas activation. Despite the increased Fas expression, the reduced Mcl-1 expression was instrumental in apoptosis sensitization. This reduced Mcl-1-mediated apoptosis sensitization was Bax and Bak dependent. MACC1 knockdown also increased TRAIL-induced apoptosis. MACC1 overexpression enhanced STAT1/3 phosphorylation and increased Mcl-1 expression, which was abrogated by ruxolitinib. The central role of Mcl-1 was strengthened by the resistance of Mcl-1 overexpressing cells to apoptosis induction. The clinical relevance of Mcl-1 regulation by MACC1 was supported by their positive expression correlation in patient-derived tumors. Altogether, we reveal a novel death receptor-mediated apoptosis regulatory mechanism by MACC1 in solid cancers through modulation of the STAT1/3-Mcl-1 axis. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Localization and role of NPC1L1 in cholesterol absorption in human intestine.

    PubMed

    Sané, Alain Théophile; Sinnett, Daniel; Delvin, Edgard; Bendayan, Moise; Marcil, Valérie; Ménard, Daniel; Beaulieu, Jean-François; Levy, Emile

    2006-10-01

    Recent studies have documented the presence of Niemann-Pick C1-Like 1 (NPC1L1) in the small intestine and its capacity to transport cholesterol in mice and rats. The current investigation was undertaken to explore the localization and function of NPC1L1 in human enterocytes. Cell fractionation experiments revealed an NPC1L1 association with apical membrane of the enterocyte in human jejunum. Signal was also detected in lysosomes, endosomes, and mitochondria. Confirmation of cellular NPC1L1 distribution was obtained by immunocytochemistry. Knockdown of NPC1L1 caused a decline in the ability of Caco-2 cells to capture micellar [(14)C]free cholesterol. Furthermore, this NPC1L1 suppression resulted in increased and decreased mRNA levels and activity of HMG-CoA reductase, the rate-limiting step in cholesterol synthesis, and of ACAT, the key enzyme in cholesterol esterification, respectively. An increase was also noted in the transcriptional factor sterol-regulatory element binding protein that modulates cholesterol homeostasis. Efforts were devoted to define the impact of NPC1L1 knockdown on other mediators of cholesterol uptake. RT-PCR evidence is presented to show the significant decrease in the levels of scavenger receptor class B type I (SR-BI) with no changes in ABCA1, ABCG5, and cluster determinant 36 in NPC1L1-deficient Caco-2 cells. Together, our data suggest that NPC1L1 contributes to intestinal cholesterol homeostasis and possibly cooperates with SR-BI to mediate cholesterol absorption in humans.

  16. N-retinylidene-phosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR).

    PubMed

    Beharry, Seelochan; Zhong, Ming; Molday, Robert S

    2004-12-24

    ABCA4, a member of the family of ATP binding cassette (ABC) proteins found in rod and cone photoreceptors, has been implicated in the transport of retinoid compounds across the outer segment disk membrane following the photoactivation of rhodopsin. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy and related retinal degenerative diseases that cause a loss in vision. To identify the retinoid substrate that interacts with ABCA4, we have isolated ABCA4 from rod outer segment disk membranes on an immunoaffinity matrix and analyzed retinoid compounds that bind to ABCA4 using high performance liquid chromatography and radiolabeling methods. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, approximately 0.9 mol of N-retinylidene-phosphatidylethanolamine and 0.3 mol of all-trans-retinal were bound per mol of ABCA4 with an apparent K(d) of 2-5 microm. ATP and GTP released these retinoids from ABCA4, whereas ADP, GDP, and nonhydrolyzable derivatives, adenosine 5'-(beta,gamma-imido)triphosphate and guanosine 5'-(beta,gamma-imido)triphosphate, were ineffective. One mole of N-retinyl-phosphatidylethanolamine, the reduced form of N-retinylidene-phosphatidylethanolamine, bound per mol of ABCA4, whereas 0.3 mol of all-trans-retinal were bound in the absence of phosphatidylethanolamine. No binding of all-trans-retinol to ABCA4 was observed. Our results indicate that ABCA4 preferentially binds N-retinylidene-phosphatidylethanolamine with high affinity in the absence of ATP. Our studies further suggest that ATP binding and hydrolysis induces a protein conformational change that causes N-retinylidene-phosphatidylethanolamine to dissociate from ABCA4.

  17. Clinical and genetic analyses reveal novel pathogenic ABCA4 mutations in Stargardt disease families

    PubMed Central

    Lin, Bing; Cai, Xue-Bi; Zheng, Zhi-Li; Huang, Xiu-Feng; Liu, Xiao-Ling; Qu, Jia; Jin, Zi-Bing

    2016-01-01

    Stargardt disease (STGD1) is a juvenile macular degeneration predominantly inherited in an autosomal recessive pattern, characterized by decreased central vision in the first 2 decades of life. The condition has a genetic basis due to mutation in the ABCA4 gene, and arises from the deposition of lipofuscin-like substance in the retinal pigmented epithelium (RPE) with secondary photoreceptor cell death. In this study, we describe the clinical and genetic features of Stargardt patients from four unrelated Chinese cohorts. The targeted exome sequencing (TES) was carried out in four clinically confirmed patients and their family members using a gene panel comprising 164 known causative inherited retinal dystrophy (IRD) genes. Genetic analysis revealed eight ABCA4 mutations in all of the four pedigrees, including six mutations in coding exons and two mutations in adjacent intronic areas. All the affected individuals showed typical manifestations consistent with the disease phenotype. We disclose two novel ABCA4 mutations in Chinese patients with STGD disease, which will expand the existing spectrum of disease-causing variants and will further aid in the future mutation screening and genetic counseling, as well as in the understanding of phenotypic and genotypic correlations. PMID:27739528

  18. Jab1 Mediates Protein Degradation of Rad9/Rad1/Hus1 Checkpoint Complex

    PubMed Central

    Huang, Jin; Yuan, Honglin; Lu, Chongyuan; Liu, Ximeng; Cao, Xu; Wan, Mei

    2009-01-01

    Summary The Rad1-Rad9-Hus1 (9-1-1) complex serves a dual role as a DNA-damage sensor in checkpoint signaling and as a mediator in DNA repair pathway. However, the intercellular mechanisms that regulate 9-1-1 complex are poorly understood. Jab1, the fifth component of the COP9 signalosome complex, plays a central role in the degradation of multiple proteins and is emerging as an important regulator in cancer development. Here, we tested the hypothesis that Jab1 controls the protein stability of the 9-1-1 complex via the proteosome pathway. We provide evidence that Jab1 physically associates with the 9-1-1 complex. This association is mediated through direct interaction between Jab1 and Rad1, one of the subunits of 9-1-1 complex. Importantly, Jab1 causes the translocation of the 9-1-1 complex from the nucleus to the cytoplasm, mediating rapid degradation of the 9-1-1 complex via 26S proteasome. Furthermore, Jab1 significantly suppresses checkpoint signaling activation, DNA synthesis recovery from blockage and cell viability after replication stresses such as UV exposure, γ radiation and hydroxyurea treatment. These results suggest that Jab1 is an important regulator for 9-1-1 protein stability control in cells, which may provide novel information on the involvement of Jab1 in checkpoint and DNA repair signaling in response to DNA damage. PMID:17583730

  19. Synonymous ABCA3 Variants Do Not Increase Risk for Neonatal Respiratory Distress Syndrome

    PubMed Central

    Wambach, Jennifer A.; Wegner, Daniel J.; Heins, Hillary B.; Druley, Todd E.; Mitra, Robi D.; Hamvas, Aaron; Cole, F. Sessions

    2014-01-01

    Objective To determine whether synonymous variants in the adenosine triphosphate-binding cassette A3 transporter (ABCA3) gene increase the risk for neonatal respiratory distress syndrome (RDS) in term and late preterm infants of European and African descent. Study design Using next-generation pooled sequencing of race-stratified DNA samples from infants of European and African descent at $34 weeks gestation with and without RDS (n = 503), we scanned all exons of ABCA3, validated each synonymous variant with an independent genotyping platform, and evaluated race-stratified disease risk associated with common synonymous variants and collapsed frequencies of rare synonymous variants. Results The synonymous ABCA3 variant frequency spectrum differs between infants of European descent and those of African descent. Using in silico prediction programs and statistical strategies, we found no potentially disruptive synonymous ABCA3 variants or evidence of selection pressure. Individual common synonymous variants and collapsed frequencies of rare synonymous variants did not increase disease risk in term and late-preterm infants of European or African descent. Conclusion In contrast to rare, nonsynonymous ABCA3 mutations, synonymous ABCA3 variants do not increase the risk for neonatal RDS among term and late-preterm infants of European or African descent. PMID:24657120

  20. Melanocortin 1 Receptor Deficiency Promotes Atherosclerosis in Apolipoprotein E-/- Mice.

    PubMed

    Rinne, Petteri; Kadiri, James J; Velasco-Delgado, Mauricio; Nuutinen, Salla; Viitala, Miro; Hollmén, Maija; Rami, Martina; Savontaus, Eriika; Steffens, Sabine

    2018-02-01

    The MC1-R (melanocortin 1 receptor) is expressed by monocytes and macrophages where it mediates anti-inflammatory actions. MC1-R also protects against macrophage foam cell formation primarily by promoting cholesterol efflux through the ABCA1 (ATP-binding cassette transporter subfamily A member 1) and ABCG1 (ATP-binding cassette transporter subfamily G member 1). In this study, we aimed to investigate whether global deficiency in MC1-R signaling affects the development of atherosclerosis. Apoe -/- (apolipoprotein E deficient) mice were crossed with recessive yellow (Mc1r e/e ) mice carrying dysfunctional MC1-R and fed a high-fat diet to induce atherosclerosis. Apoe -/- Mc1r e/e mice developed significantly larger atherosclerotic lesions in the aortic sinus and in the whole aorta compared with Apoe -/- controls. In terms of plaque composition, MC1-R deficiency was associated with less collagen and smooth muscle cells and increased necrotic core, indicative of more vulnerable lesions. These changes were accompanied by reduced Abca1 and Abcg1 expression in the aorta. Furthermore, Apoe -/- Mc1r e/e mice showed a defect in bile acid metabolism that aggravated high-fat diet-induced hypercholesterolemia and hepatic lipid accumulation. Flow cytometric analysis of leukocyte profile revealed that dysfunctional MC1-R enhanced arterial accumulation of classical Ly6C high monocytes and macrophages, effects that were evident in mice fed a normal chow diet but not under high-fat diet conditions. In support of enhanced arterial recruitment of Ly6C high monocytes, these cells had increased expression of L-selectin and P-selectin glycoprotein ligand 1. The present study highlights the importance of MC1-R in the development of atherosclerosis. Deficiency in MC1-R signaling exacerbates atherosclerosis by disturbing cholesterol handling and by increasing arterial monocyte accumulation. © 2017 The Authors.

  1. microRNA-212 promotes lipid accumulation and attenuates cholesterol efflux in THP-1 human macrophages by targeting SIRT1.

    PubMed

    Miao, Haiwei; Zeng, Honghui; Gong, Hui

    2018-02-15

    Macrophage foam cell formation is a key initiating event in the pathogenesis of atherosclerosis. This work was conducted to determine the role of microRNA (miR)-212 in the transformation of foam cells from macrophages. We examined the expression of miR-212 in atherosclerotic lesions in an apoE-deficient (apoE -/- ) mouse model. The effects of miR-212 overexpression and knockdown on lipid accumulation and cholesterol homeostasis in THP-1 macrophages after exposure to oxidized low-density lipoprotein (oxLDL). The mechanism underlying the activity of miR-212 was explored. It was found that miR-212 was downregulated in atherosclerotic lesions and macrophages from apoE -/- mice fed high-fat diet, compared to the equivalents from apoE -/- mice fed standard diet. Overexpression of miR-212 promoted lipid accumulation in oxLDL-treated THP-1 macrophages, whereas miR-212 depletion exerted an opposite effect. Macrophage cholesterol efflux to apolipoprotein A-I was significantly reduced by miR-212, which was accompanied by reduced ABCA1 expression. Mechanistically, miR-212 targeted sirtuin 1 (SIRT1) to repress the expression of ABCA1 in THP-1 macrophages. Rescue experiments confirmed that co-expression of SIRT1 attenuated lipid accumulation and restored cholesterol efflux in miR-212-overexpressing THP-1 macrophages. Collectively, miR-212 facilitates macrophage foam cell formation and suppresses ABCA1-dependent cholesterol efflux through downregulation of SIRT1. Targeting miR-212 may provide a potential therapeutic strategy for atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Effect of sulfonylurea agents on reverse cholesterol transport in vitro and vivo.

    PubMed

    Terao, Yoshio; Ayaori, Makoto; Ogura, Masatsune; Yakushiji, Emi; Uto-Kondo, Harumi; Hisada, Tetsuya; Ozasa, Hideki; Takiguchi, Shunichi; Nakaya, Kazuhiro; Sasaki, Makoto; Komatsu, Tomohiro; Iizuka, Maki; Horii, Shunpei; Mochizuki, Seibu; Yoshimura, Michihiro; Ikewaki, Katsunori

    2011-01-01

    Reverse cholesterol transport (RCT) is a critical mechanism for the anti-atherogenic property of HDL. The inhibitory effect of the sulfonylurea agent (SUA) glibenclamide on ATP binding-cassette transporter (ABC) A1 may decrease HDL function but it remains unclear whether it attenuates RCT in vivo. We therefore investigated how the SUAs glibenclamide and glimepiride affected the functionality of ABCA1/ABCG1 and scavenger receptor class B type I (SR-BI) expression in macrophages in vitro and overall RCT in vivo. RAW264.7, HEK293 and BHK-21 cells were used for in vitro studies. To investigate RCT in vivo, 3H-cholesterol-labeled and acetyl LDL-loaded RAW264.7 cells were injected into mice. High dose (500µM) of glibenclamide inhibited ABCA1 function and apolipoprotein A-I (apoA-I)-mediated cholesterol efflux, and attenuated ABCA1 expression. Although glimepiride maintained apoA-I-mediated cholesterol efflux from RAW264.7 cells, like glibenclamide, it inhibited ABCA1-mediated cholesterol efflux from transfected HEK293 cells. Similarly, the SUAs inhibited SR-BI-mediated cholesterol efflux from transfected BHK-21 cells. High doses of SUAs increased ABCG1 expression in RAW264.7 cells, promoting HDL-mediated cholesterol efflux in an ABCG1-independent manner. Low doses (0.1-100 µM) of SUAs did not affect cholesterol efflux from macrophages despite dose-dependent increases in ABCA1/G1 expression. Furthermore, they did not change RCT or plasma lipid levels in mice. High doses of SUAs inhibited the functionality of ABCA1/SR-BI, but not ABCG1. At lower doses, they had no unfavorable effects on cholesterol efflux or overall RCT in vivo. These results indicate that SUAs do not have adverse effects on atherosclerosis contrary to previous findings for glibenclamide.

  3. Atheroprotective effects of methotrexate on reverse cholesterol transport proteins and foam cell transformation in human THP-1 monocyte/macrophages.

    PubMed

    Reiss, Allison B; Carsons, Steven E; Anwar, Kamran; Rao, Soumya; Edelman, Sari D; Zhang, Hongwei; Fernandez, Patricia; Cronstein, Bruce N; Chan, Edwin S L

    2008-12-01

    To determine whether methotrexate (MTX) can overcome the atherogenic effects of cyclooxygenase 2 (COX-2) inhibitors and interferon-gamma (IFNgamma), both of which suppress cholesterol efflux protein and promote foam cell transformation in human THP-1 monocyte/macrophages. Message and protein levels of the reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFNgamma, with and without MTX. Foam cell transformation of lipid-laden THP-1 macrophages was detected with oil red O staining and light microscopy. MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean +/- SEM 112.8 +/- 13.1% for NS398 plus MTX versus 71.1 +/- 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor-mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A2A receptor-specific antagonist ZM241385. MTX also prevented NS398 and IFNgamma from increasing transformation of lipid-laden THP-1 macrophages into foam cells. This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall.

  4. RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation.

    PubMed

    Lin, Juan; Kumari, Snehlata; Kim, Chun; Van, Trieu-My; Wachsmuth, Laurens; Polykratis, Apostolos; Pasparakis, Manolis

    2016-12-01

    Receptor-interacting protein kinase 1 (RIPK1) regulates cell death and inflammation through kinase-dependent and -independent functions. RIPK1 kinase activity induces caspase-8-dependent apoptosis and RIPK3 and mixed lineage kinase like (MLKL)-dependent necroptosis. In addition, RIPK1 inhibits apoptosis and necroptosis through kinase-independent functions, which are important for late embryonic development and the prevention of inflammation in epithelial barriers. The mechanism by which RIPK1 counteracts RIPK3-MLKL-mediated necroptosis has remained unknown. Here we show that RIPK1 prevents skin inflammation by inhibiting activation of RIPK3-MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM1). ZBP1 deficiency inhibited keratinocyte necroptosis and skin inflammation in mice with epidermis-specific RIPK1 knockout. Moreover, mutation of the conserved RIP homotypic interaction motif (RHIM) of endogenous mouse RIPK1 (RIPK1 mRHIM ) caused perinatal lethality that was prevented by RIPK3, MLKL or ZBP1 deficiency. Furthermore, mice expressing only RIPK1 mRHIM in keratinocytes developed skin inflammation that was abrogated by MLKL or ZBP1 deficiency. Mechanistically, ZBP1 interacted strongly with phosphorylated RIPK3 in cells expressing RIPK1 mRHIM , suggesting that the RIPK1 RHIM prevents ZBP1 from binding and activating RIPK3. Collectively, these results show that RIPK1 prevents perinatal death as well as skin inflammation in adult mice by inhibiting ZBP1-induced necroptosis. Furthermore, these findings identify ZBP1 as a critical mediator of inflammation beyond its previously known role in antiviral defence and suggest that ZBP1 might be implicated in the pathogenesis of necroptosis-associated inflammatory diseases.

  5. Overlap between Parkinson disease and Alzheimer disease in ABCA7 functional variants

    PubMed Central

    Nuytemans, Karen; Maldonado, Lizmarie; Ali, Aleena; John-Williams, Krista; Beecham, Gary W.; Martin, Eden; Scott, William K.

    2016-01-01

    Objective: Given their reported function in phagocytosis and clearance of protein aggregates in Alzheimer disease (AD), we hypothesized that variants in ATP-binding cassette transporter A7 (ABCA7) might be involved in Parkinson disease (PD). Methods: ABCA7 variants were identified using whole-exome sequencing (WES) on 396 unrelated patients with PD and 222 healthy controls. In addition, we used the publicly available WES data from the Parkinson's Progression Markers Initiative (444 patients and 153 healthy controls) as a second, independent data set. Results: We observed a higher frequency of loss-of-function (LOF) variants and rare putative highly functional variants (Combined Annotation Dependent Depletion [CADD] >20) in clinically diagnosed patients with PD than in healthy controls in both data sets. Overall, we identified LOF variants in 11 patients and 1 healthy control (odds ratio [OR] 4.94, Fisher exact p = 0.07). Four of these variants have been previously implicated in AD risk (p.E709AfsX86, p.W1214X, p.L1403RfsX7, and rs113809142). In addition, rare variants with CADD >20 were observed in 19 patients vs 3 healthy controls (OR 2.85, Fisher exact p = 0.06). Conclusion: The presence of ABCA7 LOF variants in clinically defined PD suggests that they might be risk factors for neurodegeneration in general, especially those variants hallmarked by protein aggregation. More studies will be needed to evaluate the overall impact of this transporter in neurodegenerative disease. PMID:27066581

  6. Targeted inactivation of the murine Abca3 gene leads to respiratory failure in newborns with defective lamellar bodies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hammel, Markus; Michel, Geert; Hoefer, Christina

    2007-08-10

    Mutations in the human ABCA3 gene, encoding an ABC-transporter, are associated with respiratory failure in newborns and pediatric interstitial lung disease. In order to study disease mechanisms, a transgenic mouse model with a disrupted Abca3 gene was generated by targeting embryonic stem cells. While heterozygous animals developed normally and were fertile, individuals homozygous for the altered allele (Abca3-/-) died within one hour after birth from respiratory failure, ABCA3 protein being undetectable. Abca3-/- newborns showed atelectasis of the lung in comparison to a normal gas content in unaffected or heterozygous littermates. Electron microscopy demonstrated the absence of normal lamellar bodies inmore » type II pneumocytes. Instead, condensed structures with apparent absence of lipid content were found. We conclude that ABCA3 is required for the formation of lamellar bodies and lung surfactant function. The phenotype of respiratory failure immediately after birth corresponds to the clinical course of severe ABCA3 mutations in human newborns.« less

  7. Atheroprotective Effects of Methotrexate on Reverse Cholesterol Transport Proteins and Foam Cell Transformation in THP-1 Human Monocytes/Macrophages

    PubMed Central

    Reiss, Allison B.; Carsons, Steven E.; Anwar, Kamran; Rao, Soumya; Edelman, Sari D.; Zhang, Hongwei; Fernandez, Patricia; Cronstein, Bruce N.; Chan, Edwin S.L.

    2008-01-01

    OBJECTIVE: To determine whether MTX can overcome the atherogenic effect of COX-2 inhibitors and IFN-γ, both of which suppress cholesterol efflux protein levels and promote foam cell transformation in THP-1 human monocytes/macrophages. METHODS: Message and protein level of the reverse cholesterol transport (RCT) proteins cholesterol 27-hydroxylase and ABCA1 in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFN-γ with/without MTX. Foam cell transformation of lipid-loaded THP-1 macrophages was detected with oil red O staining and light microscopy. RESULTS: MTX increased 27-hydroxylase message and completely blocked NS398-induced downregulation of 27-hydroxylase (112.8±13.1% for NS398+MTX versus 71.1±4.3% for NS398 alone, with untreated as 100%, n=3, p<0.01). MTX also negated COX-2 inhibitor-mediated downregulation of ABCA1. Reversal of inhibitory effects on 27-hydroxylase and ABCA1 in the presence of MTX were blocked by the adenosine A2A receptor-specific antagonist ZM-241385. MTX also prevented NS398 and IFN-γ from increasing transformation of lipid-loaded THP-1 macrophages into foam cells. CONCLUSIONS: This study provides evidence supporting the atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes RCT and limits foam cell formation in THP-1 macrophages. This is the first evidence that any commonly used medication can increase expression of anti-atherogenic RCT proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular mortality in RA patients is through facilitation of cholesterol outflow from cells of the artery wall. PMID:19035488

  8. Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease

    PubMed Central

    Braun, Terry A.; Mullins, Robert F.; Wagner, Alex H.; Andorf, Jeaneen L.; Johnston, Rebecca M.; Bakall, Benjamin B.; Deluca, Adam P.; Fishman, Gerald A.; Lam, Byron L.; Weleber, Richard G.; Cideciyan, Artur V.; Jacobson, Samuel G.; Sheffield, Val C.; Tucker, Budd A.; Stone, Edwin M.

    2013-01-01

    Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10−6). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing. PMID:23918662

  9. Increased Risk of Interstitial Lung Disease in Children with a Single R288K Variant of ABCA3

    PubMed Central

    Wittmann, Thomas; Frixel, Sabrina; Höppner, Stefanie; Schindlbeck, Ulrike; Schams, Andrea; Kappler, Matthias; Hegermann, Jan; Wrede, Christoph; Liebisch, Gerhard; Vierzig, Anne; Zacharasiewicz, Angela; Kopp, Matthias Volkmar; Poets, Christian F; Baden, Winfried; Hartl, Dominik; van Kaam, Anton H; Lohse, Peter; Aslanidis, Charalampos; Zarbock, Ralf; Griese, Matthias

    2016-01-01

    The ABCA3 gene encodes a lipid transporter in type II pneumocytes critical for survival and normal respiratory function. The frequent ABCA3 variant R288K increases the risk for neonatal respiratory distress syndrome among term and late preterm neonates, but its role in children’s interstitial lung disease has not been studied in detail. In a retrospective cohort study of 228 children with interstitial lung disease related to the alveolar surfactant system, the frequency of R288K was assessed and the phenotype of patients carrying a single R288K variant further characterized by clinical course, lung histology, computed tomography and bronchoalveolar lavage phosphatidylcholine PC 32:0. Cell lines stably transfected with ABCA3-R288K were analyzed for intracellular transcription, processing and targeting of the protein. ABCA3 function was assessed by detoxification assay of doxorubicin, and the induction and volume of lamellar bodies. We found nine children with interstitial lung disease carrying a heterozygous R288K variant, a frequency significantly higher than in the general Caucasian population. All identified patients had neonatal respiratory insufficiency, recovered and developed chronic interstitial lung disease with intermittent exacerbations during early childhood. In vitro analysis showed normal transcription, processing, and targeting of ABCA3-R288K, but impaired detoxification function and smaller lamellar bodies. We propose that the R288K variant can underlie interstitial lung disease in childhood due to reduced function of ABCA3, demonstrated by decelerated detoxification of doxorubicin, reduced PC 32:0 content and decreased lamellar body volume. PMID:26928390

  10. Phenotype/genotype correlation in a case series of Stargardt's patients identifies novel mutations in the ABCA4 gene.

    PubMed

    Gemenetzi, M; Lotery, A J

    2013-11-01

    To investigate phenotypic variability in terms of best-corrected visual acuity (BCVA) in patients with Stargardt disease (STGD) and confirmed ABCA4 mutations. Entire coding region analysis of the ABCA4 gene by direct sequencing of seven patients with clinical findings of STGD seen in the Retina Clinics of Southampton Eye Unit between 2002 and 2011.Phenotypic variables recorded were BCVA, fluorescein angiographic appearance, electrophysiology, and visual fields. All patients had heterozygous amino acid-changing variants (missense mutations) in the ABCA4 gene. A splice sequence change was found in a 30-year-old patient with severly affected vision. Two novel sequence changes were identified: a missense mutation in a mildly affected 44-year-old patient and a frameshift mutation in a severly affected 34-year-old patient. The identified ABCA4 mutations were compatible with the resulting phenotypes in terms of BCVA. Higher BCVAs were recorded in patients with missense mutations. Sequence changes, predicted to have more deleterious effect on protein function, resulted in a more severe phenotype. This case series of STGD patients demonstrates novel genotype/phenotype correlations, which may be useful to counselling of patients. This information may prove useful in selection of candidates for clinical trials in ABCA4 disease.

  11. Multiple Rap1 effectors control Epac1-mediated tightening of endothelial junctions.

    PubMed

    Pannekoek, Willem-Jan; Vliem, Marjolein J; Bos, Johannes L

    2018-02-17

    Epac1 and Rap1 mediate cAMP-induced tightening of endothelial junctions. We have previously found that one of the mechanisms is the inhibition of Rho-mediated tension in radial stress fibers by recruiting the RhoGAP ArhGAP29 in a complex containing the Rap1 effectors Rasip1 and Radil. However, other mechanisms have been proposed as well, most notably the induction of tension in circumferential actin cables by Cdc42 and its GEF FGD5. Here, we have investigated how Rap1 controls FGD5/Cdc42 and how this interconnects with Radil/Rasip1/ArhGAP29. Using endothelial barrier measurements, we show that Rho inhibition is not sufficient to explain the barrier stimulating effect of Rap1. Indeed, Cdc42-mediated tension is induced at cell-cell contacts upon Rap1 activation and this is required for endothelial barrier function. Depletion of potential Rap1 effectors identifies AF6 to mediate Rap1 enhanced tension and concomitant Rho-independent barrier function. When overexpressed in HEK293T cells, AF6 is found in a complex with FGD5 and Radil. From these results we conclude that Rap1 utilizes multiple pathways to control tightening of endothelial junctions, possibly through a multiprotein effector complex, in which AF6 functions to induce tension in circumferential actin cables.

  12. Retinal-specific ATP-binding cassette transporter (ABCR/ABCA4) is expressed at the choroid plexus in rat brain.

    PubMed

    Bhongsatiern, Jiraganya; Ohtsuki, Sumio; Tachikawa, Masanori; Hori, Satoko; Terasaki, Tetsuya

    2005-03-01

    ATP-binding cassette (ABC) transporter A4 is a member of the ABC transporter subfamily A which has been reported to be exclusively expressed in the retina. In contrast, a previous report has suggested a possible relationship between ABCA4 and CNS function. The purpose of the present study was to investigate the localization of ABCA4 mRNA and protein in rat brain. In situ hybridization analysis revealed that ABCA4 mRNA was localized in the lateral ventricles. RT-PCR analysis detected ABCA4 mRNA in isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB). Furthermore, ABCA4 protein was also detected in the isolated rat choroid plexus at about 250 kDa by western blot analysis, and its apparent molecular size was reduced by N-glycosidase F treatment. These results suggest that glycosylated ABCA4 protein is expressed in rat choroid plexus epithelial cells. ABCA4 may play a role in the function of the blood-cerebrospinal fluid barrier and affect CSF conditions.

  13. Molecular and cellular characteristics of ABCA3 mutations associated with diffuse parenchymal lung diseases in children

    PubMed Central

    Flamein, Florence; Riffault, Laure; Muselet-Charlier, Céline; Pernelle, Julie; Feldmann, Delphine; Jonard, Laurence; Durand-Schneider, Anne-Marie; Coulomb, Aurore; Maurice, Michèle; Nogee, Lawrence M.; Inagaki, Nobuya; Amselem, Serge; Dubus, Jean Christophe; Rigourd, Virginie; Brémont, François; Marguet, Christophe; Brouard, Jacques; de Blic, Jacques; Clement, Annick; Epaud, Ralph; Guillot, Loïc

    2012-01-01

    ABCA3 (ATP-binding cassette subfamily A, member 3) is expressed in the lamellar bodies of alveolar type II cells and is crucial to pulmonary surfactant storage and homeostasis. ABCA3 gene mutations have been associated with neonatal respiratory distress (NRD) and pediatric interstitial lung disease (ILD). The objective of this study was to look for ABCA3 gene mutations in patients with severe NRD and/or ILD. The 30 ABCA3 coding exons were screened in 47 patients with severe NRD and/or ILD. ABCA3 mutations were identified in 10 out of 47 patients, including 2 homozygous, 5 compound heterozygous and 3 heterozygous patients. SP-B and SP-C expression patterns varied across patients. Among patients with ABCA3 mutations, five died shortly after birth and five developed ILD (including one without NRD). Functional studies of p.D253H and p.T1173R mutations revealed that p.D253H and p.T1173R induced abnormal lamellar bodies. Additionally, p.T1173R increased IL-8 secretion in vitro. In conclusion, we identified new ABCA3 mutations in patients with life-threatening NRD and/or ILD. Two mutations associated with ILD acted via different pathophysiological mechanisms despite similar clinical phenotypes. PMID:22068586

  14. Proximal tubule sphingosine kinase-1 has a critical role in A1 adenosine receptor-mediated renal protection from ischemia

    PubMed Central

    Park, Sang Won; Kim, Mihwa; Kim, Joo Yun; Brown, Kevin M.; Haase, Volker H.; D’Agati, Vivette D.; Lee, H. Thomas

    2012-01-01

    Renal ischemia reperfusion injury is a major cause of acute kidney injury. We previously found that renal A1 adenosine receptor (A1AR) activation attenuated multiple cell death pathways including necrosis, apoptosis and inflammation. Here, we tested whether induction of cytoprotective sphingosine kinase (SK)-1 and sphingosine-1 phosphate (S1P) synthesis might be the mechanism of protection. A selective A1AR agonist (CCPA) increased the synthesis of S1P and selectively induced SK-1 in mouse kidney and HK-2 cells. This agonist failed to protect SK1-knockout but protected SK2-knockout mice against renal ischemia reperfusion injury indicating a critical role of SK1 in A1AR-mediated renal protection. Inhibition of SK prevented A1AR-mediated defense against necrosis and apoptosis in HK-2 cells. A selective S1P1R antagonist (W146) and global in vivo gene knockdown of S1P1Rs with small interfering RNA completely abolished the renal protection provided by CCPA. Mice selectively deficient in renal proximal tubule S1P1Rs (S1P1Rflox/flox PEPCKCre/−) were not protected against renal ischemia reperfusion injury by CCPA. Mechanistically, CCPA increased nuclear translocation of hypoxia inducible factor-1α in HK-2 cells and selective hypoxia inducible factor-1α inhibition blocked A1AR-mediated induction of SK1. Thus, proximal tubule SK-1 has a critical role in A1AR-mediated protection against renal ischemia reperfusion injury. PMID:22695326

  15. Kimchi methanol extract and the kimchi active compound, 3'-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid, downregulate CD36 in THP-1 macrophages stimulated by oxLDL.

    PubMed

    Yun, Ye-Rang; Kim, Hyun-Ju; Song, Yeong-Ok

    2014-08-01

    Macrophage foam cell formation by oxidized low-density lipoprotein (oxLDL) is a key step in the progression of atherosclerosis, which is involved in cholesterol influx and efflux in macrophages mediated by related proteins such as peroxisome proliferator-activated receptor γ (PPARγ), CD36, PPARα, liver-X receptor α (LXRα), and ATP-binding cassette transporter A1 (ABCA1). The aim of this study was to investigate the beneficial effects of kimchi methanol extract (KME) and a kimchi active compound, 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA) on cholesterol flux in THP-1-derived macrophages treated with oxLDL. The effects of KME and HDMPPA on cell viability and lipid peroxidation were determined. Furthermore, the protein expression of PPARγ, CD36, PPARα, LXRα, and ABCA1 was examined. OxLDL strongly induced cell death and lipid peroxidation in THP-1-derived macrophages. However, KME and HDMPPA significantly improved cell viability and inhibited lipid peroxidation induced by oxLDL in THP-1-derived macrophages (P<.05). Moreover, KME and HDMPPA suppressed CD36 and PPARγ expressions, both of which participate in cholesterol influx. In contrast, KME and HDMPPA augmented LXRα, PPARα, and ABCA1 expression, which are associated with cholesterol efflux. Consequently, KME and HDMPPA suppressed lipid accumulation. These results indicate that KME and HDMPPA may inhibit lipid accumulation, in part, by regulating cholesterol influx- and efflux-related proteins. These findings will thus be useful for future prevention strategies against atherosclerosis.

  16. Estimation of Indirect Effects in the Presence of Unmeasured Confounding for the Mediator-Outcome Relationship in a Multilevel 2-1-1 Mediation Model

    ERIC Educational Resources Information Center

    Talloen, Wouter; Moerkerke, Beatrijs; Loeys, Tom; De Naeghel, Jessie; Van Keer, Hilde; Vansteelandt, Stijn

    2016-01-01

    To assess the direct and indirect effect of an intervention, multilevel 2-1-1 studies with intervention randomized at the upper (class) level and mediator and outcome measured at the lower (student) level are frequently used in educational research. In such studies, the mediation process may flow through the student-level mediator (the within…

  17. Interleukin-1 receptor (IL-1R) mediates epilepsy-induced sleep disruption.

    PubMed

    Huang, Tzu-Rung; Jou, Shuo-Bin; Chou, Yu-Ju; Yi, Pei-Lu; Chen, Chun-Jen; Chang, Fang-Chia

    2016-11-22

    Sleep disruptions are common in epilepsy patients. Our previous study demonstrates that homeostatic factors and circadian rhythm may mediate epilepsy-induced sleep disturbances when epilepsy occurs at different zeitgeber hours. The proinflammatory cytokine, interleukin-1 (IL-1), is a somnogenic cytokine and may also be involved in epileptogenesis; however, few studies emphasize the effect of IL-1 in epilepsy-induced sleep disruption. We herein hypothesized that IL-1 receptor type 1 (IL-1R1) mediates the pathogenesis of epilepsy and epilepsy-induced sleep disturbances. We determined the role of IL-1R1 by using IL-1R1 knockout (IL-1R1 -/- KO) mice. Our results elucidated the decrease of non-rapid eye movement (NREM) sleep during the light period in IL-1R -/- mice and confirmed the somnogenic role of IL-1R1. Rapid electrical amygdala kindling was performed to induce epilepsy at the particular zeitgeber time (ZT) point, ZT13. Our results demonstrated that seizure thresholds induced by kindling stimuli, such as the after-discharge threshold and successful kindling rates, were not altered in IL-1R -/- mice when compared to those obtained from the wildtype mice (IL-1R +/+ mice). This result suggests that IL-1R1 is not involved in kindling-induced epileptogenesis. During sleep, ZT13 kindling stimulation significantly enhanced NREM sleep during the subsequent 6 h (ZT13-18) in wildtype mice, and sleep returned to the baseline the following day. However, the kindling-induced sleep alteration was absent in the IL-1R -/- KO mice. These results indicate that the IL-1 signal mediates epilepsy-induced sleep disturbance, but dose not participate in kindling-induced epileptogenesis.

  18. Macular Pigment and Lutein Supplementation in ABCA4-associated Retinal Degenerations

    PubMed Central

    Aleman, Tomas S.; Cideciyan, Artur V.; Windsor, Elizabeth A. M.; Schwartz, Sharon B.; Swider, Malgorzata; Chico, John D.; Sumaroka, Alexander; Pantelyat, Alexander Y.; Duncan, Keith G.; Gardner, Leigh M.; Emmons, Jessica M.; Steinberg, Janet D.; Stone, Edwin M.; Jacobson, Samuel G.

    2008-01-01

    PURPOSE To determine macular pigment (MP) optical density (OD) in patients with ABCA4-associated retinal degenerations (ABCA4-RD) and the response of MP and vision to supplementation with lutein. METHODS Stargardt disease or cone-rod dystrophy patients with foveal fixation and with known or suspected disease-causing mutations in the ABCA4 gene were included. MPOD profiles were measured with heterochromatic flicker photometry. Serum carotenoids, visual acuity, foveal sensitivity and retinal thickness were quantified. Changes in MPOD and central vision were determined in a subset of patients receiving oral supplementation with lutein for 6 months. RESULTS MPOD in patients ranged from normal to markedly abnormal. As a group, ABCA4-RD patients had reduced foveal MPOD and there was strong correlation with retinal thickness. Average foveal tissue concentration of MP, estimated by dividing MPOD by retinal thickness, was normal in patients whereas serum concentration of lutein and zeaxanthin was significantly lower than normal. After oral lutein supplementation for 6 months, 91% of the patients showed significant increases in serum lutein and 63% of the patient eyes showed a significant augmentation in MPOD. The retinal responders tended to be female, and have lower serum lutein and zeaxanthin, lower MPOD and greater retinal thickness at baseline. Responding eyes had significantly lower baseline MP concentration compared to non-responding eyes. Central vision was unchanged after the period of supplementation. CONCLUSIONS MP is strongly affected by the stage of ABCA4 disease leading to abnormal foveal architecture. MP could be augmented by supplemental lutein in some patients. There was no change in central vision after 6 months of lutein supplementation. Long-term influences on the natural history of this supplement on macular degenerations require further study. PMID:17325179

  19. Code-Switching: L1-Coded Mediation in a Kindergarten Foreign Language Classroom

    ERIC Educational Resources Information Center

    Lin, Zheng

    2012-01-01

    This paper is based on a qualitative inquiry that investigated the role of teachers' mediation in three different modes of coding in a kindergarten foreign language classroom in China (i.e. L2-coded intralinguistic mediation, L1-coded cross-lingual mediation, and L2-and-L1-mixed mediation). Through an exploratory examination of the varying effects…

  20. Nfatc1 Is a Functional Transcriptional Factor Mediating Nell-1-Induced Runx3 Upregulation in Chondrocytes.

    PubMed

    Li, Chenshuang; Zheng, Zhong; Zhang, Xinli; Asatrian, Greg; Chen, Eric; Song, Richard; Culiat, Cymbeline; Ting, Kang; Soo, Chia

    2018-01-06

    Neural EGFL like 1 (Nell-1) is essential for chondrogenic differentiation, maturation, and regeneration. Our previous studies have demonstrated that Nell-1's pro-chondrogenic activities are predominantly reliant upon runt-related transcription factor 3 (Runx3)-mediated Indian hedgehog (Ihh) signaling. Here, we identify the nuclear factor of activated T-cells 1 (Nfatc1) as the key transcriptional factor mediating the Nell-1 → Runx3 signal transduction in chondrocytes. Using chromatin immunoprecipitation assay, we were able to determine that Nfatc1 binds to the -833--810 region of the Runx3 -promoter in response to Nell-1 treatment. By revealing the Nell-1 → Nfatc1 → Runx3 → Ihh cascade, we demonstrate the involvement of Nfatc1, a nuclear factor of activated T-cells, in chondrogenesis, while providing innovative insights into developing a novel therapeutic strategy for cartilage regeneration and other chondrogenesis-related conditions.

  1. Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

    PubMed Central

    Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

    2011-01-01

    Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

  2. HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis

    PubMed Central

    2018-01-01

    ABSTRACT Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments. IMPORTANCE Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the

  3. A mediator methylation mystery: JMJD1C demethylates MDC1 to regulate DNA repair.

    PubMed

    Lu, Jian; Matunis, Michael J

    2013-12-01

    Mediator of DNA-damage checkpoint 1 (MDMDC1) has a central role in repair of DNA double-strand breaks (DSBs) by both homologous recombination and nonhomologous end joining, and its function is regulated by post-translational phosphorylation, ubiquitylation and sumoylation. In this issue, a new study by Watanabe et al. reveals that methylation of MDMDC1 is also critical for its function in DSB repair and specifically affects repair through BRCA1-dependent homologous recombination.

  4. LDB1-mediated enhancer looping can be established independent of mediator and cohesin.

    PubMed

    Krivega, Ivan; Dean, Ann

    2017-08-21

    Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/β-globin proximity. CRISPR/Cas9 editing of the β-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/β-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/β-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the β-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  5. Platelet-derived HMGB1 is a critical mediator of thrombosis.

    PubMed

    Vogel, Sebastian; Bodenstein, Rebecca; Chen, Qiwei; Feil, Susanne; Feil, Robert; Rheinlaender, Johannes; Schäffer, Tilman E; Bohn, Erwin; Frick, Julia-Stefanie; Borst, Oliver; Münzer, Patrick; Walker, Britta; Markel, Justin; Csanyi, Gabor; Pagano, Patrick J; Loughran, Patricia; Jessup, Morgan E; Watkins, Simon C; Bullock, Grant C; Sperry, Jason L; Zuckerbraun, Brian S; Billiar, Timothy R; Lotze, Michael T; Gawaz, Meinrad; Neal, Matthew D

    2015-12-01

    Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

  6. Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint.

    PubMed

    London, Nitobe; Biggins, Sue

    2014-01-15

    The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint.

  7. The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters.

    PubMed

    Salinero, Alicia C; Knoll, Elisabeth R; Zhu, Z Iris; Landsman, David; Curcio, M Joan; Morse, Randall H

    2018-02-01

    The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting non-essential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility do not appear to effect genomic Ty1 RNA levels, Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.

  8. The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters

    PubMed Central

    Salinero, Alicia C.; Knoll, Elisabeth R.; Zhu, Z. Iris

    2018-01-01

    The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting non-essential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility do not appear to effect genomic Ty1 RNA levels, Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility. PMID:29462141

  9. U(1) mediation of flux supersymmetry breaking

    NASA Astrophysics Data System (ADS)

    Grimm, Thomas W.; Klemm, Albrecht

    2008-10-01

    We study the mediation of supersymmetry breaking triggered by background fluxes in Type II string compactifications with Script N = 1 supersymmetry. The mediation arises due to an U(1) vector multiplet coupling to both a hidden supersymmetry breaking flux sector and a visible D-brane sector. The required internal manifolds can be constructed by non-Kähler resolutions of singular Calabi-Yau manifolds. The effective action encoding the U(1) coupling is then determined in terms of the global topological properties of the internal space. We investigate suitable local geometries for the hidden and visible sector in detail. This includes a systematic study of orientifold symmetries of del Pezzo surfaces realized in compact geometries after geometric transition. We construct compact examples admitting the key properties to realize flux supersymmetry breaking and U(1) mediation. Their toric realization allows us to analyze the geometry of curve classes and confirm the topological connection between the hidden and visible sector.

  10. Mediator-regulated transcription through the +1 nucleosome.

    PubMed

    Nock, Adam; Ascano, Janice M; Barrero, Maria J; Malik, Sohail

    2012-12-28

    Many genes are regulated at the level of a Pol II that is recruited to a nucleosome-free region upstream of the +1 nucleosome. How the Mediator coactivator complex, which functions at multiple steps, affects transcription through the promoter proximal region, including this nucleosome, remains largely unaddressed. We have established a fully defined in vitro assay system to delineate mechanisms for Pol II transit across the +1 nucleosome. Our results reveal cooperative functions of multiple cofactors, particularly of Mediator and elongation factor SII, in transcribing into this nucleosome. This is achieved, in part, through an unusual activity of SII that alters the intrinsic catalytic properties of promoter-proximal Pol II and, in concert with the Mediator, leads to enhancement in transcription of nucleosomal DNA. Our data provide additional mechanistic bases for Mediator function after recruitment of Pol II and, potentially, for regulation of genes controlled via nucleosome-mediated promoter-proximal pausing. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Stargardt macular dystrophy: common ABCA4 mutations in South Africa—establishment of a rapid genetic test and relating risk to patients

    PubMed Central

    Nossek, Christel A.; Greenberg, L. Jacquie; Ramesar, Rajkumar S.

    2012-01-01

    Purpose Based on the previous indications of founder ATP-binding cassette sub-family A member 4 gene (ABCA4) mutations in a South African subpopulation, the purpose was to devise a mechanism for identifying common disease-causing mutations in subjects with ABCA4-associated retinopathies (AARs). Facilitating patient access to this data and determining the frequencies of the mutations in the South African population would enhance the current molecular diagnostic service offered. Methods The majority of subjects in this study were of Caucasian ancestry and affected with Stargardt macular dystrophy. The initial cohort consisted of DNA samples from 181 patients, and was screened using the ABCR400 chip. An assay was then designed to screen a secondary cohort of 72 patients for seven of the most commonly occurring ABCA4 mutations in this population. A total of 269 control individuals were also screened for the seven ABCA4 mutations. Results Microarray screening results from a cohort of 181 patients affected with AARs revealed that seven ABCA4 mutations (p.Arg152*, c.768G>T, p.Arg602Trp, p.Gly863Ala, p.Cys1490Tyr, c.5461–10T>C, and p.Leu2027Phe) occurred at a relatively high frequency. The newly designed genetic assay identified two of the seven disease-associated mutations in 28/72 patients in a secondary patient cohort. In the control cohort, 12/269 individuals were found to be heterozygotes, resulting in an estimated background frequency of these mutations in this particular population of 4.46 per 100 individuals. Conclusions The relatively high detection rate of seven ABCA4 mutations in the primary patient cohort led to the design and subsequent utility of a multiplex assay. This assay can be used as a viable screening tool and to reduce costs and laboratory time. The estimated background frequency of the seven ABCA4 mutations, together with the improved diagnostic service, could be used by counselors to facilitate clinical and genetic management of South African

  12. The ABCA4 2588G>C Stargardt mutation: single origin and increasing frequency from South-West to North-East Europe.

    PubMed

    Maugeri, Alessandra; Flothmann, Kris; Hemmrich, Nadine; Ingvast, Sofie; Jorge, Paula; Paloma, Eva; Patel, Reshma; Rozet, Jean-Michel; Tammur, Jaana; Testa, Francesco; Balcells, Susana; Bird, Alan C; Brunner, Han G; Hoyng, Carel B; Metspalu, Andres; Simonelli, Francesca; Allikmets, Rando; Bhattacharya, Shomi S; D'Urso, Michele; Gonzàlez-Duarte, Roser; Kaplan, Josseline; te Meerman, Gerard J; Santos, Rosário; Schwartz, Marianne; Van Camp, Guy; Wadelius, Claes; Weber, Bernhard H F; Cremers, Frans P M

    2002-03-01

    Inherited retinal dystrophies represent the most important cause of vision impairment in adolescence, affecting approximately 1 out of 3000 individuals. Mutations of the photoreceptor-specific gene ABCA4 (ABCR) are a common cause of retinal dystrophy. A number of mutations have been repeatedly reported for this gene, notably the 2588G>C mutation which is frequent in both patients and controls. Here we ascertained the frequency of the 2588G>C mutation in a total of 2343 unrelated random control individuals from 11 European countries and 241 control individuals from the US, as well as in 614 patients with STGD both from Europe and the US. We found an overall carrier frequency of 1 out of 54 in Europe, compared with 1 out of 121 in the US, confirming that the 2588G>C ABCA4 mutation is one of the most frequent autosomal recessive mutations in the European population. Carrier frequencies show an increasing gradient in Europe from South-West to North-East. The lowest carrier frequency, 0 out of 199 (0%), was found in Portugal; the highest, 11 out of 197 (5.5%), was found in Sweden. Haplotype analysis in 16 families segregating the 2588G>C mutation showed four intragenic polymorphisms invariably present in all 16 disease chromosomes and sharing of the same allele for several markers flanking the ABCA4 locus in most of the disease chromosomes. These results indicate a single origin of the 2588G>C mutation which, to our best estimate, occurred between 2400 and 3000 years ago.

  13. Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons.

    PubMed

    Kim, Jae-Ick; Ganesan, Subhashree; Luo, Sarah X; Wu, Yu-Wei; Park, Esther; Huang, Eric J; Chen, Lu; Ding, Jun B

    2015-10-02

    Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that γ-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here, we show that GABA co-release in dopamine neurons does not use the conventional GABA-synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol (EtOH) at concentrations seen in blood alcohol after binge drinking, and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction. Copyright © 2015, American Association for the Advancement of Science.

  14. Flavonoids Are Inhibitors of Human Organic Anion Transporter 1 (OAT1)–Mediated Transport

    PubMed Central

    An, Guohua; Wang, Xiaodong

    2014-01-01

    Organic anion transporter 1 (OAT1) has been reported to be involved in the nephrotoxicity of many anionic xenobiotics. As current clinically used OAT1 inhibitors are often associated with safety issues, identifying potent OAT1 inhibitors with little toxicity is of great value in reducing OAT1-mediated drug nephrotoxicity. Flavonoids are a class of polyphenolic compounds with exceptional safety records. Our objective was to evaluate the effects of 18 naturally occurring flavonoids, and some of their glycosides, on the uptake of para-aminohippuric acid (PAH) in both OAT1-expressing and OAT1-negative LLC-PK1 cells. Most flavonoid aglycones produced substantial decreases in PAH uptake in OAT1-expressing cells. Among the flavonoids screened, fisetin, luteolin, morin, and quercetin exhibited the strongest effect and produced complete inhibition of OAT1-mediated PAH uptake at a concentration of 50 μM. Further concentration-dependent studies revealed that both morin and luteolin are potent OAT1 inhibitors, with IC50 values of <0.3 and 0.47 μM, respectively. In contrast to the tested flavonoid aglycones, all flavonoid glycosides had negligible or small effects on OAT1. In addition, the role of OAT1 in the uptake of fisetin, luteolin, morin, and quercetin was investigated and fisetin was found to be a substrate of OAT1. Taken together, our results indicate that flavonoids are a novel class of OAT1 modulators. Considering the high consumption of flavonoids in the diet and in herbal products, OAT1-mediated flavonoid-drug interactions may be clinically relevant. Further investigation is warranted to evaluate the nephroprotective role of flavonoids in relation to drug-induced nephrotoxicity mediated by the OAT1 pathway. PMID:25002746

  15. NOS1 mediates AP1 nuclear translocation and inflammatory response.

    PubMed

    Srivastava, Mansi; Baig, Mirza S

    2018-06-01

    A hallmark of the AP1 functioning is its nuclear translocation, which induces proinflammatory cytokine expression and hence the inflammatory response. After endotoxin shock AP1 transcription factor, which comprises Jun, ATF2, and Fos family of proteins, translocates into the nucleus and induces proinflammatory cytokine expression. In the current study, we found, NOS1 inhibition prevents nuclear translocation of the AP1 transcription factor subunits. Pharmacological inhibition of NOS1 impedes translocation of subunits into the nucleus, suppressing the transcription of inflammatory genes causing a diminished inflammatory response. In conclusion, the study shows the novel mechanism of NOS1- mediated AP1 nuclear translocation, which needs to be further explored. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  16. DDB1-Mediated CRY1 Degradation Promotes FOXO1-Driven Gluconeogenesis in Liver.

    PubMed

    Tong, Xin; Zhang, Deqiang; Charney, Nicholas; Jin, Ethan; VanDommelen, Kyle; Stamper, Kenneth; Gupta, Neil; Saldate, Johnny; Yin, Lei

    2017-10-01

    Targeted protein degradation through ubiquitination is an important step in the regulation of glucose metabolism. Here, we present evidence that the DDB1-CUL4A ubiquitin E3 ligase functions as a novel metabolic regulator that promotes FOXO1-driven hepatic gluconeogenesis. In vivo, hepatocyte-specific Ddb1 deletion leads to impaired hepatic gluconeogenesis in the mouse liver but protects mice from high-fat diet-induced hyperglycemia. Lack of Ddb1 downregulates FOXO1 protein expression and impairs FOXO1-driven gluconeogenic response. Mechanistically, we discovered that DDB1 enhances FOXO1 protein stability via degrading the circadian protein cryptochrome 1 (CRY1), a known target of DDB1 E3 ligase. In the Cry1 depletion condition, insulin fails to reduce the nuclear FOXO1 abundance and suppress gluconeogenic gene expression. Chronic depletion of Cry1 in the mouse liver not only increases FOXO1 protein but also enhances hepatic gluconeogenesis. Thus, we have identified the DDB1-mediated CRY1 degradation as an important target of insulin action on glucose homeostasis. © 2017 by the American Diabetes Association.

  17. Regulation of Organic Anion Transporting Polypeptides (OATP) 1B1- and OATP1B3-Mediated Transport: An Updated Review in the Context of OATP-Mediated Drug-Drug Interactions.

    PubMed

    Alam, Khondoker; Crowe, Alexandra; Wang, Xueying; Zhang, Pengyue; Ding, Kai; Li, Lang; Yue, Wei

    2018-03-14

    Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important hepatic transporters that mediate the uptake of many clinically important drugs, including statins from the blood into the liver. Reduced transport function of OATP1B1 and OATP1B3 can lead to clinically relevant drug-drug interactions (DDIs). Considering the importance of OATP1B1 and OATP1B3 in hepatic drug disposition, substantial efforts have been given on evaluating OATP1B1/1B3-mediated DDIs in order to avoid unwanted adverse effects of drugs that are OATP substrates due to their altered pharmacokinetics. Growing evidences suggest that the transport function of OATP1B1 and OATP1B3 can be regulated at various levels such as genetic variation, transcriptional and post-translational regulation. The present review summarizes the up to date information on the regulation of OATP1B1 and OATP1B3 transport function at different levels with a focus on potential impact on OATP-mediated DDIs.

  18. Regulation of Organic Anion Transporting Polypeptides (OATP) 1B1- and OATP1B3-Mediated Transport: An Updated Review in the Context of OATP-Mediated Drug-Drug Interactions

    PubMed Central

    Alam, Khondoker; Crowe, Alexandra; Wang, Xueying; Zhang, Pengyue; Ding, Kai; Li, Lang; Yue, Wei

    2018-01-01

    Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important hepatic transporters that mediate the uptake of many clinically important drugs, including statins from the blood into the liver. Reduced transport function of OATP1B1 and OATP1B3 can lead to clinically relevant drug-drug interactions (DDIs). Considering the importance of OATP1B1 and OATP1B3 in hepatic drug disposition, substantial efforts have been given on evaluating OATP1B1/1B3-mediated DDIs in order to avoid unwanted adverse effects of drugs that are OATP substrates due to their altered pharmacokinetics. Growing evidences suggest that the transport function of OATP1B1 and OATP1B3 can be regulated at various levels such as genetic variation, transcriptional and post-translational regulation. The present review summarizes the up to date information on the regulation of OATP1B1 and OATP1B3 transport function at different levels with a focus on potential impact on OATP-mediated DDIs. PMID:29538325

  19. Association of a homozygous nonsense mutation in the ABCA4 (ABCR) gene with cone-rod dystrophy phenotype in an Italian family.

    PubMed

    Simonelli, Francesca; Testa, Francesco; Zernant, Jana; Nesti, Anna; Rossi, Settimio; Rinaldi, Ernesto; Allikmets, Rando

    2004-01-01

    Genetic variation in the ABCA4 (ABCR) gene has been associated with several distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), retinitis pigmentosa (RP) and age-related macular degeneration. The current model of genotype/phenotype association suggests that patients harboring deleterious mutations in both ABCR alleles would develop RP-like retinal pathology. Here we describe ABCA4-associated phenotypes, including a proband with a homozygous nonsense mutation in a family from Southern Italy. The proband had been originally diagnosed with STGD. Ophthalmologic examination included kinetic perimetry, electrophysiological studies and fluorescein angiography. DNA of the affected individual and family members was analyzed for variants in all 50 exons of the ABCA4 gene by screening on the ABCR400 microarray. A homozygous nonsense mutation 2971G>T (G991X) was detected in a patient initially diagnosed with STGD based on funduscopic evidence, including bull's eye depigmentation of the fovea and flecks at the posterior pole extending to the mid-peripheral retina. Since this novel nucleotide substitution results in a truncated, nonfunctional, ABCA4 protein, the patient was examined in-depth for the severity of the disease phenotype. Indeed, subsequent electrophysiological studies determined severely reduced cone amplitude as compared to the rod amplitude, suggesting the diagnosis of CRD. ABCR400 microarray is an efficient tool for determining causal genetic variation, including new mutations. A homozygous protein-truncating mutation in ABCA4 can cause a phenotype ranging from STGD to CRD as diagnosed at an early stage of the disease. Only a combination of comprehensive genotype/phenotype correlation studies will determine the proper diagnosis and prognosis of ABCA4-associated pathology. Copyright 2004 S. Karger AG, Basel

  20. Rme1 is necessary for Mi-1-mediated resistance and acts early in the resistance pathway.

    PubMed

    Martinez de Ilarduya, Oscar; Nombela, Gloria; Hwang, Chin-Feng; Williamson, Valerie M; Muñiz, Mariano; Kaloshian, Isgouhi

    2004-01-01

    The tomato gene Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphid, and whitefly. Using genetic screens, we have isolated a mutant, rme1 (resistance to Meloidogyne spp.), compromised in resistance to M. javanica and potato aphid. Here, we show that the rme1 mutant is also compromised in resistance to M. incognita, M. arenaria, and whitefly. In addition, using an Agrobacterium-mediated transient assay in leaves to express constitutive gain-of-function mutant Pto(L205D), we demonstrated that the rme1 mutation is not compromised in Pto-mediated hypersensitive response. Moreover, the mutation in rme1 does not result in increased virulence of pathogenic Pseudomonas syringae or Mi-1-virulent M. incognita. Using a chimeric Mi-1 construct, Mi-DS4, which confers constitutive cell death phenotype and A. rhizogenes root transformation, we showed that the Mi-1-mediated cell death pathway is intact in this mutant. Our results indicate that Rme1 is required for Mi-1-mediated resistance and acts either at the same step in the signal transduction pathway as Mi-1 or upstream of Mi-1.

  1. GPER1-mediated IGFBP-1 induction modulates IGF-1-dependent signaling in tamoxifen-treated breast cancer cells.

    PubMed

    Vaziri-Gohar, Ali; Houston, Kevin D

    2016-02-15

    Tamoxifen, a selective estrogen receptor modulator, is a commonly prescribed adjuvant therapy for estrogen receptor-α (ERα)-positive breast cancer patients. To determine if extracellular factors contribute to the modulation of IGF-1 signaling after tamoxifen treatment, MCF-7 cells were treated with IGF-1 in conditioned medium (CM) obtained from 4-OHT-treated MCF-7 cells and the accumulation of phospho-Akt (S473) was measured. CM inhibited IGF-1-dependent cell signaling and suggesting the involvement of extracellular factors (ie. IGFBPs). A significant increase in IGFBP-1 mRNA and extracellular IGFBP-1 protein was observed in 4-OHT-treated MCF-7 cells. Knockdown experiments demonstrated that both GPER1 and CREB mediate IGFBP-1 induction. Furthermore, experiments showed that 4-OHT-dependent IGFBP-1 transcription is downstream of GPER1-activation in breast cancer cells. Additionally, neutralization and knockdown experiments demonstrated a role for IGFBP-1 in the observed inhibition of IGF-1 signaling. These results suggested that 4-OHT inhibits IGF-1 signaling via GPER1 and CREB mediated extracellular IGFBP-1 accumulation in breast cancer cells. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  2. A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY -cholesterol

    USDA-ARS?s Scientific Manuscript database

    Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantifying cholesterol efflux involves labeling cells with [(3)H]cholesterol and measuring release of the labeled sterol. Using [(3)H]cholesterol is not ideal for...

  3. A Genetic Screen Identifies a Requirement for Cysteine-Rich-Receptor-Like Kinases in Rice NH1 (OsNPR1)-Mediated Immunity.

    PubMed

    Chern, Mawsheng; Xu, Qiufang; Bart, Rebecca S; Bai, Wei; Ruan, Deling; Sze-To, Wing Hoi; Canlas, Patrick E; Jain, Rashmi; Chen, Xuewei; Ronald, Pamela C

    2016-05-01

    Systemic acquired resistance, mediated by the Arabidopsis NPR1 gene and the rice NH1 gene, confers broad-spectrum immunity to diverse pathogens. NPR1 and NH1 interact with TGA transcription factors to activate downstream defense genes. Despite the importance of this defense response, the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined. Here we report the identification of a rice mutant, snim1, which suppresses NH1-mediated immunity and demonstrate that two genes encoding previously uncharacterized cysteine-rich-receptor-like kinases (CRK6 and CRK10), complement the snim1 mutant phenotype. Silencing of CRK6 and CRK10 genes individually in the parental genetic background recreates the snim1 phenotype. We identified a rice mutant in the Kitaake genetic background with a frameshift mutation in crk10; this mutant also displays a compromised immune response highlighting the important role of crk10. We also show that elevated levels of NH1 expression lead to enhanced CRK10 expression and that the rice TGA2.1 protein binds to the CRK10 promoter. These experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression.

  4. A genetic screen identifies a requirement for cysteine-rich–receptor-like kinases in rice NH1 (OsNPR1)-mediated immunity

    DOE PAGES

    Chern, Mawsheng; Xu, Qiufang; Bart, Rebecca S.; ...

    2016-05-13

    Systemic acquired resistance, mediated by the Arabidopsis NPR1 gene and the rice NH1 gene, confers broad-spectrum immunity to diverse pathogens. NPR1 and NH1 interact with TGA transcription factors to activate downstream defense genes. Despite the importance of this defense response, the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined. Here we report the identification of a rice mutant, snim1, which suppresses NH1-mediated immunity and demonstrate that two genes encoding previously uncharacterized cysteine-rich-receptor-like kinases ( CRK6 and CRK10), complement the snim1 mutant phenotype. Silencing of CRK6 and CRK10 genes individually in the parental genetic background recreates the snim1more » phenotype. We identified a rice mutant in the Kitaake genetic background with a frameshift mutation in crk10; this mutant also displays a compromised immune response highlighting the important role of crk10. We also show that elevated levels of NH1 expression lead to enhanced CRK10 expression and that the rice TGA2.1 protein binds to the CRK10 promoter. Furthermore, these experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression.« less

  5. Mutations in ABCA12 Underlie the Severe Congenital Skin Disease Harlequin Ichthyosis

    PubMed Central

    Kelsell, David P.; Norgett, Elizabeth E.; Unsworth, Harriet; Teh, Muy-Teck; Cullup, Thomas; Mein, Charles A.; Dopping-Hepenstal, Patricia J.; Dale, Beverly A.; Tadini, Gianluca; Fleckman, Philip; Stephens, Karen G.; Sybert, Virginia P.; Mallory, Susan B.; North, Bernard V.; Witt, David R.; Sprecher, Eli; E. M. Taylor, Aileen; Ilchyshyn, Andrew; Kennedy, Cameron T.; Goodyear, Helen; Moss, Celia; Paige, David; Harper, John I.; Young, Bryan D.; Leigh, Irene M.; Eady, Robin A. J.; O’Toole, Edel A.

    2005-01-01

    Harlequin ichthyosis (HI) is the most severe and frequently lethal form of recessive congenital ichthyosis. Although defects in lipid transport, protein phosphatase activity, and differentiation have been described, the genetic basis underlying the clinical and cellular phenotypes of HI has yet to be determined. By use of single-nucleotide–polymorphism chip technology and homozygosity mapping, a common region of homozygosity was observed in five patients with HI in the chromosomal region 2q35. Sequencing of the ABCA12 gene, which maps within the minimal region defined by homozygosity mapping, revealed disease-associated mutations, including large intragenic deletions and frameshift deletions in 11 of the 12 screened individuals with HI. Since HI epidermis displays abnormal lamellar granule formation, ABCA12 may play a critical role in the formation of lamellar granules and the discharge of lipids into the intercellular spaces, which would explain the epidermal barrier defect seen in this disorder. This finding paves the way for early prenatal diagnosis. In addition, functional studies of ABCA12 will lead to a better understanding of epidermal differentiation and barrier formation. PMID:15756637

  6. DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1

    EPA Science Inventory


    DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1. R A Pegram1 and M K Ross2. 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC; 1Pharmacokinetics Branch, NHEERL, ORD, United States Environmental Protection Ag...

  7. Procarcinogenic effects of cyclosporine A are mediated through the activation of TAK1/TAB1 signaling pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu, Jianmin; Walsh, Stephanie B.; Verney, Zoe M.

    Research highlights: {yields} Organ transplant recipients are highly susceptible to early skin cancer development. {yields} CsA-mediated TGFB1-dependent TAK1/TAB1 signaling augments invasive tumor growth. {yields} CsA enhances accumulation of upstream kinases, ZMP, AMPK and IRAK to activate TAK1. {yields} TAK1 mediates enhanced proliferation and reduced apoptosis via CsA-dependent NF{kappa}B. -- Abstract: Cyclosporine A (CsA) is an immunosuppressive drug commonly used for maintaining chronic immune suppression in organ transplant recipients. It is known that patients receiving CsA manifest increased growth of aggressive non-melanoma skin cancers. However, the underlying mechanism by which CsA augments tumor growth is not fully understood. Here, we showmore » that CsA augments the growth of A431 epidermoid carcinoma xenograft tumors by activating tumor growth factor {beta}-activated kinase1 (TAK1). The activation of TAK1 by CsA occurs at multiple levels by kinases ZMP, AMPK and IRAK. TAK1 forms heterodimeric complexes with TAK binding protein 1 and 2 (TAB1/TAB2) which in term activate nuclear factor {kappa}B (NF{kappa}B) and p38 MAP kinase. Transcriptional activation of NF{kappa}B is evidenced by IKK{beta}-mediated phosphorylation-dependent degradation of I{kappa}B and consequent nuclear translocation of p65. This also leads to enhancement in the expression of its transcriptional target genes cyclin D1, Bcl2 and COX-2. Similarly, activation of p38 leads to enhanced inflammation-related signaling shown by increased phosphorylation of MAPKAPK2 and which in turn phosphorylates its substrate HSP27. Activation of both NF{kappa}B and p38 MAP kinase provide mitogenic stimuli to augment the growth of SCCs.« less

  8. Aflatoxin B1 and M1 Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators

    PubMed Central

    Loi, Martina; Fanelli, Francesca; Zucca, Paolo; Liuzzi, Vania C.; Quintieri, Laura; Cimmarusti, Maria T.; Monaci, Linda; Haidukowski, Miriam; Logrieco, Antonio F.; Sanjust, Enrico; Mulè, Giuseppina

    2016-01-01

    Laccases (LCs) are multicopper oxidases that find application as versatile biocatalysts for the green bioremediation of environmental pollutants and xenobiotics. In this study we elucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxin B1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps and identified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performed in vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidation was 23%. Toxin degradation was also investigated in the presence of three redox mediators, (2,2′-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols, acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediated action of Lac2 with redox mediators univocally proved the correlation between Lac2 activity and aflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at 10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded by Lac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pure enzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that the mechanism of an effective degradation occurs via the mediation of natural phenolic compounds. These results opened new perspective for Lac2 application in the food and feed supply chains as a biotransforming agent of AFB1 and AFM1. PMID:27563923

  9. Outcome of ABCA4 disease-associated alleles in autosomal recessive retinal dystrophies: retrospective analysis in 420 Spanish families.

    PubMed

    Riveiro-Alvarez, Rosa; Lopez-Martinez, Miguel-Angel; Zernant, Jana; Aguirre-Lamban, Jana; Cantalapiedra, Diego; Avila-Fernandez, Almudena; Gimenez, Ascension; Lopez-Molina, Maria-Isabel; Garcia-Sandoval, Blanca; Blanco-Kelly, Fiona; Corton, Marta; Tatu, Sorina; Fernandez-San Jose, Patricia; Trujillo-Tiebas, Maria-Jose; Ramos, Carmen; Allikmets, Rando; Ayuso, Carmen

    2013-11-01

    To provide a comprehensive overview of all detected mutations in the ABCA4 gene in Spanish families with autosomal recessive retinal disorders, including Stargardt's disease (arSTGD), cone-rod dystrophy (arCRD), and retinitis pigmentosa (arRP), and to assess genotype-phenotype correlation and disease progression in 10 years by considering the type of variants and age at onset. Case series. A total of 420 unrelated Spanish families: 259 arSTGD, 86 arCRD, and 75 arRP. Spanish families were analyzed through a combination of ABCR400 genotyping microarray, denaturing high-performance liquid chromatography, and high-resolution melting scanning. Direct sequencing was used as a confirmation technique for the identified variants. Screening by multiple ligation probe analysis was used to detect possible large deletions or insertions in the ABCA4 gene. Selected families were analyzed further by next generation sequencing. DNA sequence variants, mutation detection rates, haplotypes, age at onset, central or peripheral vision loss, and night blindness. Overall, we detected 70.5% and 36.6% of all expected ABCA4 mutations in arSTGD and arCRD patient cohorts, respectively. In the fraction of the cohort where the ABCA4 gene was sequenced completely, the detection rates reached 73.6% for arSTGD and 66.7% for arCRD. However, the frequency of possibly pathogenic ABCA4 alleles in arRP families was only slightly higher than that in the general population. Moreover, in some families, mutations in other known arRP genes segregated with the disease phenotype. An increasing understanding of causal ABCA4 alleles in arSTGD and arCRD facilitates disease diagnosis and prognosis and also is paramount in selecting patients for emerging clinical trials of therapeutic interventions. Because ABCA4-associated diseases are evolving retinal dystrophies, assessment of age at onset, accurate clinical diagnosis, and genetic testing are crucial. We suggest that ABCA4 mutations may be associated with a

  10. Diosgenin inhibits atherosclerosis via suppressing the MiR-19b-induced downregulation of ATP-binding cassette transporter A1.

    PubMed

    Lv, Yun-cheng; Yang, Jing; Yao, Feng; Xie, Wei; Tang, Yan-yan; Ouyang, Xin-ping; He, Ping-ping; Tan, Yu-lin; Li, Liang; Zhang, Min; Liu, Dan; Cayabyab, Francisco S; Zheng, Xi-Long; Tang, Chao-ke

    2015-05-01

    Diosgenin (Dgn), a structural analogue of cholesterol, has been reported to have the hypolipidemic and antiatherogenic properties, but the underlying mechanisms are not fully understood. Given the key roles of macrophages in cholesterol metabolism and atherogenesis, it is critical to investigate macrophage cholesterol efflux and development of atherosclerotic lesion after Dgn treatment. This study was designed to evaluate the potential effects of Dgn on macrophage cholesterol metabolism and the development of aortic atherosclerosis, and to explore its underlying mechanisms. Dgn significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) protein, but didn't affect liver X receptor α levels in foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by western blotting. The miR-19b levels were markedly down-regulated in Dgn-treated THP-1 macrophages/MPM-derived foam cells. Cholesterol transport assays revealed that treatment with Dgn alone or together with miR-19b inhibitor notably enhanced ABCA1-dependent cholesterol efflux, resulting in the reduced levels of total cholesterol, free cholesterol and cholesterol ester as determined by high-performance liquid chromatography. The fecal 3H-sterol originating from cholesterol-laden MPMs was increased in apolipoprotein E knockout mice treated with Dgn or both Dgn and antagomiR-19b. Treatment with Dgn alone or together with antagomiR-19b elevated plasma high-density lipoprotein levels, but reduced plasma low-density lipoprotein levels. Accordingly, aortic lipid deposition and plaque area were reduced, and collagen content and ABCA1 expression were increased in mice treated with Dgn alone or together with antagomiR-19b. However, miR-19b overexpression abrogated the lipid-lowering and atheroprotective effects induced by Dgn. The present study demonstrates that Dgn enhances ABCA1-dependent cholesterol efflux and inhibits aortic atherosclerosis

  11. 2,3,7, 8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)-MEDIATED OXIDATIVE STRESS IN FEMALE CYP1A-2 KNOCKOUT (CYP1A2-/-) MICE

    EPA Science Inventory

    2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD)-Mediated Oxidative Stress in Female CYP1A2 Knockout (CYP1A2-/-) Mice

    Deborah Burgin1, Janet Diliberto2, Linda Birnbaum2
    1UNC Toxicology; 2USEPA/ORD/NHEERL, RTP, NC

    Most of the effects due to TCDD exposure are mediated via...

  12. AP-11B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory

    PubMed Central

    Glyvuk, Nataliya; Tsytsyura, Yaroslav; Geumann, Constanze; D'Hooge, Rudi; Hüve, Jana; Kratzke, Manuel; Baltes, Jennifer; Böning, Daniel; Klingauf, Jürgen; Schu, Peter

    2010-01-01

    Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1–σ1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation. PMID:20203623

  13. SESN2 facilitates mitophagy by helping Parkin translocation through ULK1 mediated Beclin1 phosphorylation.

    PubMed

    Kumar, Ashish; Shaha, Chandrima

    2018-01-12

    Mitophagy, the selective degradation of mitochondria by autophagy, is crucial for the maintenance of healthy mitochondrial pool in cells. The critical event in mitophagy is the translocation of cytosolic Parkin, a ubiquitin ligase, to the surface of defective mitochondria. This study elucidates a novel role of SESN2/Sestrin2, a stress inducible protein, in mitochondrial translocation of PARK2/Parkin during mitophagy. The data demonstrates that SESN2 downregulation inhibits BECN1/Beclin1 and Parkin interaction, thereby preventing optimum mitochondrial accumulation of Parkin. SESN2 interacts with ULK1 (unc-51 like kinase 1) and assists ULK1 mediated phosphorylation of Beclin1 at serine-14 position required for binding with Parkin prior to mitochondrial translocation. The trigger for SESN2 activation and regulation of Parkin translocation is the generation of mitochondrial superoxide. Scavenging of mitochondrial superoxide lower the levels of SESN2, resulting in retardation of Parkin translocation. Importantly, we observe that SESN2 mediated cytosolic interaction of Parkin and Beclin1 is PINK1 independent but mitochondrial translocation of Parkin is PINK1 dependent. Together, these findings suggest the role of SESN2 as a positive regulator of Parkin mediated mitophagy.

  14. GATA3 acts upstream of FOXA1 in mediating ESR1 binding by shaping enhancer accessibility.

    PubMed

    Theodorou, Vasiliki; Stark, Rory; Menon, Suraj; Carroll, Jason S

    2013-01-01

    Estrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors; however, its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of cofactors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1-binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3-mediated redistribution of ESR1 binding. The GATA3-mediated redistributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1-bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen-ESR1-mediated interactions between cis-regulatory elements. Together, these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility, and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer.

  15. GATA3 acts upstream of FOXA1 in mediating ESR1 binding by shaping enhancer accessibility

    PubMed Central

    Theodorou, Vasiliki; Stark, Rory; Menon, Suraj; Carroll, Jason S.

    2013-01-01

    Estrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors; however, its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of cofactors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1-binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3-mediated redistribution of ESR1 binding. The GATA3-mediated redistributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1-bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen–ESR1-mediated interactions between cis-regulatory elements. Together, these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility, and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer. PMID:23172872

  16. Novel Mutation in the ATP-Binding Cassette Transporter A3 (ABCA3) Encoding Gene Causes Respiratory Distress Syndrome in A Term Newborn in Southwest Iran

    PubMed Central

    Rezaei, Farideh; Shafiei, Mohammad; Shariati, Gholamreza; Dehdashtian, Ali; Mohebbi, Maryam; Galehdari, Hamid

    2016-01-01

    Introduction ABCA3 glycoprotein belongs to the ATP-binding cassette (ABC) superfamily of transporters, which utilize the energy derived from hydrolysis of ATP for the translocation of a wide variety of substrates across the plasma membrane. Mutations in the ABCA3 gene are knowingly causative for fatal surfactant deficiency, particularly respiratory distress syndrome (RDS) in term babies. Case Presentation In this study, Sanger sequencing of the whole ABCA3 gene (NCBI NM_001089) was performed in a neonatal boy with severe RDS. A homozygous mutation has been identified in the patient. Parents were heterozygous for the same missense mutation GGA > AGA at position 202 in exon 6 of the ABCA3 gene (c.604G > A; p.G202R). Furthermore, 70 normal individuals have been analyzed for the mentioned change with negative results. Conclusions Regarding Human Genome Mutation Database (HGMD) and other literature recherche, the detected change is a novel mutation and has not been reported before. Bioinformatics mutation predicting tools prefer it as pathogenic. PMID:27437095

  17. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    PubMed

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  18. Evidence for a role of MRCK in mediating HeLa cell elongation induced by the C1 domain ligand HMI-1a3.

    PubMed

    Talman, Virpi; Gateva, Gergana; Ahti, Marja; Ekokoski, Elina; Lappalainen, Pekka; Tuominen, Raimo K

    2014-05-13

    Diacylglycerol (DAG) is a central mediator of signaling pathways that regulate cell proliferation, survival and apoptosis. Therefore, C1 domain, the DAG binding site within protein kinase C (PKC) and other DAG effector proteins, is considered a potential cancer drug target. Derivatives of 5-(hydroxymethyl)isophthalic acid are a novel group of C1 domain ligands with antiproliferative and differentiation-inducing effects. Our previous work showed that these isophthalate derivatives exhibit antiproliferative and elongation-inducing effects in HeLa human cervical cancer cells. In this study we further characterized the effects of bis(3-trifluoromethylbenzyl) 5-(hydroxymethyl)isophthalate (HMI-1a3) on HeLa cell proliferation and morphology. HMI-1a3-induced cell elongation was accompanied with loss of focal adhesions and actin stress fibers, and exposure to HMI-1a3 induced a prominent relocation of cofilin-1 into the nucleus regardless of cell phenotype. The antiproliferative and morphological responses to HMI-1a3 were not modified by pharmacological inhibition or activation of PKC, or by RNAi knock-down of specific PKC isoforms, suggesting that the effects of HMI-1a3 were not mediated by PKC. Genome-wide gene expression microarray and gene set enrichment analysis suggested that, among others, HMI-1a3 induces changes in small GTPase-mediated signaling pathways. Our experiments revealed that the isophthalates bind also to the C1 domains of β2-chimaerin, protein kinase D (PKD) and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), which are potential mediators of small GTPase signaling and cytoskeletal reorganization. Pharmacological inhibition of MRCK, but not that of PKD attenuated HMI-1a3-induced cell elongation, suggesting that MRCK participates in mediating the effects of HMI-1a3 on HeLa cell morphology. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. HEAT-INDUCED TAS1 TARGET1 Mediates Thermotolerance via HEAT STRESS TRANSCRIPTION FACTOR A1a–Directed Pathways in Arabidopsis[C][W

    PubMed Central

    Li, Shuxia; Liu, Jinxin; Liu, Zhongyuan; Li, Xiaorong; Wu, Feijie; He, Yuke

    2014-01-01

    Many heat stress transcription factors (Hsfs) and heat shock proteins (Hsps) have been identified to play important roles in the heat tolerance of plants. However, many of the key factors mediating the heat response pathways remain unknown. Here, we report that two genes, which are targets of TAS1 (trans-acting siRNA precursor 1)–derived small interfering RNAs that we named HEAT-INDUCED TAS1 TARGET1 (HTT1) and HTT2, are involved in thermotolerance. Microarray analysis revealed that the HTT1 and HTT2 genes were highly upregulated in Arabidopsis thaliana seedlings in response to heat shock. Overexpression of TAS1a, whose trans-acting small interfering RNAs target the HTT genes, elevated accumulation of TAS1-siRNAs and reduced expression levels of the HTT genes, causing weaker thermotolerance. By contrast, overexpression of HTT1 and HTT2 upregulated several Hsf genes, leading to stronger thermotolerance. In heat-tolerant plants overexpressing HsfA1a, the HTT genes were upregulated, especially at high temperatures. Meanwhile, HsfA1a directly activated HTT1 and HTT2 through binding to their promoters. HTT1 interacted with the heat shock proteins Hsp70-14 and Hsp40 and NUCLEAR FACTOR Y, SUBUNIT C2. Taken together, these results suggest that HTT1 mediates thermotolerance pathways because it is targeted by TAS1a, mainly activated by HsfA1a, and acts as cofactor of Hsp70-14 complexes. PMID:24728648

  20. The Role of Nuclear Receptor Coactivator A1B1 in Growth Factor-Mediated Mammary Tumorigenesis

    DTIC Science & Technology

    2007-03-01

    study display dwarfism and the retardation of mammary gland growth [9]. At the 4-month time point, I similarly observed an overall decrease in mammary...Coactivator A1B1 in Growth Factor- Mediated Mammary Tumorigenesis PRINCIPAL INVESTIGATOR: Mark P Fereshteh (BS) CONTRACTING ORGANIZATION...U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION

  1. Deducing the pathogenic contribution of recessive ABCA4 alleles in an outbred population.

    PubMed

    Schindler, Emily I; Nylen, Erik L; Ko, Audrey C; Affatigato, Louisa M; Heggen, Andrew C; Wang, Kai; Sheffield, Val C; Stone, Edwin M

    2010-10-01

    Accurate prediction of the pathogenic effects of specific genotypes is important for the design and execution of clinical trials as well as for meaningful counseling of individual patients. However, for many autosomal recessive diseases, it can be difficult to deduce the relative pathogenic contribution of individual alleles because relatively few affected individuals share the same two disease-causing variations. In this study, we used multiple regression analysis to estimate the pathogenicity of specific alleles of ABCA4 in patients with retinal phenotypes ranging from Stargardt disease to retinitis pigmentosa. This analysis revealed quantitative allelic effects on two aspects of the visual phenotype, visual acuity (P < 10(-3)) and visual field (P < 10(-7)). Discordance between visual acuity and visual field in individual patients suggests the existence of at least two non-ABCA4 modifying factors. The findings of this study will facilitate the discovery of factors that modify ABCA4 disease and will also aid in the optimal selection of subjects for clinical trials of new therapies.

  2. Celecoxib sensitizes imatinib-resistant K562 cells to imatinib by inhibiting MRP1-5, ABCA2 and ABCG2 transporters via Wnt and Ras signaling pathways.

    PubMed

    Dharmapuri, Gangappa; Doneti, Ravinder; Philip, Gundala Harold; Kalle, Arunasree M

    2015-07-01

    Imatinib mesylate, a tyrosine kinase inhibitor, is very effective in the treatment of chronic myeloid leukemia (CML). However, development of resistance to imatinib therapy is also a very common mechanism observed with long-term administration of the drug. Our previous studies have highlighted the role of cyclooxygenase-2 (COX-2) in regulating the expression of multidrug resistant protein-1 (MDR1), P-gp, in imatinib-resistant K562 cells (IR-K562) via PGE2-cAMP-PKC-NF-κB pathway and inhibition of COX-2 by celecoxib, a COX-2 specific inhibitor, inhibits this pathway and reverses the drug resistance. Studies have identified that not only MDR1 but other ATP-binding cassette transport proteins (ABC transporters) are involved in the development of imatinib resistance. Here, we tried to study the role of COX-2 in the regulation of other ABC transporters such as MRP1, MRP2, MRP3, ABCA2 and ABCG2 that have been already implicated in imatinib resistance development. The results of the study clearly indicated that overexpression of COX-2 lead to upregulation of MRP family proteins in IR-K562 cells and celecoxib down-regulated the ABC transporters through Wnt and MEK signaling pathways. The study signifies that celecoxib in combination with the imatinib can be a good alternate treatment strategy for the reversal of imatinib resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Alpha-1 Antitrypsin Attenuates M1 Microglia-Mediated Neuroinflammation in Retinal Degeneration

    PubMed Central

    Zhou, Tian; Huang, Zijing; Zhu, Xiaowei; Sun, Xiaowei; Liu, Yan; Cheng, Bing; Li, Mei; Liu, Yizhi; He, Chang; Liu, Xialin

    2018-01-01

    Neurodegenerative diseases are a set of disorders characterized by progressive neuronal death and are associated with microglia-mediated neuroinflammation. Recently, neuroinflammation is proposed as a promising therapeutic target for many neurodegenerative diseases. Alpha-1 antitrypsin (AAT) is recognized as a novel immunomodulatory agent in autoimmune diseases and transplantation, however, its impact on neuroinflammation and neurodegeneration remains unknown. This study aims to explore the effects of AAT on microglia-mediated neuroinflammation and retinal degeneration in rd1 mouse model. We found reduced expression of AAT in rd1 retina, and AAT supplement exhibited certain protective effect on retinal degeneration, presenting with increased amount of photoreceptor nuclei, and amplified wave amplitudes in electroretinogram analysis. Of note, AAT shifted microglia phenotype from pro-inflammatory M1 (CD16/CD32+, iNOS+) to anti-inflammatory M2 (CD206+, Arg1+) both in vivo and in vitro, underscoring the concept of immunomodulation on microglia polarization by AAT during neurodegeneration. Furthermore, AAT suppressed the activation of STAT1, promoted the expression of IRF4 while inhibited IRF8 expression, indicating the involvement of these signaling pathways in AAT immunomodulation. Collectively, our data provided evidence for a novel protective role of AAT through immunomodulation on microglia polarization. Attenuating neuroinflammation by AAT may be beneficial to retard neurodegeneration in rd1 mice. PMID:29899745

  4. Preventive effects of heregulin-beta1 on macrophage foam cell formation and atherosclerosis.

    PubMed

    Xu, Gang; Watanabe, Takuya; Iso, Yoshitaka; Koba, Shinji; Sakai, Tetsuo; Nagashima, Masaharu; Arita, Shigeko; Hongo, Shigeki; Ota, Hidekazu; Kobayashi, Youichi; Miyazaki, Akira; Hirano, Tsutomu

    2009-08-28

    Human heregulins, neuregulin-1 type I polypeptides that activate proliferation, differentiation, and survival of glial cells, neurons, and myocytes, are expressed in macrophage foam cells within human coronary atherosclerotic lesions. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor class A (SR-A), acyl-coenzyme A:cholesterol acyltransferase (ACAT)1, and ATP-binding cassette transporter (ABC)A1. The present study clarified the roles of heregulins in macrophage foam cell formation and atherosclerosis. Plasma heregulin-beta(1) levels were significantly decreased in 31 patients with acute coronary syndrome and 33 patients with effort angina pectoris compared with 34 patients with mild hypertension and 40 healthy volunteers (1.3+/-0.3, 2.0+/-0.4 versus 7.6+/-1.4, 8.2+/-1.2 ng/mL; P<0.01). Among all patients with acute coronary syndrome and effort angina pectoris, plasma heregulin-beta(1) levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-beta(1) was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-beta(1), but not heregulin-alpha, significantly reduced acetylated low-density lipoprotein-induced cholesterol ester accumulation in primary cultured human monocyte-derived macrophages by reducing SR-A and ACAT1 expression and by increasing ABCA1 expression at both mRNA and protein levels. Heregulin-beta(1) significantly decreased endocytic uptake of [(125)I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-beta(1) into ApoE(-/-) mice significantly suppressed the development of atherosclerotic lesions. This study provided the first evidence that heregulin-beta(1) inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A

  5. Addition of glycosylation to influenza A virus hemagglutinin modulates antibody-mediated recognition of H1N1 2009 pandemic viruses.

    PubMed

    Job, Emma R; Deng, Yi-Mo; Barfod, Kenneth K; Tate, Michelle D; Caldwell, Natalie; Reddiex, Scott; Maurer-Stroh, Sebastian; Brooks, Andrew G; Reading, Patrick C

    2013-03-01

    Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the viral hemagglutinin (HA). Glycans on the head of HA promote virus survival by shielding antigenic sites, but highly glycosylated seasonal IAV are inactivated by soluble lectins of the innate immune system. In 2009, human strains of pandemic H1N1 [A(H1N1)pdm] expressed a single glycosylation site (Asn(104)) on the head of HA. Since then, variants with additional glycosylation sites have been detected, and the location of these sites has been distinct to those of recent seasonal H1N1 strains. We have compared wild-type and reverse-engineered A(H1N1)pdm IAV with differing potential glycosylation sites on HA for sensitivity to collectins and to neutralizing Abs. Addition of a glycan (Asn(136)) to A(H1N1)pdm HA was associated with resistance to neutralizing Abs but did not increase sensitivity to collectins. Moreover, variants expressing Asn(136) showed enhanced growth in A(H1N1)pdm-vaccinated mice, consistent with evasion of Ab-mediated immunity in vivo. Thus, a fine balance exists regarding the optimal pattern of HA glycosylation to facilitate evasion of Ab-mediated immunity while maintaining resistance to lectin-mediated defenses of the innate immune system.

  6. A novel method for determination of aflatoxin B1 mediated by FCLA + BSA

    NASA Astrophysics Data System (ADS)

    Chen, WenLi; Xing, Da

    2005-02-01

    As a chemiluminescence (CL) probe, 3,7-dihydro-6-{4-{2-(N"-(5-fluoresceinyl) thioureido)ethoxy}phenyl}-2-met -hylimi-dazo{1,2-a}pyrazin-3-one dosium salt (FCLA) can sensitively and specifically react with singlet oxygen (1O2 ) and superoxide(O2""). BSA (Bovine Serum Albumin) can enlarge the CL intensity of FCLA to 860%. This report presents a novel method for determination of Aflatoxin B1 (AfB1) mediated by FCLA+BSA. The concentration of AFB1 showed an obvious positive correlation with the CL intensity mediated by FCLA+BSA. This method could measure accurately ng/ml of AfB1 concentration. At the same time, the fluorescence spectrum of FCLA+BSA and FCLA+BSA+AfB1 were measured respectively, which showed that the fluorescence intensity of FCLA+BSA+AfB1 was higher than FCLA+BSA. Comparing the peak value of FCLA, FCLA+BSA and FCLA+BSA+AfB1 had a 6nm Einstein shift (red shift). The study suggested that CL method mediated by FCLA+BSA might be applicable to the determination of AfB1 concentration.

  7. Glucose Elevates NITRATE TRANSPORTER2.1 Protein Levels and Nitrate Transport Activity Independently of Its HEXOKINASE1-Mediated Stimulation of NITRATE TRANSPORTER2.1 Expression1[W][OPEN

    PubMed Central

    de Jong, Femke; Thodey, Kate; Lejay, Laurence V.; Bevan, Michael W.

    2014-01-01

    Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth. PMID:24272701

  8. Multidrug Resistance-Associated Protein 1 (MRP1) mediated vincristine resistance: effects of N-acetylcysteine and Buthionine Sulfoximine

    PubMed Central

    Akan, Ilhan; Akan, Selma; Akca, Hakan; Savas, Burhan; Ozben, Tomris

    2005-01-01

    Background Multidrug resistance mediated by the multidrug resistance-associated protein 1 (MRP1) decreases cellular drug accumulation. The exact mechanism of MRP1 involved multidrug resistance has not been clarified yet, though glutathione (GSH) is likely to have a role for the resistance to occur. N-acetylcysteine (NAC) is a pro-glutathione drug. DL-Buthionine (S,R)-sulfoximine (BSO) is an inhibitor of GSH synthesis. The aim of our study was to investigate the effect of NAC and BSO on MRP1-mediated vincristine resistance in Human Embryonic Kidney (HEK293) and its MRP1 transfected 293MRP cells. Human Embryonic Kidney (HEK293) cells were transfected with a plasmid encoding whole MRP1 gene. Both cells were incubated with vincristine in the presence or absence of NAC and/or BSO. The viability of both cells was determined under different incubation conditions. GSH, Glutathione S-Transferase (GST) and glutathione peroxidase (GPx) levels were measured in the cell extracts obtained from both cells incubated with different drugs. Results N-acetylcysteine increased the resistance of both cells against vincristine and BSO decreased NAC-enhanced MRP1-mediated vincristine resistance, indicating that induction of MRP1-mediated vincristine resistance depends on GSH. Vincristine decreased cellular GSH concentration and increased GPx activity. Glutathione S-Transferase activity was decreased by NAC. Conclusion Our results demonstrate that NAC and BSO have opposite effects in MRP1 mediated vincristine resistance and BSO seems a promising chemotherapy improving agent in MRP1 overexpressing tumor cells. PMID:16042792

  9. An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk-dependent repression of macrophage cholesterol efflux

    PubMed Central

    Gillespie, Mark A; Gold, Elizabeth S; Ramsey, Stephen A; Podolsky, Irina; Aderem, Alan; Ranish, Jeffrey A

    2015-01-01

    LXR–cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment-quantitative mass spectrometry (PE-QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand- and binding-dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll-like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3–LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro-inflammatory and anti-inflammatory pathways that results in repression of macrophage cholesterol efflux. PMID:25755249

  10. Why childhood-onset type 1 diabetes impacts labour market outcomes: a mediation analysis.

    PubMed

    Persson, Sofie; Dahlquist, Gisela; Gerdtham, Ulf-G; Steen Carlsson, Katarina

    2018-02-01

    Previous studies show a negative effect of type 1 diabetes on labour market outcomes such as employment and earnings later in life. However, little is known about the mechanisms underlying these effects. This study aims to analyse the mediating role of adult health, education, occupation and family formation. A total of 4179 individuals from the Swedish Childhood Diabetes Register and 16,983 individuals forming a population control group born between 1962 and 1979 were followed between 30 and 50 years of age. The total effect of having type 1 diabetes was broken down into a direct effect and an indirect (mediating) effect using statistical mediation analysis. We also analysed whether type 1 diabetes has different effects on labour market outcome between the sexes and across socioeconomic status. Childhood-onset type 1 diabetes had a negative impact on employment (OR 0.68 [95% CI 0.62, 0.76] and OR 0.76 [95% CI 0.67, 0.86]) and earnings (-6%, p < 0.001 and -8%, p < 0.001) for women and men, respectively. Each of the mediators studied contributed to the total effect with adult health and occupational field accounting for the largest part. However, some of the effect could not be attributed to any of the mediators studied and was therefore likely related to other characteristics of the disease that hamper career opportunities. The effect of type 1 diabetes on employment and earnings did not vary significantly according to socioeconomic status of the family (parental education and earnings). A large part of the effect of type 1 diabetes on the labour market is attributed to adult health but there are other important mediating factors that need to be considered to reduce this negative effect.

  11. EPAS-1 mediates SP-1-dependent FBI-1 expression and regulates tumor cell survival and proliferation.

    PubMed

    Wang, Xiaogang; Cao, Peng; Li, Zhiqing; Wu, Dongyang; Wang, Xi; Liang, Guobiao

    2014-09-04

    Factor binding IST-1 (FBI-1) plays an important role in oncogenic transformation and tumorigenesis. As FBI-1 is over-expressed in multiple human cancers, the regulation of itself would provide new effective options for cancer intervention. In this work, we aimed to study the role that EPAS-1 plays in regulating FBI-1. We use the fact that specificity protein-1 (SP-1) is one of the crucial transcription factors of FBI-1, and that SP-1 can interact with the endothelial pas domain protein-1 (EPAS-1) for the induction of hypoxia related genes. The study showed that EPAS-1 plays an indispensible role in SP-1 transcription factor-mediated FBI-1 induction, and participated in tumor cell survival and proliferation. Thus, EPAS-1 could be a novel target for cancer therapeutics.

  12. Uncaria alkaloids reverse ABCB1-mediated cancer multidrug resistance

    PubMed Central

    Huang, Bao-Yuan; Zeng, Yu; Li, Ying-Jie; Huang, Xiao-Jun; Hu, Nan; Yao, Nan; Chen, Min-Feng; Yang, Zai-Gang; Chen, Zhe-Sheng; Zhang, Dong-Mei; Zeng, Chang-Qing

    2017-01-01

    The overexpression of ATP-binding cassette (ABC) transporters is the main cause of cancer multidrug resistance (MDR), which leads to chemotherapy failure. Uncaria alkaloids are the major active components isolated from uncaria, which is a common Chinese herbal medicine. In this study, the MDR-reversal activities of uncaria alkaloids, including rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine (Icory), hirsutine and hirsuteine, were screened; they all exhibited potent reversal efficacy when combined with doxorubicin. Among them, Icory significantly sensitized ABCB1-overexpressing HepG2/ADM and MCF-7/ADR cells to vincristine, doxorubicin and paclitaxel, but not to the non-ABCB1 substrate cisplatin. Noteworthy, Icory selectively reversed ABCB1-overexpressing MDR cancer cells but not ABCC1- or ABCG2-mediated MDR. Further mechanistic study revealed that Icory increased the intracellular accumulation of doxorubicin in ABCB1-overexpressing cells by blocking the efflux function of ABCB1. Instead of inhibiting ABCB1 expression and localization, Icory acts as a substrate of the ABCB1 transporter by competitively binding to substrate binding sites. Collectively, these results indicated that Icory reversed ABCB1-mediated MDR by suppressing its efflux function, and it would be beneficial to increase the efficacy of these types of uncaria alkaloids and develop them to be selective ABCB1-mediated MDR-reversal agents. PMID:28534954

  13. Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.

    PubMed

    Hadjighassem, Mahmoud R; Galaraga, Kimberly; Albert, Paul R

    2011-01-01

    The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A-DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A-DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2-DRE complexes were distinguished from Freud-1-DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter-reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A-DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  14. Preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1.

    PubMed

    Ma, Min; Zhou, Qiong-Jie; Xiong, Yu; Li, Bin; Li, Xiao-Tian

    2018-01-01

    Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson's correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn't change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia.

  15. Preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1

    PubMed Central

    Ma, Min; Zhou, Qiong-Jie; Xiong, Yu; Li, Bin; Li, Xiao-Tian

    2018-01-01

    Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson’s correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn’t change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia. PMID:29422991

  16. Acylation of Superoxide Dismutase 1 (SOD1) at K122 Governs SOD1-Mediated Inhibition of Mitochondrial Respiration

    PubMed Central

    Banks, Courtney J.; Rodriguez, Nathan W.; Gashler, Kyle R.; Pandya, Rushika R.; Mortenson, Jeffrey B.; Whited, Matthew D.; Soderblom, Erik J.; Thompson, J. Will; Moseley, M. Arthur; Reddi, Amit R.; Tessem, Jeffery S.; Torres, Matthew P.; Bikman, Benjamin T.

    2017-01-01

    ABSTRACT In this study, we employed proteomics to identify mechanisms of posttranslational regulation on cell survival signaling proteins. We focused on Cu-Zn superoxide dismutase (SOD1), which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked the suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a previously unknown interplay between SOD1 acylation, metabolic regulation, and SOD1-mediated cell survival. PMID:28739857

  17. Mediator Tail Module Is Required for Tac1-Activated CDR1 Expression and Azole Resistance in Candida albicans.

    PubMed

    Liu, Zhongle; Myers, Lawrence C

    2017-11-01

    The human fungal pathogen Candida albicans develops drug resistance after long-term exposure to azole drugs in the treatment of chronic candidiasis. Gain-of-function (GOF) mutations in the transcription factor Tac1 and the consequent expression of its targets, drug efflux pumps Cdr1 and Cdr2, are a common mechanism by which C. albicans acquires fluconazole resistance. The mechanism by which GOF mutations hyperactivate Tac1 is currently unknown. Here, we define a transcriptional activation domain (TAD) at the C terminus of Tac1. GOF mutations within the Tac1 TAD, outside the context of full-length Tac1, generally do not enhance its absolute potential as a transcriptional activator. Negative regulation of the Tac1 TAD by the Tac1 middle region is necessary for the activating effect of GOF mutations or fluphenazine to be realized. We have found that full-length Tac1, when hyperactivated by xenobiotics or GOF mutations, facilitates the recruitment of the Mediator coactivator complex to the CDR1 promoter. Azole resistance and the activation of Tac1 target genes, such as CDR1 , are dependent on the Tac1 TAD and subunits of the Mediator tail module. The dependence of different Tac1 target promoters on the Mediator tail module, however, varies widely. Lastly, we show that hyperactivation of Tac1 is correlated with its Mediator-dependent phosphorylation, a potentially useful biomarker for Tac1 hyperactivation. The role of Mediator in events downstream of Tac1 hyperactivation in fluconazole-resistant clinical isolates is complex and provides opportunities and challenges for therapeutic intervention. Copyright © 2017 American Society for Microbiology.

  18. Circadian clocks govern calorie restriction-mediated life span extension through BMAL1- and IGF-1-dependent mechanisms.

    PubMed

    Patel, Sonal A; Chaudhari, Amol; Gupta, Richa; Velingkaar, Nikkhil; Kondratov, Roman V

    2016-04-01

    Calorie restriction (CR) increases longevity in many species by unknown mechanisms. The circadian clock was proposed as a potential mediator of CR. Deficiency of the core component of the circadian clock-transcriptional factor BMAL1 (brain and muscle ARNT [aryl hydrocarbon receptor nuclear translocator]-like protein 1)-results in accelerated aging. Here we investigated the role of BMAL1 in mechanisms of CR. The 30% CR diet increased the life span of wild-type (WT) mice by 20% compared to mice on anad libitum(AL) diet but failed to increase life span ofBmal1(-/-)mice. BMAL1 deficiency impaired CR-mediated changes in the plasma levels of IGF-1 and insulin. We detected a statistically significantly reduction of IGF-1 in CRvs.AL by 50 to 70% in WT mice at several daily time points tested, while inBmal1(-/-)the reduction was not significant. Insulin levels in WT were reduced by 5 to 9%, whileBmal1(-/-)induced it by 10 to 35% at all time points tested. CR up-regulated the daily average expression ofBmal1(by 150%) and its downstream target genesPeriods(by 470% forPer1and by 130% forPer2). We propose that BMAL1 is an important mediator of CR, and activation of BMAL1 might link CR mechanisms with biologic clocks.-Patel, S. A., Chaudhari, A., Gupta, R., Velingkaar, N., Kondratov, R. V. Circadian clocks govern calorie restriction-mediated life span extension through BMAL1- and IGF-1-dependent mechanisms. © FASEB.

  19. Fundus Autofluorescence in the Abca4−/− Mouse Model of Stargardt Disease—Correlation With Accumulation of A2E, Retinal Function, and Histology

    PubMed Central

    Charbel Issa, Peter; Barnard, Alun R.; Singh, Mandeep S.; Carter, Emma; Jiang, Zhichun; Radu, Roxana A.; Schraermeyer, Ulrich; MacLaren, Robert E.

    2013-01-01

    Purpose. To investigate fundus autofluorescence (AF) characteristics in the Abca4−/− mouse, an animal model for AMD and Stargardt disease, and to correlate findings with functional, structural, and biochemical assessments. Methods. Blue (488 nm) and near-infrared (790 nm) fundus AF images were quantitatively and qualitatively analyzed in pigmented Abca4−/− mice and wild type (WT) controls in vivo. Functional, structural, and biochemical assessments included electroretinography (ERG), light and electron microscopic analysis, and A2E quantification. All assessments were performed across age groups. Results. In Abca4−/− mice, lipofuscin-related 488 nm AF increased early in life with a ceiling effect after 6 months. This increase was first paralleled by an accumulation of typical lipofuscin granules in the retinal pigment epithelium (RPE). Later, lipofuscin and melanin granules decreased in number, whereas melanolipofuscin granules increased. This increase in melanolipofuscin granules paralleled an increase in melanin-related 790 nm AF. Old Abca4−/− mice revealed a flecked fundus AF pattern at both excitation wavelengths. The amount of A2E, a major lipofuscin component, increased 10- to 12-fold in 6- to 9-month-old Abca4−/− mice compared with controls, while 488 nm AF intensity only increased 2-fold. Despite pronounced lipofuscin accumulation in the RPE of Abca4−/− mice, ERG and histology showed a slow age-related thinning of the photoreceptor layer similar to WT controls up to 12 months. Conclusions. Fundus AF can be used to monitor lipofuscin accumulation and melanin-related changes in vivo in mouse models of retinal disease. High RPE lipofuscin may not adversely affect retinal structure or function over prolonged time intervals, and melanin-related changes (melanolipofuscin formation) may occur before the decline in retinal function. PMID:23761084

  20. Cholesterol Accumulation in Dendritic Cells Links the Inflammasome to Acquired Immunity.

    PubMed

    Westerterp, Marit; Gautier, Emmanuel L; Ganda, Anjali; Molusky, Matthew M; Wang, Wei; Fotakis, Panagiotis; Wang, Nan; Randolph, Gwendalyn J; D'Agati, Vivette D; Yvan-Charvet, Laurent; Tall, Alan R

    2017-06-06

    Autoimmune diseases such as systemic lupus erythematosus (SLE) are associated with increased cardiovascular disease and reduced plasma high-density lipoprotein (HDL) levels. HDL mediates cholesterol efflux from immune cells via the ATP binding cassette transporters A1 and G1 (ABCA1/G1). The significance of impaired cholesterol efflux pathways in autoimmunity is unknown. We observed that Abca1/g1-deficient mice develop enlarged lymph nodes (LNs) and glomerulonephritis suggestive of SLE. This lupus-like phenotype was recapitulated in mice with knockouts of Abca1/g1 in dendritic cells (DCs), but not in macrophages or T cells. DC-Abca1/g1 deficiency increased LN and splenic CD11b + DCs, which displayed cholesterol accumulation and inflammasome activation, increased cell surface levels of the granulocyte macrophage-colony stimulating factor receptor, and enhanced inflammatory cytokine secretion. Consequently, DC-Abca1/g1 deficiency enhanced T cell activation and T h 1 and T h 17 cell polarization. Nlrp3 inflammasome deficiency diminished the enlarged LNs and enhanced T h 1 cell polarization. These findings identify an essential role of DC cholesterol efflux pathways in maintaining immune tolerance. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. EPAS-1 Mediates SP-1-Dependent FBI-1 Expression and Regulates Tumor Cell Survival and Proliferation

    PubMed Central

    Wang, Xiaogang; Cao, Peng; Li, Zhiqing; Wu, Dongyang; Wang, Xi; Liang, Guobiao

    2014-01-01

    Factor binding IST-1 (FBI-1) plays an important role in oncogenic transformation and tumorigenesis. As FBI-1 is over-expressed in multiple human cancers, the regulation of itself would provide new effective options for cancer intervention. In this work, we aimed to study the role that EPAS-1 plays in regulating FBI-1. We use the fact that specificity protein-1 (SP-1) is one of the crucial transcription factors of FBI-1, and that SP-1 can interact with the endothelial pas domain protein-1 (EPAS-1) for the induction of hypoxia related genes. The study showed that EPAS-1 plays an indispensible role in SP-1 transcription factor-mediated FBI-1 induction, and participated in tumor cell survival and proliferation. Thus, EPAS-1 could be a novel target for cancer therapeutics. PMID:25192290

  2. Phenotypic spectrum of autosomal recessive cone-rod dystrophies caused by mutations in the ABCA4 (ABCR) gene.

    PubMed

    Klevering, B Jeroen; Blankenagel, Anita; Maugeri, Alessandra; Cremers, Frans P M; Hoyng, Carel B; Rohrschneider, Klaus

    2002-06-01

    To describe the phenotype of 12 patients with autosomal recessive or isolated cone-rod types of progressive retinal degeneration (CRD) caused by mutations in the ABCA4 gene. The charts of patients who had originally received a diagnosis of isolated or autosomal recessive CRD were reviewed after molecular analysis revealed mutations in the ABCA4 gene. In two of the patients both the photopic and scotopic electroretinogram were nonrecordable. In the remainder, the photopic cone b-wave amplitudes appeared to be more seriously affected than the scotopic rod b-wave amplitudes. Although the clinical presentation was heterogeneous, all patients experienced visual loss early in life, impaired color vision, and a central scotoma. Fundoscopy revealed evidence of early-onset maculopathy, sometimes accompanied by involvement of the retinal periphery in the later stages of the disease. Mutations in the ABCA4 gene are the pathologic cause of the CRD-like dystrophy in these patients, and the resultant clinical pictures are complex and heterogeneous. Given this wide clinical spectrum of CRD-like phenotypes associated with ABCA4 mutations, detailed clinical subclassifications are difficult and may not be very useful.

  3. An Auxilin-Like J-Domain Protein, JAC1, Regulates Phototropin-Mediated Chloroplast Movement in Arabidopsis1[w

    PubMed Central

    Suetsugu, Noriyuki; Kagawa, Takatoshi; Wada, Masamitsu

    2005-01-01

    The ambient-light conditions mediate chloroplast relocation in plant cells. Under the low-light conditions, chloroplasts accumulate in the light (accumulation response), while under the high-light conditions, they avoid the light (avoidance response). In Arabidopsis (Arabidopsis thaliana), the accumulation response is mediated by two blue-light receptors, termed phototropins (phot1 and phot2) that act redundantly, and the avoidance response is mediated by phot2 alone. A mutant, J-domain protein required for chloroplast accumulation response 1 (jac1), lacks the accumulation response under weak blue light but shows a normal avoidance response under strong blue light. In dark-adapted wild-type cells, chloroplasts accumulate on the bottom of cells. Both the jac1 and phot2 mutants are defective in this chloroplast movement in darkness. Positional cloning of JAC1 reveals that this gene encodes a J-domain protein, resembling clathrin-uncoating factor auxilin at its C terminus. The amounts of JAC1 transcripts and JAC1 proteins are not regulated by light and by phototropins. A green fluorescent protein-JAC1 fusion protein showed a similar localization pattern to green fluorescent protein alone in a transient expression assay using Arabidopsis mesophyll cells and onion (Allium cepa) epidermal cells, suggesting that the JAC1 protein may be a soluble cytosolic protein. Together, these results suggest that JAC1 is an essential component of phototropin-mediated chloroplast movement. PMID:16113208

  4. Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo.

    PubMed

    Yang, Ting; Xie, Zhongping; Li, Hua; Yue, Lei; Pang, Zheng; MacNeil, Adam J; Tremblay, Michel L; Tang, Jin-Tian; Lin, Tong-Jun

    2016-01-01

    Mast cells play a critical role in allergic reactions. The cross-linking of FcεRI-bound IgE with multivalent antigen initiates a cascade of signaling events leading to mast cell activation. It has been well-recognized that cross linking of FcεRI mediates tyrosine phosphorylation. However, the mechanism involved in tyrosine dephosphorylation in mast cells is less clear. Here we demonstrated that protein tyrosine phosphatase 1B (PTP1B)-deficient mast cells showed increased IgE-mediated phosphorylation of the signal transducer and activator of transcription 5 (STAT5) and enhanced production of CCL9 (MIP-1γ) and IL-6 in IgE-mediated mast cells activation in vitro. However, IgE-mediated calcium mobilization, β-hexaosaminidase release (degranulation), and phosphorylation of IκB and MAP kinases were not affected by PTP1B deficiency. Furthermore, PTP1B deficient mice showed normal IgE-dependent passive cutaneous anaphylaxis and late phase cutaneous reactions in vivo. Thus, PTP1B specifically regulates IgE-mediated STAT5 pathway, but is redundant in influencing mast cell function in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. [HSV-1 based vector mediated IL-1Rα gene for knee osteoarthritis in rabbits].

    PubMed

    Wu, Yi; Li, Jianming; Kong, Ying; Chen, Ding; Liu, Bo; Wang, Wanchun

    2013-06-01

    To investigate the effect and mechanism of herpes simplex virus type 1 (HSV-1) based vector mediated interlukin-1 receptor antagonist (IL-1Rα) gene for knee osteoarthritis in rabbits. HSV-1 vectors containing IL-1Rα genes were constructed and injected into the joint space of the osteoarthritis knee in rabbits for 4 weeks. The rabbits were sacrificed, and the knees were lavaged, dissected and the effect of transgene expression was analyzed. Levels of IL-1Rα and IL-1 expression in the recovered lavage fluids were measured with a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were observed with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The blank control group was injected pHSV-LacZ vector into rabbit knees. Intra-articular delivery of pHSV-IL-1Rα-LacZ resulted in a significant inhibition of IL-1 level and cartilage degradation compared with those in the blank control group (P<0.05). pHSV-LacZ is an ideal vector to mediate intra-articular gene delivery in the rabbit model of osteoarthritis. Continuous intra-articular expression of IL-1Rα can treat knee osteoarthritis by inhibiting IL-1.

  6. Enhanced Disease Susceptibility1 Mediates Pathogen Resistance and Virulence Function of a Bacterial Effector in Soybean1[C][W][OPEN

    PubMed Central

    Wang, Jialin; Shine, M.B.; Gao, Qing-Ming; Navarre, Duroy; Jiang, Wei; Liu, Chunyan; Chen, Qingshan; Hu, Guohua; Kachroo, Aardra

    2014-01-01

    Enhanced disease susceptibility1 (EDS1) and phytoalexin deficient4 (PAD4) are well-known regulators of both basal and resistance (R) protein-mediated plant defense. We identified two EDS1-like (GmEDS1a/GmEDS1b) proteins and one PAD4-like (GmPAD4) protein that are required for resistance signaling in soybean (Glycine max). Consistent with their significant structural conservation to Arabidopsis (Arabidopsis thaliana) counterparts, constitutive expression of GmEDS1 or GmPAD4 complemented the pathogen resistance defects of Arabidopsis eds1 and pad4 mutants, respectively. Interestingly, however, the GmEDS1 and GmPAD4 did not complement pathogen-inducible salicylic acid accumulation in the eds1/pad4 mutants. Furthermore, the GmEDS1a/GmEDS1b proteins were unable to complement the turnip crinkle virus coat protein-mediated activation of the Arabidopsis R protein Hypersensitive reaction to Turnip crinkle virus (HRT), even though both interacted with HRT. Silencing GmEDS1a/GmEDS1b or GmPAD4 reduced basal and pathogen-inducible salicylic acid accumulation and enhanced soybean susceptibility to virulent pathogens. The GmEDS1a/GmEDS1b and GmPAD4 genes were also required for Resistance to Pseudomonas syringae pv glycinea2 (Rpg2)-mediated resistance to Pseudomonas syringae. Notably, the GmEDS1a/GmEDS1b proteins interacted with the cognate bacterial effector AvrA1 and were required for its virulence function in rpg2 plants. Together, these results show that despite significant structural similarities, conserved defense signaling components from diverse plants can differ in their functionalities. In addition, we demonstrate a role for GmEDS1 in regulating the virulence function of a bacterial effector. PMID:24872380

  7. TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus.

    PubMed

    Brouillette, Rachel B; Phillips, Elisabeth K; Patel, Radhika; Mahauad-Fernandez, Wadie; Moller-Tank, Sven; Rogers, Kai J; Dillard, Jacob A; Cooney, Ashley L; Martinez-Sobrido, Luis; Okeoma, Chioma; Maury, Wendy

    2018-06-06

    Lassa virus (LASV) is an Old World arenavirus responsible for hundreds of thousands of infections in West Africa every year. LASV entry into a variety of cell types is mediated by interactions with glycosyltransferase LARGE-modified O-linked glycans present on the ubiquitous receptor, α-dystroglycan (αDG). Yet, cells lacking αDG are permissive to LASV infection, suggesting that alternative receptors exist. Previous studies demonstrate that phosphatidylserine (PtdSer)-binding receptors, Axl and Tyro3 along with C-type lectin receptors, mediate αDG-independent entry. Here, we demonstrate that another PtdSer receptor, TIM-1, mediates LASV glycoprotein (GP) pseudotyped virions entry into αDG knocked out HEK 293T and wild-type (WT) Vero which express αDG lacking appropriate glycosylation. To investigate the mechanism by which TIM-1 mediates enhancement of entry, we demonstrate that mutagenesis of the TIM-1 IgV domain PtdSer-binding pocket abrogated transduction. Further, the human TIM-1 IgV domain binding monoclonal antibody, ARD5, blocked transduction of pseudovirions bearing LASV GP in a dose-dependent manner. Finally, as we showed previously for other viruses that use TIM-1 for entry, a chimeric TIM-1 protein that substitutes the proline rich region (PRR) from murine leukemia virus envelope (Env) for the mucin-like domain served as a competent receptor. These studies provide evidence that, in the absence of a functional αDG, TIM-1 mediates entry of LASV pseudoviral particles through interactions of virions with the IgV PtdSer binding pocket of TIM-1. Importance PtdSer receptors, such as TIM-1, are emerging as critical entry factors for many enveloped viruses. Most recently, Hepatitis C virus and Zika virus have been added to a growing list. PtdSer receptors engage with enveloped viruses through binding of PtdSer embedded in the viral envelope, defining them as GP-independent receptors. This GP-independent entry mechanism should effectively mediate entry of all

  8. Hypoxia-inducible factor 1mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions

    PubMed Central

    Zhang, Feng-Lin; Shen, Guo-Min; Liu, Xiao-Ling; Wang, Fang; Zhao, Ying-Ze; Zhang, Jun-Wu

    2012-01-01

    Abstract Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type–specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl2 induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. PMID:22050843

  9. JIP1-Mediated JNK Activation Negatively Regulates Synaptic Plasticity and Spatial Memory.

    PubMed

    Morel, Caroline; Sherrin, Tessi; Kennedy, Norman J; Forest, Kelly H; Avcioglu Barutcu, Seda; Robles, Michael; Carpenter-Hyland, Ezekiel; Alfulaij, Naghum; Standen, Claire L; Nichols, Robert A; Benveniste, Morris; Davis, Roger J; Todorovic, Cedomir

    2018-04-11

    The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JNK-interacting protein 1 (JIP1) scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDAR currents, increased NMDAR-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDAR-mediated synaptic plasticity and learning. SIGNIFICANCE STATEMENT The results of this study demonstrate that c-Jun N-terminal kinase (JNK) activation induced by the JNK-interacting protein 1 (JIP1) scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study identifies JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDAR-dependent synaptic plasticity and memory. Copyright © 2018 the authors 0270-6474/18/383708-21$15.00/0.

  10. β-dystroglycan is regulated by a balance between WWP1-mediated degradation and protection from WWP1 by dystrophin and utrophin.

    PubMed

    Cho, Eun-Bee; Yoo, Wonjin; Yoon, Sungjoo Kim; Yoon, Jong-Bok

    2018-06-01

    Dystroglycan is a ubiquitous membrane protein that functions as a mechanical connection between the extracellular matrix and cytoskeleton. In skeletal muscle, dystroglycan plays an indispensable role in regulating muscle regeneration; a malfunction in dystroglycan is associated with muscular dystrophy. The regulation of dystroglycan stability is poorly understood. Here, we report that WWP1, a member of NEDD4 E3 ubiquitin ligase family, promotes ubiquitination and subsequent degradation of β-dystroglycan. Our results indicate that dystrophin and utrophin protect β-dystroglycan from WWP1-mediated degradation by competing with WWP1 for the shared binding site at the cytosolic tail of β-dystroglycan. In addition, we show that a missense mutation (arginine 440 to glutamine) in WWP1-which is known to cause muscular dystrophy in chickens-increases the ubiquitin ligase-mediated ubiquitination of both β-dystroglycan and WWP1. The R440Q missense mutation in WWP1 decreases HECT domain-mediated intramolecular interactions to relieve autoinhibition of the enzyme. Our results provide new insight into the regulation of β-dystroglycan degradation by WWP1 and other Nedd4 family members and improves our understanding of dystroglycan-related disorders. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu, Xiaolin; Li, Qian; Pang, Liewen

    Highlights: •Arctigenin enhanced cholesterol efflux in oxLDL-loaded THP-1 macrophages. •The expression of ABCA1, ABCG1 and apoE was upregulated in arctigenin-treated cells. •Arctigenin promoted the expression of PPAR-γ and LXR-α. •Inhibition of PPAR-γ or LXR-α reversed arctigenin-mediated biological effects. •Arctigenin promotes cholesterol efflux via activation of PPAR-γ/LXR-α/ABCA1 pathway. -- Abstract: Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-densitymore » lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.« less

  12. A role for POR1, a Rac1-interacting protein, in ARF6-mediated cytoskeletal rearrangements.

    PubMed Central

    D'Souza-Schorey, C; Boshans, R L; McDonough, M; Stahl, P D; Van Aelst, L

    1997-01-01

    The ARF6 GTPase, the least conserved member of the ADP ribosylation factor (ARF) family, associates with the plasma membrane and intracellular endosome vesicles. Mutants of ARF6 defective in GTP binding and hydrolysis have a marked effect on endocytic trafficking and the gross morphology of the peripheral membrane system. Here we report that expression of the GTPase-defective mutant of ARF6, ARF6(Q67L), remodels the actin cytoskeleton by inducing actin polymerization at the cell periphery. This cytoskeletal rearrangement was inhibited by co-expression of ARF6(Q67L) with deletion mutants of POR1, a Rac1-interacting protein involved in membrane ruffling, but not with the dominant-negative mutant of Rac1, Rac1(S17N). A synergistic effect between POR1 and ARF6 for the induction of actin polymerization was detected. Furthermore, we observed that ARF6 interacts directly with POR1 and that this interaction was GTP dependent. These findings indicate that ARF6 and Rac1 function on distinct signaling pathways to mediate cytoskeletal reorganization, and suggest a role for POR1 as an important regulatory element in orchestrating cytoskeletal rearrangements at the cell periphery induced by ARF6 and Rac1. PMID:9312003

  13. Regulated efflux of photoreceptor outer segment-derived cholesterol by human RPE cells.

    PubMed

    Storti, Federica; Raphael, Gabriele; Griesser, Vera; Klee, Katrin; Drawnel, Faye; Willburger, Carolin; Scholz, Rebecca; Langmann, Thomas; von Eckardstein, Arnold; Fingerle, Jürgen; Grimm, Christian; Maugeais, Cyrille

    2017-12-01

    Genetic studies have linked age-related macular degeneration (AMD) to genes involved in high-density lipoprotein (HDL) metabolism, including ATP-binding cassette transporter A1 (ABCA1). The retinal pigment epithelium (RPE) handles large amounts of lipids, among others cholesterol, partially derived from internalized photoreceptor outer segments (OS) and lipids physiologically accumulate in the aging eye. To analyze the potential function of ABCA1 in the eye, we measured cholesterol efflux, the first step of HDL generation, in RPE cells. We show the expression of selected genes related to HDL metabolism in mouse and human eyecups as well as in ARPE-19 and human primary RPE cells. Immunofluorescence staining revealed localization of ABCA1 on both sides of polarized RPE cells. This was functionally confirmed by directional efflux to apolipoprotein AI (ApoA-I) of 3 H-labeled cholesterol given to the cells via serum or via OS. ABCA1 expression and activity was modulated using a liver-X-receptor (LXR) agonist and an ABCA1 neutralizing antibody, demonstrating that the efflux was ABCA1-dependent. We concluded that the ABCA1-mediated lipid efflux pathway, and hence HDL biosynthesis, is functional in RPE cells towards both the basal (choroidal) and apical (subretinal) space. Impaired activity of the pathway might cause age-related perturbations of lipid homeostasis in the outer retina and thus may contribute to disease development and/or progression. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. HDL-cholesterol concentration in pregnant Chinese Han women of late second trimester associated with genetic variants in CETP, ABCA1, APOC3, and GALNT2.

    PubMed

    Cui, Mingxuan; Li, Wei; Ma, Liangkun; Ping, Fan; Liu, Juntao; Wu, Xueyan; Mao, Jiangfeng; Wang, Xi; Nie, Min

    2017-08-22

    To investigate whether HDL-C level in pregnant Chinese Han women of late second trimester correlated with loci in high-density lipoprotein-cholesterol (HDL-C)-related genes found in genome-wide association studies (GWAS). Seven single-nucleotide polymorphisms (rs3764261 in CETP , rs1532085 in LIPC , rs7241918 in LIPG , rs1883025 in ABCA1 , rs4225 in APOC3 , rs1059611 in LPL , and rs16851339 in GALNT2 ) were genotyped using the Sequenom MassArray system for 1,884 pregnant women. The following polymorphisms were statistically associated with HDL-C level after adjusting for age, gestational week, pre-pregnancy BMI and state of GDM or HOMAIR: (i) rs3764261 (b = -0.055 mmol/L, 95% CI -0.101 to -0.008, p = 0.021), (ii) rs1883025 (b = -0.054 mmol/L, 95% CI -0.097 to -0.012, p = 0.013), (iii) rs4225 (b = -0.071 mmol/L, 95% CI -0.116 to -0.027, p = 1.79E-3) and (iv) rs16851339 (b = -0.064 mmol/L, 95% CI -0.120 to -0.008, p = 0.025). The more risk alleles the pregnant women have, the lower the plasma HDL-C levels of the subjects are. Several risk alleles found to be related to HDL-C in GWAS are also associated with HDL-C levels in pregnant Chinese Han women and these risk loci contribute additively to low HDL-C levels.

  15. HDL-cholesterol concentration in pregnant Chinese Han women of late second trimester associated with genetic variants in CETP, ABCA1, APOC3, and GALNT2

    PubMed Central

    Cui, Mingxuan; Li, Wei; Ma, Liangkun; Ping, Fan; Liu, Juntao; Wu, Xueyan; Mao, Jiangfeng; Wang, Xi; Nie, Min

    2017-01-01

    Objective To investigate whether HDL-C level in pregnant Chinese Han women of late second trimester correlated with loci in high-density lipoprotein-cholesterol (HDL-C)-related genes found in genome-wide association studies (GWAS). Methods Seven single-nucleotide polymorphisms (rs3764261 in CETP, rs1532085 in LIPC, rs7241918 in LIPG, rs1883025 in ABCA1, rs4225 in APOC3, rs1059611 in LPL, and rs16851339 in GALNT2) were genotyped using the Sequenom MassArray system for 1,884 pregnant women. Results The following polymorphisms were statistically associated with HDL-C level after adjusting for age, gestational week, pre-pregnancy BMI and state of GDM or HOMAIR: (i) rs3764261 (b = -0.055 mmol/L, 95% CI -0.101 to -0.008, p = 0.021), (ii) rs1883025 (b = -0.054 mmol/L, 95% CI -0.097 to -0.012, p = 0.013), (iii) rs4225 (b = -0.071 mmol/L, 95% CI -0.116 to -0.027, p = 1.79E-3) and (iv) rs16851339 (b = -0.064 mmol/L, 95% CI -0.120 to -0.008, p = 0.025). The more risk alleles the pregnant women have, the lower the plasma HDL-C levels of the subjects are. Conclusions Several risk alleles found to be related to HDL-C in GWAS are also associated with HDL-C levels in pregnant Chinese Han women and these risk loci contribute additively to low HDL-C levels. PMID:28915626

  16. Lubiprostone Increases Small Intestinal Smooth Muscle Contractions Through a Prostaglandin E Receptor 1 (EP1)-mediated Pathway.

    PubMed

    Chan, Walter W; Mashimo, Hiroshi

    2013-07-01

    Lubiprostone, a chloride channel type 2 (ClC-2) activator, was thought to treat constipation by enhancing intestinal secretion. It has been associated with increased intestinal transit and delayed gastric emptying. Structurally similar to prostones with up to 54% prostaglandin E2 activity on prostaglandin E receptor 1 (EP1), lubiprostone may also exert EP1-mediated procontractile effect on intestinal smooth muscles. We investigated lubiprostone's effects on intestinal smooth muscle contractions and pyloric sphincter tone. Isolated murine small intestinal (longitudinal and circular) and pyloric tissues were mounted in organ baths with modified Krebs solution for isometric recording. Basal muscle tension and response to electrical field stimulation (EFS; 2 ms pulses/10 V/6 Hz/30 sec train) were measured with lubiprostone (10(-10)-10(-5) M) ± EP1 antagonist. Significance was established using Student t test and P < 0.05. Lubiprostone had no effect on the basal tension or EFS-induced contractions of longitudinal muscles. With circular muscles, lubiprostone caused a dose-dependent increase in EFS-induced contractions (2.11 ± 0.88 to 4.43 ± 1.38 N/g, P = 0.020) that was inhibited by pretreatment with EP1 antagonist (1.69 ± 0.70 vs. 4.43 ± 1.38 N/g, P = 0.030). Lubiprostone had no effect on circular muscle basal tension, but it induced a dose-dependent increase in pyloric basal tone (1.07 ± 0.01 to 1.97 ± 0.86 fold increase, P < 0.05) that was inhibited by EP1 antagonist. In mice, lubiprostone caused a dose-dependent and EP1-mediated increase in contractility of circular but not longitudinal small intestinal smooth muscles, and in basal tone of the pylorus. These findings suggest another mechanism for lubiprostone's observed clinical effects on gastrointestinal motility.

  17. Inhibition of soluble epoxide hydrolase in mice promotes reverse cholesterol transport and regression of atherosclerosis.

    PubMed

    Shen, Li; Peng, Hongchun; Peng, Ran; Fan, Qingsong; Zhao, Shuiping; Xu, Danyan; Morisseau, Christophe; Chiamvimonvat, Nipavan; Hammock, Bruce D

    2015-04-01

    Adipose tissue is the body largest free cholesterol reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), which maintains plasma high-density lipoprotein (HDL) levels. HDLs have a protective role in atherosclerosis by mediating reverse cholesterol transport (RCT). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition has various beneficial effects on cardiovascular disease. The sEH is highly expressed in adipocytes, and it converts epoxyeicosatrienoic acids (EETs) into less bioactive dihydroxyeicosatrienoic acids. We previously showed that increasing EETs levels with a sEH inhibitor (sEHI) (t-AUCB) resulted in elevated ABCA1 expression and promoted ABCA1-mediated cholesterol efflux from 3T3-L1 adipocytes. The present study investigates the impacts of t-AUCB in mice deficient for the low density lipoprotein (LDL) receptor (Ldlr(-/-) mice) with established atherosclerotic plaques. The sEH inhibitor delivered in vivo for 4 weeks decreased the activity of sEH in adipose tissue, enhanced ABCA1 expression and cholesterol efflux from adipose depots, and consequently increased HDL levels. Furthermore, t-AUCB enhanced RCT to the plasma, liver, bile and feces. It also showed the reduction of plasma LDL-C levels. Consistently, t-AUCB-treated mice showed reductions in the size of atherosclerotic plaques. These studies establish that raising adipose ABCA1 expression, cholesterol efflux, and plasma HDL levels with t-AUCB treatment promotes RCT, decreasing LDL-C and atherosclerosis regression, suggesting that sEH inhibition may be a promising strategy to treat atherosclerotic vascular disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Phenotypic characteristics of Alzheimer patients carrying an ABCA7 mutation.

    PubMed

    Van den Bossche, Tobi; Sleegers, Kristel; Cuyvers, Elise; Engelborghs, Sebastiaan; Sieben, Anne; De Roeck, Arne; Van Cauwenberghe, Caroline; Vermeulen, Steven; Van den Broeck, Marleen; Laureys, Annelies; Peeters, Karin; Mattheijssens, Maria; Vandenbulcke, Mathieu; Vandenberghe, Rik; Martin, Jean-Jacques; De Deyn, Peter P; Cras, Patrick; Van Broeckhoven, Christine

    2016-06-07

    To generate a clinical and pathologic phenotype of patients carrying rare loss-of-function mutations in ABCA7, identified in a Belgian Alzheimer patient cohort and in an autosomal dominant family. We performed a retrospective review of available data records, medical records, results of CSF analyses and neuroimaging studies, and neuropathology data. The mean onset age of the mutation carriers (n = 22) was 73.4 ± 8.4 years with a wide age range of 36 (54-90) years, which was independent of APOE genotype and cerebrovascular disease. The mean disease duration was 5.7 ± 3.0 years (range 2-12 years). A positive family history was recorded for 10 carriers (45.5%). All patient carriers except one presented with memory complaints. The 4 autopsied brains showed typical immunohistochemical changes of late-onset Alzheimer disease. All patients carrying a loss-of-function mutation in ABCA7 exhibited a classical Alzheimer disease phenotype, though with a striking wide onset age range, suggesting the influence of unknown modifying factors. © 2016 American Academy of Neurology.

  19. Phenotypic characteristics of Alzheimer patients carrying an ABCA7 mutation

    PubMed Central

    Van den Bossche, Tobi; Sleegers, Kristel; Cuyvers, Elise; Engelborghs, Sebastiaan; Sieben, Anne; De Roeck, Arne; Van Cauwenberghe, Caroline; Vermeulen, Steven; Van den Broeck, Marleen; Laureys, Annelies; Peeters, Karin; Mattheijssens, Maria; Vandenbulcke, Mathieu; Vandenberghe, Rik; Martin, Jean-Jacques; De Deyn, Peter P.; Cras, Patrick

    2016-01-01

    Objective: To generate a clinical and pathologic phenotype of patients carrying rare loss-of-function mutations in ABCA7, identified in a Belgian Alzheimer patient cohort and in an autosomal dominant family. Methods: We performed a retrospective review of available data records, medical records, results of CSF analyses and neuroimaging studies, and neuropathology data. Results: The mean onset age of the mutation carriers (n = 22) was 73.4 ± 8.4 years with a wide age range of 36 (54–90) years, which was independent of APOE genotype and cerebrovascular disease. The mean disease duration was 5.7 ± 3.0 years (range 2–12 years). A positive family history was recorded for 10 carriers (45.5%). All patient carriers except one presented with memory complaints. The 4 autopsied brains showed typical immunohistochemical changes of late-onset Alzheimer disease. Conclusions: All patients carrying a loss-of-function mutation in ABCA7 exhibited a classical Alzheimer disease phenotype, though with a striking wide onset age range, suggesting the influence of unknown modifying factors. PMID:27037232

  20. [Hepatotoxicity of emodin based on UGT1A1 enzyme-mediated bilirubin in liver microsomes].

    PubMed

    Wang, Qi; Dai, Zhong; Zhang, Yu-Jie; Ma, Shuang-Cheng

    2016-12-01

    To study the hepatotoxicity of emodin based on bilirubin metabolism mediated by glucuronidation of UGT1A1 enzyme. In this study, three different incubation systems were established by using RLM, HLM, and rUGT1A1, with bilirubin as the substrate. Different concentrations of bilirubin and emodin were added in the incubation systems. The double reciprocal Michaelis equation was drawn based on the total amount of bilirubin glucuronidation. The apparent inhibition constant Ki was then calculated with the slope curve to predict the hepatotoxicity. The results indicated that emodin had a significant inhibition to the UGT1A1 enzyme in all of the three systems, with Ki=5.400±0.956(P<0.05) in HLM system, Ki =10.020±0.611(P<0.05) in RLM system, Ki=4.850±0.528(P<0.05) in rUGT1A1 system. Meanwhile, emodin had no significant difference between rat and human in terms of inhibition of UGT1A1 enzyme. Emodin had a potential risk of the hepatotoxicity by inhibiting the UGT1A1 enzyme activity. And the method established in this study provides a new thought and new method to evaluate hepatotoxicity and safety of traditional Chinese medicines. Copyright© by the Chinese Pharmaceutical Association.

  1. Dexras1 mediates glucocorticoid-associated adipogenesis and diet-induced obesity

    PubMed Central

    Cha, Jiyoung Y.; Kim, Hyo Jung; Yu, Jung Hwan; Xu, Jing; Kim, Daham; Paul, Bindu D.; Choi, Hyeonjin; Kim, Seyun; Lee, Yoo Jeong; Ho, Gary P.; Rao, Feng; Snyder, Solomon H.; Kim, Jae-woo

    2013-01-01

    Adipogenesis, the conversion of precursor cells into adipocytes, is associated with obesity and is mediated by glucocorticoids acting via hitherto poorly characterized mechanisms. Dexras1 is a small G protein of the Ras family discovered on the basis of its marked induction by the synthetic glucocorticoid dexamethasone. We show that Dexras1 mediates adipogenesis and diet-induced obesity. Adipogenic differentiation of 3T3-L1 cells is abolished with Dexras1 depletion, whereas overexpression of Dexras1 elicits adipogenesis. Adipogenesis is markedly reduced in mouse embryonic fibroblasts from Dexras1-deleted mice, whereas adiposity and diet-induced weight gain are diminished in the mutant mice. PMID:24297897

  2. Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation

    NASA Astrophysics Data System (ADS)

    Cheng, Jingdong; Yang, Huirong; Fang, Jian; Ma, Lixiang; Gong, Rui; Wang, Ping; Li, Ze; Xu, Yanhui

    2015-05-01

    DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1-USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1-USP7 interaction surface for therapeutic applications.

  3. The organic anion transport polypeptide 1d1 (Oatp1d1) mediates hepatocellular uptake of phalloidin and microcystin into skate liver.

    PubMed

    Meier-Abt, F; Hammann-Hänni, A; Stieger, B; Ballatori, N; Boyer, J L

    2007-02-01

    Organic anion transporting polypeptides (rodent Oatp; human OATP) mediate cellular uptake of numerous organic compounds including xenobiotic toxins into mammalian hepatocytes. In the little skate Leucoraja erinacea a liver-specific Oatp (Oatp1d1, also called sOatp) has been identified and suggested to represent an evolutionarily ancient precursor of the mammalian liver OATP1B1 (human), Oatp1b2 (rat), and OATP1B3 (human). The present study tested whether Oatp1d1 shares functional transport activity of the xenobiotic oligopeptide toxins phalloidin and microcystin with the mammalian liver Oatps/OATPs. The phalloidin analogue [(3)H]-demethylphalloin was taken up into skate hepatocytes with high affinity (Km approximately 0.4 microM), and uptake could be inhibited by phalloidin and a variety of typical Oatp/OATP substrates such as bromosulfophthalein, bile salts, estrone-3-sulfate, cyclosporine A and high concentrations of microcystin-LR (Ki approximately 150 microM). When expressed in Xenopus laevis oocytes Oatp1d1 increased uptake of demethylphalloin (Km approximately 2.2 microM) and microcystin-LR (Km approximately 27 microM) 2- to 3-fold over water-injected oocytes, whereas the alternative skate liver organic anion transporter, the dimeric Ostalpha/beta, exhibited no phalloidin and only minor microcystin-LR transport. Also, the closest mammalian Oatp1d1 orthologue, the human brain and testis OATP1C1, did not show any phalloidin transport activity. These results demonstrate that the evolutionarily ancient Oatp1d1 is able to mediate uptake of cyclic oligopeptide toxins into skate liver. The findings support the notion that Oatp1d1 is a precursor of the liver-specific mammalian Oatps/OATPs and that its transport properties are closely associated with certain forms of toxic liver injury such as for example protein phosphatase inhibition by the water-borne toxin microcystin.

  4. Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sakamaki, Jun-ichi; Daitoku, Hiroaki; Yoshimochi, Kenji

    2009-05-08

    Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27{sup Kip1} gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependentmore » on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27{sup Kip1} gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27{sup Kip1} gene expression.« less

  5. Resolving TRPV1 and TNF-α Mediated Spinal Cord Synaptic Plasticity and Inflammatory Pain with Neuroprotectin D1

    PubMed Central

    Park, Chul-Kyu; Lü, Ning; Xu, Zhen-Zhong; Liu, Tong; Serhan, Charles N.; Ji, Ru-Rong

    2011-01-01

    Mechanisms of inflammatory pain are not fully understood. We investigated the role of TRPV1 and TNF-α, two critical mediators for inflammatory pain, in regulating spinal cord synaptic transmission. We found in mice lacking Trpv1 the frequency but not the amplitude of spontaneous EPSCs (sEPSCs) in lamina II neurons of spinal cord slices is reduced. Further, C-fiber-induced spinal long-term potentiation (LTP) in vivo is abolished in Trpv1 knockout mice. TNF-α also increases sEPSC frequency but not amplitude in spinal lamina IIo neurons, and this increase is abolished in Trpv1 knockout mice. Single-cell PCR analysis revealed that TNF-α-responding neurons in lamina IIo are exclusively excitatory (vGluT2+) neurons. Notably, neuroprotectin-1 (NPD1), an anti-inflammatory lipid mediator derived from omega-3 polyunsaturated fatty acid (docosahexaenoic acid) blocks TNF-α- and capsaicin-evoked sEPSC frequency increases but has no effect on basal synaptic transmission. Strikingly, NPD1 potently inhibits capsaicin-induced TRPV1 current (IC50=0.4 nM) in dissociated dorsal root ganglion neurons, and this IC50 is ≈ 500 times lower than that of AMG9810, a commonly used TRPV1 antagonist. NPD1 inhibition of TRPV1 is mediated by GPCRs, since the effects were blocked by pertussis toxin. In contrast, NPD1 had not effect on mustard oil-induced TRPA1 currents. Spinal injection of NPD1, at very low doses (0.1–10 ng), blocks spinal LTP and reduces TRPV1-dependent inflammatory pain, without affecting baseline pain. NPD1 also reduces TRPV1-independent but TNF-α-dependent pain hypersensitivity. Our findings demonstrate a novel role of NPD1 in regulating TRPV1/TNF-α-mediated spinal synaptic plasticity and identify NPD1 as a novel analgesic for treating inflammatory pain. PMID:22016541

  6. Sphingosine-1-Phosphate Receptor-1 Promotes Environment-Mediated and Acquired Chemoresistance.

    PubMed

    Lifshitz, Veronica; Priceman, Saul J; Li, Wenzhao; Cherryholmes, Gregory; Lee, Heehyoung; Makovski-Silverstein, Adar; Borriello, Lucia; DeClerck, Yves A; Yu, Hua

    2017-11-01

    Drug resistance is a major barrier for the development of effective and durable cancer therapies. Overcoming this challenge requires further defining the cellular and molecular mechanisms underlying drug resistance, both acquired and environment-mediated drug resistance (EMDR). Here, using neuroblastoma (NB), a childhood cancer with high incidence of recurrence due to resistance to chemotherapy, as a model we show that human bone marrow-mesenchymal stromal cells induce tumor expression of sphingosine-1-phosphate receptor-1 (S1PR1), leading to their resistance to chemotherapy. Targeting S1PR1 by shRNA markedly enhances etoposide-induced apoptosis in NB cells and abrogates EMDR, while overexpression of S1PR1 significantly protects NB cells from multidrug-induced apoptosis via activating JAK-STAT3 signaling. Elevated S1PR1 expression and STAT3 activation are also observed in human NB cells with acquired resistance to etoposide. We show in vitro and in human NB xenograft models that treatment with FTY720, an FDA-approved drug and antagonist of S1PR1, dramatically sensitizes drug-resistant cells to etoposide. In summary, we identify S1PR1 as a critical target for reducing both EMDR and acquired chemoresistance in NB. Mol Cancer Ther; 16(11); 2516-27. ©2017 AACR . ©2017 American Association for Cancer Research.

  7. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zaja, Ivan; Bai, Xiaowen, E-mail: xibai@mcw.edu; Liu, Yanan

    Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1more » (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

  8. HER1 signaling mediates extravillous trophoblast differentiation in humans.

    PubMed

    Wright, J K; Dunk, C E; Amsalem, H; Maxwell, C; Keating, S; Lye, S J

    2010-12-01

    This study examines the role of HER1 signaling in the differentiation of proliferative extravillous trophoblast (EVT) into invasive EVT. Using the JAR choriocarcinoma cell line and placental villous explants as experimental models and immunohistochemical assessment of protein markers of EVT differentiation (downregulation of HER1 and Cx40 and upregulation of HER2 and alpha1 integrin), we show that the ability of decidual conditioned medium (DCM) to induce HER1/2 switching was abrogated in the presence of the HER1 antagonist, AG1478. Similarly, epidermal growth factor (EGF) treatment resulted in the downregulation of HER1 and an upregulation of HER2 expression, whereas co-incubation of EGF with AG1478 inhibited this response. However, EGF did not downregulate Cx40 or induce migration of EVT. In contrast, heparin-binding epidermal-like growth factor (HBEGF) stimulated dose-dependent JAR cell migration, which was inhibited by both AG1478 and AG825 (HER2 antagonist). Western blot analysis of HER1 activation demonstrated that HBEGF-mediated phosphorylation of the HER1 Tyr992 and Tyr1068 sites, while EGF activated the Tyr1045 site. Moreover, HBEGF induced a stronger and more sustained activation of both the mitogen-activated protein kinase and phosphoinositol 3 kinase (PIK3) signaling pathways. Migration assays using a panel of signaling pathway inhibitors demonstrated that the HBEGF-mediated migration was dependent on the PIK3 pathway. These results demonstrate that HBEGF-mediated HER1 signaling through PIK3 is an important component of EVT invasion.

  9. Role of APOE Isforms in the Pathogenesis of TBI Induced Alzheimer’s Disease

    DTIC Science & Technology

    2014-10-01

    the inheritance of APOe4 is the only proven genetic risk factor for sporadic Alzheimer disease (AD). Importantly, TBI is a risk factor for the...mice on human APOE genetic background were exceptionally difficult to generate. We are considering changes in the genotype of those particular groups...mediated through ABCA1. 2 Keywords Traumatic brain injury, APOE isoforms, ABCA1, Alzheimer disease, APPmice, amyloid beta, axonal injury, inflamma

  10. I-domain of lymphocyte function-associated antigen-1 mediates rolling of polystyrene particles on ICAM-1 under flow.

    PubMed

    Eniola, A Omolola; Krasik, Ellen F; Smith, Lee A; Song, Gang; Hammer, Daniel A

    2005-11-01

    In their active state, beta(2)-integrins, such as LFA-1, mediate the firm arrest of leukocytes by binding intercellular adhesion molecules (ICAMs) expressed on endothelium. Although the primary function of LFA-1 is assumed to be the ability to mediate firm adhesion, recent work has shown that LFA-1 can contribute to cell tethering and rolling under hydrodynamic flow, a role previously largely attributed to the selectins. The inserted (I) domain of LFA-1 has recently been crystallized in the wild-type (wt) and locked-open conformations and has been shown to, respectively, support rolling and firm adhesion under flow when expressed in alpha(L)beta(2) heterodimers or as isolated domains on cells. Here, we report results from cell-free adhesion assays where wt I-domain-coated polystyrene particles were allowed to interact with ICAM-1-coated surfaces in shear flow. We show that wt I-domain can independently mediate the capture of particles from flow and support their rolling on ICAM-1 surfaces in a manner similar to how carbohydrate-selectin interactions mediate rolling. Adhesion is specific and blocked by appropriate antibodies. We also show that the rolling velocity of I-domain-coated particles depends on the wall shear stress in flow chamber, I-domain site density on microsphere surfaces, and ICAM-1 site density on substrate surfaces. Furthermore, we show that rolling is less sensitive to wall shear stress and ICAM-1 substrate density at high density of I-domain on the microsphere surface. Computer simulations using adhesive dynamics can recreate bead rolling dynamics and show that the mechanochemical properties of ICAM-1-I-domain interactions are similar to those of carbohydrate-selectin interactions. Understanding the biophysics of adhesion mediated by the I-domain of LFA-1 can elucidate the complex roles this integrin plays in leukocyte adhesion in inflammation.

  11. MACC1 mediates chemotherapy sensitivity of 5-FU and cisplatin via regulating MCT1 expression in gastric cancer.

    PubMed

    Wang, Chunlin; Wen, Zhaowei; Xie, Jianming; Zhao, Yang; Zhao, Liang; Zhang, Shuyi; Liu, Yajing; Xue, Yan; Shi, Min

    2017-04-08

    Chemotherapeutic insensitivity is a main obstacle for effective treatment of gastric cancer (GC), the underlying mechanism remains to be investigated. Metastasis-associated in colon cancer-1 (MACC1), a transcription factor highly expressed in GC, is found to be related to chemotherapy sensitivity. Monocarboxylate transporter 1 (MCT1), a plasma membrane protein co-transporting lactate and H + , mediates drug sensitivity by regulating lactate metabolism. Targeting MCT1 has recently been regarded as a promising way to treat cancers and MCT1 inhibitor has entered the clinical trial for GC treatment. However, the correlation of these two genes and their combined effects on chemotherapy sensitivity has not been clarified. In this study, we found that MACC1 and MCT1 were both highly expressed in GC and exhibited a positive correlation in clinical samples. Further, we demonstrated that MACC1 could mediate sensitivity of 5-FU and cisplatin in GC cells, and MACC1 mediated MCT1 regulation was closely related to this sensitivity. A MCT1 inhibitor AZD3965 recovered the sensitivity of 5-FU and cisplatin in GC cells which overexpressed MACC1. These results suggested that MACC1 could influence the chemotherapy sensitivity by regulating MCT1 expression, providing new ideas and strategy for GC treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Brain angiogenesis inhibitor 1 (BAI1) is a pattern recognition receptor that mediates macrophage binding and engulfment of Gram-negative bacteria

    PubMed Central

    Das, Soumita; Owen, Katherine A.; Ly, Kim T.; Park, Daeho; Black, Steven G.; Wilson, Jeffrey M.; Sifri, Costi D.; Ravichandran, Kodi S.; Ernst, Peter B.; Casanova, James E.

    2011-01-01

    Bacterial recognition by host cells is essential for initiation of infection and the host response. Bacteria interact with host cells via multiple pattern recognition receptors that recognize microbial products or pathogen-associated molecular patterns. In response to this interaction, host cell signaling cascades are activated that lead to inflammatory responses and/or phagocytic clearance of attached bacteria. Brain angiogenesis inhibitor 1 (BAI1) is a receptor that recognizes apoptotic cells through its conserved type I thrombospondin repeats and triggers their engulfment through an ELMO1/Dock/Rac1 signaling module. Because thrombospondin repeats in other proteins have been shown to bind bacterial surface components, we hypothesized that BAI1 may also mediate the recognition and clearance of pathogenic bacteria. We found that preincubation of bacteria with recombinant soluble BAI1 ectodomain or knockdown of endogenous BAI1 in primary macrophages significantly reduced binding and internalization of the Gram-negative pathogen Salmonella typhimurium. Conversely, overexpression of BAI1 enhanced attachment and engulfment of Salmonella in macrophages and in heterologous nonphagocytic cells. Bacterial uptake is triggered by the BAI1-mediated activation of Rac through an ELMO/Dock-dependent mechanism, and inhibition of the BAI1/ELMO1 interaction prevents both Rac activation and bacterial uptake. Moreover, inhibition of ELMO1 or Rac function significantly impairs the proinflammatory response to infection. Finally, we show that BAI1 interacts with a variety of Gram-negative, but not Gram-positive, bacteria through recognition of their surface lipopolysaccharide. Together these findings identify BAI1 as a pattern recognition receptor that mediates nonopsonic phagocytosis of Gram-negative bacteria by macrophages and directly affects the host response to infection. PMID:21245295

  13. GPER signalling in both cancer-associated fibroblasts and breast cancer cells mediates a feedforward IL1β/IL1R1 response

    PubMed Central

    De Marco, Paola; Lappano, Rosamaria; Francesco, Ernestina Marianna De; Cirillo, Francesca; Pupo, Marco; Avino, Silvia; Vivacqua, Adele; Abonante, Sergio; Picard, Didier; Maggiolini, Marcello

    2016-01-01

    Cancer-associated fibroblasts (CAFs) contribute to the malignant aggressiveness through secreted factors like IL1β, which may drive pro-tumorigenic inflammatory phenotypes mainly acting via the cognate receptor named IL1R1. Here, we demonstrate that signalling mediated by the G protein estrogen receptor (GPER) triggers IL1β and IL1R1 expression in CAFs and breast cancer cells, respectively. Thereby, ligand-activation of GPER generates a feedforward loop coupling IL1β induction by CAFs to IL1R1 expression by cancer cells, promoting the up-regulation of IL1β/IL1R1 target genes such as PTGES, COX2, RAGE and ABCG2. This regulatory interaction between the two cell types induces migration and invasive features in breast cancer cells including fibroblastoid cytoarchitecture and F-actin reorganization. A better understanding of the mechanisms involved in the regulation of pro-inflammatory cytokines by GPER-integrated estrogen signals may be useful to target these stroma-cancer interactions. PMID:27072893

  14. LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice

    PubMed Central

    Geister, Krista A.; Brinkmeier, Michelle L.; Cheung, Leonard Y.; Wendt, Jennifer; Oatley, Melissa J.; Burgess, Daniel L.; Kozloff, Kenneth M.; Cavalcoli, James D.; Oatley, Jon M.; Camper, Sally A.

    2015-01-01

    Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility. PMID:26496357

  15. LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice.

    PubMed

    Geister, Krista A; Brinkmeier, Michelle L; Cheung, Leonard Y; Wendt, Jennifer; Oatley, Melissa J; Burgess, Daniel L; Kozloff, Kenneth M; Cavalcoli, James D; Oatley, Jon M; Camper, Sally A

    2015-10-01

    Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility.

  16. Astrocytes Regulate GLP-1 Receptor-Mediated Effects on Energy Balance

    PubMed Central

    Reiner, David J.; Mietlicki-Baase, Elizabeth G.; McGrath, Lauren E.; Zimmer, Derek J.; Bence, Kendra K.; Sousa, Gregory L.; Konanur, Vaibhav R.; Krawczyk, Joanna; Burk, David H.; Kanoski, Scott E.; Hermann, Gerlinda E.; Rogers, Richard C.

    2016-01-01

    Astrocytes are well established modulators of extracellular glutamate, but their direct influence on energy balance-relevant behaviors is largely understudied. As the anorectic effects of glucagon-like peptide-1 receptor (GLP-1R) agonists are partly mediated by central modulation of glutamatergic signaling, we tested the hypothesis that astrocytic GLP-1R signaling regulates energy balance in rats. Central or peripheral administration of a fluorophore-labeled GLP-1R agonist, exendin-4, localizes within astrocytes and neurons in the nucleus tractus solitarius (NTS), a hindbrain nucleus critical for energy balance control. This effect is mediated by GLP-1R, as the uptake of systemically administered fluorophore-tagged exendin-4 was blocked by central pretreatment with the competitive GLP-1R antagonist exendin-(9–39). Ex vivo analyses show prolonged exendin-4-induced activation (live cell calcium signaling) of NTS astrocytes and neurons; these effects are also attenuated by exendin-(9–39), indicating mediation by the GLP-1R. In vitro analyses show that the application of GLP-1R agonists increases cAMP levels in astrocytes. Immunohistochemical analyses reveal that endogenous GLP-1 axons form close synaptic apposition with NTS astrocytes. Finally, pharmacological inhibition of NTS astrocytes attenuates the anorectic and body weight-suppressive effects of intra-NTS GLP-1R activation. Collectively, data demonstrate a role for NTS astrocytic GLP-1R signaling in energy balance control. SIGNIFICANCE STATEMENT Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce food intake and are approved by the Food and Drug Administration for the treatment of obesity, but the cellular mechanisms underlying the anorectic effects of GLP-1 require further investigation. Astrocytes represent a major cellular population in the CNS that regulates neurotransmission, yet the role of astrocytes in mediating energy balance is largely unstudied. The current data provide novel evidence that

  17. Distinct signalling properties of insulin receptor substrate (IRS)-1 and IRS-2 in mediating insulin/IGF-1 action.

    PubMed

    Rabiee, Atefeh; Krüger, Marcus; Ardenkjær-Larsen, Jacob; Kahn, C Ronald; Emanuelli, Brice

    2018-07-01

    Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type, IRS-1 -/- and IRS-2 -/- mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1. These regulated sites included previously reported substrates of the insulin/IGF-1 signalling pathway, as well as novel substrates including Nuclear Factor I X and Semaphorin-4B. In silico prediction suggests the protein kinase B (PKB), protein kinase C (PKC), and cyclin-dependent kinase (CDK) as the main mediators of these phosphorylation events. Importantly, we found preferential phosphorylation patterns depending on the presence of either IRS-1 or IRS-2, which was associated with specific sets of kinases involved in signal transduction downstream of these substrates such as PDHK1, MAPK3, and PKD1 for IRS-1, and PIN1 and PKC beta for IRS-2. Overall, by generating a comprehensive phosphoproteomic profile from brown preadipocyte cells in response to IGF-1 stimulation, we reveal both common and distinct insulin/IGF-1 signalling events mediated by specific IRS proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Altered surfactant homeostasis and recurrent respiratory failure secondary to TTF-1 nuclear targeting defect.

    PubMed

    Peca, Donatella; Petrini, Stefania; Tzialla, Chryssoula; Boldrini, Renata; Morini, Francesco; Stronati, Mauro; Carnielli, Virgilio P; Cogo, Paola E; Danhaive, Olivier

    2011-08-25

    Mutations of genes affecting surfactant homeostasis, such as SFTPB, SFTPC and ABCA3, lead to diffuse lung disease in neonates and children. Haploinsufficiency of NKX2.1, the gene encoding the thyroid transcription factor-1 (TTF-1)--critical for lung, thyroid and central nervous system morphogenesis and function--causes a rare form of progressive respiratory failure designated brain-lung-thyroid syndrome. Molecular mechanisms involved in this syndrome are heterogeneous and poorly explored. We report a novel TTF-1 molecular defect causing recurrent respiratory failure episodes in an infant. The subject was an infant with severe neonatal respiratory distress syndrome followed by recurrent respiratory failure episodes, hypopituitarism and neurological abnormalities. Lung histology and ultrastructure were assessed by surgical biopsy. Surfactant-related genes were studied by direct genomic DNA sequencing and array chromatine genomic hybridization (aCGH). Surfactant protein expression in lung tissue was analyzed by confocal immunofluorescence microscopy. For kinetics studies, surfactant protein B and disaturated phosphatidylcholine (DSPC) were isolated from serial tracheal aspirates after intravenous administration of stable isotope-labeled (2)H(2)O and (13)C-leucine; fractional synthetic rate was derived from gas chromatography/mass spectrometry (2)H and (13)C enrichment curves. Six intubated infants with no primary lung disease were used as controls. Lung biopsy showed desquamative interstitial pneumonitis and lamellar body abnormalities suggestive of genetic surfactant deficiency. Genetic studies identified a heterozygous ABCA3 mutation, L941P, previously unreported. No SFTPB, SFTPC or NKX2.1 mutations or deletions were found. However, immunofluorescence studies showed TTF-1 prevalently expressed in type II cell cytoplasm instead of nucleus, indicating defective nuclear targeting. This pattern has not been reported in human and was not found in two healthy controls and

  19. Altered surfactant homeostasis and recurrent respiratory failure secondary to TTF-1 nuclear targeting defect

    PubMed Central

    2011-01-01

    Background Mutations of genes affecting surfactant homeostasis, such as SFTPB, SFTPC and ABCA3, lead to diffuse lung disease in neonates and children. Haploinsufficiency of NKX2.1, the gene encoding the thyroid transcription factor-1 (TTF-1) - critical for lung, thyroid and central nervous system morphogenesis and function - causes a rare form of progressive respiratory failure designated brain-lung-thyroid syndrome. Molecular mechanisms involved in this syndrome are heterogeneous and poorly explored. We report a novel TTF-1 molecular defect causing recurrent respiratory failure episodes in an infant. Methods The subject was an infant with severe neonatal respiratory distress syndrome followed by recurrent respiratory failure episodes, hypopituitarism and neurological abnormalities. Lung histology and ultrastructure were assessed by surgical biopsy. Surfactant-related genes were studied by direct genomic DNA sequencing and array chromatine genomic hybridization (aCGH). Surfactant protein expression in lung tissue was analyzed by confocal immunofluorescence microscopy. For kinetics studies, surfactant protein B and disaturated phosphatidylcholine (DSPC) were isolated from serial tracheal aspirates after intravenous administration of stable isotope-labeled 2H2O and 13C-leucine; fractional synthetic rate was derived from gas chromatography/mass spectrometry 2H and 13C enrichment curves. Six intubated infants with no primary lung disease were used as controls. Results Lung biopsy showed desquamative interstitial pneumonitis and lamellar body abnormalities suggestive of genetic surfactant deficiency. Genetic studies identified a heterozygous ABCA3 mutation, L941P, previously unreported. No SFTPB, SFTPC or NKX2.1 mutations or deletions were found. However, immunofluorescence studies showed TTF-1 prevalently expressed in type II cell cytoplasm instead of nucleus, indicating defective nuclear targeting. This pattern has not been reported in human and was not found in two

  20. A novel requirement for C. elegans Alix/ALX-1 in RME-1 mediated membrane transport

    PubMed Central

    Shi, Anbing; Pant, Saumya; Balklava, Zita; Chen, Carlos Chih-Hsiung; Figueroa, Vanesa; Grant, Barth D.

    2007-01-01

    Summary Background Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition Alix is associated with the actin cytoskeleton and may regulate cytoskeletal dynamics. Results Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane called RME-1. Analysis of alx-1 mutants indicates that ALX-1 is required for endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by analysis of rme-1 mutants. Expression of truncated human Alix in HeLa cells disrupts recycling of MHCI, a known Ehd1/RME-1 dependent transport step, suggesting phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears dispensable for ALX-1 function in MVEs/late endosomes. Conclusions This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1. PMID:17997305

  1. RACK1-mediated translation control promotes liver fibrogenesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Min; Peng, Peike; Wang, Jiajun

    Activation of quiescent hepatic stellate cells (HSCs) is the central event of liver fibrosis. The translational machinery is an optimized molecular network that affects cellular homoeostasis and diseases, whereas the role of protein translation in HSCs activation and liver fibrosis is little defined. Our previous report suggests that up-regulation of receptor for activated C-kinase 1(RACK1) in HSCs is critical for liver fibrogenesis. In this study, we found that RACK1 promoted macrophage conditioned medium (MCM)-induced assembly of eIF4F and phosphorylation of eIF4E in primary HSCs. RACK1 enhanced the translation and expression of pro-fibrogenic factors collagen 1α1, snail and cyclin E1 inducedmore » by MCM. Administration of PP242 or knock-down of eIF4E suppressed RACK1-stimulated collagen 1α1 production, proliferation and migration in primary HSCs. In addition, depletion of eIF4E attenuated thioacetamide (TAA)-induced liver fibrosis in vivo. Our data suggest that RACK1-mediated stimulation of cap-dependent translation plays crucial roles in HSCs activation and liver fibrogenesis, and targeting translation initiation could be a promising strategy for the treatment of liver fibrosis. - Highlights: • RACK1 induces the assembly of eIF4F and phosphorylation of eIF4E in primary HSCs. • RACK1 stimulates the translation of collagen 1α1, snail and cyclin E1 in HSCs. • RACK1 promotes HSCs activation via cap-mediated translation. • Depletion of eIF4E suppresses liver fibrogenesis in vivo.« less

  2. A Cytogenetic Abnormality and Rare Coding Variants Identify ABCA13 as a Candidate Gene in Schizophrenia, Bipolar Disorder, and Depression

    PubMed Central

    Knight, Helen M.; Pickard, Benjamin S.; Maclean, Alan; Malloy, Mary P.; Soares, Dinesh C.; McRae, Allan F.; Condie, Alison; White, Angela; Hawkins, William; McGhee, Kevin; van Beck, Margaret; MacIntyre, Donald J.; Starr, John M.; Deary, Ian J.; Visscher, Peter M.; Porteous, David J.; Cannon, Ronald E.; St Clair, David; Muir, Walter J.; Blackwood, Douglas H.R.

    2009-01-01

    Schizophrenia and bipolar disorder are leading causes of morbidity across all populations, with heritability estimates of ∼80% indicating a substantial genetic component. Population genetics and genome-wide association studies suggest an overlap of genetic risk factors between these illnesses but it is unclear how this genetic component is divided between common gene polymorphisms, rare genomic copy number variants, and rare gene sequence mutations. We report evidence that the lipid transporter gene ABCA13 is a susceptibility factor for both schizophrenia and bipolar disorder. After the initial discovery of its disruption by a chromosome abnormality in a person with schizophrenia, we resequenced ABCA13 exons in 100 cases with schizophrenia and 100 controls. Multiple rare coding variants were identified including one nonsense and nine missense mutations and compound heterozygosity/homozygosity in six cases. Variants were genotyped in additional schizophrenia, bipolar, depression (n > 1600), and control (n > 950) cohorts and the frequency of all rare variants combined was greater than controls in schizophrenia (OR = 1.93, p = 0.0057) and bipolar disorder (OR = 2.71, p = 0.00007). The population attributable risk of these mutations was 2.2% for schizophrenia and 4.0% for bipolar disorder. In a study of 21 families of mutation carriers, we genotyped affected and unaffected relatives and found significant linkage (LOD = 4.3) of rare variants with a phenotype including schizophrenia, bipolar disorder, and major depression. These data identify a candidate gene, highlight the genetic overlap between schizophrenia, bipolar disorder, and depression, and suggest that rare coding variants may contribute significantly to risk of these disorders. PMID:19944402

  3. Cofilin1-dependent actin dynamics control DRP1-mediated mitochondrial fission

    PubMed Central

    Rehklau, Katharina; Hoffmann, Lena; Gurniak, Christine B; Ott, Martin; Witke, Walter; Scorrano, Luca; Culmsee, Carsten; Rust, Marco B

    2017-01-01

    Mitochondria form highly dynamic networks in which organelles constantly fuse and divide. The relevance of mitochondrial dynamics is evident from its implication in various human pathologies, including cancer or neurodegenerative, endocrine and cardiovascular diseases. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission that oligomerizes at the mitochondrial outer membrane and hydrolyzes GTP to drive mitochondrial fragmentation. Previous studies demonstrated that DRP1 recruitment and mitochondrial fission is promoted by actin polymerization at the mitochondrial surface, controlled by the actin regulatory proteins inverted formin 2 (INF2) and Spire1C. These studies suggested the requirement of additional actin regulatory activities to control DRP1-mediated mitochondrial fission. Here we show that the actin-depolymerizing protein cofilin1, but not its close homolog actin-depolymerizing factor (ADF), is required to maintain mitochondrial morphology. Deletion of cofilin1 caused mitochondrial DRP1 accumulation and fragmentation, without altering mitochondrial function or other organelles’ morphology. Mitochondrial morphology in cofilin1-deficient cells was restored upon (i) re-expression of wild-type cofilin1 or a constitutively active mutant, but not of an actin-binding-deficient mutant, (ii) pharmacological destabilization of actin filaments and (iii) genetic depletion of DRP1. Our work unraveled a novel function for cofilin1-dependent actin dynamics in mitochondrial fission, and identified cofilin1 as a negative regulator of mitochondrial DRP1 activity. We conclude that cofilin1 is required for local actin dynamics at mitochondria, where it may balance INF2/Spire1C-induced actin polymerization. PMID:28981113

  4. Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction.

    PubMed

    Mathew, Stephen O; Chaudhary, Pankaj; Powers, Sheila B; Vishwanatha, Jamboor K; Mathew, Porunelloor A

    2016-10-18

    Prostate cancer is the most common type of cancer diagnosed and the second leading cause of cancer-related death in American men. Natural Killer (NK) cells are the first line of defense against cancer and infections. NK cell function is regulated by a delicate balance between signals received through activating and inhibitory receptors. Previously, we identified Lectin-like transcript-1 (LLT1/OCIL/CLEC2D) as a counter-receptor for the NK cell inhibitory receptor NKRP1A (CD161). Interaction of LLT1 expressed on target cells with NKRP1A inhibits NK cell activation. In this study, we have found that LLT1 was overexpressed on prostate cancer cell lines (DU145, LNCaP, 22Rv1 and PC3) and in primary prostate cancer tissues both at the mRNA and protein level. We further showed that LLT1 is retained intracellularly in normal prostate cells with minimal cell surface expression. Blocking LLT1 interaction with NKRP1A by anti-LLT1 mAb on prostate cancer cells increased the NK-mediated cytotoxicity of prostate cancer cells. The results indicate that prostate cancer cells may evade immune attack by NK cells by expressing LLT1 to inhibit NK cell-mediated cytolytic activity through LLT1-NKRP1A interaction. Blocking LLT1-NKRP1A interaction will make prostate cancer cells susceptible to killing by NK cells and therefore may be a new therapeutic option for treatment of prostate cancer.

  5. Tangeretin inhibits the proliferation of human breast cancer cells via CYP1A1/CYP1B1 enzyme induction and CYP1A1/CYP1B1-mediated metabolism to the product 4' hydroxy tangeretin.

    PubMed

    Surichan, Somchaiya; Arroo, Randolph R; Tsatsakis, Aristidis M; Androutsopoulos, Vasilis P

    2018-04-04

    Tangeretin is a polymethoxylated flavone with multifaceted anticancer activity. In the present study, the metabolism of tangeretin was evaluated in the CYP1 expressing human breast cancer cell lines MCF7 and MDA-MB-468 and in the normal breast cell line MCF10A. Tangeretin was converted to 4' OH tangeretin by recombinant CYP1 enzymes and by CYP1 enzymes expressed in MCF7 and MDA-MB-468 cells. This metabolite was absent in MCF10A cells that did not express CYP1 enzymes. Tangeretin exhibited submicromolar IC50 (0.25 ± 0.15 μM) in MDA-MB-468 cells, whereas it was less active in MCF7 cells (39.3 ± 1.5 μM) and completely inactive in MCF10A cells (>100 μM). In MDA-MB-468 cells that were coincubated with the CYP1 inhibitor acacetin, an approximately 70-fold increase was noted in the IC50 (18 ± 1.6 μM) of tangeretin. In the presence of the CYP1 inhibitor acacetin, the conversion of tangeretin to 4' OH tangeretin was significantly reduced in MDA-MB-468 cells (2.55 ± 0.19 μM vs. 6.33 ± 0.12 μM). The mechanism of antiproliferative action involved cell cycle arrest at the G1 phase for MCF7 and MDA-MB-468 cells. Tangeretin was further shown to induce CYP1 enzyme activity and CYP1A1/CYP1B1 protein expression in MCF7 and MDA-MB-468 cells. These results suggest that tangeretin inhibits the proliferation of breast cancer cells via CYP1A1/CYP1B1-mediated metabolism to the product 4' hydroxy tangeretin. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Andrographolide Inhibits Oxidized LDL-Induced Cholesterol Accumulation and Foam Cell Formation in Macrophages.

    PubMed

    Lin, Hung-Chih; Lii, Chong-Kuei; Chen, Hui-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Chen, Haw-Wen

    2018-01-01

    oxLDL is involved in the pathogenesis of atherosclerotic lesions through cholesterol accumulation in macrophage foam cells. Andrographolide, the bioactive component of Andrographis paniculata, possesses several biological activities such as anti-inflammatory, anti-oxidant, and anticancer functions. Scavenger receptors (SRs), including class A SR (SR-A) and CD36, are responsible for the internalization of oxLDL. In contrast, receptors for reverse cholesterol transport, including ABCA1 and ABCG1, mediate the efflux of cholesterol from macrophage foam cells. Transcription factor liver X receptor [Formula: see text] (LXR[Formula: see text] plays a key role in lipid metabolism and inflammation as well as in the regulation of ABCA1 and ABCG1 expression. Because of the contribution of inflammation to macrophage foam cell formation and the potent anti-inflammatory activity of andrographolide, we hypothesized that andrographolide might inhibit oxLDL-induced macrophage foam cell formation. The results showed that andrographolide reduced oxLDL-induced lipid accumulation in macrophage foam cells. Andrographolide decreased the mRNA and protein expression of CD36 by inducing the degradation of CD36 mRNA; however, andrographolide had no effect on SR-A expression. In contrast, andrographolide increased the mRNA and protein expression of ABCA1 and ABCG1, which were dependent on LXR[Formula: see text]. Andrographolide enhanced LXR[Formula: see text] nuclear translocation and DNA binding activity. Treatment with the LXR[Formula: see text] antagonist GGPP and transfection with LXR[Formula: see text] siRNA reversed the ability of andrographolide to stimulate ABCA1 and ABCG1 protein expression. In conclusion, inhibition of CD36-mediated oxLDL uptake and induction of ABCA1- and ABCG1-dependent cholesterol efflux are two working mechanisms by which andrographolide inhibits macrophage foam cell formation, which suggests that andrographolide could be a potential candidate to prevent

  7. Tanshindiol C inhibits oxidized low-density lipoprotein induced macrophage foam cell formation via a peroxiredoxin 1 dependent pathway.

    PubMed

    Yang, Yuyu; Li, Xueyan; Peng, Liying; An, Lin; Sun, Ningyuan; Hu, Xuewen; Zhou, Ping; Xu, Yong; Li, Ping; Chen, Jun

    2018-03-01

    NF-E2-related factor 2 (Nrf2) has been shown to be protective in atherosclerosis. The loss of Nrf2 in macrophages enhances foam cell formation and promotes early atherogenesis. Tanshindiol C (Tan C) is isolated from the root of Salvia miltiorrhiza Bge., a traditional Chinese medicine that has been used for the treatment of several cardiovascular diseases for many years. This study was aimed to test the potential role of Tan C against macrophage foam cell formation and to explore the underlying mechanism. Firstly, we observed that Tan C markedly suppressed oxidized low-density lipoprotein (oxLDL) induced macrophage foam cell formation. Then, we found that Tan C was an activator of both Nrf2 and Sirtuin 1 (Sirt1) in macrophages. Nrf2 and Sirt1 synergistically activated the transcription of anti-oxidant peroxiredoxin 1 (Prdx1) after Tan C treatment. More important, we demonstrated that silencing of Prdx1 promoted oxLDL-induced macrophage foam cell formation. Prdx1 upregulated adenosine triphosphate-binding cassette (ABC) transporter A1 (ABCA1) expression and decreased intracellular lipid accumulation. Furthermore, Tan C ameliorated oxLDL induced macrophage foam cell formation in a Prdx1-dependent manner. These observations suggest that Tan C protects macrophages from oxLDL induced foam cell formation via activation of Prdx1/ABCA1 signaling and that Prdx1 may be a novel target for therapeutic intervention of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. WISP1 mediates IL-6-dependent proliferation in primary human lung fibroblasts.

    PubMed

    Klee, S; Lehmann, M; Wagner, D E; Baarsma, H A; Königshoff, M

    2016-02-12

    Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. IPF is characterized by epithelial cell injury and reprogramming, increases in (myo)fibroblasts, and altered deposition of extracellular matrix. The Wnt1-inducible signaling protein 1 (WISP1) is involved in impaired epithelial-mesenchymal crosstalk in pulmonary fibrosis. Here, we aimed to further investigate WISP1 regulation and function in primary human lung fibroblasts (phLFs). We demonstrate that WISP1 is directly upregulated by Transforming growth factor β1 (TGFβ1) and Tumor necrosis factor α (TNFα) in phLFs, using a luciferase-based reporter system. WISP1 mRNA and protein secretion increased in a time- and concentration-dependent manner by TGFβ1 and TNFα in phLFs, as analysed by qPCR and ELISA, respectively. Notably, WISP1 is required for TGFβ1- and TNFα-dependent induction of interleukin 6 (IL-6), a mechanism that is conserved in IPF phLFs. The siRNA-mediated WISP1 knockdown led to a significant IL-6 reduction after TGFβ1 or TNFα stimulation. Furthermore, siRNA-mediated downregulation or antibody-mediated neutralization of WISP1 reduced phLFs proliferation, a process that was in part rescued by IL-6. Taken together, these results strongly indicate that WISP1-induced IL-6 expression contributes to the pro-proliferative effect on fibroblasts, which is likely orchestrated by a variety of profibrotic mediators, including Wnts, TGFβ1 and TNFα.

  9. Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

    PubMed

    Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

    2015-12-01

    Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

  10. TRPA1 channel mediates organophosphate-induced delayed neuropathy

    PubMed Central

    Ding, Qiang; Fang, Sui; Chen, Xueqin; Wang, Youxin; Li, Jian; Tian, Fuyun; Xu, Xiang; Attali, Bernard; Xie, Xin; Gao, Zhaobing

    2017-01-01

    The organophosphate-induced delayed neuropathy (OPIDN), often leads to paresthesias, ataxia and paralysis, occurs in the late-stage of acute poisoning or after repeated exposures to organophosphate (OP) insecticides or nerve agents, and may contribute to the Gulf War Syndrome. The acute phase of OP poisoning is often attributed to acetylcholinesterase inhibition. However, the underlying mechanism for the delayed neuropathy remains unknown and no treatment is available. Here we demonstrate that TRPA1 channel (Transient receptor potential cation channel, member A1) mediates OPIDN. A variety of OPs, exemplified by malathion, activates TRPA1 but not other neuronal TRP channels. Malathion increases the intracellular calcium levels and upregulates the excitability of mouse dorsal root ganglion neurons in vitro. Mice with repeated exposures to malathion also develop local tissue nerve injuries and pain-related behaviors, which resembles OPIDN. Both the neuropathological changes and the nocifensive behaviors can be attenuated by treatment of TRPA1 antagonist HC030031 or abolished by knockout of Trpa1 gene. In the classic hens OPIDN model, malathion causes nerve injuries and ataxia to a similar level as the positive inducer tri-ortho-cresyl phosphate (TOCP), which also activates TRPA1 channel. Treatment with HC030031 reduces the damages caused by malathion or tri-ortho-cresyl phosphate. Duloxetine and Ketotifen, two commercially available drugs exhibiting TRPA1 inhibitory activity, show neuroprotective effects against OPIDN and might be used in emergency situations. The current study suggests TRPA1 is the major mediator of OPIDN and targeting TRPA1 is an effective way for the treatment of OPIDN. PMID:28894590

  11. Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies

    PubMed Central

    Awata, Hiroko; Wakuda, Ryo; Ishimaru, Yoshiyasu; Matsuoka, Yuji; Terao, Kanta; Katata, Satomi; Matsumoto, Yukihisa; Hamanaka, Yoshitaka; Noji, Sumihare; Mito, Taro; Mizunami, Makoto

    2016-01-01

    Revealing reinforcing mechanisms in associative learning is important for elucidation of brain mechanisms of behavior. In mammals, dopamine neurons are thought to mediate both appetitive and aversive reinforcement signals. Studies using transgenic fruit-flies suggested that dopamine neurons mediate both appetitive and aversive reinforcements, through the Dop1 dopamine receptor, but our studies using octopamine and dopamine receptor antagonists and using Dop1 knockout crickets suggested that octopamine neurons mediate appetitive reinforcement and dopamine neurons mediate aversive reinforcement in associative learning in crickets. To fully resolve this issue, we examined the effects of silencing of expression of genes that code the OA1 octopamine receptor and Dop1 and Dop2 dopamine receptors by RNAi in crickets. OA1-silenced crickets exhibited impairment in appetitive learning with water but not in aversive learning with sodium chloride solution, while Dop1-silenced crickets exhibited impairment in aversive learning but not in appetitive learning. Dop2-silenced crickets showed normal scores in both appetitive learning and aversive learning. The results indicate that octopamine neurons mediate appetitive reinforcement via OA1 and that dopamine neurons mediate aversive reinforcement via Dop1 in crickets, providing decisive evidence that neurotransmitters and receptors that mediate appetitive reinforcement indeed differ among different species of insects. PMID:27412401

  12. Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies.

    PubMed

    Awata, Hiroko; Wakuda, Ryo; Ishimaru, Yoshiyasu; Matsuoka, Yuji; Terao, Kanta; Katata, Satomi; Matsumoto, Yukihisa; Hamanaka, Yoshitaka; Noji, Sumihare; Mito, Taro; Mizunami, Makoto

    2016-07-14

    Revealing reinforcing mechanisms in associative learning is important for elucidation of brain mechanisms of behavior. In mammals, dopamine neurons are thought to mediate both appetitive and aversive reinforcement signals. Studies using transgenic fruit-flies suggested that dopamine neurons mediate both appetitive and aversive reinforcements, through the Dop1 dopamine receptor, but our studies using octopamine and dopamine receptor antagonists and using Dop1 knockout crickets suggested that octopamine neurons mediate appetitive reinforcement and dopamine neurons mediate aversive reinforcement in associative learning in crickets. To fully resolve this issue, we examined the effects of silencing of expression of genes that code the OA1 octopamine receptor and Dop1 and Dop2 dopamine receptors by RNAi in crickets. OA1-silenced crickets exhibited impairment in appetitive learning with water but not in aversive learning with sodium chloride solution, while Dop1-silenced crickets exhibited impairment in aversive learning but not in appetitive learning. Dop2-silenced crickets showed normal scores in both appetitive learning and aversive learning. The results indicate that octopamine neurons mediate appetitive reinforcement via OA1 and that dopamine neurons mediate aversive reinforcement via Dop1 in crickets, providing decisive evidence that neurotransmitters and receptors that mediate appetitive reinforcement indeed differ among different species of insects.

  13. Ephrin-A1/EphA4-mediated adhesion of monocytes to endothelial cells.

    PubMed

    Jellinghaus, Stefanie; Poitz, David M; Ende, Georg; Augstein, Antje; Weinert, Sönke; Stütz, Beryl; Braun-Dullaeus, Rüdiger C; Pasquale, Elena B; Strasser, Ruth H

    2013-10-01

    The Eph receptors represent the largest family of receptor tyrosine kinases. Both Eph receptors and their ephrin ligands are cell-surface proteins, and they typically mediate cell-to-cell communication by interacting at sites of intercellular contact. The major aim of the present study was to investigate the involvement of EphA4-ephrin-A1 interaction in monocyte adhesion to endothelial cells, as this process is a crucial step during the initiation and progression of the atherosclerotic plaque. Immunohistochemical analysis of human atherosclerotic plaques revealed expression of EphA4 receptor and ephrin-A1 ligand in major cell types within the plaque. Short-time stimulation of endothelial cells with the soluble ligand ephrin-A1 leads to a fourfold increase in adhesion of human monocytes to endothelial cells. In addition, ephrin-A1 further increases monocyte adhesion to already inflamed endothelial cells. EphrinA1 mediates its effect on monocyte adhesion via the activated receptor EphA4. This ephrinA1/EphA4 induced process involves the activation of the Rho signaling pathway and does not require active transcription. Rho activation downstream of EphA4 leads to increased polymerization of actin filaments in endothelial cells. This process was shown to be crucial for the proadhesive effect of ephrin-A1. The results of the present study show that ephrin-A1-induced EphA4 forward signaling promotes monocyte adhesion to endothelial cells via activation of RhoA and subsequent stress-fiber formation by a non-transcriptional mechanism. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Epithelial estrogen receptor 1 intrinsically mediates squamous differentiation in the mouse vagina.

    PubMed

    Miyagawa, Shinichi; Iguchi, Taisen

    2015-10-20

    Estrogen-mediated actions in female reproductive organs are tightly regulated, mainly through estrogen receptor 1 (ESR1). The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen and provides a unique model for analyzing the homeostasis of stratified squamous epithelia. To address the role of ESR1-mediated tissue events during homeostasis, we analyzed mice with a vaginal epithelium-specific knockout of Esr1 driven by keratin 5-Cre (K5-Esr1KO). We show here that loss of epithelial ESR1 in the vagina resulted in aberrant epithelial cell proliferation in the suprabasal cell layers and led to failure of keratinized differentiation. Gene expression analysis showed that several known estrogen target genes, including erbB growth factor ligands, were not induced by estrogen in the K5-Esr1KO mouse vagina. Organ culture experiments revealed that the addition of erbB growth factor ligands, such as amphiregulin, could activate keratinized differentiation in the absence of epithelial ESR1. Thus, epithelial ESR1 integrates estrogen and growth factor signaling to mediate regulation of cell proliferation in squamous differentiation, and our results provide new insights into estrogen-mediated homeostasis in female reproductive organs.

  15. Key mediators of intracellular amino acids signaling to mTORC1 activation.

    PubMed

    Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

    2015-05-01

    Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

  16. The microRNA-let-7b-mediated attenuated strain of influenza A (H1N1) virus in a mouse model.

    PubMed

    Tan, Mingming; Sun, Wenkui; Feng, Chunlai; Xia, Di; Shen, Xiaoyue; Ding, Yuan; Liu, Zhicheng; Xing, Zheng; Su, Xin; Shi, Yi

    2016-09-30

    Evaluating the attenuation of influenza viruses in animal studies is important in developing safe and effective vaccines. This study aimed to demonstrate that the microRNA (miRNA)-let-7b-mediated attenuated influenza viruses (miRT-H1N1) are sufficiently attenuated and safe in mice. The pathogenicity of the miRT-H1N1virus was investigated in a mouse model, evaluated with median lethal dose (LD50). The replicative dynamics of the miRT-H1N1, wild type (wt)-H1N1, and scramble (scbl)-H1N1 viruses in the lungs of infected mice were compared. The degrees of lesions and the expression levels of IL-6, TNF-α, and IFN-β in the lungs of mice infected with different viruses were also analyzed. In miRT-H1N1 virus-infected mice, 100% of mice survived, and a lower pathogenicity was characterized with non-significant weight loss when compared to mice infected with the control wt virus. The miRT-H1N1 virus was not fatal for mice, even at the highest dose administered. The viral load in the lungs of miRT-H1N1-infected mice was significantly lower than that of the wild-type virus-infected mice. Fewer pulmonary lesions and lower levels of selected pro-inflammatory cytokines in the lungs of the mice infected with the miRT-H1N1 virus were also observed. The virulence of the miRT-H1N1 virus reduced significantly, suggesting that the miRT-H1N1 virus was safe for mice. Our study demonstrated that the miRNA-mediated gene silencing is an alternative approach to attenuating the pathogenicity of wt influenza viruses that have potential in the development of influenza vaccines.

  17. SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1

    PubMed Central

    Côte, Marjorie; Fos, Camille; Canonigo-Balancio, Ann J.; Ley, Klaus; Bécart, Stéphane; Altman, Amnon

    2015-01-01

    ABSTRACT SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca2+ signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4+ T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4+ T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6−/−) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling. PMID:26483383

  18. SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1.

    PubMed

    Côte, Marjorie; Fos, Camille; Canonigo-Balancio, Ann J; Ley, Klaus; Bécart, Stéphane; Altman, Amnon

    2015-12-01

    SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca(2+) signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4(+) T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4(+) T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6(-/-)) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling. © 2015. Published by The Company of Biologists Ltd.

  19. VEZF1 Elements Mediate Protection from DNA Methylation

    PubMed Central

    Strogantsev, Ruslan; Gaszner, Miklos; Hair, Alan; Felsenfeld, Gary; West, Adam G.

    2010-01-01

    There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state. PMID:20062523

  20. Oxysterols decrease apical-to-basolateral transport of Aß peptides via an ABCB1-mediated process in an in vitro Blood-brain barrier model constituted of bovine brain capillary endothelial cells.

    PubMed

    Saint-Pol, Julien; Candela, Pietra; Boucau, Marie-Christine; Fenart, Laurence; Gosselet, Fabien

    2013-06-23

    It is known that activation of the liver X receptors (LXRs) by natural or synthetic agonists decreases the amyloid burden and enhances cognitive function in transgenic murine models of Alzheimer's disease (AD). Recent evidence suggests that LXR activation may affect the transport of amyloid ß (Aß) peptides across the blood-brain barrier (the BBB, which isolates the brain from the peripheral circulation). By using a well-characterized in vitro BBB model, we demonstrated that LXR agonists (24S-hydroxycholesterol, 27-hydroxycholesterol and T0901317) modulated the expression of target genes involved in cholesterol homeostasis (such as ATP-binding cassette sub-family A member 1 (ABCA1)) and promoted cellular cholesterol efflux to apolipoprotein A-I and high density lipoproteins. Interestingly, we also observed a decrease in Aß peptide influx across brain capillary endothelial cells, although ABCA1 did not appear to be directly involved in this process. By focusing on others receptors and transporters that are thought to have major roles in Aß peptide entry into the brain, we then demonstrated that LXR stimulation provoked an increase in expression of the ABCB1 transporter (also named P-glycoprotein (P-gp)). Further investigations confirmed ABCB1's involvement in the restriction of Aß peptide influx. Taken as a whole, our results not only reinforce the BBB's key role in cerebral cholesterol homeostasis but also demonstrate the importance of the LXR/ABCB1 axis in Aß peptide influx-highlighting an attractive new therapeutic approach whereby the brain could be protected from peripheral Aß peptide entry. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    PubMed

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  2. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses

    PubMed Central

    McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T.; Dennison, S. Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S. Munir; Haynes, Barton F.; Tomaras, Georgia D.

    2016-01-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  3. p21WAF1/CIP1 Upregulation through the Stress Granule-Associated Protein CUGBP1 Confers Resistance to Bortezomib-Mediated Apoptosis

    PubMed Central

    Gareau, Cristina; Fournier, Marie-Josée; Filion, Christine; Coudert, Laetitia; Martel, David; Labelle, Yves; Mazroui, Rachid

    2011-01-01

    Background p21WAF1/CIP1 is a well known cyclin-dependent kinase inhibitor induced by various stress stimuli. Depending on the stress applied, p21 upregulation can either promote apoptosis or prevent against apoptotic injury. The stress-mediated induction of p21 involves not only its transcriptional activation but also its posttranscriptional regulation, mainly through stabilization of p21 mRNA levels. We have previously reported that the proteasome inhibitor MG132 induces the stabilization of p21 mRNA, which correlates with the formation of cytoplasmic RNA stress granules. The mechanism underlying p21 mRNA stabilization, however, remains unknown. Methodology/Principal Findings We identified the stress granules component CUGBP1 as a factor required for p21 mRNA stabilization following treatment with bortezomib ( =  PS-341/Velcade). This peptide boronate inhibitor of the 26S proteasome is very efficient for the treatment of myelomas and other hematological tumors. However, solid tumors are sometimes refractory to bortezomib treatment. We found that depleting CUGBP1 in cancer cells prevents bortezomib-mediated p21 upregulation. FISH experiments combined to mRNA stability assays show that this effect is largely due to a mistargeting of p21 mRNA in stress granules leading to its degradation. Altering the expression of p21 itself, either by depleting CUGBP1 or p21, promotes bortezomib-mediated apoptosis. Conclusions/Significance We propose that one key mechanism by which apoptosis is inhibited upon treatment with chemotherapeutic drugs might involve upregulation of the p21 protein through CUGBP1. PMID:21637851

  4. BDNF mediates improvements in executive function following a 1-year exercise intervention

    PubMed Central

    Leckie, Regina L.; Oberlin, Lauren E.; Voss, Michelle W.; Prakash, Ruchika S.; Szabo-Reed, Amanda; Chaddock-Heyman, Laura; Phillips, Siobhan M.; Gothe, Neha P.; Mailey, Emily; Vieira-Potter, Victoria J.; Martin, Stephen A.; Pence, Brandt D.; Lin, Mingkuan; Parasuraman, Raja; Greenwood, Pamela M.; Fryxell, Karl J.; Woods, Jeffrey A.; McAuley, Edward; Kramer, Arthur F.; Erickson, Kirk I.

    2014-01-01

    Executive function declines with age, but engaging in aerobic exercise may attenuate decline. One mechanism by which aerobic exercise may preserve executive function is through the up-regulation of brain-derived neurotropic factor (BDNF), which also declines with age. The present study examined BDNF as a mediator of the effects of a 1-year walking intervention on executive function in 90 older adults (mean age = 66.82). Participants were randomized to a stretching and toning control group or a moderate intensity walking intervention group. BDNF serum levels and performance on a task-switching paradigm were collected at baseline and follow-up. We found that age moderated the effect of intervention group on changes in BDNF levels, with those in the highest age quartile showing the greatest increase in BDNF after 1-year of moderate intensity walking exercise (p = 0.036). The mediation analyses revealed that BDNF mediated the effect of the intervention on task-switch accuracy, but did so as a function of age, such that exercise-induced changes in BDNF mediated the effect of exercise on task-switch performance only for individuals over the age of 71. These results demonstrate that both age and BDNF serum levels are important factors to consider when investigating the mechanisms by which exercise interventions influence cognitive outcomes, particularly in elderly populations. PMID:25566019

  5. Regulation of ATP-binding Cassette Transporters and Cholesterol Efflux by Glucose in Primary Human Monocytes and Murine Bone Marrow-derived Macrophages

    PubMed Central

    Spartano, N. L.; Lamon-Fava, S.; Matthan, N. R.; Ronxhi, J.; Greenberg, A. S.; Obin, M. S.; Lichtenstein, A. H.

    2014-01-01

    Purpose Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. 2 models were used to assess this potential relationship: human monocytes/leukocytes and murine bone marrow-derived macrophages (BMDM). Methods 10 subjects (4 F/6 M, 50–85 years, BMI 25–35 kg/m2) underwent an oral glucose challenge. Baseline and 1- and 2-h post-challenge ABC-transporter mRNA expression was determined in monocytes, leukocytes and peripheral blood mononuclear cells (PBMC). In a separate study, murine-BMDM were exposed to 5 mmol/L D-glucose (control) or additional 20 mmol/L D-or L-glucose and 25 ug/mL oxidized low density lipoprotein (oxLDL). High density lipoprotein (HDL)-mediated cholesterol efflux and ABC-transporter (ABCA1 and ABCG1) expression were determined. Results Baseline ABCA1and ABCG1 expression was lower (> 50 %) in human monocytes and PBMC than leukocytes (p < 0.05). 1 h post-challenge leukocyte ABCA1 and ABCG1 expression increased by 37 % and 30 %, respectively (p < 0.05), and began to return to baseline thereafter. There was no significant change in monocyte ABC-transporter expression. In murine BMDM, higher glucose concentrations suppressed HDL-mediated cholesterol efflux (10 %; p < 0.01) without significantly affecting ABCA1 and ABCG1 expression. Data demonstrate that leukocytes are not a reliable indicator of monocyte ABC-transporter expression. Conclusions Human monocyte ABC-transporter gene expression was unresponsive to a glucose challenge. Correspondingly, in BMDM, hyperglycemia attenuated macrophage cholesterol efflux in the absence of altered ABC-transporter expression, suggesting that hyperglycemia, per se, suppresses cholesterol transporter activity. This glucose-related impairment in cholesterol efflux may potentially contribute to

  6. Hypericin-mediated sonodynamic therapy induces autophagy and decreases lipids in THP-1 macrophage by promoting ROS-dependent nuclear translocation of TFEB.

    PubMed

    Li, Xuesong; Zhang, Xin; Zheng, Longbin; Kou, Jiayuan; Zhong, Zhaoyu; Jiang, Yueqing; Wang, Wei; Dong, Zengxiang; Liu, Zhongni; Han, Xiaobo; Li, Jing; Tian, Ye; Zhao, Yajun; Yang, Liming

    2016-12-22

    Lipid catabolism disorder is the primary cause of atherosclerosis. Transcription factor EB (TFEB) prevents atherosclerosis by activating macrophage autophagy to promote lipid degradation. Hypericin-mediated sonodynamic therapy (HY-SDT) has been proved non-invasively inducing THP-1-derived macrophage apoptosis; however, it is unknown whether macrophage autophagy could be triggered by HY-SDT to influence cellular lipid catabolism via regulating TFEB. Here, we report that HY-SDT resulted in the time-dependent THP-1-derived macrophage autophagy activation through AMPK/AKT/mTOR pathway. Besides, TFEB nuclear translocation in macrophage was triggered by HY-SDT to promote autophagy activation and lysosome regeneration which enhanced lipid degradation in response to atherogenic lipid stressors. Moreover, following HY-SDT, the ABCA1 expression level was increased to promote lipid efflux in macrophage, and the expression levels of CD36 and SR-A were decreased to inhibit lipid uptake, both of which were prevented by TFEB knockdown. These results indicated that TFEB nuclear translocation activated by HY-SDT was not only the key regulator of autophagy activation and lysosome regeneration in macrophage to promote lipolysis, but also had a crucial role in reverse cholesterol transporters to decrease lipid uptake and increase lipid efflux. Reactive oxygen species (ROS) were adequately generated in macrophage by HY-SDT. Further, ROS scavenger N-acetyl-l-cysteine abolished HY-SDT-induced TFEB nuclear translocation and autophagy activation, implying that ROS were the primary upstream factors responsible for these effects during HY-SDT. In summary, our data indicate that HY-SDT decreases lipid content in macrophage by promoting ROS-dependent nuclear translocation of TFEB to influence consequent autophagy activation and cholesterol transporters. Thus, HY-SDT may be beneficial for atherosclerosis via TFEB regulation to ameliorate lipid overload in atherosclerotic plaques.

  7. Activity-Dependent Ubiquitination of GluA1 Mediates a Distinct AMPAR Endocytosis and Sorting Pathway

    PubMed Central

    Schwarz, Lindsay A.; Hall, Benjamin J.; Patrick, Gentry N.

    2010-01-01

    The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, while dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer’s disease. Previous work has shown that ubiquitination of integral membrane proteins is a common post-translational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its carboxy-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA, but not for internalization of AMPARs in response to the NMDA receptor (NMDAR) agonist NMDA. Through over-expression or RNAi-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1, is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues, and suggest that changes to this pathway may occur as neurons mature. PMID:21148011

  8. Submergence tolerance conferred by Sub1A is mediated by SLR1 and SLRL1 restriction of gibberellin responses in rice

    PubMed Central

    Fukao, Takeshi; Bailey-Serres, Julia

    2008-01-01

    Submergence-tolerant rice maintains viability during complete submergence by limiting underwater elongation until floodwaters recede. Acclimation responses to submergence are coordinated by the submergence-inducible Sub1A, which encodes an ethylene-responsive factor-type transcription factor (ERF). Sub1A is limited to tolerant genotypes and sufficient to confer submergence tolerance to intolerant accessions. Here we evaluated the role of Sub1A in the integration of ethylene, abscisic acid (ABA), and gibberellin (GA) signaling during submergence. The submergence-stimulated decrease in ABA content was Sub1A-independent, whereas GA-mediated underwater elongation was significantly restricted by Sub1A. Transgenics that ectopically express Sub1A displayed classical GA-insensitive phenotypes, leading to the hypothesis that Sub1A limits the response to GA. Notably Sub1A increased the accumulation of the GA signaling repressors Slender Rice-1 (SLR1) and SLR1 Like-1 (SLRL1) and concomitantly diminished GA-inducible gene expression under submerged conditions. In the Sub1A overexpression line, SLR1 protein levels declined under prolonged submergence but were accompanied by an increase in accumulation of SLRL1, which lacks the DELLA domain. In the presence of Sub1A, the increase in these GA signaling repressors and decrease in GA responsiveness were stimulated by ethylene, which promotes Sub1A expression. Conversely, ethylene promoted GA responsiveness and shoot elongation in submergence-intolerant lines. Together, these results demonstrate that Sub1A limits ethylene-promoted GA responsiveness during submergence by augmenting accumulation of the GA signaling repressors SLR1 and SLRL1. PMID:18936491

  9. Epithelial estrogen receptor 1 intrinsically mediates squamous differentiation in the mouse vagina

    PubMed Central

    Miyagawa, Shinichi; Iguchi, Taisen

    2015-01-01

    Estrogen-mediated actions in female reproductive organs are tightly regulated, mainly through estrogen receptor 1 (ESR1). The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen and provides a unique model for analyzing the homeostasis of stratified squamous epithelia. To address the role of ESR1-mediated tissue events during homeostasis, we analyzed mice with a vaginal epithelium-specific knockout of Esr1 driven by keratin 5-Cre (K5-Esr1KO). We show here that loss of epithelial ESR1 in the vagina resulted in aberrant epithelial cell proliferation in the suprabasal cell layers and led to failure of keratinized differentiation. Gene expression analysis showed that several known estrogen target genes, including erbB growth factor ligands, were not induced by estrogen in the K5-Esr1KO mouse vagina. Organ culture experiments revealed that the addition of erbB growth factor ligands, such as amphiregulin, could activate keratinized differentiation in the absence of epithelial ESR1. Thus, epithelial ESR1 integrates estrogen and growth factor signaling to mediate regulation of cell proliferation in squamous differentiation, and our results provide new insights into estrogen-mediated homeostasis in female reproductive organs. PMID:26438838

  10. Bruton's tyrosine kinase regulates B cell antigen receptor-mediated JNK1 response through Rac1 and phospholipase C-gamma2 activation.

    PubMed

    Inabe, Kazunori; Miyawaki, Toshio; Longnecker, Richard; Matsukura, Hiroyoshi; Tsukada, Satoshi; Kurosaki, Tomohiro

    2002-03-13

    Bruton's tyrosine kinase (Btk) is essential for B cell development and B cell antigen receptor (BCR) function. Recent studies have shown that Btk plays an important role in BCR-mediated c-Jun NH(2)-terminal kinase (JNK) 1 activation; however, the mechanism by which Btk participates in the JNK1 response remains elusive. Here we show that the BCR-mediated Rac1 activation is significantly inhibited by loss of Btk, while this Rac1 activation is not affected by loss of phospholipase C-gamma2 (PLC-gamma2). Since PLC-gamma2 is also required for BCR-mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC-gamma2 pathway, both of which contribute to the BCR-mediated JNK1 response.

  11. Reduced-illuminance autofluorescence imaging in ABCA4-associated retinal degenerations

    NASA Astrophysics Data System (ADS)

    Cideciyan, Artur V.; Swider, Malgorzata; Aleman, Tomas S.; Roman, Marisa I.; Sumaroka, Alexander; Schwartz, Sharon B.; Stone, Edwin M.; Jacobson, Samuel G.

    2007-05-01

    The health of the retinal pigment epithelium (RPE) can be estimated with autofluorescence (AF) imaging of lipofuscin, which accumulates as a byproduct of retinal exposure to light. Lipofuscin may be toxic to the RPE, and its toxicity may be enhanced by short-wavelength (SW) illumination. The high-intensity and SW excitation light used in conventional AF imaging could, at least in principle, increase the rate of lipofuscin accumulation and/or increase its toxicity. We considered two reduced-illuminance AF imaging (RAFI) methods as alternatives to conventional AF imaging. RAFI methods use either near-infrared (NIR) light or reduced-radiance SW illumination for excitation of fluorophores. We quantified the distribution of RAFI signals in relation to retinal structure and function in patients with the prototypical lipofuscin accumulation disease caused by mutations in ABCA4. There was evidence for two subclinical stages of macular ABCA4 disease involving hyperautofluorescence of both SW- and NIR-RAFI with and without associated loss of visual function. Use of RAFI methods and microperimetry in future clinical trials involving lipofuscinopathies should allow quantification of subclinical disease expression and progression without subjecting the diseased retina/RPE to undue light exposure.

  12. An RLP23-SOBIR1-BAK1 complex mediates NLP-triggered immunity.

    PubMed

    Albert, Isabell; Böhm, Hannah; Albert, Markus; Feiler, Christina E; Imkampe, Julia; Wallmeroth, Niklas; Brancato, Caterina; Raaymakers, Tom M; Oome, Stan; Zhang, Heqiao; Krol, Elzbieta; Grefen, Christopher; Gust, Andrea A; Chai, Jijie; Hedrich, Rainer; Van den Ackerveken, Guido; Nürnberger, Thorsten

    2015-10-05

    Plants and animals employ innate immune systems to cope with microbial infection. Pattern-triggered immunity relies on the recognition of microbe-derived patterns by pattern recognition receptors (PRRs). Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) constitute plant immunogenic patterns that are unique, as these proteins are produced by multiple prokaryotic (bacterial) and eukaryotic (fungal, oomycete) species. Here we show that the leucine-rich repeat receptor protein (LRR-RP) RLP23 binds in vivo to a conserved 20-amino-acid fragment found in most NLPs (nlp20), thereby mediating immune activation in Arabidopsis thaliana. RLP23 forms a constitutive, ligand-independent complex with the LRR receptor kinase (LRR-RK) SOBIR1 (Suppressor of Brassinosteroid insensitive 1 (BRI1)-associated kinase (BAK1)-interacting receptor kinase 1), and recruits a second LRR-RK, BAK1, into a tripartite complex upon ligand binding. Stable, ectopic expression of RLP23 in potato (Solanum tuberosum) confers nlp20 pattern recognition and enhanced immunity to destructive oomycete and fungal plant pathogens, such as Phytophthora infestans and Sclerotinia sclerotiorum. PRRs that recognize widespread microbial patterns might be particularly suited for engineering immunity in crop plants.

  13. ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease.

    PubMed

    Sangermano, Riccardo; Khan, Mubeen; Cornelis, Stéphanie S; Richelle, Valerie; Albert, Silvia; Garanto, Alejandro; Elmelik, Duaa; Qamar, Raheel; Lugtenberg, Dorien; van den Born, L Ingeborgh; Collin, Rob W J; Cremers, Frans P M

    2018-01-01

    Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire ABCA4 gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on ABCA4 pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in ABCA4 allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes. © 2018 Sangermano et al.; Published by Cold Spring Harbor Laboratory Press.

  14. SIAH1-induced p34SEI-1 polyubiquitination/degradation mediates p53 preferential vitamin C cytotoxicity.

    PubMed

    Lee, Soonduck; Kim, Jinsun; Jung, Samil; Li, Chengping; Yang, Young; Kim, Keun Il; Lim, Jong-Seok; Kim, Yonghwan; Cheon, Choong-Il; Lee, Myeong-Sok

    2015-03-01

    Vitamin C is considered as an important anticancer therapeutic agent although this view is debatable. In this study, we introduce a physiological mechanism demonstrating how vitamin C exerts anticancer activity that induces cell cycle arrest and apoptosis. Our previous and current data reveal that p53 tumor suppressor is the prerequisite factor for stronger anticancer effects of vitamin C. In addition, vitamin C-mediated cancer cell cytotoxicity appears to be achieved at least partly through the downregulation of the p34SEI-1 oncoprotein. Our previous study showed that p34SEI-1 increases the survival of various types of cancer cells by inhibiting their apoptosis. Present data suggest that vitamin C treatment decreases the p34SEI-1 expression at the protein level and therefore alleviates its anti-apoptotic activity. Of note, SIAH1, E3 ubiquitin ligase, appears to be responsible for the p34SEI-1 polyubiquitination and its subsequent degradation, which is dependent on p53. In summary, vitamin C increases cancer cell death by inducing SIAH1-mediated polyubiquitination/degradation of the p34SEI-1 oncoprotein in a p53-dependent manner.

  15. Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene.

    PubMed

    Yoshizaki, Keigo; Hu, Lizhi; Nguyen, Thai; Sakai, Kiyoshi; Ishikawa, Masaki; Takahashi, Ichiro; Fukumoto, Satoshi; DenBesten, Pamela K; Bikle, Daniel D; Oda, Yuko; Yamada, Yoshihiko

    2017-08-18

    Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1 -deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

  16. Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1.

    PubMed

    Alver, Robert C; Chadha, Gaganmeet Singh; Gillespie, Peter J; Blow, J Julian

    2017-03-07

    Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. A CONSTITUTIVELY ACTIVE FORM OF NEUROKININ 1 RECEPTOR AND NEUROKININ 1 RECEPTOR-MEDIATED APOPTOSIS IN GLIOBLASTOMAS

    PubMed Central

    Akazawa, Toshimasa; Kwatra, Shawn G.; Goldsmith, Laura E.; Richardson, Mark D.; Cox, Elizabeth A.; Sampson, John H.; Kwatra, Madan M.

    2009-01-01

    Previous studies have shown that neurokinin 1 receptor (NK1R) occurs naturally in human glioblastomas and its stimulation causes cell proliferation. In the present study we show that stimulation of NK1R in human U373 glioblastoma cells by substance P (SP) increases Akt phosphorylation by 2.5-fold, with an EC50 of 57 nM. Blockade of NK1R lowers basal phosphorylation of Akt, indicating the presence of a constitutively active form of NK1R; similar results are seen in U251 MG and DBTRG-05 glioblastoma cells. Linkage of NK1R to Akt implicates NK1R in apoptosis of glioblastoma cells. Indeed, treatment of serum-starved U373 cells with SP reduces apoptosis by 53 ± 1% (P < 0.05), and treatment with NK1R antagonist L-733,060 increases apoptosis by 64 ± 16 % (P < 0.01). Further, the blockade of NK1R in human glioblastoma cells with L-733,060 causes cleavage of Caspase-3 and proteolysis of poly (ADP-ribose) polymerase (PARP). Experiments designed to elucidate the mechanism of NK1R-mediated Akt phosphorylation revealed total involvement of non-receptor tyrosine kinase Src and PI-3-kinase, a partial involvement of epidermal growth factor receptor (EGFR), and no involvement of MEK. Taken together, the results of the present study indicate a key role for NK1R in glioblastoma apoptosis. PMID:19519779

  18. Myeloid HIF-1 is protective in Helicobacter pylori-mediated gastritis.

    PubMed

    Matak, Pavle; Heinis, Mylène; Mathieu, Jacques R R; Corriden, Ross; Cuvellier, Sylvain; Delga, Stéphanie; Mounier, Rémi; Rouquette, Alexandre; Raymond, Josette; Lamarque, Dominique; Emile, Jean-François; Nizet, Victor; Touati, Eliette; Peyssonnaux, Carole

    2015-04-01

    Helicobacter pylori infection triggers chronic inflammation of the gastric mucosa that may progress to gastric cancer. The hypoxia-inducible factors (HIFs) are the central mediators of cellular adaptation to low oxygen levels (hypoxia), but they have emerged recently as major transcriptional regulators of immunity and inflammation. No studies have investigated whether H. pylori affects HIF signaling in immune cells and a potential role for HIF in H. pylori-mediated gastritis. HIF-1 and HIF-2 expression was examined in human H. pylori-positive gastritis biopsies. Subsequent experiments were performed in naive and polarized bone marrow-derived macrophages from wild-type (WT) and myeloid HIF-1α-null mice (HIF-1(Δmyel)). WT and HIF-1(Δmyel) mice were inoculated with H. pylori by oral gavage and sacrificed 6 mo postinfection. HIF-1 was specifically expressed in macrophages of human H. pylori-positive gastritis biopsies. Macrophage HIF-1 strongly contributed to the induction of proinflammatory genes (IL-6, IL-1β) and inducible NO synthase in response to H. pylori. HIF-2 expression and markers of M2 macrophage differentiation were decreased in response to H. pylori. HIF-1(Δmyel) mice inoculated with H. pylori for 6 mo presented with a similar bacterial colonization than WT mice but, surprisingly, a global increase of inflammation, leading to a worsening of the gastritis, measured by an increased epithelial cell proliferation. In conclusion, myeloid HIF-1 is protective in H. pylori-mediated gastritis, pointing to the complex counterbalancing roles of innate immune and inflammatory phenotypes in driving this pathology. Copyright © 2015 by The American Association of Immunologists, Inc.

  19. Activation of HIV Transcription by the Viral Tat Protein Requires a Demethylation Step Mediated by Lysine-specific Demethylase 1 (LSD1/KDM1)

    PubMed Central

    Sakane, Naoki; Kwon, Hye-Sook; Pagans, Sara; Kaehlcke, Katrin; Mizusawa, Yasuhiro; Kamada, Masafumi; Lassen, Kara G.; Chan, Jonathan; Greene, Warner C.; Schnoelzer, Martina; Ott, Melanie

    2011-01-01

    The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear. We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells. Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation

  20. Kinetic Analysis of Rhodamines Efflux Mediated by the Multidrug Resistance Protein (MRP1)

    PubMed Central

    Saengkhae, Chantarawan; Loetchutinat, Chatchanok; Garnier-Suillerot, Arlette

    2003-01-01

    Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, ka = VM/km, was very similar for the four rhodamine analogs but ∼10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes. PMID:12944313

  1. IGF-1 mediated phosphorylation of specific IRS-1 serines in Ames dwarf fibroblasts is associated with longevity.

    PubMed

    Papaconstantinou, John; Hsieh, Ching-Chyuan

    2015-11-03

    Insulin/IGF-1 signaling involves phosphorylation/dephosphorylation of serine/threonine or tyrosine residues of the insulin receptor substrate (IRS) proteins and is associated with hormonal control of longevity determination of certain long-lived mice. The stimulation of serine phosphorylations by IGF-1 suggests there is insulin/IGF-1 crosstalk that involves the phosphorylation of the same serine residues. By this mechanism, insulin and IGF-1 mediated phosphorylation of specific IRS-1 serines could play a role in longevity determination.We used fibroblasts from WT and Ames dwarf mice to examine whether: (a) IGF-1 stimulates phosphorylation of IRS-1 serines targeted by insulin; (b) the levels of serine phosphorylation differ in WT vs. Ames fibroblasts; and (c) aging affects the levels of these serine phosphorylations which are altered in the Ames dwarf mutant. We have shown that IRS-1 is a substrate for IGF-1 induced phosphorylation of Ser307, Ser612, Ser636/639, and Ser1101; that the levels of phosphorylation of these serines are significantly lower in Ames vs. WT cells; that IGF-1 mediated phosphorylation of these serines increases with age in WT cells. We propose that insulin/IGF-1 cross talk and level of phosphorylation of specific IRS-1 serines may promote the Ames dwarf longevity phenotype.

  2. Estrogen receptor ESR1 mediates activation of ERK1/2, CREB, and ELK1 in the corpus of the epididymis.

    PubMed

    Cavalcanti, Fernanda N; Lucas, Thais F G; Lazari, Maria Fatima M; Porto, Catarina S

    2015-06-01

    Expression of the estrogen receptor ESR1 is higher in the corpus than it is in the initial segment/caput and cauda of the epididymis. ESR1 immunostaining in the corpus has been localized not only in the nuclei but also in the cytoplasm and apical membrane, which indicates that ESR1 plays a role in membrane-initiated signaling. The present study investigated whether ESR1 mediates the activation of rapid signaling pathways by estradiol (E2) in the epididymis. We investigated the effect of E2 and the ESR1-selective agonist (4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT) on the activation of extracellular signal-regulated protein kinases (ERK1/2), CREB protein, and ETS oncogene-related protein (ELK1). Treatment with PPT did not affect ERK1/2 phosphorylation in the cauda, but it rapidly increased ERK1/2 phosphorylation in the initial segment/caput and corpus of the epididymis. PPT also activated CREB and ELK1 in the corpus of the epididymis. The PPT-induced phosphorylation of ERK1/2, CREB, and ELK1 was blocked by the ESR1-selective antagonist MPP and by pretreatment with a non-receptor tyrosine kinase SRC inhibitor, an EGFR kinase inhibitor, an MEK1/2 inhibitor, and a phosphatidylinositol-3-kinase inhibitor. In conclusion, these results indicate that the corpus, which is a region with high expression of the estrogen receptor ESR1, is a major target in the epididymis for the activation of rapid signaling by E2. The sequence of events that follow E2 interaction with ESR1 includes the SRC-mediated transactivation of EGFR and the phosphorylation of ERK1/2, CREB, and ELK1. This rapid estrogen signaling may modulate gene expression in the corpus of the epididymis, and it may play a role in the dynamic microenvironment of the epididymal lumen. © 2015 Society for Endocrinology.

  3. Vitamin E Secretion by Caco-2 Monolayers to APOA1, but Not to HDL, Is Vitamer Selective12

    PubMed Central

    Nicod, Nathalie; Parker, Robert S.

    2013-01-01

    The aim of this study was to characterize the pathways of basolateral secretion of common dietary tocopherols from polarized Caco-2 monolayers, a model of intestinal absorption. Given differences in structure and physical properties, we hypothesized that secretion may differ between different forms of vitamin E, thus potentially contribute to the selectivity seen in vivo. Monolayers were incubated apically and simultaneously with 10 μmol/L α-, γ-, and δ-tocopherol (1:1:1) in lipid micelles. Treatment with the microsomal triglyceride transfer protein inhibitor BMS201038 revealed that the triglyceride-rich particle secretory pathway (apolipoprotein B–dependent pathway) accounted for ∼80% of total tocopherol secretion, without selectivity among the three forms of vitamin E. Apolipoprotein B–independent secretion of tocopherols (and cholesterol) was greatly enhanced by the liver X receptor agonist T0901317. T0901317 induced ATP-binding cassette transporter A1 (ABCA1) protein expression and basolateral secretion of tocopherols to apolipoprotein A1. ABCA1-dependent secretion demonstrated vitamer selectivity such that efficiency of secretion of α- and γ-tocopherols exceeded that of δ-tocopherol. Basal addition of HDL stimulated vitamin E secretion but without selectivity among the three forms, whereas LDL had no effect. Basal addition of scavenger receptor class B member I (SR-BI) blocking antibody, which inhibits the interaction between SR-BI and HDL, increased basal accumulation of all tocopherols, demonstrating a role for SR-BI in cellular re-uptake of secreted vitamin E. These findings demonstrated that vitamin E and cholesterol utilize common pathways of secretion and that secretion via the ABCA1 pathway favors certain forms of vitamin E. PMID:23946344

  4. HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-Mediated Innate Immune Response.

    PubMed

    Morchikh, Mehdi; Cribier, Alexandra; Raffel, Raoul; Amraoui, Sonia; Cau, Julien; Severac, Dany; Dubois, Emeric; Schwartz, Olivier; Bennasser, Yamina; Benkirane, Monsef

    2017-08-03

    The DNA-mediated innate immune response underpins anti-microbial defenses and certain autoimmune diseases. Here we used immunoprecipitation, mass spectrometry, and RNA sequencing to identify a ribonuclear complex built around HEXIM1 and the long non-coding RNA NEAT1 that we dubbed the HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein complex (HDP-RNP). The HDP-RNP contains DNA-PK subunits (DNAPKc, Ku70, and Ku80) and paraspeckle proteins (SFPQ, NONO, PSPC1, RBM14, and MATRIN3). We show that binding of HEXIM1 to NEAT1 is required for its assembly. We further demonstrate that the HDP-RNP is required for the innate immune response to foreign DNA, through the cGAS-STING-IRF3 pathway. The HDP-RNP interacts with cGAS and its partner PQBP1, and their interaction is remodeled by foreign DNA. Remodeling leads to the release of paraspeckle proteins, recruitment of STING, and activation of DNAPKc and IRF3. Our study establishes the HDP-RNP as a key nuclear regulator of DNA-mediated activation of innate immune response through the cGAS-STING pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Biogenesis of non-structural protein 1 (nsp1) and nsp1-mediated type I interferon modulation in arteriviruses

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Han, Mingyuan; Kim, Chi Yong; Rowland, Raymond R.R.

    2014-06-15

    Type I interferons (IFNs-α/β) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (PRRSV), has been shown to down-regulate the production of IFNs during infection. Non-structural protein (nsp) 1 of PRRSV has been identified as a viral IFN antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the CREB-binding protein (CBP) and to inhibit the formation of enhanceosome thus resulting in the suppression of IFN production. The study was expanded to other member viruses in the family Arteriviridae: equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virusmore » (LDV), and simian hemorrhagic fever virus (SHFV). While PRRSV–nsp1 and LDV–nsp1 were auto-cleaved to produce the nsp1α and nsp1β subunits, EAV–nsp1 remained uncleaved. SHFV–nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1β, and nsp1γ), but only two subunits were generated as SHFV–nsp1αβ and SHFV–nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1β motif of nsp1β was functional to generate SHFV–nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV–nsp1β, LDV–nsp1β, EAV–nsp1, and SHFV–nsp1γ were predominantly found in the nucleus. All subunits of arterivirus nsp1 contained the IFN suppressive activity and inhibited both interferon regulatory factor 3 (IRF3) and NF-κB mediated IFN promoter activities. Similar to PRRSV–nsp1α, CBP degradation was evident in cells expressing LDV–nsp1α and SHFV–nsp1γ, but no such degradation was observed for EAV–nsp1. Regardless of CBP degradation, all subunits of arterivirus nsp1 suppressed the IFN-sensitive response element (ISRE)-promoter activities. Our data show that the nsp1-mediated IFN modulation is a common strategy for all arteriviruses but their mechanism of action may

  6. AKAP150 mediates TRPV1 sensitivity to phosphatidylinositol-4, 5-bisphosphate

    PubMed Central

    Jeske, Nathaniel A.; Por, Elaine D.; Belugin, Sergei; Chaudhury, Sraboni; Berg, Kelly A.; Akopian, Armen N.; Henry, Michael A.; Gomez, Ruben

    2011-01-01

    A-kinase anchoring protein 150 (AKAP150) is a scaffolding protein that controls protein kinase A- and C-mediated phosphorylation of the transient receptor potential family V type 1 (TRPV1), dictating receptor response to nociceptive stimuli. The phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) anchors AKAP150 to the plasma membrane in naïve conditions, and also affects TRPV1 activity. In the present study, we sought to determine whether the effects of PIP2 on TRPV1 are mediated through AKAP150. In trigeminal neurons and CHO cells, the manipulation of cellular PIP2 led to significant changes in the association of AKAP150 and TRPV1. Following PIP2 degradation, increased TRPV1:AKAP150 co-immunoprecipitation was observed, resulting in increased receptor response to capsaicin treatment. Phospholipase C activation in neurons isolated from AKAP150−/− animals indicated that PIP2 -mediated inhibition of TRPV1 in the whole cell environment requires expression of the scaffolding protein. Furthermore, the addition of PIP2 to neurons isolated from AKAP150 wild-type mice reduced PKA-sensitization of TRPV1 compared to isolated neurons from AKAP150−/− mice. These findings suggest that PIP2 degradation increases AKAP150 association with TRPV1 in the whole cell environment, leading to sensitization of the receptor to nociceptive stimuli. PMID:21653872

  7. Identification of a Novel Mutation in the ABCA4 Gene in a Chinese Family with Retinitis Pigmentosa Using Exome Sequencing.

    PubMed

    Huang, Xiangjun; Yuan, Lamei; Xu, Hongbo; Zheng, Wen; Cao, Yanna; Yi, Junhui; Guo, Yi; Yang, Zhijian; Li, Yu; Deng, Hao

    2018-02-05

    Retinitis pigmentosa (RP) is a group of hereditary, degenerative retinal disorders characterized by progressive retinal dysfunction, outer retina cell loss, and retinal tissue atrophy. It eventually leads to tunnel vision and legal, or total blindness. Here we aimed to reveal the causal gene and mutation contributing to the development of autosomal recessive RP (arRP) in a consanguineous family. A novel homozygous mutation, c.4845delT (p.K1616Rfs*46), in the ATP-binding cassette subfamily A member 4gene ( ABCA4 ) was identified. It may reduce ABCA4 protein activity, leading to progressive degeneration of both rod and cone photoreceptors. The study extends the arRP genotypic spectrum and confirms a genotype-phenotype relationship. This study may also disclose some new clues for RP genetic causes and pathogenesis, as well as clinical and genetic diagnosis. The research findings may contribute to improvement in clinical care, therapy, genetic screening, and counseling. ©2018 The Author(s).

  8. Heme oxygenase-1 mediates BAY 11-7085 induced ferroptosis.

    PubMed

    Chang, Ling-Chu; Chiang, Shih-Kai; Chen, Shuen-Ei; Yu, Yung-Luen; Chou, Ruey-Hwang; Chang, Wei-Chao

    2018-03-01

    Ferroptosis is a form of oxidative cell death and has become a chemotherapeutic target for cancer treatment. BAY 11-7085 (BAY), which is a well-known IκBα inhibitor, suppressed viability in cancer cells via induction of ferroptotic death in an NF-κB-independent manner. Reactive oxygen species scavenging, relief of lipid peroxidation, replenishment of glutathione and thiol-containing agents, as well as iron chelation, rescued BAY-induced cell death. BAY upregulated a variety of Nrf2 target genes related to redox regulation, particularly heme oxygenase-1 (HO-1). Studies with specific inhibitors and shRNA interventions suggested that the hierarchy of induction is Nrf2-SLC7A11-HO-1. SLC7A11 inhibition by erastin, sulfasalazine, or shRNA interference sensitizes BAY-induced cell death. Overexperession of SLC7A11 attenuated BAY-inhibited cell viability. The ferroptotic process induced by hHO-1 overexpression further indicated that HO-1 is a key mediator of BAY-induced ferroptosis that operates through cellular redox regulation and iron accumulation. BAY causes compartmentalization of HO-1 into the nucleus and mitochondrion, and followed mitochondrial dysfunctions, leading to lysosome targeting for mitophagy. In this study, we first discovered that BAY induced ferroptosis via Nrf2-SLC7A11-HO-1 pathway and HO-1 is a key mediator by responding to the cellular redox status. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Adiponectin-Mediated Heme Oxygenase-1 Induction Protects Against Iron-Induced Liver Injury via a PPARα-Dependent Mechanism

    PubMed Central

    Lin, Heng; Yu, Chun-Hsien; Jen, Chih-Yu; Cheng, Ching-Feng; Chou, Ying; Chang, Chih-Cheng; Juan, Shu-Hui

    2010-01-01

    Protective effects of adiponectin (APN; an adipocytokine) were shown against various oxidative challenges; however, its therapeutic implications and the mechanisms underlying hepatic iron overload remain unclear. Herein, we show that the deleterious effects of iron dextran on liver function and iron deposition were significantly reversed by adiponectin gene therapy, which was accompanied by AMP-activated protein kinase (AMPK) phosphorylation and heme oxygenase (HO)-1 induction. Furthermore, AMPK-mediated peroxisome proliferator-activated receptor-α (PPARα) activation by APN was ascribable to HO-1 induction. Additionally, we revealed direct transcriptional regulation of HO-1 by the binding of PPARα to a PPAR-responsive element (PPRE) by various experimental assessments. Interestingly, overexpression of HO-1 in hepatocytes mimicked the protective effect of APN in attenuating iron-mediated injury, whereas it was abolished by SnPP and small interfering HO-1. Furthermore, bilirubin, the end-product of the HO-1 reaction, but not CO, protected hepatocytes from iron dextran-mediated caspase activation. Herein, we demonstrate a novel functional PPRE in the promoter regions of HO-1, and APN-mediated HO-1 induction elicited an antiapoptotic effect and a decrease in iron deposition in hepatocytes subjected to iron challenge. PMID:20709802

  10. Histone methyltransferase Ash1L mediates activity-dependent repression of neurexin-1α

    PubMed Central

    Zhu, Τao; Liang, Chen; Li, Dongdong; Tian, Miaomiao; Liu, Sanxiong; Gao, Guanjun; Guan, Ji-Song

    2016-01-01

    Activity-dependent transcription is critical for the regulation of long-term synaptic plasticity and plastic rewiring in the brain. Here, we report that the transcription of neurexin1α (nrxn1α), a presynaptic adhesion molecule for synaptic formation, is regulated by transient neuronal activation. We showed that 10 minutes of firing at 50 Hz in neurons repressed the expression of nrxn1α for 24 hours in a primary cortical neuron culture through a transcriptional repression mechanism. By performing a screening assay using a synthetic zinc finger protein (ZFP) to pull down the proteins enriched near the nrxn1α promoter region in vivo, we identified that Ash1L, a histone methyltransferase, is enriched in the nrxn1α promoter. Neuronal activity triggered binding of Ash1L to the promoter and enriched the histone marker H3K36me2 at the nrxn1α promoter region. Knockout of Ash1L in mice completely abolished the activity-dependent repression of nrxn1α. Taken together, our results reveal that a novel process of activity-dependent transcriptional repression exists in neurons and that Ash1L mediates the long-term repression of nrxn1α, thus implicating an important role for epigenetic modification in brain functioning. PMID:27229316

  11. Proresolving lipid mediators resolvin D1, resolvin D2, and maresin 1 are critical in modulating T cell responses.

    PubMed

    Chiurchiù, Valerio; Leuti, Alessandro; Dalli, Jesmond; Jacobsson, Anders; Battistini, Luca; Maccarrone, Mauro; Serhan, Charles N

    2016-08-24

    Resolution of inflammation is a finely regulated process mediated by specialized proresolving lipid mediators (SPMs), including docosahexaenoic acid (DHA)-derived resolvins and maresins. The immunomodulatory role of SPMs in adaptive immune cells is of interest. We report that D-series resolvins (resolvin D1 and resolvin D2) and maresin 1 modulate adaptive immune responses in human peripheral blood lymphocytes. These lipid mediators reduce cytokine production by activated CD8(+) T cells and CD4(+) T helper 1 (TH1) and TH17 cells but do not modulate T cell inhibitory receptors or abrogate their capacity to proliferate. Moreover, these SPMs prevented naïve CD4(+) T cell differentiation into TH1 and TH17 by down-regulating their signature transcription factors, T-bet and Rorc, in a mechanism mediated by the GPR32 and ALX/FPR2 receptors; they concomitantly enhanced de novo generation and function of Foxp3(+) regulatory T (Treg) cells via the GPR32 receptor. These results were also supported in vivo in a mouse deficient for DHA synthesis (Elovl2(-/-)) that showed an increase in TH1/TH17 cells and a decrease in Treg cells compared to wild-type mice. Additionally, either DHA supplementation in Elovl2(-/-) mice or in vivo administration of resolvin D1 significantly reduced cytokine production upon specific stimulation of T cells. These findings demonstrate actions of specific SPMs on adaptive immunity and provide a new avenue for SPM-based approaches to modulate chronic inflammation. Copyright © 2016, American Association for the Advancement of Science.

  12. Pathways by which reconstituted high-density lipoprotein mobilizes free cholesterol from whole body and from macrophages.

    PubMed

    Cuchel, Marina; Lund-Katz, Sissel; de la Llera-Moya, Margarita; Millar, John S; Chang, David; Fuki, Ilia; Rothblat, George H; Phillips, Michael C; Rader, Daniel J

    2010-03-01

    Reconstituted high-density lipoprotein (rHDL) is of interest as a potential novel therapy for atherosclerosis because of its ability to promote free cholesterol (FC) mobilization after intravenous administration. We performed studies to identify the underlying molecular mechanisms by which rHDL promote FC mobilization from whole body in vivo and macrophages in vitro. Wild-type (WT), SR-BI knockout (KO), ABCA1 KO, and ABCG1 KO mice received either rHDL or phosphate-buffered saline intravenously. Blood was drawn before and at several time points after injection for apolipoprotein A-I, phosphatidylcholine, and FC measurement. In WT mice, serum FC peaked at 20 minutes and rapidly returned toward baseline levels by 24 hours. Unexpectedly, ABCA1 KO and ABCG1 KO mice did not differ from WT mice regarding the kinetics of FC mobilization. In contrast, in SR-BI KO mice the increase in FC level at 20 minutes was only 10% of that in control mice (P<0.01). Bone marrow-derived macrophages from WT, SR-BI O, ABCA1 KO, and ABCG1 KO mice were incubated in vitro with rHDL and cholesterol efflux was determined. Efflux from SR-BI KO and ABCA1 KO macrophages was not different from WT macrophages. In contrast, efflux from ABCG1 KO macrophages was approximately 50% lower as compared with WT macrophages (P<0.001). The bulk mobilization of FC observed in circulation after rHDL administration is primarily mediated by SR-BI. However, cholesterol mobilization from macrophages to rHDL is primarily mediated by ABCG1.

  13. β-Arrestin-1 mediates thyrotropin-enhanced osteoblast differentiation.

    PubMed

    Boutin, Alisa; Eliseeva, Elena; Gershengorn, Marvin C; Neumann, Susanne

    2014-08-01

    Thyrotropin (TSH) activation of the TSH receptor (TSHR), a 7-transmembrane-spanning receptor (7TMR), may have osteoprotective properties by direct effects on bone. TSHR activation by TSH phosphorylates protein kinases AKT1, p38α, and ERK1/2 in some cells. We found TSH-induced phosphorylation of these kinases in 2 cell lines engineered to express TSHRs, human embryonic kidney HEK-TSHR cells and human osteoblastic U2OS-TSHR cells. In U2OS-TSHR cells, TSH up-regulated pAKT1 (7.1±0.5-fold), p38α (2.9±0.4-fold), and pERK1/2 (3.1±0.2-fold), whereas small molecule TSHR agonist C2 had no or little effect on pAKT1 (1.8±0.08-fold), p38α (1.2±0.09-fold), and pERK1/2 (1.6±0.19-fold). Furthermore, TSH increased expression of osteoblast marker genes ALPL (8.2±4.6-fold), RANKL (21±5.9-fold), and osteopontin (OPN; 17±5.3-fold), whereas C2 had little effect (ALPL, 1.7±0.5-fold; RANKL, 1.3±0.6-fold; and OPN, 2.2±0.7-fold). β-Arrestin-1 and -2 can mediate activatory signals by 7TMRs. TSH stimulated translocation of β-arrestin-1 and -2 to TSHR, whereas C2 failed to translocate either β-arrestin. Down-regulation of β-arrestin-1 by siRNA inhibited TSH-stimulated phosphorylation of ERK1/2, p38α, and AKT1, whereas down-regulation of β-arrestin-2 increased phosphorylation of AKT1 in both cell types and of ERK1/2 in HEK-TSHR cells. Knockdown of β-arrestin-1 inhibited TSH-stimulated up-regulation of mRNAs for OPN by 87 ± 1.7% and RANKL by 73 ± 2.4%, and OPN secretion by 74 ± 10%. We conclude that TSH enhances osteoblast differentiation in U2OS cells that is, in part, caused by activatory signals mediated by β-arrestin-1. © FASEB.

  14. ADP-Ribosylation Factor 6 and a Functional PIX/p95-APP1 Complex Are Required for Rac1B-mediated Neurite Outgrowth

    PubMed Central

    Albertinazzi, Chiara; Za, Lorena; Paris, Simona; de Curtis, Ivan

    2003-01-01

    The mechanisms coordinating adhesion, actin organization, and membrane traffic during growth cone migration are poorly understood. Neuritogenesis and branching from retinal neurons are regulated by the Rac1B/Rac3 GTPase. We have identified a functional connection between ADP-ribosylation factor (Arf) 6 and p95-APP1 during the regulation of Rac1B-mediated neuritogenesis. P95-APP1 is an ADP-ribosylation factor GTPase-activating protein (ArfGAP) of the GIT family expressed in the developing nervous system. We show that Arf6 has a predominant role in neurite extension compared with Arf1 and Arf5. Cotransfection experiments indicate a specific and cooperative potentiation of neurite extension by Arf6 and the carboxy-terminal portion of p95-APP1. Localization studies in neurons expressing different p95-derived constructs show a codistribution of p95-APP1 with Arf6, but not Arf1. Moreover, p95-APP1–derived proteins with a mutated or deleted ArfGAP domain prevent Rac1B-induced neuritogenesis, leading to PIX-mediated accumulation at large Rab11-positive endocytic vesicles. Our data support a role of p95-APP1 as a specific regulator of Arf6 in the control of membrane trafficking during neuritogenesis. PMID:12686588

  15. Activity-dependent ubiquitination of GluA1 mediates a distinct AMPA receptor endocytosis and sorting pathway.

    PubMed

    Schwarz, Lindsay A; Hall, Benjamin J; Patrick, Gentry N

    2010-12-08

    The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, whereas dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer's disease. Previous work has shown that ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its C-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA but not for internalization of AMPARs in response to the NMDA receptor agonist NMDA. Through overexpression or RNA interference-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1 (neural-precursor cell-expressed developmentally downregulated gene 4-1), is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues and suggest that changes to this pathway may occur as neurons mature.

  16. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Jing-Min, E-mail: wjm730222@163.com; Wang, Dong, E-mail: 8888dd@163.com; Tan, Yu-Yan, E-mail: tyytyz@sina.com

    Highlights: • Cholesterosis is a metabolic disease characterized by excessive lipid droplets. • Lipid droplet efflux is mediated by the ABCA1 transporter. • 22(R)-hydroxycholesterol can activate LXRα and up-regulate ABCA1. • Pioglitazone up-regulates ABCA1 in a PPARγ–LXRα–ABCA1-dependent manner. • 22(R)-hydroxycholesterol and pioglitazone synergistically decrease lipid droplets. - Abstract: Cholesterosis is a disease of cholesterol metabolism characterized by the presence of excessive lipid droplets in the cytoplasm. These lipid droplets are mainly composed of cholesterol esters derived from free cholesterol. The removal of excess cholesterol from gallbladder epithelial cells (GBECs) is very important for the maintenance of intracellular cholesterol homeostasis andmore » the preservation of gallbladder function. Several lines of evidence have indicated that the activation of either peroxisome proliferator-activated receptor gamma (PPARγ) or liver X receptor α (LXRα) relates to cholesterol efflux. While pioglitazone can regulate the activation of PPARγ, 22(R)-hydroxycholesterol can activate LXRα and is a metabolic intermediate in the biosynthesis of steroid hormones. However, the effect of 22(R)-hydroxycholesterol in combination with pioglitazone on cholesterosis of the gallbladder is unclear. GBECs were treated with pioglitazone, 22(R)-hydroxycholesterol or PPARγ siRNA followed by Western blot analysis for ATP-binding cassette transporter A1 (ABCA1), PPARγ and LXRα. Cholesterol efflux to apoA-I was determined, and Oil Red O staining was performed to monitor variations in lipid levels in treated GBECs. Our data showed that 22(R)-hydroxycholesterol can modestly up-regulate LXRα while simultaneously increasing ABCA1 by 56%. The combination of 22(R)-hydroxycholesterol and pioglitazone resulted in a 3.64-fold increase in ABCA1 expression and a high rate of cholesterol efflux. Oil Red O staining showed an obvious reduction in the lipid

  17. Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance.

    PubMed

    Liu, Zhongle; Myers, Lawrence C

    2017-11-01

    Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1 , a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We identified the Swi/Snf chromatin remodeling complex as a key coactivator for Mrr1, which is required to maintain basal and induced open chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2 , the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1 GOF mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1 GOF mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1 GOF strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple coactivators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity-restoring small molecules more complicated. Copyright © 2017 American Society for Microbiology.

  18. LANP mediates neuritic pathology in Spinocerebellar ataxia type 1

    PubMed Central

    Cvetanovic, Marija; Kular, Rupinder K.; Opal, Puneet

    2014-01-01

    Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease that results from a pathogenic glutamine-repeat expansion in the protein ataxin-1 (ATXN1). Although the functions of ATXN1 are still largely unknown, there is evidence to suggest that ATXN1 plays a role in regulating gene expression, the earliest process known to go awry in SCA1 mouse models. In this study, we show that ATXN1 reduces histone acetylation, a post-translational modification of histones associated with enhanced transcription, and represses histone acetyl transferase-mediated transcription. In addition, we find that depleting the Leucine-rich Acidic Nuclear Protein (LANP)—an ATXN1 binding inhibitor of histone acetylation—reverses aspects of SCA1 neuritic pathology. PMID:22884877

  19. The cognitive mediation model: factors influencing public knowledge of the H1N1 pandemic and intention to take precautionary behaviors.

    PubMed

    Ho, Shirley S; Peh, Xianghong; Soh, Veronica W L

    2013-01-01

    This study uses the cognitive mediation model as the theoretical framework to examine the influence of motivations, communication, and news elaboration on public knowledge of the H1N1 pandemic and the intention to take precautionary behaviors in Singapore. Using a nationally representative random digit dialing telephone survey of 1,055 adult Singaporeans, the authors' results show that the cognitive mediation model can be applied to health contexts, in which motivations (surveillance gratification, guidance, and need for cognition) were positively associated with news attention, elaboration, and interpersonal communication. News attention, elaboration, and interpersonal communication in turn positively influence public knowledge about the H1N1 influenza. In addition, results show that the motivations have significant indirect effects on behavioral intentions, as partially mediated by communication (media attention and interpersonal communication), elaboration, and knowledge. The authors conclude that the cognitive mediation model can be extended to behavioral outcomes, above and beyond knowledge. Implications for theory and practice for health communication were discussed.

  20. Inflammation induction of Dickkopf-1 mediates chondrocyte apoptosis in osteoarthritic joint.

    PubMed

    Weng, L-H; Wang, C-J; Ko, J-Y; Sun, Y-C; Su, Y-S; Wang, F-S

    2009-07-01

    Dysregulated Wnt signaling appears to modulate chondrocyte fate and joint disorders. Dickkopf-1 (DKK1) regulates the pathogenesis of skeletal tissue by inhibiting Wnt actions. This study examined whether DKK1 expression is linked to chondrocyte fate in osteoarthritis (OA). Articular cartilage specimens harvested from nine patients with knee OA and from six controls with femoral neck fracture were assessed for DKK1, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), Bad, Bax, Bcl2 and caspase-3 expression by real time-polymerase chain reaction (RT-PCR) and immunohistochemistry. Apoptotic chondrocytes were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) and 4', 6-dianidino-2-phenylindole dihydrochloride (DAPI) staining. Human chondrocyte cultures were treated with recombinant IL-1beta and monoclonal DKK1 antibody to determine whether DKK1 impairs chondrocyte survival. Expression of DKK1 correlated with inflammatory cytokine levels (IL-1beta and TNF-alpha expressions), proapoptosis regulators (Bad and caspase-3 expressions) and TUNEL staining in OA cartilage tissues. The IL-1beta induced expressions of DKK1, Bax, Bad and caspase-3-dependent apoptosis of chondrocyte cultures. Neutralization of DKK1 by monoclonal DKK1 antibody significantly abrogated IL-1beta-mediated caspase-3 cleavage and apoptosis and reversed chondrocyte proliferation. Recombinant DKK1 treatment impaired chondrocyte growth and promoted apoptosis. By suppressing nuclear beta-catenin accumulation and Akt phosphorylation, DKK1 mediated IL-1beta promotion of chondrocyte apoptosis. Chondrocyte apoptosis correlates with joint OA. Expression of DKK1 contributes to cartilage deterioration and is a potent factor in OA pathogenesis. Attenuating DKK1 may reduce cartilage deterioration in OA.

  1. Tanshinone IIA suppresses FcεRI-mediated mast cell signaling and anaphylaxis by activation of the Sirt1/LKB1/AMPK pathway.

    PubMed

    Li, Xian; Park, Soon Jin; Jin, Fansi; Deng, Yifeng; Yang, Ju Hye; Chang, Jae-Hoon; Kim, Dong-Young; Kim, Jung-Ae; Lee, Youn Ju; Murakami, Makoto; Son, Kun Ho; Chang, Hyeun Wook

    2018-06-01

    AMP-activated protein kinase (AMPK) and its upstream mediators liver kinase B1 (LKB1) and sirtuin 1 (Sirt1) are generally known as key regulators of metabolism. We have recently reported that the AMPK pathway negatively regulates mast cell activation and anaphylaxis. Tanshinone IIA (Tan IIA), an active component of Salvia miltiorrhiza extract that is currently used for the treatment of cardiovascular and cerebrovascular diseases, shows anti-diabetic activity and improves insulin resistance in db/db mice through activation of AMPK. The aim of this study was to evaluate the anti-allergic activity of Tan IIA in vivo and to investigate the underlying mechanism in vitro in the context of AMPK signaling. The anti-allergic effect of Tan IIA was evaluated using mouse bone marrow-derived mast cells (BMMCs) from AMPKα2 -/- or Sirt1 -/- mice, or BMMCs transfected with siRNAs specific for AMPKα2, LKB1, or Sirt1. AMPKα2 -/- and Sirt1 -/- mice were used to confirm the anti-allergic effect of Tan IIA in anaphylaxis in vivo. Tan IIA dose-dependently inhibited FcεRI-mediated degranulation and production of eicosanoids and cytokines in BMMCs. These inhibitory effects were diminished by siRNA-mediated knockdown or genetic deletion of AMPKα2 or Sirt1. Moreover, Tan IIA inhibited a mast cell-mediated local passive anaphylactic reaction in wild-type mice, but not in AMPKα2 -/- or Sirt1 -/- mice. In conclusion, Tan IIA suppresses FcεRI-mediated mast cell activation and anaphylaxis through activation of the inhibitory Sirt1-LKB1-AMPK pathway. Thus, Tan IIA may be useful as a new therapeutic agent for mast cell-mediated allergic diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. O-GlcNAc modification of Sp1 mediates hyperglycaemia-induced ICAM-1 up-regulation in endothelial cells.

    PubMed

    Zhang, Yuan; Qu, Yuan; Niu, Tian; Wang, Haiyan; Liu, Kun

    2017-02-26

    Intracellular adhesion molecule 1 (ICAM-1) is an important inflammatory factor that causes retinal damage during diabetic retinopathy. Hyperglycaemia can increase ICAM-1 expression in endothelial cells and the ICAM-1 promoter is responsive to the transcription factor specificity protein 1 (Sp1). O-GlcNAc modification is driven by the glucose concentration and has a profound effect on Sp1 activity. In this study, we investigated the underlying mechanism through which hyperglycaemia triggers ICAM-1 expression, which is mediated by O-GlcNAc modification of Sp1 in human umbilical vein endothelial cells (HUVECs) and rat retinal capillary endothelial cells (RRCECs). We showed that hyperglycaemia (30 mM) increased ICAM-1 expression compared to control conditions (5 mM). The addition of an OGT inhibitor decreased ICAM-1 expression and addition of an OGA inhibitor enhanced ICAM-1 expression. Furthermore, cells transduced with siSp1 exhibited dramatically decreased ICAM-1 expression. These results proved that the up-regulation of ICAM-1 with hyperglycaemia is mediated by O-GlcNAc modification of Sp1. It helps to explain the mechanism of ICAM-1 processing in HUVECs and RRCECs. Understanding how this inflammatory factor is modulated during diabetic retinopathy will ultimately help to design novel therapeutics to treat this condition. Copyright © 2017. Published by Elsevier Inc.

  3. CCAR1 is required for Ngn3-mediated endocrine differentiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lu, Chung-Kuang; Lai, Yi-Chyi; Lin, Yung-Fu

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer We identify CCAR1 to directly interact with Ngn3. Black-Right-Pointing-Pointer CCAR1 is co-localized with Ngn3 in the nucleus. Black-Right-Pointing-Pointer CCAR1 cooperates with Ngn3 in activating NeuroD expression. Black-Right-Pointing-Pointer CCAR1 is required for Ngn3-mediated PANC-1 transdifferentiation. -- Abstract: Neurogenin3 (Ngn3) is a basic helix-loop-helix transcription factor that specifies pancreatic endocrine cell fates during pancreas development. It can also initiate a transdifferentiation program when expressed in pancreatic exocrine and ductal cells. However, how Ngn3 initiates a transcriptional cascade to achieve endocrine differentiation is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which is a transcriptionalmore » coactivator for nuclear receptors, also interacts with Ngn3. The association between Ngn3 and CCAR1 was verified by pull-down assays and co-immunoprecipitation analyses. Using gene reporter assays, we found that CCAR1 is essential for Ngn3 to activate the expression of the reporter genes containing the NeuroD promoter. Moreover, down-regulation of endogenous CCAR1 in the PANC-1 pancreatic ductal cell line inhibits the transdifferentiation program initiated by Ngn3. CCAR1 is, therefore, a novel partner of Ngn3 in mediating endocrine differentiation.« less

  4. The inhibition of the potassium channel TASK-1 in rat cardiac muscle by endothelin-1 is mediated by phospholipase C.

    PubMed

    Schiekel, Julia; Lindner, Moritz; Hetzel, Andrea; Wemhöner, Konstantin; Renigunta, Vijay; Schlichthörl, Günter; Decher, Niels; Oliver, Dominik; Daut, Jürgen

    2013-01-01

    The two-pore-domain potassium channel TASK-1 is robustly inhibited by the activation of receptors coupled to the Gα(q) subgroup of G-proteins, but the signal transduction pathway is still unclear. We have studied the mechanisms by which endothelin receptors inhibit the current carried by TASK-1 channels (I(TASK)) in cardiomyocytes. Patch-clamp measurements were carried out in isolated rat cardiomyocytes. I(TASK) was identified by extracellular acidification to pH 6.0 and by the application of the TASK-1 blockers A293 and A1899. Endothelin-1 completely inhibited I(TASK) with an EC(50) of <10 nM; this effect was mainly mediated by endothelin-A receptors. Application of 20 nM endothelin-1 caused a significant increase in action potential duration under control conditions; this was significantly reduced after pre-incubation of the cardiomyocytes with 200 nM A1899. The inhibition of I(TASK) by endothelin-1 was not affected by inhibitors of protein kinase C or rho kinase, but was strongly reduced by U73122, an inhibitor of phospholipase C (PLC). The ability of endothelin-1 to activate PLC-mediated signalling pathways was examined in mammalian cells transfected with TASK-1 and the endothelin-A receptor using patch-clamp measurements and total internal reflection microscopy. U73122 prevented the inhibition of I(TASK) by endothelin-1 and blocked PLC-mediated signalling, as verified with a fluorescent probe for phosphatidylinositol-(4,5)-bisphosphate hydrolysis. Our results show that I(TASK) in rat cardiomyocytes is controlled by endothelin-1 and suggest that the inhibition of TASK-1 via endothelin receptors is mediated by the activation of PLC. The prolongation of the action potential observed with 20 nM endothelin-1 was mainly due to the inhibition of I(TASK).

  5. The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

    PubMed

    Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

    2016-07-01

    The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  6. UNC93B1 mediates differential trafficking of endosomal TLRs

    PubMed Central

    Lee, Bettina L; Moon, Joanne E; Shu, Jeffrey H; Yuan, Lin; Newman, Zachary R; Schekman, Randy; Barton, Gregory M

    2013-01-01

    UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases. DOI: http://dx.doi.org/10.7554/eLife.00291.001 PMID:23426999

  7. Febuxostat, an Inhibitor of Xanthine Oxidase, Suppresses Lipopolysaccharide-Induced MCP-1 Production via MAPK Phosphatase-1-Mediated Inactivation of JNK

    PubMed Central

    Nomura, Johji; Busso, Nathalie; Ives, Annette; Tsujimoto, Syunsuke; Tamura, Mizuho; So, Alexander; Yamanaka, Yoshihiro

    2013-01-01

    Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes. PMID:24086554

  8. Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53.

    PubMed

    Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

    2015-11-02

    Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.

  9. Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    PubMed Central

    Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

    2015-01-01

    Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway. PMID:26522181

  10. Complement component 1, q subcomponent binding protein (C1QBP) in lipid rafts mediates hepatic metastasis of pancreatic cancer by regulating IGF-1/IGF-1R signaling.

    PubMed

    Shi, Haojun; Fang, Winston; Liu, Minda; Fu, Deliang

    2017-10-01

    Pancreatic cancer shows a remarkable predilection for hepatic metastasis. Complement component 1, q subcomponent binding protein (C1QBP) can mediate growth factor-induced cancer cell chemotaxis and distant metastasis by activation of receptor tyrosine kinases. Coincidentally, insulin-like growth factor-1 (IGF-1) derived from the liver and cancer cells itself has been recognized as a critical inducer of hepatic metastasis. However, the mechanism underlying IGF-1-dependent hepatic metastasis of pancreatic cancer, in which C1QBP may be involved, remains unknown. In the study, we demonstrated a significant association between C1QBP expression and hepatic metastasis in patients with pancreatic cancer. IGF-1 induced the translocation of C1QBP from cytoplasm to lipid rafts and further drove the formation of CD44 variant 6 (CD44v6)/C1QBP complex in pancreatic cancer cells. C1QBP interacting with CD44v6 in lipid rafts promoted phosphorylation of IGF-1R and thus activated downstream PI3K and MAPK signaling pathways which mediated metastatic potential of pancreatic cancer cells including proliferation, apoptosis, invasion, adhesion and energy metabolism. Furthermore, C1QBP knockdown suppressed hepatic metastasis of pancreatic cancer cells in nude mice. We therefore conclude that C1QBP in lipid rafts serves a key regulator of IGF-1/IGF-1R-induced hepatic metastasis from pancreatic cancer. Our findings about C1QBP in lipid rafts provide a novel strategy to block IGF-1/IGF-1R signaling in pancreatic cancer and a reliable premise for more efficient combined modality therapies. © 2017 UICC.

  11. Fomiroid A, a Novel Compound from the Mushroom Fomitopsis nigra, Inhibits NPC1L1-Mediated Cholesterol Uptake via a Mode of Action Distinct from That of Ezetimibe

    PubMed Central

    Chiba, Tomohiro; Sakurada, Tsuyoshi; Watanabe, Rie; Yamaguchi, Kohji; Kimura, Yasuhisa; Kioka, Noriyuki; Kawagishi, Hirokazu; Matsuo, Michinori; Ueda, Kazumitsu

    2014-01-01

    Hypercholesterolemia is one of the key risk factors for coronary heart disease, a major cause of death in developed countries. Suppression of NPC1L1-mediated dietary and biliary cholesterol absorption is predicted to be one of the most effective ways to reduce the risk of hypercholesterolemia. In a screen for natural products that inhibit ezetimibe glucuronide binding to NPC1L1, we found a novel compound, fomiroid A, in extracts of the mushroom Fomitopsis nigra. Fomiroid A is a lanosterone derivative with molecular formula C30H48O3. Fomiroid A inhibited ezetimibe glucuronide binding to NPC1L1, and dose-dependently prevented NPC1L1-mediated cholesterol uptake and formation of esterified cholesterol in NPC1L1-expressing Caco2 cells. Fomiroid A exhibited a pharmacological chaperone activity that corrected trafficking defects of the L1072T/L1168I mutant of NPC1L1. Because ezetimibe does not have such an activity, the binding site and mode of action of fomiroid A are likely to be distinct from those of ezetimibe. PMID:25551765

  12. APPL1 Mediates Adiponectin-Induced LKB1 Cytosolic Localization Through the PP2A-PKCζ Signaling Pathway

    PubMed Central

    Deepa, Sathyaseelan S.; Zhou, Lijun; Ryu, Jiyoon; Wang, Changhua; Mao, Xuming; Li, Cai; Zhang, Ning; Musi, Nicolas; DeFronzo, Ralph A.; Liu, Feng

    2011-01-01

    We recently found that the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL)1 is essential for mediating adiponectin signal to induce liver kinase B (LKB)1 cytosloic translocation, an essential step for activation of AMP-activated protein kinase (AMPK) in cells. However, the underlying molecular mechanisms remain unknown. Here, we demonstrate that treating C2C12 myotubes with adiponectin promoted APPL1 interaction with protein phosphatase 2A (PP2A) and protein kinase Cζ (PKCζ), leading to the activation of PP2A and subsequent dephosphorylation and inactivation of PKCζ. The adiponectin-induced inactivation of PKCζ results in dephosphorylation of LKB1 at Ser307 and its subsequent translocation to the cytosol, where it stimulates AMPK activity. Interestingly, we found that metformin also induces LKB1 cytosolic translocation, but the stimulation is independent of APPL1 and the PP2A-PKCζ pathway. Together, our study uncovers a new mechanism underlying adiponectin-stimulated AMPK activation in muscle cells and shed light on potential targets for prevention and treatment of insulin resistance and its associated diseases. PMID:21835890

  13. ARRB1/β-arrestin-1 mediates neuroprotection through coordination of BECN1-dependent autophagy in cerebral ischemia

    PubMed Central

    Wang, Pei; Xu, Tian-Ying; Wei, Kai; Guan, Yun-Feng; Wang, Xia; Xu, Hui; Su, Ding-Feng; Pei, Gang; Miao, Chao-Yu

    2014-01-01

    Autophagy, a highly conserved process conferring cytoprotection against stress, contributes to the progression of cerebral ischemia. β-arrestins are multifunctional proteins that mediate receptor desensitization and serve as important signaling scaffolds involved in numerous physiopathological processes. Here, we show that both ARRB1 (arrestin, β 1) and ARRB2 (arrestin, β 2) were upregulated by cerebral ischemic stress. Knockout of Arrb1, but not Arrb2, aggravated the mortality, brain infarction, and neurological deficit in a mouse model of cerebral ischemia. Accordingly, Arrb1-deficient neurons exhibited enhanced cell injury upon oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Deletion of Arrb1 did not affect the cerebral ischemia-induced inflammation, oxidative stress, and nicotinamide phosphoribosyltransferase upregulation, but markedly suppressed autophagy and induced neuronal apoptosis/necrosis in vivo and in vitro. Additionally, we found that ARRB1 interacted with BECN1/Beclin 1 and PIK3C3/Vps34, 2 major components of the BECN1 autophagic core complex, under the OGD condition but not normal conditions in neurons. Finally, deletion of Arrb1 impaired the interaction between BECN1 and PIK3C3, which is a critical event for autophagosome formation upon ischemic stress, and markedly reduced the kinase activity of PIK3C3. These findings reveal a neuroprotective role for ARRB1, in the context of cerebral ischemia, centered on the regulation of BECN1-dependent autophagosome formation. PMID:24988431

  14. Reduced syncytin-1 expression in chriocarcinoma BeWo cells activates the calpain1-AIF-mediated apoptosis, implication for preeclampsia

    PubMed Central

    Huang, Qiang; Chen, Haibin; Wang, Fengchao; Brost, Brian C.; Li, Jinping; Gao, Yu; Li, Zongfang; Gao, Ya; Jiang, Shi-Wen

    2015-01-01

    Placentas associated with preeclampsia are characterized by extensive apoptosis in trophoblast lineages. Syncytin-1 (HERVWE1) mediates the fusion of cytotrophoblasts to form syncytiotrophoblasts, which assume the placental barrier, fetal-maternal exchange and endocrine functions. While decreased syncytin-1 expression has been observed in preeclamptic placentas, it is not clear if this alteration is involved in trophoblast apoptosis. In the current study we found that siRNA-mediated knockdown of syncytin-1 led to apoptosis in choriocarcinoma BeWo, a cell line of trophoblastic origin. Characterization of the apoptotic pathways indicated that this effect does not rely on the activation of caspases. Rather, decreased syncytin-1 levels activated the AIF apoptotic pathway by inducing the expression, cleavage, and nuclear translocation of AIF. Moreover, calpain1, the cysteine protease capable of cleaving AIF, was upregulated by syncytin-1 knockdown. Furthermore, treatment with calpain1 inhibitor MDL28170 effectively reversed AIF cleavage, AIF nuclear translocation, and cell apoptosis triggered by syncytin-1 downregulation, verifying the specific action of calpain1-AIF pathway in trophoblast apoptosis. We confirmed that preeclamptic placentas express lower levels of syncytin-1 than normal placentas, and observed an inverse correlation between syncytin-1 and AIF/calpain1 mRNA levels, a result consistent with the in vitro findings. Immunohistochemistry analyses indicated decreased syncytin-1, increased AIF and calpain1 protein levels in apoptotic cells of preeclamptic placentas. These findings have for the first time revealed that decreased levels of syncytin-1 can trigger the AIF-mediated apoptosis pathway in BeWo cells. This novel mechanism may contribute to the structural and functional deficiencies of syncytium frequently observed in preeclamptic placentas. PMID:24413738

  15. Molecular pathways of pannexin1-mediated neurotoxicity

    PubMed Central

    Shestopalov, Valery I.; Slepak, Vladlen Z.

    2014-01-01

    Pannexin1 (Panx1) forms non-selective membrane channels, structurally similar to gap junction hemichannels, and are permeable to ions, nucleotides, and other small molecules below 900 Da. Panx1 activity has been implicated in paracrine signaling and inflammasome regulation. Recent studies in different animal models showed that overactivation of Panx1 correlates with a selective demise of several types of neurons, including retinal ganglion cells, brain pyramidal, and enteric neurons. The list of Panx1 activators includes extracellular ATP, glutamate, high K+, Zn2+, fibroblast growth factors (FGFs),pro-inflammatory cytokines, and elevation of intracellular Ca2+. Most of these molecules are released following mechanical, ischemic, or inflammatory injury of the CNS, and rapidly activate the Panx1 channel. Prolonged opening of Panx1 channel induced by these “danger signals” triggers a cascade of neurotoxic events capable of killing cells. The most vulnerable cell type are neurons that express high levels of Panx1. Experimental evidence suggests that Panx1 channels mediate at least two distinct neurotoxic processes: increased permeability of the plasma membrane and activation of the inflammasome in neurons and glia. Importantly, both pharmacological and genetic inactivation of Panx1 suppresses both these processes, providing a marked protection in several disease and injury models. These findings indicate that external danger signals generated after diverse types of injuries converge to activate Panx1. In this review we discuss molecular mechanisms associated with Panx1 toxicity and the crosstalk between different pathways. PMID:24575045

  16. Hypoxia-inducible factor 1α is a critical downstream mediator for hypoxia-induced mitogenic factor (FIZZ1/RELMα)-induced pulmonary hypertension

    PubMed Central

    Johns, Roger A.; Takimoto, Eiki; Meuchel, Lucas W.; Elsaigh, Esra; Zhang, Ailan; Heller, Nicola M.; Semenza, Gregg L.; Yamaji-Kegan, Kazuyo

    2017-01-01

    Objective Pulmonary hypertension (PH) is characterized by progressive elevation of pulmonary vascular resistance, right ventricular failure, and ultimately death. We have shown that in rodents, hypoxia-induced mitogenic factor (HIMF; also known as FIZZ1 or RELMα) causes PH by initiating lung vascular inflammation. We hypothesized that hypoxia-inducible factor-1 (HIF-1) is a critical downstream signal mediator of HIMF during PH development. Approach and Results In this study, we compared the degree of HIMF-induced pulmonary vascular remodeling and PH development in wild-type (HIF-1α+/+) and HIF-1α heterozygous null (HIF-1α+/−) mice. HIMF-induced PH was significantly diminished in HIF-1α+/− mice and was accompanied by a dysregulated VEGF-A–VEGF receptor 2 pathway. HIF-1α was critical for bone marrow-derived cell migration and vascular tube formation in response to HIMF. Furthermore, HIMF and its human homolog, resistin-like molecule-β (RELMβ), significantly increased IL-6 in macrophages and lung resident cells through a mechanism dependent on HIF-1α and, at least to some extent, on nuclear factor κB. Conclusions Our results suggest that HIF-1α is a critical downstream transcription factor for HIMF-induced pulmonary vascular remodeling and PH development. Importantly, both HIMF and human RELMβ significantly increased IL-6 in lung resident cells and increased perivascular accumulation of IL-6–expressing macrophages in the lungs of mice. These data suggest that HIMF can induce HIF-1, VEGF-A, and interleukin-6, which are critical mediators of both hypoxic inflammation and PH pathophysiology. PMID:26586659

  17. Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory.

    PubMed

    D'Urso, Agustina; Takahashi, Yoh-Hei; Xiong, Bin; Marone, Jessica; Coukos, Robert; Randise-Hinchliff, Carlo; Wang, Ji-Ping; Shilatifard, Ali; Brickner, Jason H

    2016-06-23

    In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8(-) Mediator, during memory, Cdk8(+) Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism.

  18. Opposite effects of dihydrosphingosine 1-phosphate and sphingosine 1-phosphate on transforming growth factor-beta/Smad signaling are mediated through the PTEN/PPM1A-dependent pathway.

    PubMed

    Bu, Shizhong; Kapanadze, Bagrat; Hsu, Tien; Trojanowska, Maria

    2008-07-11

    Transforming growth factor-beta (TGF-beta) is an important regulator of physiological connective tissue biosynthesis and plays a central role in pathological tissue fibrosis. Previous studies have established that a biologically active lipid mediator, sphingosine 1-phosphate (S1P), mimics some of the profibrotic functions of TGF-beta through cross-activation of Smad signaling. Here we report that another product of sphingosine kinase, dihydrosphingosine 1-phosphate (dhS1P), has an opposite role in the regulation of TGF-beta signaling. In contrast to S1P, dhS1P inhibits TGF-beta-induced Smad2/3 phosphorylation and up-regulation of collagen synthesis. The effects of dhS1P require a lipid phosphatase, PTEN, a key modulator of cell growth and survival. dhS1P stimulates phosphorylation of the C-terminal domain of PTEN and its subsequent translocation into the nucleus. We demonstrate a novel function of nuclear PTEN as a co-factor of the Smad2/3 phosphatase, PPM1A. Complex formation of PTEN with PPM1A does not require the lipid phosphatase activity but depends on phosphorylation of the serine/threonine residues located in the C-terminal domain of PTEN. Upon complex formation with PTEN, PPM1A is protected from degradation induced by the TGF-beta signaling. Consequently, overexpression of PTEN abrogates TGF-beta-induced Smad2/3 phosphorylation. This study establishes a novel role for nuclear PTEN in the stabilization of PPM1A. PTEN-mediated cross-talk between the sphingolipid and TGF-beta signaling pathways may play an important role in physiological and pathological TGF-beta signaling.

  19. RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice.

    PubMed

    Gutiérrez, Jaime; Droppelmann, Cristian A; Contreras, Osvaldo; Takahashi, Chiaki; Brandan, Enrique

    2015-01-01

    Fibroblasts are critical for wound contraction; a pivotal step in wound healing. They produce and modify the extracellular matrix (ECM) required for the proper tissue remodeling. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a key regulator of ECM homeostasis and turnover. However, its role in wound contraction is presently unknown. Here we describe that Transforming growth factor type β1 (TGF-β1), one of the main pro-fibrotic wound-healing promoting factors, decreases RECK expression in fibroblasts through the Smad and JNK dependent pathways. This TGF-β1 dependent downregulation of RECK occurs with the concomitant increase of β1-integrin, which is required for fibroblasts adhesion and wound contraction through the activation of focal adhesion kinase (FAK). Loss and gain RECK expression experiments performed in different types of fibroblasts indicate that RECK downregulation mediates TGF-β1 dependent β1-integrin expression. Also, reduced levels of RECK potentiate TGF-β1 effects over fibroblasts FAK-dependent contraction, without affecting its cognate signaling. The above results were confirmed on fibroblasts derived from the Reck+/- mice compared to wild type-derived fibroblasts. We observed that Reck+/- mice heal dermal wounds more efficiently than wild type mice. Our results reveal a critical role for RECK in skin wound contraction as a key mediator in the axis: TGF-β1-RECK-β1-integrin.

  20. PPARgamma is not a critical mediator of primary monocyte differentiation or foam cell formation.

    PubMed

    Patel, Lisa; Charlton, Steven J; Marshall, Ian C; Moore, Gary B T; Coxon, Phil; Moores, Kitty; Clapham, John C; Newman, Suzanna J; Smith, Stephen A; Macphee, Colin H

    2002-01-18

    In the present report we clarify the role of PPARgamma in differentiation and function of human-derived monocyte/macrophages in vitro. Rosiglitazone, a selective PPARgamma activator, had no effect on the kinetics of appearance of monocyte/macrophage differentiation markers or on cell size or granularity. Depletion of PPARgamma by more than 90% using antisense oligonucleotides did not influence accumulation of oxidized LDL or prevent the upregulation of CD36 that normally accompanies oxLDL treatment. In contrast, PPARgamma depletion reduced the expression of ABCA1 and LXRalpha mRNAs. Metalloproteinase-9 expression, a marker of atherosclerotic plaque vulnerability, was suppressed by rosiglitazone. We conclude that activation of PPARgamma does not affect monocyte/macrophage differentiation. In addition, PPARgamma is not absolutely required for oxLDL-driven lipid accumulation, but is required for full expression of ABCA1 and LXRalpha. Our data support a role for rosiglitazone as a potential directly acting antiatherosclerotic agent.

  1. Regular aerobic exercise reduces endothelin-1-mediated vasoconstrictor tone in overweight and obese adults.

    PubMed

    Dow, Caitlin A; Stauffer, Brian L; Brunjes, Danielle L; Greiner, Jared J; DeSouza, Christopher A

    2017-09-01

    What is the central question of this study? Does aerobic exercise training reduce endothelin-1 (ET-1)-mediated vasoconstrictor tone in overweight/obese adults? And, if so, does lower ET-1 vasoconstriction underlie the exercise-related enhancement in endothelium-dependent vasodilatation in overweight/obese adults? What is the main finding and its importance? Regular aerobic exercise reduces ET-1-mediated vasoconstrictor tone in previously sedentary overweight/obese adults, independent of weight loss. Decreased ET-1 vasoconstriction is an important mechanism underlying the aerobic exercise-induced improvement in endothelium-dependent vasodilator function in overweight/obese adults. Endothelin-1 (ET-1)-mediated vasoconstrictor tone is elevated in overweight and obese adults, contributing to vasomotor dysfunction and increased cardiovascular disease risk. Although the effects of habitual aerobic exercise on endothelium-dependent vasodilatation in overweight/obese adults have been studied, little is known regarding ET-1-mediated vasoconstriction. Accordingly, the aims of the present study were to determine the following: (i) whether regular aerobic exercise training reduces ET-1-mediated vasoconstrictor tone in overweight and obese adults; and, if so, (ii) whether the reduction in ET-1-mediated vasoconstriction contributes to exercise-induced improvement in endothelium-dependent vasodilatation in this population. Forearm blood flow (FBF) in response to intra-arterial infusion of selective ET A receptor blockade (BQ-123, 100 nmol min -1 for 60 min), acetylcholine [4.0, 8.0 and 16.0 μg (100 ml tissue) -1  min -1 ] in the absence and presence of ET A receptor blockade and sodium nitroprusside [1.0, 2.0 and 4.0 μg (100 ml tissue) -1  min -1 ] were determined before and after a 3 month aerobic exercise training intervention in 25 (16 men and nine women) overweight/obese (body mass index 30.1 ± 0.5 kg m -2 ) adults. The vasodilator response to BQ-123 was

  2. Independent of 5-HT1A receptors, neurons in the paraventricular hypothalamus mediate ACTH responses from MDMA

    PubMed Central

    Zaretsky, Dmitry V.; Zaretskaia, Maria V.; DiMicco, Joseph A.; Durant, Pamela J.; Ross, Christian T.; Rusyniak, Daniel E.

    2013-01-01

    Acute and chronic complications from the substituted amphetamine 3,4-methylenedioxymethamphetamine (MDMA) are linked to activation of the hypothalamic-pituitary-adrenal (HPA) axis. How MDMA activates the HPA axis is not known. HPA responses to stress are known to be mediated through the paraventricular (PVH) hypothalamus and to involve serotonin-1a (5-HT1A) receptors. We sought to determine if the PVH and 5-HT1A receptors were also involved in mediating HPA responses to MDMA. Rats were pretreated with either saline or a 5-HT1A antagonist, WAY-100635 (WAY), followed by a systemic dose of MDMA (7.5 mg/kg i.v.). Animals pretreated with WAY had significantly lower plasma ACTH concentrations after MDMA. To determine if neurons in the PVH were involved, and if their involvement was mediated by 5-HT1A receptors, rats implanted with guide cannulas targeting the PVH were microinjected with the GABAA receptor agonist muscimol, aCSF, or WAY followed by MDMA. Compared to aCSF microinjections of muscimol significantly attenuated the MDMA-induced rise in plasma ACTH (126 vs. 588 pg/ml, P=<0.01). WAY had no effect. Our data demonstrates that neurons in the PVH, independent of 5-HT1A receptors, mediate ACTH responses to MDMA. PMID:23933156

  3. 53BP1 promotes microhomology-mediated end-joining in G1-phase cells

    PubMed Central

    Xiong, Xiahui; Du, Zhanwen; Wang, Ying; Feng, Zhihui; Fan, Pan; Yan, Chunhong; Willers, Henning; Zhang, Junran

    2015-01-01

    Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair. PMID:25586219

  4. Resveratrol counteracts lipopolysaccharide-mediated microglial inflammation by modulating a SOCS-1 dependent signaling pathway.

    PubMed

    Dragone, Teresa; Cianciulli, Antonia; Calvello, Rosa; Porro, Chiara; Trotta, Teresa; Panaro, Maria Antonietta

    2014-09-01

    Brain damage or exposure to inflammatory agents provokes the activation of microglia and secretion of pro-inflammatory and neurotoxic mediators responsible for neuronal loss. Several lines of evidence show that resveratrol, a natural non-flavonoid polyphenol, may exert a neuroprotective action in neurodegenerative diseases. Suppressor of cytokine signaling (SOCS) proteins are a family of eight members expressed by immune cells and the central nervous system (CNS) cells, that regulate immune processes within the CNS, including microglia activation. We demonstrate that resveratrol had anti-inflammatory effects in murine N13 microglial cells stimulated with lipopolysaccharide (LPS), through up-regulating SOCS-1 expression. Interestingly, in SOCS-1-silenced cells resveratrol failed to play a protective role after LPS treatment. Our data demonstrate that resveratrol can impair microglia activation by activating a SOCS-1 mediated signaling pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep

    2010-04-25

    Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFalpha leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNAmore » Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.« less

  6. Human ABCB1 (P-glycoprotein) and ABCG2 mediate resistance to BI 2536, a potent and selective inhibitor of Polo-like kinase 1.

    PubMed

    Wu, Chung-Pu; Hsiao, Sung-Han; Sim, Hong-May; Luo, Shi-Yu; Tuo, Wei-Cherng; Cheng, Hsing-Wen; Li, Yan-Qing; Huang, Yang-Hui; Ambudkar, Suresh V

    2013-10-01

    The overexpression of the serine/threonine specific Polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. BI 2536 is the first selective inhibitor of Plk1 that inhibits cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to BI 2536 is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that overexpressing of either ABCB1 or ABCG2 is a novel mechanism of acquired resistance to BI 2536 in human cancer cells. Moreover, BI 2536 stimulates the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner, and inhibits the drug substrate transport mediated by these transporters. More significantly, the reduced chemosensitivity and BI 2536-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor or other chemotherapeutic agents that also interact with ABCB1 and ABCG2, such as tyrosine kinase inhibitors nilotinib and lapatinib. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to BI 2536, a combined regimen of BI 2536 and inhibitors or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Insulin-like growth factor 1: common mediator of multiple enterotrophic hormones and growth factors.

    PubMed

    Bortvedt, Sarah F; Lund, P Kay

    2012-03-01

    To summarize the recent evidence that insulin-like growth factor 1 (IGF1) mediates growth effects of multiple trophic factors and discuss clinical relevance. Recent reviews and original reports indicate benefits of growth hormone (GH) and long-acting glucagon-like peptide 2 (GLP2) analogs in short bowel syndrome and Crohn's disease. This review highlights the evidence that biomarkers of sustained small intestinal growth or mucosal healing and evaluation of intestinal epithelial stem cell biomarkers may improve clinical measures of intestinal growth or response to trophic hormones. Compelling evidence that IGF1 mediates growth effects of GH and GLP2 on intestine or linear growth in preclinical models of resection or Crohn's disease is presented, along with a concept that these hormones or IGF1 may enhance sustained growth if given early after bowel resection. Evidence that suppressor of cytokine signaling protein induction by GH or GLP2 in normal or inflamed intestine may limit IGF1-induced growth, but protect against risk of dysplasia or fibrosis, is reviewed. Whether IGF1 receptor mediates IGF1 action and potential roles of insulin receptors are addressed. IGF1 has a central role in mediating trophic hormone action in small intestine. Better understanding of benefits and risks of IGF1, receptors that mediate IGF1 action, and factors that limit undesirable growth are needed.

  8. APOL1 Oligomerization as the Key Mediator of Kidney Disease in African Americans

    DTIC Science & Technology

    2016-10-01

    AWARD NUMBER: W81XWH-14-1-0334 TITLE: APOL1 Oligomerization as the Key Mediator of Kidney Disease in African Americans PRINCIPAL INVESTIGATOR...SUBTITLE APOL1 Oligomerization as the Key Mediator of Kidney Disease in African Americans 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT...treating, kidney disease, in particular the APOL1- associated form of kidney disease that accounts for the high rate of kidney disease in African Americans

  9. APOL1 Oligomerization as the Key Mediator of Kidney Disease in African Americans

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBER: W81XWH-14-1-0334 TITLE: APOL1 Oligomerization as the Key Mediator of Kidney Disease in African Americans PRINCIPAL...29 Sep 2015 4. TITLE AND SUBTITLE APOL1 Oligomerization as the Key Mediator of Kidney Disease in African Americans 5a. CONTRACT NUMBER 5b. GRANT...work we are conducting is aimed at understanding, and eventually preventing and treating, kidney disease, in particular the APOL1- associated form of

  10. Identification of critical regions in human SAMHD1 required for nuclear localization and Vpx-mediated degradation.

    PubMed

    Guo, Haoran; Wei, Wei; Wei, Zhenhong; Liu, Xianjun; Evans, Sean L; Yang, Weiming; Wang, Hong; Guo, Ying; Zhao, Ke; Zhou, Jian-Ying; Yu, Xiao-Fang

    2013-01-01

    The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-β1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation.

  11. Palmatine suppresses glutamine-mediated interaction between pancreatic cancer and stellate cells through simultaneous inhibition of survivin and COL1A1

    PubMed Central

    Chakravarthy, Divya; Muñoz, Amanda R.; Su, Angel; Hwang, Rosa F.; Keppler, Brian R.; Chan, Daniel E.; Halff, Glenn; Ghosh, Rita; Kumar, Addanki P.

    2018-01-01

    Reciprocal interaction between pancreatic stellate cells (PSCs) and cancer cells (PCCs) in the tumor microenvironment (TME) promotes tumor cell survival and progression to lethal, therapeutically resistant pancreatic cancer. The goal of this study was to test the ability of Palmatine (PMT) to disrupt this reciprocal interaction in vitro and examine the underlying mechanism of interaction. We show that PSCs secrete glutamine into the extracellular environment under nutrient deprivation. PMT suppresses glutamine-mediated changes in GLI signaling in PCCs resulting in the inhibition of growth and migration while inducing apoptosis by inhibition of survivin. PMT-mediated inhibition of (glioma-associated oncogene 1) GLI activity in stellate cells leads to suppression (collagen type 1 alpha 1) COL1A1 activation. Remarkably, PMT potentiated gemcitabine’s growth inhibitory activity in PSCs, PCCs and inherently gemcitabine-resistant pancreatic cancer cells. This is the first study that shows the ability of PMT to inhibit growth of PSCs and PCCs either alone or in combination with gemcitabine. These studies warrant additional investigations using preclinical models to develop PMT as an agent for clinical management of pancreatic cancer. PMID:29414301

  12. SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes.

    PubMed

    Calapre, Leslie; Gray, Elin S; Kurdykowski, Sandrine; David, Anthony; Descargues, Pascal; Ziman, Mel

    2017-06-10

    Exposure to heat stress after UVB irradiation induces a reduction of apoptosis, resulting in survival of DNA damaged human keratinocytes. This heat-mediated evasion of apoptosis appears to be mediated by activation of SIRT1 and inactivation of p53 signalling. In this study, we assessed the role of SIRT1 in the inactivation of p53 signalling and impairment of DNA damage response in UVB plus heat exposed keratinocytes. Activation of SIRT1 after multiple UVB plus heat exposures resulted in increased p53 deacetylation at K382, which is known to affect its binding to specific target genes. Accordingly, we noted decreased apoptosis and down regulation of the p53 targeted pro-apoptotic gene BAX and the DNA repair genes ERCC1 and XPC after UVB plus heat treatments. In addition, UVB plus heat induced increased expression of the cell survival gene Survivin and the proliferation marker Ki67. Notably, keratinocytes exposed to UVB plus heat in the presence of the SIRT1 inhibitor, Ex-527, showed a similar phenotype to those exposed to UV alone; i.e. an increase in p53 acetylation, increased apoptosis and low levels of Survivin. This study demonstrate that heat-induced SIRT1 activation mediates survival of DNA damaged keratinocytes through deacetylation of p53 after exposure to UVB plus heat.

  13. Sirtuin 1 (SIRT1) activation mediates sildenafil induced delayed cardioprotection against ischemia-reperfusion injury in mice.

    PubMed

    Shalwala, Mona; Zhu, Shu-Guang; Das, Anindita; Salloum, Fadi N; Xi, Lei; Kukreja, Rakesh C

    2014-01-01

    It has been well documented that phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. SIRT1 is part of the class III Sirtuin family of histone deacetylases that deacetylates proteins involved in cellular stress response including those related to I-R injury. We tested the hypothesis that SIL-induced cardioprotection may be mediated through activation of SIRT1. Adult male ICR mice were treated with SIL (0.7 mg/kg, i.p.), Resveratrol (RSV, 5 mg/kg, a putative activator of SIRT1 used as the positive control), or saline (0.2 mL). The hearts were harvested 24 hours later and homogenized for SIRT1 activity analysis. Both SIL- and RSV-treated mice had increased cardiac SIRT1 activity (P<0.001) as compared to the saline-treated controls 24 hours after drug treatment. In isolated ventricular cardiomyocytes, pretreatment with SIL (1 µM) or RSV (1 µM) for one hour in vitro also upregulated SIRT1 activity (P<0.05). We further examined the causative relationship between SIRT1 activation and SIL-induced late cardioprotection. Pretreatment with SIL (or RSV) 24 hours prior to 30 min ischemia and 24 hours of reperfusion significantly reduced infarct size, which was associated with a significant increase in SIRT1 activity (P<0.05). Moreover, sirtinol (a SIRT1 inhibitor, 5 mg/kg, i.p.) given 30 min before I-R blunted the infarct-limiting effect of SIL and RSV (P<0.001). Our study shows that activation of SIRT1 following SIL treatment plays an essential role in mediating the SIL-induced cardioprotection against I-R injury. This newly identified SIRT1-activating property of SIL may have enormous therapeutic implications.

  14. PKA, novel PKC isoforms, and ERK is mediating PACAP auto-regulation via PAC1R in human neuroblastoma NB-1 cells.

    PubMed

    Georg, Birgitte; Falktoft, Birgitte; Fahrenkrug, Jan

    2016-12-01

    The neuropeptide PACAP is expressed throughout the central and peripheral nervous system where it modulates diverse physiological functions including neuropeptide gene expression. We here report that in human neuroblastoma NB-1 cells PACAP transiently induces its own expression. Maximal PACAP mRNA expression was found after stimulation with PACAP for 3h. PACAP auto-regulation was found to be mediated by activation of PACAP specific PAC 1 Rs as PACAP had >100-fold higher efficacy than VIP, and the PAC 1 R selective agonist Maxadilan potently induced PACAP gene expression. Experiments with pharmacological kinase inhibitors revealed that both PKA and novel but not conventional PKC isozymes were involved in the PACAP auto-regulation. Inhibition of MAPK/ERK kinase (MEK) also impeded the induction, and we found that PKA, novel PKC and ERK acted in parallel and were thus not part of the same pathways. The expression of the transcription factor EGR1 previously ascribed as target of PACAP signalling was found to be transiently induced by PACAP and pharmacological inhibition of either PKC or MEK1/2 abolished PACAP mediated EGR1 induction. In contrast, inhibition of PKA mediated increased PACAP mediated EGR1 induction. Experiments using siRNA against EGR1 to lower the expression did however not affect the PACAP auto-regulation indicating that this immediate early gene product is not part of PACAP auto-regulation in NB-1 cells. We here reveal that in NB-1 neuroblastoma cells, PACAP induces its own expression by activation of PAC 1 R, and that the signalling is different from the PAC 1 R signalling mediating induction of VIP in the same cells. PACAP auto-regulation depends on parallel activation of PKA, novel PKC isoforms, and ERK, while EGR1 does not seem to be part of the PACAP auto-regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Transient Receptor Potential Vanilloid 1 Expression Mediates Capsaicin-Induced Cell Death.

    PubMed

    Ramírez-Barrantes, Ricardo; Córdova, Claudio; Gatica, Sebastian; Rodriguez, Belén; Lozano, Carlo; Marchant, Ivanny; Echeverria, Cesar; Simon, Felipe; Olivero, Pablo

    2018-01-01

    The transient receptor potential (TRP) ion channel family consists of a broad variety of non-selective cation channels that integrate environmental physicochemical signals for dynamic homeostatic control. Involved in a variety of cellular physiological processes, TRP channels are fundamental to the control of the cell life cycle. TRP channels from the vanilloid (TRPV) family have been directly implicated in cell death. TRPV1 is activated by pain-inducing stimuli, including inflammatory endovanilloids and pungent exovanilloids, such as capsaicin (CAP). TRPV1 activation by high doses of CAP (>10 μM) leads to necrosis, but also exhibits apoptotic characteristics. However, CAP dose-response studies are lacking in order to determine whether CAP-induced cell death occurs preferentially via necrosis or apoptosis. In addition, it is not known whether cytosolic Ca 2+ and mitochondrial dysfunction participates in CAP-induced TRPV1-mediated cell death. By using TRPV1-transfected HeLa cells, we investigated the underlying mechanisms involved in CAP-induced TRPV1-mediated cell death, the dependence of CAP dose, and the participation of mitochondrial dysfunction and cytosolic Ca 2+ increase. Together, our results contribute to elucidate the pathophysiological steps that follow after TRPV1 stimulation with CAP. Low concentrations of CAP (1 μM) induce cell death by a mechanism involving a TRPV1-mediated rapid and transient intracellular Ca 2+ increase that stimulates plasma membrane depolarization, thereby compromising plasma membrane integrity and ultimately leading to cell death. Meanwhile, higher doses of CAP induce cell death via a TRPV1-independent mechanism, involving a slow and persistent intracellular Ca 2+ increase that induces mitochondrial dysfunction, plasma membrane depolarization, plasma membrane loss of integrity, and ultimately, cell death.

  16. Epithelial Cell TRPV1-Mediated Airway Sensitivity as a Mechanism for Respiratory Symptoms Associated with Gulf War Illness?

    DTIC Science & Technology

    2010-06-01

    TITLE: “Epithelial Cell TRPV1 -Mediated Airway Sensitivity as a Mechanism for Respiratory Symptoms Associated with Gulf War Illness” PRINCIPAL...66,),&$7,212) E7(/(3+21(180%(5 ,QFOXGHDUHDFRGH 01-06-2010 Annual Report 1 JUN 2009 - 31 MAY 2010 Epithelial Cell TRPV1 -Mediated Airway...express functional TRPV1 . More recently we found that these cells also express another important irritant receptor, namely TRPA1. Activation of

  17. Teaching resources. Model of the TIR1 pathway for auxin-mediated gene expression.

    PubMed

    Laskowski, Marta

    2006-02-14

    Auxin mediates numerous plant responses, some of which have been shown to require transcriptional regulation. One auxin response pathway, which depends on the relief of transcriptional repression, is mediated by TIR1 (transport inhibitor response protein 1). TIR1 is an auxin receptor and also a subunit of an SCF-type ubiquitin ligase. In the presence of a low concentration of auxin in the nucleus, members of the Aux/IAA family of transcriptional repressors bind to ARF proteins and inhibit the transcription of specific auxin response genes. Increased nuclear concentrations of auxin promote auxin binding to TIR1, causing the Aux/IAA proteins to associate with TIR1 and leading to their degradation by a proteasome-mediated pathway. This decreases the concentration of Aux/IAA proteins in the nucleus and thereby enables the expression of certain auxin response genes.

  18. TRPV1, but not TRPA1, in primary sensory neurons contributes to cutaneous incision-mediated hypersensitivity

    PubMed Central

    2013-01-01

    Background Mechanisms underlying postoperative pain remain poorly understood. In rodents, skin-only incisions induce mechanical and heat hypersensitivity similar to levels observed with skin plus deep incisions. Therefore, cutaneous injury might drive the majority of postoperative pain. TRPA1 and TRPV1 channels are known to mediate inflammatory and nerve injury pain, making them key targets for pain therapeutics. These channels are also expressed extensively in cutaneous nerve fibers. Therefore, we investigated whether TRPA1 and TRPV1 contribute to mechanical and heat hypersensitivity following skin-only surgical incision. Results Behavioral responses to mechanical and heat stimulation were compared between skin-incised and uninjured, sham control groups. Elevated mechanical responsiveness occurred 1 day post skin-incision regardless of genetic ablation or pharmacological inhibition of TRPA1. To determine whether functional changes in TRPA1 occur at the level of sensory neuron somata, we evaluated cytoplasmic calcium changes in sensory neurons isolated from ipsilateral lumbar 3–5 DRGs of skin-only incised and sham wild type (WT) mice during stimulation with the TRPA1 agonist cinnamaldehyde. There were no changes in the percentage of neurons responding to cinnamaldehyde or in their response amplitudes. Likewise, the subpopulation of DRG somata retrogradely labeled specifically from the incised region of the plantar hind paw showed no functional up-regulation of TRPA1 after skin-only incision. Next, we conducted behavior tests for heat sensitivity and found that heat hypersensitivity peaked at day 1 post skin-only incision. Skin incision-induced heat hypersensitivity was significantly decreased in TRPV1-deficient mice. In addition, we conducted calcium imaging with the TRPV1 agonist capsaicin. DRG neurons from WT mice exhibited sensitization to TRPV1 activation, as more neurons (66%) from skin-incised mice responded to capsaicin compared to controls (46%), and the

  19. Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis.

    PubMed

    Chekeni, Faraaz B; Elliott, Michael R; Sandilos, Joanna K; Walk, Scott F; Kinchen, Jason M; Lazarowski, Eduardo R; Armstrong, Allison J; Penuela, Silvia; Laird, Dale W; Salvesen, Guy S; Isakson, Brant E; Bayliss, Douglas A; Ravichandran, Kodi S

    2010-10-14

    Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the 'selective' plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

  20. The Mediator Complex Subunit PFT1 Is a Key Regulator of Jasmonate-Dependent Defense in Arabidopsis[C][W

    PubMed Central

    Kidd, Brendan N.; Edgar, Cameron I.; Kumar, Krish K.; Aitken, Elizabeth A.; Schenk, Peer M.; Manners, John M.; Kazan, Kemal

    2009-01-01

    Jasmonate signaling plays an important role in both plant defense and development. Here, we have identified a subunit of the Mediator complex as a regulator of the jasmonate signaling pathway in Arabidopsis thaliana. The Mediator complex is a conserved multiprotein complex that acts as a universal adaptor between transcription factors and the RNA polymerase II transcriptional machinery. We report that the PHYTOCHROME AND FLOWERING TIME1 (PFT1) gene, which encodes the MEDIATOR25 subunit of Mediator, is required for jasmonate-dependent defense gene expression and resistance to leaf-infecting necrotrophic fungal pathogens. Conversely, PFT1 appears to confer susceptibility to Fusarium oxysporum, a root-infecting hemibiotrophic fungal pathogen known to hijack jasmonate responses for disease development. Consistent with this, jasmonate gene expression was suppressed in the pft1 mutant during infection with F. oxysporum. In addition, a wheat (Triticum aestivum) homolog of PFT1 complemented the defense and the developmental phenotypes of the pft1 mutant, suggesting that the jasmonate signaling functions of PFT1 may be conserved in higher plants. Overall, our results identify an important control point in the regulation of the jasmonate signaling pathway within the transcriptional machinery. PMID:19671879

  1. Mediator Recruitment to Heat Shock Genes Requires Dual Hsf1 Activation Domains and Mediator Tail Subunits Med15 and Med16*

    PubMed Central

    Kim, Sunyoung; Gross, David S.

    2013-01-01

    The evolutionarily conserved Mediator complex is central to the regulation of gene transcription in eukaryotes because it serves as a physical and functional interface between upstream regulators and the Pol II transcriptional machinery. Nonetheless, its role appears to be context-dependent, and the detailed mechanism by which it governs the expression of most genes remains unknown. Here we investigate Mediator involvement in HSP (heat shock protein) gene regulation in the yeast Saccharomyces cerevisiae. We find that in response to thermal upshift, subunits representative of each of the four Mediator modules (Head, Middle, Tail, and Kinase) are rapidly, robustly, and selectively recruited to the promoter regions of HSP genes. Their residence is transient, returning to near-background levels within 90 min. Hsf1 (heat shock factor 1) plays a central role in recruiting Mediator, as indicated by the fact that truncation of either its N- or C-terminal activation domain significantly reduces Mediator occupancy, whereas removal of both activation domains abolishes it. Likewise, ablation of either of two Mediator Tail subunits, Med15 or Med16, reduces Mediator recruitment to HSP promoters, whereas deletion of both abolishes it. Accompanying the loss of Mediator, recruitment of RNA polymerase II is substantially diminished. Interestingly, Mediator antagonizes Hsf1 occupancy of non-induced promoters yet facilitates enhanced Hsf1 association with activated ones. Collectively, our observations indicate that Hsf1, via its dual activation domains, recruits holo-Mediator to HSP promoters in response to acute heat stress through cooperative physical and/or functional interactions with the Tail module. PMID:23447536

  2. The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy.

    PubMed

    Smilansky, Angela; Dangoor, Liron; Nakdimon, Itay; Ben-Hail, Danya; Mizrachi, Dario; Shoshan-Barmatz, Varda

    2015-12-25

    The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼ 2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. APOL1 Oligomerization as the Key Mediator of Kidney Disease in African Americans

    DTIC Science & Technology

    2016-10-01

    AWARD NUMBER: W81XWH-14-1-0333 TITLE: APOL1 Oligomerization as the Key Mediator of Kidney Disease in African Americans PRINCIPAL INVESTIGATOR...SUBTITLE APOL1 Oligomerization as the Key Mediator of Kidney Disease in African Americans 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER...treating, kidney disease, in particular the APOL1- associated form of kidney disease that accounts for the high rate of kidney disease in African Americans

  4. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yu, Teng, E-mail: tengyu33@yahoo.com; Ji, Jiang; Guo, Yong-li

    2013-11-08

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen speciesmore » (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.« less

  5. Pin1-FADD interactions regulate Fas-mediated apoptosis in activated eosinophils#

    PubMed Central

    Oh, Jiyoung; Malter, James S.

    2013-01-01

    Abnormally long-lived eosinophils (Eos) are the major inflammatory component of allergic responses in the lungs of active asthmatics. Eos recruited to the airways after allergen exposure produce and respond to IL-5 and GM-CSF, enhancing their survival. Pro-survival signaling activates Pin1, a cis-trans peptidyl isomerase (PPIase) that binds to Bax and prevents it activation. How long-lived Eos, despite the continued presence of GM-CSF or IL-5, eventually undergo apoptosis to end allergic inflammation remains unclear. Here we show that Pin1 location, activity and protein interactions are jointly influenced by Fas and pro-survival cytokine IL-5. Fas signaling strongly induced the phosphorylation of FADD at Ser194 and Pin1 at Ser16 as well as their nuclear accumulation. Phospho-mimic Ser194Glu FADD mutants accelerated Eos apoptosis compared to WT or Ser194Ala mutants. Downstream of FADD phosphorylation, Caspase 8, 9 and 3 cleavage as well as Eos apoptosis induced by Fas were reduced by constitutively active Pin1 and enhanced by Pin1 inhibition. Pin1 was activated by IL-5 while simultaneous IL-5 and anti-Fas treatment modestly reduced PPIase activity but induced Pin1 to associate with FADD after its phosphorylation at Ser194. Mechanistically, Pin1 mediated isomerization facilitated the subsequent dephosphorylation of Ser194 FADD and maintenance of cytoplasmic location. In vivo activated bronchoalvelolar (BAL) Eos obtained after allergen challenge showed elevated survival and Pin1 activity that could be reversed by anti-Fas. Therefore, our data suggest that Pin1 is a critical link between FADD mediated cell death and IL-5 mediated pro-survival signaling. PMID:23606538

  6. FYCO1 mediates clearance of α-synuclein aggregates through a Rab7-dependent mechanism.

    PubMed

    Saridaki, Theodora; Nippold, Markus; Dinter, Elisabeth; Roos, Andreas; Diederichs, Leonie; Fensky, Luisa; Schulz, Jörg B; Falkenburger, Björn H

    2018-05-10

    Parkinson disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. We have previously shown that overexpression of the small GTPase Rab7 can induce clearance of α-synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson disease we expressed the pathogenic A53T mutant of α-synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP). FYCO1-EGFP decorated vesicles containing α-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain α-synuclein. FYCO1 overexpression reduced the number of cells with α-synuclein aggregates, defined as visible particles of EGFP-tagged α-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α-synuclein protein as assayed by western blot, increased the disappearance of α-synuclein aggregates in time-lapse microscopy, and decreased α-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7 induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7 induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by the dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α-synuclein aggregates. Extracellular α-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-α-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates

  7. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Lipin-1 regulates Bnip3-mediated mitophagy in glycolytic muscle.

    PubMed

    Alshudukhi, Abdullah A; Zhu, Jing; Huang, Dengtong; Jama, Abdulrahman; Smith, Jeffrey D; Wang, Qing Jun; Esser, Karyn A; Ren, Hongmei

    2018-06-25

    Autophagy of mitochondria (mitophagy) is essential for maintaining muscle mass and healthy skeletal muscle. Patients with heritable phosphatidic acid phosphatase lipin-1-null mutations present with severe rhabdomyolysis and muscle atrophy in glycolytic muscle fibers, which are accompanied with mitochondrial aggregates and reduced mitochondrial cytochrome c oxidase activity. However, the underlying mechanisms leading to muscle atrophy as a result of lipin-1 deficiency are still not clear. In this study, we found that lipin-1 deficiency in mice is associated with a marked accumulation of abnormal mitochondria and autophagic vacuoles in glycolytic muscle fibers. Our studies using lipin-1-deficient myoblasts suggest that lipin-1 participates in B-cell leukemia (BCL)-2 adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3)-regulated mitophagy by interacting with microtubule-associated protein 1A/1B-light chain (LC)3, which is an important step in the recruitment of mitochondria to nascent autophagosomes. The requirement of lipin-1 for Bnip3-mediated mitophagy was further verified in vivo in lipin-1-deficient green fluorescent protein-LC3 transgenic mice (lipin-1 -/- -GFP-LC3). Finally, we showed that lipin-1 deficiency in mice resulted in defective mitochondrial adaptation to starvation-induced metabolic stress and impaired contractile muscle force in glycolytic muscle fibers. In summary, our study suggests that deregulated mitophagy arising from lipin-1 deficiency is associated with impaired muscle function and may contribute to muscle rhabdomyolysis in humans.-Alshudukhi, A. A., Zhu, J., Huang, D., Jama, A., Smith, J. D., Wang, Q. J., Esser, K. A., Ren, H. Lipin-1 regulates Bnip3-mediated mitophagy in glycolytic muscle.

  9. A Comprehensive Survey of Sequence Variation in the ABCA4 (ABCR) Gene in Stargardt Disease and Age-Related Macular Degeneration

    PubMed Central

    Rivera, Andrea; White, Karen; Stöhr, Heidi; Steiner, Klaus; Hemmrich, Nadine; Grimm, Timo; Jurklies, Bernhard; Lorenz, Birgit; Scholl, Hendrik P. N.; Apfelstedt-Sylla, Eckhart; Weber, Bernhard H. F.

    2000-01-01

    Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of ∼58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required. PMID:10958763

  10. A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration.

    PubMed

    Rivera, A; White, K; Stöhr, H; Steiner, K; Hemmrich, N; Grimm, T; Jurklies, B; Lorenz, B; Scholl, H P; Apfelstedt-Sylla, E; Weber, B H

    2000-10-01

    Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of approximately 58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required.

  11. Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum.

    PubMed

    Le, Phuong U; Guay, Ginette; Altschuler, Yoram; Nabi, Ivan R

    2002-02-01

    Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expression further inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in untransformed and transformed NIH-3T3 cells. Adenoviral expression of caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to levels observed in untransformed NIH-3T3 cells. Cholesterol-rich detergent-resistant membrane domains or glycolipid rafts therefore invaginate independently of caveolin-1 expression to form endocytosis-competent caveolar vesicles via rapid dynamin-dependent detachment from the plasma membrane. Caveolin-1 stabilizes the plasma membrane association of caveolae and thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum.

  12. TMC-1 mediates alkaline sensation in C. elegans through nociceptive neurons

    PubMed Central

    Wang, Xiang; Li, Guang; Liu, Jie; Liu, Jianfeng; Xu, X.Z. Shawn

    2016-01-01

    Noxious pH triggers pungent taste and nocifensive behavior. While the mechanisms underlying acidic pH sensation has been extensively characterized, little is known about how animals sense alkaline pH in the environment. TMC genes encode a family of evolutionarily conserved membrane proteins, whose functions are largely unknown. Here, we characterize C. elegans TMC-1 which was suggested to form a Na+-sensitive channel mediating salt chemosensation. Interestingly, we find that TMC-1 is required for worms to avoid noxious alkaline environment. Alkaline pH evokes an inward current in nociceptive neurons, which is primarily mediated by TMC-1 and to a lesser extent by the TRP channel OSM-9. However, unlike OSM-9 which is sensitive to both acidic and alkaline pH, TMC-1 is only required for alkali-activated current, revealing a specificity for alkaline sensation. Ectopic expression of TMC-1 confers alkaline sensitivity to alkali-insensitive cells. Our results identify an unexpected role for TMCs in alkaline sensation and nociception. PMID:27321925

  13. TRPA1 mediates the hypothermic action of acetaminophen

    PubMed Central

    Gentry, Clive; Andersson, David A.; Bevan, Stuart

    2015-01-01

    Acetaminophen (APAP) is an effective antipyretic and one of the most commonly used analgesic drugs. Unlike antipyretic non-steroidal anti-inflammatory drugs, APAP elicits hypothermia in addition to its antipyretic effect. Here we have examined the mechanisms responsible for the hypothermic activity of APAP. Subcutaneous, but not intrathecal, administration of APAP elicited a dose dependent decrease in body temperature in wildtype mice. Hypothermia was abolished in mice pre-treated with resiniferatoxin to destroy or defunctionalize peripheral TRPV1-expressing terminals, but resistant to inhibition of cyclo-oxygenases. The hypothermic activity was independent of TRPV1 since APAP evoked hypothermia was identical in wildtype and Trpv1−/− mice, and not reduced by administration of a maximally effective dose of a TRPV1 antagonist. In contrast, a TRPA1 antagonist inhibited APAP induced hypothermia and APAP was without effect on body temperature in Trpa1−/− mice. In a model of yeast induced pyrexia, administration of APAP evoked a marked hypothermia in wildtype and Trpv1−/− mice, but only restored normal body temperature in Trpa1−/− and Trpa1−/−/Trpv1−/− mice. We conclude that TRPA1 mediates APAP evoked hypothermia. PMID:26227887

  14. Variants in the ATP-Binding Cassette Transporter (ABCA7), Apolipoprotein E ε4, and the Risk of Late-Onset Alzheimer Disease in African Americans

    PubMed Central

    Reitz, Christiane; Jun, Gyungah; Naj, Adam; Rajbhandary, Ruchita; Vardarajan, Badri Narayan; Wang, Li-San; Valladares, Otto; Lin, Chiao-Feng; Larson, Eric B.; Graff-Radford, Neill R.; Evans, Denis; De Jager, Philip L.; Crane, Paul K.; Buxbaum, Joseph D.; Murrell, Jill R.; Raj, Towfique; Ertekin-Taner, Nilufer; Logue, Mark; Baldwin, Clinton T.; Green, Robert C.; Barnes, Lisa L.; Cantwell, Laura B.; Fallin, M. Daniele; Go, Rodney C. P.; Griffith, Patrick; Obisesan, Thomas O.; Manly, Jennifer J.; Lunetta, Kathryn L.; Kamboh, M. Ilyas; Lopez, Oscar L.; Bennett, David A.; Hendrie, Hugh; Hall, Kathleen S.; Goate, Alison M.; Byrd, Goldie S.; Kukull, Walter A.; Foroud, Tatiana M.; Haines, Jonathan L.; Farrer, Lindsay A.; Pericak-Vance, Margaret A.; Schellenberg, Gerard D.; Mayeux, Richard

    2013-01-01

    Importance Genetic variants associated with susceptibility to late-onset Alzheimer disease are known for individuals of European ancestry, but whether the same or different variants account for the genetic risk of Alzheimer disease in African American individuals is unknown. Identification of disease-associated variants helps identify targets for genetic testing, prevention, and treatment. Objective To identify genetic loci associated with late-onset Alzheimer disease in African Americans. Design, Setting, and Participants The Alzheimer Disease Genetics Consortium (ADGC) assembled multiple data sets representing a total of 5896 African Americans (1968 case participants, 3928 control participants) 60 years or older that were collected between 1989 and 2011 at multiple sites. The association of Alzheimer disease with genotyped and imputed single-nucleotide polymorphisms (SNPs) was assessed in case-control and in family-based data sets. Results from individual data sets were combined to perform an inverse variance–weighted meta-analysis, first with genome-wide analyses and subsequently with gene-based tests for previously reported loci. Main Outcomes and Measures Presence of Alzheimer disease according to standardized criteria. Results Genome-wide significance in fully adjusted models (sex, age, APOE genotype, population stratification) was observed for a SNP in ABCA7 (rs115550680, allele = G; frequency, 0.09 cases and 0.06 controls; odds ratio [OR], 1.79 [95% CI, 1.47-2.12]; P = 2.2 × 10–9), which is in linkage disequilibrium with SNPs previously associated with Alzheimer disease in Europeans (0.8ABCA7 was comparable with that of the APOE ε4–determining SNP rs429358 (allele = C; frequency, 0.30 cases and 0.18 controls; OR, 2.31 [95% CI, 2.19-2.42]; P = 5.5 × 10–47). Several loci previously associated with Alzheimer disease but not reaching significance in genome-wide analyses were replicated in gene-based analyses

  15. Variants in the ATP-binding cassette transporter (ABCA7), apolipoprotein E ϵ4,and the risk of late-onset Alzheimer disease in African Americans.

    PubMed

    Reitz, Christiane; Jun, Gyungah; Naj, Adam; Rajbhandary, Ruchita; Vardarajan, Badri Narayan; Wang, Li-San; Valladares, Otto; Lin, Chiao-Feng; Larson, Eric B; Graff-Radford, Neill R; Evans, Denis; De Jager, Philip L; Crane, Paul K; Buxbaum, Joseph D; Murrell, Jill R; Raj, Towfique; Ertekin-Taner, Nilufer; Logue, Mark; Baldwin, Clinton T; Green, Robert C; Barnes, Lisa L; Cantwell, Laura B; Fallin, M Daniele; Go, Rodney C P; Griffith, Patrick; Obisesan, Thomas O; Manly, Jennifer J; Lunetta, Kathryn L; Kamboh, M Ilyas; Lopez, Oscar L; Bennett, David A; Hendrie, Hugh; Hall, Kathleen S; Goate, Alison M; Byrd, Goldie S; Kukull, Walter A; Foroud, Tatiana M; Haines, Jonathan L; Farrer, Lindsay A; Pericak-Vance, Margaret A; Schellenberg, Gerard D; Mayeux, Richard

    2013-04-10

    Genetic variants associated with susceptibility to late-onset Alzheimer disease are known for individuals of European ancestry, but whether the same or different variants account for the genetic risk of Alzheimer disease in African American individuals is unknown. Identification of disease-associated variants helps identify targets for genetic testing, prevention, and treatment. To identify genetic loci associated with late-onset Alzheimer disease in African Americans. The Alzheimer Disease Genetics Consortium (ADGC) assembled multiple data sets representing a total of 5896 African Americans (1968 case participants, 3928 control participants) 60 years or older that were collected between 1989 and 2011 at multiple sites. The association of Alzheimer disease with genotyped and imputed single-nucleotide polymorphisms (SNPs) was assessed in case-control and in family-based data sets. Results from individual data sets were combined to perform an inverse variance-weighted meta-analysis, first with genome-wide analyses and subsequently with gene-based tests for previously reported loci. Presence of Alzheimer disease according to standardized criteria. Genome-wide significance in fully adjusted models (sex, age, APOE genotype, population stratification) was observed for a SNP in ABCA7 (rs115550680, allele = G; frequency, 0.09 cases and 0.06 controls; odds ratio [OR], 1.79 [95% CI, 1.47-2.12]; P = 2.2 × 10(-9)), which is in linkage disequilibrium with SNPs previously associated with Alzheimer disease in Europeans (0.8 < D' < 0.9). The effect size for the SNP in ABCA7 was comparable with that of the APOE ϵ4-determining SNP rs429358 (allele = C; frequency, 0.30 cases and 0.18 controls; OR, 2.31 [95% CI, 2.19-2.42]; P = 5.5 × 10(-47)). Several loci previously associated with Alzheimer disease but not reaching significance in genome-wide analyses were replicated in gene-based analyses accounting for linkage disequilibrium between markers and correcting for number of tests

  16. Assembly of high-density lipoprotein.

    PubMed

    Yokoyama, Shinji

    2006-01-01

    Mammalian somatic cells do not catabolize cholesterol and need to export it for its homeostasis at the levels of cells and whole bodies. This reaction may reduce intracellularly accumulated cholesterol in excess and would contribute to prevention or regression of the initial stage of atherosclerosis. High-density lipoprotein (HDL) is thought to play a main role in this reaction, and 2 independent mechanisms are proposed for this reaction. First, cholesterol is exchanged in a nonspecific physicochemical manner between cell surface and extracellular lipoproteins, and cholesterol esterification on HDL provides a driving force for net removal of cell cholesterol. Second, apolipoproteins directly interact with cells and generate HDL by removing cellular phospholipid and cholesterol. This reaction is a major source of plasma HDL and is mediated by a membrane protein, ABCA1. Lipid-free or lipid-poor helical apolipoproteins primarily recruit cellular phospholipid to assemble HDL particles, and cholesterol enrichment in these particles is regulated independently. ABCA1 is a rate-limiting factor of the HDL assembly and is regulated by transcriptional factors and posttranscriptional factors. Posttranscriptional regulation of ABCA1 includes modulation of its calpain-mediated degradation.

  17. A masked NES in INI1/hSNF5 mediates hCRM1-dependent nuclear export: implications for tumorigenesis

    PubMed Central

    Craig, Errol; Zhang, Zhi-Kai; Davies, Kelvin P.; Kalpana, Ganjam V.

    2002-01-01

    INI1 (integrase interactor 1)/hSNF5 is a component of the mammalian SWI/SNF complex and a tumor suppressor mutated in malignant rhabdoid tumors (MRT). We have identified a nuclear export signal (NES) in the highly conserved repeat 2 domain of INI1 that is unmasked upon deletion of a downstream sequence. Mutation of conserved hydrophobic residues within the NES, as well as leptomycin B treatment abrogated the nuclear export. Full-length INI1 specifically associated with hCRM1/exportin1 in vivo and in vitro. A mutant INI1 [INI1(1–319) delG950] found in MRT lacking the 66 C-terminal amino acids mislocalized to the cytoplasm. Full-length INI1 but not the INI1(1–319 delG950) mutant caused flat cell formation and cell cycle arrest in cell lines derived from MRT. Disruption of the NES in the delG950 mutant caused nuclear localization of the protein and restored its ability to cause cell cycle arrest. These observations demonstrate that INI1 has a masked NES that mediates regulated hCRM1/exportin1-dependent nuclear export and we propose that mutations that cause deregulated nuclear export of the protein could lead to tumorigenesis. PMID:11782423

  18. DNA-HMGB1 interaction: The nuclear aggregates of polyamine mediation.

    PubMed

    Iacomino, Giuseppe; Picariello, Gianluca; Sbrana, Francesca; Raiteri, Roberto; D'Agostino, Luciano

    2016-10-01

    Nuclear aggregates of polyamines (NAPs) are supramolecular compounds generated by the self-assembly of protonated nuclear polyamines (spermine, spermidine and putrescine) and phosphate ions. In the presence of genomic DNA, the hierarchical process of self-structuring ultimately produces nanotube-like polymers that envelop the double helix. Because of their modular nature and their aggregation-disaggregation dynamics, NAPs confer plasticity and flexibility to DNA. Through the disposition of charges, NAPs also enable a bidirectional stream of information between the genome and interacting moieties. High mobility group (HMG) B1 is a non-histone chromosomal protein that binds to DNA and that influences multiple nuclear processes. Because genomic DNA binds to either NAPs or HMGB1 protein, we explored the ability of in vitro self-assembled NAPs (ivNAPs) to mediate the DNA-HMGB1 interaction. To this end, we structured DNA-NAPs-HMGB1 and DNA-HMGB1-NAPs ternary complexes in vitro through opportune sequential incubations. Mobility shift electrophoresis and atomic force microscopy showed that the DNA-ivNAPs-HGMB1 complex had conformational assets supposedly more suitable those of the DNA-HGMB1-ivNAPs to comply with the physiological and functional requirements of DNA. Our findings indicated that ivNAPs act as mediators of the DNA-HMGB1 interaction. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Association between IgE-mediated allergies and diabetes mellitus type 1 in children and adolescents.

    PubMed

    Klamt, Sabine; Vogel, Mandy; Kapellen, Thomas M; Hiemisch, Andreas; Prenzel, Freerk; Zachariae, Silke; Ceglarek, Uta; Thiery, Joachim; Kiess, Wieland

    2015-11-01

    Type 1 diabetes mellitus (T1DM) is characterized by an immunological reaction that is dominated by type-1 T helper (Th1) cells, whereas immunoglobulin E (IgE)-mediated allergies are associated with Th2 cell. According to the Th1/Th2-hypothesis, the immune system is said to either develop into the direction of Th1 or Th2 cells. This would mean that a child developing T1DM is unlikely to develop an IgE-mediated allergy and vice versa. The aim of the study was to investigate the association between the prevalence of T1DM and IgE-mediated allergies. We designed a prospective case control study with 94 children and adolescents with T1DM and 188 age- and sex-matched control children. The basis of our investigations was a questionnaire concerning the family and children's history as to the presence of IgE-mediated allergies. Moreover, the following blood investigations were done: total serum IgE, specific IgE antibodies to major inhalant allergens, and a multiplex cytokine analysis measuring levels of specific cytokines representing either Th1- or Th2- cytokines. Children with T1DM reported the presence of IgE-mediated allergies significantly more often than children of the control group. Children with T1DM had significantly higher tumor necrosis factor alpha (TNFα) levels than healthy controls. Levels of interleukin-2 (IL-2) and IL-6 were higher in the groups of children with the presence of a personal history of allergies, regardless of the presence of T1DM. Our results suggest that T1DM is associated with a higher risk of a self-reported presence of IgE-mediated allergies and that the Th1/Th2-hypothesis may be an oversimplification. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Martin, Claire; Universite de Toulouse, UPS, IPBS, Toulouse F-31000; Lafosse, Jean-Michel

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  1. Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy.

    PubMed

    Chen, Cheng; Gong, Wenyan; Li, Changzheng; Xiong, Fengxiao; Wang, Shaogui; Huang, Junying; Wang, Yu; Chen, Zhiquan; Chen, Qiuhong; Liu, Peiqing; Lan, Tian; Huang, Heqing

    2017-10-03

    Activation of sphingosine kinase 1 (SphK1) signaling pathway mediates fibronectin (FN) upregulation in glomerular mesangial cells (GMCs) under high glucose (HG) condition. However, the roles of SphK1 in advanced glycation end products (AGEs)-induced DN have not been elucidated. Here we show that AGEs upregulated FN and SphK1 and SphK1 activity. Inhibition of SphK1 signaling attenuated AGEs-induced FN synthesis in GMCs. Inhibition of AGE receptor (RAGE) signaling reduced the upregulation of FN and SphK1 and SphK1 activity in GMCs induced by AGEs. Treatment of aminoguanidine ameliorates the renal injury and fibrosis in STZ-induced diabetic mice and attenuated SphK1 expression and activity in diabetic mouse kidneys. The renal injury and fibrosis in diabetic SphK1 -/- mice was significantly attenuated than WT mice. Furthermore, AGEs upregulated SphK1 by reducing its degradation and prolonging its half-life. SphK1 mediates AGEs-induced FN synthesis in GMCs and diabetic mice under hyperglycemic condition .

  2. Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy

    PubMed Central

    Chen, Cheng; Gong, Wenyan; Li, Changzheng; Xiong, Fengxiao; Wang, Shaogui; Huang, Junying; Wang, Yu; Chen, Zhiquan; Chen, Qiuhong; Liu, Peiqing; Lan, Tian; Huang, Heqing

    2017-01-01

    Activation of sphingosine kinase 1 (SphK1) signaling pathway mediates fibronectin (FN) upregulation in glomerular mesangial cells (GMCs) under high glucose (HG) condition. However, the roles of SphK1 in advanced glycation end products (AGEs)-induced DN have not been elucidated. Here we show that AGEs upregulated FN and SphK1 and SphK1 activity. Inhibition of SphK1 signaling attenuated AGEs-induced FN synthesis in GMCs. Inhibition of AGE receptor (RAGE) signaling reduced the upregulation of FN and SphK1 and SphK1 activity in GMCs induced by AGEs. Treatment of aminoguanidine ameliorates the renal injury and fibrosis in STZ-induced diabetic mice and attenuated SphK1 expression and activity in diabetic mouse kidneys. The renal injury and fibrosis in diabetic SphK1-/- mice was significantly attenuated than WT mice. Furthermore, AGEs upregulated SphK1 by reducing its degradation and prolonging its half-life. Conclusion: SphK1 mediates AGEs-induced FN synthesis in GMCs and diabetic mice under hyperglycemic condition. PMID:29108256

  3. Opposite Effects of Dihydrosphingosine 1-Phosphate and Sphingosine 1-Phosphate on Transforming Growth Factor-β/Smad Signaling Are Mediated through the PTEN/PPM1A-dependent Pathway*S⃞

    PubMed Central

    Bu, Shizhong; Kapanadze, Bagrat; Hsu, Tien; Trojanowska, Maria

    2008-01-01

    Transforming growth factor-β (TGF-β) is an important regulator of physiological connective tissue biosynthesis and plays a central role in pathological tissue fibrosis. Previous studies have established that a biologically active lipid mediator, sphingosine 1-phosphate (S1P), mimics some of the profibrotic functions of TGF-β through cross-activation of Smad signaling. Here we report that another product of sphingosine kinase, dihydrosphingosine 1-phosphate (dhS1P), has an opposite role in the regulation of TGF-β signaling. In contrast to S1P, dhS1P inhibits TGF-β-induced Smad2/3 phosphorylation and up-regulation of collagen synthesis. The effects of dhS1P require a lipid phosphatase, PTEN, a key modulator of cell growth and survival. dhS1P stimulates phosphorylation of the C-terminal domain of PTEN and its subsequent translocation into the nucleus. We demonstrate a novel function of nuclear PTEN as a co-factor of the Smad2/3 phosphatase, PPM1A. Complex formation of PTEN with PPM1A does not require the lipid phosphatase activity but depends on phosphorylation of the serine/threonine residues located in the C-terminal domain of PTEN. Upon complex formation with PTEN, PPM1A is protected from degradation induced by the TGF-β signaling. Consequently, overexpression of PTEN abrogates TGF-β-induced Smad2/3 phosphorylation. This study establishes a novel role for nuclear PTEN in the stabilization of PPM1A. PTEN-mediated cross-talk between the sphingolipid and TGF-β signaling pathways may play an important role in physiological and pathological TGF-β signaling. PMID:18482992

  4. Phytochrome-mediated agravitropism in Arabidopsis hypocotyls requires GIL1 and confers a fitness advantage.

    PubMed

    Allen, Trudie; Ingles, Patricia J; Praekelt, Uta; Smith, Harry; Whitelam, Garry C

    2006-05-01

    Plants use specialized photoreceptors to detect the amount, quality, periodicity and direction of light and to modulate their growth and development accordingly. These regulatory light signals often interact with other environmental cues. Exposure of etiolated Arabidopsis seedlings to red (R) or far-red (FR) light causes hypocotyls to grow in random orientations with respect to the gravitational vector, thus overcoming the signal from gravity to grow upwards. This light response, mediated by either phytochrome A or phytochrome B, represents a prime example of cross-talk between environmental signalling systems. Here, we report the isolation the mutant gil1 (for gravitropic in the light) in which hypocotyls continue to grow upwards after exposure of seedlings to R or FR light. The gil1 mutant displays no other phenotypic alterations in response to gravity or light. Cloning of GIL1 has identified a novel gene that is necessary for light-dependent randomization of hypocotyl growth orientation. Using gil1, we have demonstrated that phytochrome-mediated randomization of Arabidopsis hypocotyl orientation provides a fitness advantage to seedlings developing in patchy, low-light environments.

  5. von Hippel–Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

    PubMed Central

    Mousnier, Aurélie; Kubat, Nicole; Massias-Simon, Aurélie; Ségéral, Emmanuel; Rain, Jean-Christophe; Benarous, Richard; Emiliani, Stéphane; Dargemont, Catherine

    2007-01-01

    HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin–proteasome pathway. Here, we identify von Hippel–Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel–Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin–VHL–proteasome pathway in the integration–transcription transition of the viral replication cycle. PMID:17698809

  6. Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.

    PubMed

    Khomane, Kailas S; Nandekar, Prajwal P; Wahlang, Banrida; Bagul, Pravin; Shaikh, Naeem; Pawar, Yogesh B; Meena, Chhuttan Lal; Sangamwar, Abhay T; Jain, Rahul; Tikoo, K; Bansal, Arvind K

    2012-09-04

    The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 μM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.

  7. Osimertinib (AZD9291), a Mutant-Selective EGFR Inhibitor, Reverses ABCB1-Mediated Drug Resistance in Cancer Cells.

    PubMed

    Zhang, Xiao-Yu; Zhang, Yun-Kai; Wang, Yi-Jun; Gupta, Pranav; Zeng, Leli; Xu, Megan; Wang, Xiu-Qi; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-09-15

    In recent years, tyrosine kinase inhibitors (TKIs) have been shown capable of inhibiting the ATP-binding cassette (ABC) transporter-mediated multidrug resistance (MDR). In this study, we determine whether osimertinib, a novel selective, irreversible EGFR (epidermal growth factor receptor) TKI, could reverse ABC transporter-mediated MDR. The results showed that, at non-toxic concentrations, osimertinib significantly sensitized both ABCB1-transfected and drug-selected cell lines to substrate anticancer drugs colchicine, paclitaxel, and vincristine. Osimertinib significantly increased the accumulation of [³H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant alteration in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 0.3 µM osimertinib for 72 h. In addition, ATPase assay showed osimertinib stimulated ABCB1 ATPase activity. Molecular docking and molecular dynamic simulations showed osimertinib has strong and stable interactions at the transmembrane domain of human homology ABCB1. Taken together, our findings suggest that osimertinib, a clinically-approved third-generation EGFR TKI, can reverse ABCB1-mediated MDR, which supports the combination therapy with osimertinib and ABCB1 substrates may potentially be a novel therapeutic stategy in ABCB1-positive drug resistant cancers.

  8. Insulin-like growth factor-1 is a mediator of age-related decline of bone health status in men.

    PubMed

    Chin, Kok-Yong; Ima-Nirwana, Soelaiman; Mohamed, Isa Naina; Hanapi Johari, Mohamad; Ahmad, Fairus; Mohamed Ramli, Elvy Suhana; Wan Ngah, Wan Zurinah

    2014-06-01

    The role of insulin-like growth factor-1 (IGF-1) in bone health in men is debatable. This study aimed to determine whether IGF-1 is a mediator in age-related decline of bone health status measured by calcaneal speed of sound (SOS) in Malaysian men. The study recruited 279 Chinese and Malay men. Their demographic data, weight, height, calcaneal SOS were taken and fasting blood was collected for total testosterone, sex-hormone binding globulin and IGF-1 assays. The associations between the studied variables were assessed using multiple linear regression (MLR) analysis. Mediator analysis was performed using Sobel test. There was a significant and parallel decrease of IGF-1 and SOS with age (p < 0.05). Serum IGF-1 was significantly and positively associated with SOS (p < 0.05) but after further adjustment for age, the significance was lost (p > 0.05). The strength of the association between age and SOS decreased after adjusting for IGF-1 level but it remained significant (p < 0.05). Sobel test revealed that IGF-1 was a significant partial mediator in the relationship between age and SOS (z = -4.3). Serum IGF-1 is a partial mediator in the age-related decline of bone health in men as determined by calcaneal ultrasound. A prospective study should be performed to validate this relationship.

  9. The transcriptional regulator BZR1 mediates trade-off between plant innate immunity and growth.

    PubMed

    Lozano-Durán, Rosa; Macho, Alberto P; Boutrot, Freddy; Segonzac, Cécile; Somssich, Imre E; Zipfel, Cyril

    2013-12-31

    The molecular mechanisms underlying the trade-off between plant innate immunity and steroid-mediated growth are controversial. Here, we report that activation of the transcription factor BZR1 is required and sufficient for suppression of immune signaling by brassinosteroids (BR). BZR1 induces the expression of several WRKY transcription factors that negatively control early immune responses. In addition, BZR1 associates with WRKY40 to mediate the antagonism between BR and immune signaling. We reveal that BZR1-mediated inhibition of immunity is particularly relevant when plant fast growth is required, such as during etiolation. Thus, BZR1 acts as an important regulator mediating the trade-off between growth and immunity upon integration of environmental cues. DOI: http://dx.doi.org/10.7554/eLife.00983.001.

  10. PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1.

    PubMed

    Iida, Satoshi; Chen, Wei; Nakadai, Tomoyoshi; Ohkuma, Yoshiaki; Roeder, Robert G

    2015-02-01

    PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex. © 2015 Iida et al.; Published by Cold Spring Harbor Laboratory Press.

  11. CgA1AR-1 acts as an alpha-1 adrenergic receptor in oyster Crassostrea gigas mediating both cellular and humoral immune response.

    PubMed

    Liu, Zhaoqun; Zhou, Zhi; Wang, Lingling; Qiu, Limei; Zhang, Huan; Wang, Hao; Song, Linsheng

    2016-11-01

    We have now cloned an alpha-1 adrenergic receptor (A1AR) from the cDNA library of oyster Crassostrea gigas, designating as CgA1AR-1. The full length of CgA1AR-1 was 1149 bp and it encodes a protein of 382 amino acids containing a 7 transmembrane domain, whose putative topology was similar to the A1ARs in higher organisms and shared similarity of 19% with mammalian A1ARs according to the phylogenic analysis. After cell transfection of CgA1AR-1 into HEK293T cells and the incubation with its specific agonist norepinephrine (NE), the concentration of second messenger Ca 2+ increased significantly (p < 0.05). But, this increasing of Ca 2+ could be inhibited by adding A1AR antagonist DOX. Tissue distribution assays using qRT-PCR suggested that CgA1AR-1 mRNA was ubiquitously expressed in all the major tissues of oyster. LPS stimulation could induce the up-regulation of CgA1AR-1 mRNA in haemocytes from 12 h to 24 h post stimulation. Moreover, the blocking of CgA1AR-1 by DOX before LPS stimulation affected the mRNA expression of oyster TNF (CGI_10005109 and CGI_10006440) in haemocytes, resulting in the rise of haemocyte phagocytic rate and apoptosis index. In addition to cellular immunity, CgA1AR-1 was also involved in humoral immunity of oyster. Inhibition of CgA1AR-1 with DOX could repress the up-regulation of LZY and SOD activities caused by LPS stimulation. These results suggested that CgA1AR-1 acted as an α-1 adrenergic receptor in cetacholaminergic neuroendocrine-immune network mediating both cellular and humoral immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Qiliqiangxin Rescues Mouse Cardiac Function by Regulating AGTR1/TRPV1-Mediated Autophagy in STZ-Induced Diabetes Mellitus.

    PubMed

    Tong, Jing; Lai, Yan; Yao, Yi-An; Wang, Xue-Jun; Shi, Yu-Shuang; Hou, Han-Jin; Gu, Jian-Yun; Chen, Fei; Liu, Xue-Bo

    2018-06-19

    To explore the potential role of qiliqiangxin (QLQX) A traditional Chinese medicine and the involvement of angiotensin II receptor type 1 (AGTR1) and transient receptor potential vanilloid 1 (TRPV1) in diabetic mouse cardiac function. Intragastric QLQX was administered for 5 weeks after streptozotocin (STZ) treatment. Additionally, Intraperitoneal injections of angiotensin II (Ang II) or intragastric losartan (Los) were administered to assess the activities of AGTR1 and TRPV1. Two-dimensional echocardiography and tissue histopathology were used to assess cardiac function Western blot was used to detect the autophagic biomarkers Such as light chain 3 P62 and lysosomal-associated membrane protein 2 And transmission electron microscopy was used to count the number of autophagosomes. Decreased expression of TRPV1 and autophagic hallmarks and reduced numbers of autophagolysosomes as well as increased expression of angiotensin converting enzyme 1 and AGTR1 were observed in diabetic hearts. Blocking AGTR1 with Los mimicked the QLQX-mediated improvements in cardiac function Alleviated myocardial fibrosis and enabled autophagy Whereas Ang II abolished the beneficial effects of QLQX in wild type diabetic mice but not in TRPV1-/- diabetic mice. QLQX may improve diabetic cardiac function by regulating AGTR1/ TRPV1-mediated autophagy in STZ-induced diabetic mice. © 2018 The Author(s). Published by S. Karger AG, Basel.

  13. Copper regulates primary root elongation through PIN1-mediated auxin redistribution.

    PubMed

    Yuan, Hong-Mei; Xu, Heng-Hao; Liu, Wen-Cheng; Lu, Ying-Tang

    2013-05-01

    The heavy metal copper (Cu) is an essential microelement required for normal plant growth and development, but it inhibits primary root growth when in excess. The mechanism underlying how excess Cu functions in this process remains to be further elucidated. Here, we report that a higher concentration of CuSO4 inhibited primary root elongation of Arabidopsis seedlings by affecting both the elongation and meristem zones. In the meristem zone, meristematic cell division potential was reduced by excess Cu. Further experiments showed that Cu can modulate auxin distribution, resulting in higher auxin activities in both the elongation and meristem zones of Cu-treated roots based on DR5::GUS expression patterns. This Cu-mediated auxin redistribution was shown to be responsible for Cu-mediated inhibition of primary root elongation. Additional genetic and physiological data demonstrated that it was PINFORMED1 (PIN1), but not PIN2 or AUXIN1 (AUX1), that regulated this process. However, Cu-induced hydrogen peroxide accumulation did not contribute to Cu-induced auxin redistribution for inhibition of root elongation. When the possible role of ethylene in this process was analyzed, Cu had a similar impact on the root elongation of both the wild type and the ein2-1 mutant, implying that Cu-mediated inhibition of primary root elongation was not due to the ethylene signaling pathway.

  14. Protective effect of A/H1N1 vaccination in immune-mediated disease--a prospectively controlled vaccination study.

    PubMed

    Adler, Sabine; Krivine, Anne; Weix, Janine; Rozenberg, Flore; Launay, Odile; Huesler, Juerg; Guillevin, Loïc; Villiger, Peter M

    2012-04-01

    To assess the 2009 influenza vaccine A/H1N1 on antibody response, side effects and disease activity in patients with immune-mediated diseases. Patients with RA, SpA, vasculitis (VAS) or CTD (n = 149) and healthy individuals (n = 40) received a single dose of adjuvanted A/H1N1 influenza vaccine. Sera were obtained before vaccination, and 3 weeks, 6 weeks and 6 months thereafter. A/H1N1 antibody titres were measured by haemagglutination inhibition (HAI) assay. Seroprotection was defined as specific antibody titre ≥ 1 : 40, seroconversion as 4-fold increase in antibody titre. Titres increased significantly in patients and controls with a maximum at Week 3, declining to levels below protection at Month 6 (P < 0.001). Seroprotection was more frequently reached in SpA and CTD than in RA and VAS (80 and 82% and 57 and 47%, respectively). There was a significantly negative impact by MTX (P < 0.001), rituximab (P = 0.0031) and abatacept (P = 0.045). Other DMARDs, glucocorticoids and TNF blockers did not significantly suppress response (P = 0.06, 0.11 and 0.81, respectively). A linear decline in response was noted in patients with increasing age (P < 0.001). Disease reactivation possibly related to vaccination was suspected in 8/149 patients. No prolonged side effects or A/H1N1 infections were noted. The results show that vaccination response is a function of disease type, intensity and character of medication and age. A single injection of adjuvanted influenza vaccine is sufficient to protect a high percentage of patients. Therefore, differential vaccination recommendations might in the future reduce costs and increase vaccination acceptance.

  15. Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis.

    PubMed

    Han, Jie; Goldstein, Leslie A; Gastman, Brian R; Rabinowich, Hannah

    2006-04-14

    The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1. Direct degradation of Mcl-1 by TRAIL-activated caspase-8 or caspase-3 produces Mcl-1-free Bim that mediates a Bax-dependent apoptotic cascade. Using Mcl-1 or Bim RNAi, we demonstrate that a loss in Mcl-1 expression significantly enhances the mitochondrial apoptotic response to TRAIL that is now mediated by freed Bim. Whereas overexpression of Mcl-1 contributes to the preservation of the mitochondrial membrane potential, Mcl-1 knockdown facilitates the Bim-mediated dissipation of this potential. Loss of Mcl-1 contributes to an increased level of caspase activity downstream of the mitochondrial response to TRAIL. Furthermore, the Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome c, Smac, and HtrA2 in response to Bim. These are the first findings to demonstrate the involvement of Bim in the TRAIL-mediated mitochondrial cascade. They also suggest that Mcl-1 may serve as a direct substrate for TRAIL-activated caspases implying the existence of a novel TRAIL/caspase-8/Mcl-1/Bim communication mechanism between the extrinsic and the intrinsic apoptotic pathways.

  16. γ-Oryzanol suppresses COX-2 expression by inhibiting reactive oxygen species-mediated Erk1/2 and Egr-1 signaling in LPS-stimulated RAW264.7 macrophages.

    PubMed

    Shin, Soon Young; Kim, Heon-Woong; Jang, Hwan-Hee; Hwang, Yu-Jin; Choe, Jeong-Sook; Kim, Jung-Bong; Lim, Yoongho; Lee, Young Han

    2017-09-16

    Cyclooxygenase (COX)-2 produces prostanoids, which contribute to inflammatory responses. Nuclear factor (NF)-κB is a key transcription factor mediating COX-2 expression. γ-Oryzanol is an active component in rice bran oil, which inhibits lipopolysaccharide (LPS)-mediated COX-2 expression by inhibiting NF-κB. However, the inhibition of COX-2 expression by γ-oryzanol independently of NF-κB is poorly understood. We found that LPS upregulated Egr-1 expression at the transcriptional level. Forced expression of Egr-1 trans-activated the Cox-2 promoter independently of NF-κB. In contrast, silencing of Egr-1 abrogated LPS-mediated COX-2 expression. LPS produced reactive oxygen species (ROS), which, in turn, induced Egr-1 expression via the Erk1/2 MAPK pathway. ROS scavenging activity of γ-oryzanol suppressed Egr-1 expression by inhibiting the Erk1/2 MAPK pathway. Our results suggest that γ-oryzanol inhibits LPS-mediated COX-2 expression by suppressing Erk1/2-mediated Egr-1 expression. This study supports that γ-oryzanol may be useful for ameliorating LPS-mediated inflammatory responses. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis

    PubMed Central

    Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

    2016-01-01

    Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax–at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax–HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax−CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1– cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax–CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus. PMID:27105228

  18. Deficiency of activated STAT1 in head and neck cancer cells mediates TAP1-dependent escape from cytotoxic T lymphocytes

    PubMed Central

    Leibowitz, Michael S.; Filho, Pedro A. Andrade; Ferrone, Soldano; Ferris, Robert L.

    2012-01-01

    Squamous cell carcinoma of the head and neck (SCCHN) cells can escape recognition by tumor antigen (TA)-specific cytotoxic T lymphocytes (CTL) by downregulation of antigen processing machinery (APM) components, such as the transporter associated with antigen processing (TAP)-1/2 heterodimer. APM component upregulation by interferon gamma (IFN-γ) restores SCCHN cell recognition and susceptibility to lysis by CTL, but the mechanism underlying TAP1/2 downregulation in SCCHN cells is not known. Because IFN-γ activates signal transducer and activator of transcription (STAT)-1, we investigated phosphorylated (p)-STAT1 as a mediator of low basal TAP1/2 expression in SCCHN cells. SCCHN cells were found to express basal total STAT1 but low to undetectable levels of activated STAT1. The association of increased pSTAT1 levels and APM components likely reflects a cause–effect relationship, since STAT1 knockdown significantly reduced both IFN-γ-mediated APM component expression and TA-specific CTL recognition of IFN-γ-treated SCCHN cells. On the other hand, since oncogenic pSTAT3 is overexpressed in SCCHN cells and was found to heterodimerize with pSTAT1, we also tested whether pSTAT3 and pSTAT1:pSTAT3 heterodimers inhibited IFN-γ-induced STAT1 activation and APM component expression. First, STAT3 activation or depletion did not affect basal or IFN-γ-induced expression of pSTAT1 and APM components or recognition of SCCHN cells by TA-specific CTL. Second, pSTAT1:pSTAT3 heterodimers did not interfere with IFN-γ-induced STAT1 binding to the TAP1 promoter or APM protein expression. These findings demonstrate that APM component downregulation is regulated primarily by an IFN-γ-pSTAT1-mediated signaling pathway, independent of oncogenic STAT3 overexpression in SCCHN cells. PMID:21207025

  19. Regulated nuclear trafficking of rpL10A mediated by NIK1 represents a defense strategy of plant cells against virus.

    PubMed

    Carvalho, Claudine M; Santos, Anésia A; Pires, Silvana R; Rocha, Carolina S; Saraiva, Daniela I; Machado, João Paulo B; Mattos, Eliciane C; Fietto, Luciano G; Fontes, Elizabeth P B

    2008-12-01

    The NSP-interacting kinase (NIK) receptor-mediated defense pathway has been identified recently as a virulence target of the geminivirus nuclear shuttle protein (NSP). However, the NIK1-NSP interaction does not fit into the elicitor-receptor model of resistance, and hence the molecular mechanism that links this antiviral response to receptor activation remains obscure. Here, we identified a ribosomal protein, rpL10A, as a specific partner and substrate of NIK1 that functions as an immediate downstream effector of NIK1-mediated response. Phosphorylation of cytosolic rpL10A by NIK1 redirects the protein to the nucleus where it may act to modulate viral infection. While ectopic expression of normal NIK1 or a hyperactive NIK1 mutant promotes the accumulation of phosphorylated rpL10A within the nuclei, an inactive NIK1 mutant fails to redirect the protein to the nuclei of co-transfected cells. Likewise, a mutant rpL10A defective for NIK1 phosphorylation is not redirected to the nucleus. Furthermore, loss of rpL10A function enhances susceptibility to geminivirus infection, resembling the phenotype of nik1 null alleles. We also provide evidence that geminivirus infection directly interferes with NIK1-mediated nuclear relocalization of rpL10A as a counterdefensive measure. However, the NIK1-mediated defense signaling neither activates RNA silencing nor promotes a hypersensitive response but inhibits plant growth and development. Although the virulence function of the particular geminivirus NSP studied here overcomes this layer of defense in Arabidopsis, the NIK1-mediated signaling response may be involved in restricting the host range of other viruses.

  20. Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

    PubMed Central

    Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

    2011-01-01

    Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

  1. Yeast Pah1p Phosphatidate Phosphatase Is Regulated by Proteasome-mediated Degradation*

    PubMed Central

    Pascual, Florencia; Hsieh, Lu-Sheng; Soto-Cardalda, Aníbal; Carman, George M.

    2014-01-01

    Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation. PMID:24563465

  2. Novel Ambler class A beta-lactamase LAP-1 and its association with the plasmid-mediated quinolone resistance determinant QnrS1.

    PubMed

    Poirel, Laurent; Cattoir, Vincent; Soares, Ana; Soussy, Claude-James; Nordmann, Patrice

    2007-02-01

    The plasmid-mediated quinolone resistance determinant QnrS1 was identified in non-clonally related Enterobacter cloacae isolates in association with a transferable narrow-spectrum beta-lactam resistance marker. Cloning experiments allowed the identification of a novel Ambler class A beta-lactamase, named LAP-1. It shares 62 and 61% amino acid identity with the most closely related beta-lactamases, TEM-1 and SHV-1, respectively. It has a narrow-spectrum hydrolysis of beta-lactams and is strongly inhibited by clavulanic acid and sulbactam and, to a lesser extent, by tazobactam. Association of the blaLAP-1 gene with the qnrS1 gene was identified in E. cloacae isolates from France and Vietnam. These genes were plasmid located and associated with similar insertion sequences but were not associated with sul1-type class 1 integrons, as opposed to the qnrA genes.

  3. Maresin 1, a Proresolving Lipid Mediator, Mitigates Carbon Tetrachloride-Induced Liver Injury in Mice

    PubMed Central

    Li, Ruidong; Wang, Yaxin; Zhao, Ende; Wu, Ke; Li, Wei; Shi, Liang; Wang, Di; Xie, Gengchen; Yin, Yuping; Deng, Meizhou; Zhang, Peng; Tao, Kaixiong

    2016-01-01

    Maresin 1 (MaR 1) was recently reported to have protective properties in several different animal models of acute inflammation by inhibiting inflammatory response. However, its function in acute liver injury is still unknown. To address this question, we induced liver injury in BALB/c mice with intraperitoneal injection of carbon tetrachloride with or without treatment of MaR 1. Our data showed that MaR 1 attenuated hepatic injury, oxidative stress, and lipid peroxidation induced by carbon tetrachloride, as evidenced by increased thiobarbituric acid reactive substances and reactive oxygen species levels were inhibited by treatment of MaR 1. Furthermore, MaR 1 increased activities of antioxidative mediators in carbon tetrachloride-treated mice liver. MaR 1 decreased indices of inflammatory mediators such as tumor necrosis factor-α, interleukin-6, interleukin-1β, monocyte chemotactic protein 1, myeloperoxidase, cyclooxygenase-2, and inducible nitric oxide synthase. Administration of MaR 1 inhibited activation of nuclear factor kappa B (NF-κb) and mitogen-activated protein kinases (MAPKs) in the liver of CCl4 treated mice. In conclusion, these results suggested the antioxidative, anti-inflammatory properties of MaR 1 in CCl4 induced liver injury. The possible mechanism is partly implicated in its abilities to inhibit ROS generation and activation of NF-κb and MAPK pathway. PMID:26881046

  4. Discoidin Domain Receptor-1 Deficiency Attenuates Atherosclerotic Calcification and Smooth Muscle Cell-Mediated Mineralization

    PubMed Central

    Ahmad, Pamela J.; Trcka, Daniel; Xue, Siming; Franco, Christopher; Speer, Mei Y.; Giachelli, Cecilia M.; Bendeck, Michelle P.

    2009-01-01

    Intimal calcification is a feature of advanced atherosclerotic disease that predicts a two- to eightfold increase in the risk of coronary events. Type I collagen promotes vascular smooth muscle cell-mediated calcification, although the mechanism by which this occurs is unknown. The discoidin domain receptor 1 (DDR1) is a collagen receptor that is emerging as a critical mediator of atherosclerosis. To determine whether DDR1 is involved in intimal calcification, we fed male Ddr1−/−;Ldlr−/− and Ddr1+/+;Ldlr−/− mice an atherogenic diet for 6, 12, or 24 weeks. DDR1 deficiency significantly reduced the calcium content of the aortic arch, and microcomputed tomography demonstrated a significant decrease in hydroxyapatite deposition after 24 weeks of atherogenic diet. Reduced calcification was correlated with decreases in macrophage accumulation and tumor necrosis factor α staining, suggesting that the reduction in calcification was in part due to decreased inflammation. The chondrogenic markers type II collagen, type X collagen, and Sox-9 were expressed within the mineralized foci. An in vitro assay performed with vascular smooth muscle cells revealed that DDR1 was required for cell-mediated calcification of the matrix, and Ddr1+/+ smooth muscle cells expressed more alkaline phosphatase activity, whereas Ddr1−/− smooth muscle cells expressed elevated levels of mRNA for nucleotide pyrophosphatase phosphodiesterase 1, an inhibitor of tissue mineralization. Taken together, our results demonstrate that DDR1 mediates an important mechanism for atherosclerotic calcification. PMID:19893047

  5. Discoidin domain receptor-1 deficiency attenuates atherosclerotic calcification and smooth muscle cell-mediated mineralization.

    PubMed

    Ahmad, Pamela J; Trcka, Daniel; Xue, Siming; Franco, Christopher; Speer, Mei Y; Giachelli, Cecilia M; Bendeck, Michelle P

    2009-12-01

    Intimal calcification is a feature of advanced atherosclerotic disease that predicts a two- to eightfold increase in the risk of coronary events. Type I collagen promotes vascular smooth muscle cell-mediated calcification, although the mechanism by which this occurs is unknown. The discoidin domain receptor 1 (DDR1) is a collagen receptor that is emerging as a critical mediator of atherosclerosis. To determine whether DDR1 is involved in intimal calcification, we fed male Ddr1(-/-);Ldlr(-/-) and Ddr1(+/+);Ldlr(-/-) mice an atherogenic diet for 6, 12, or 24 weeks. DDR1 deficiency significantly reduced the calcium content of the aortic arch, and microcomputed tomography demonstrated a significant decrease in hydroxyapatite deposition after 24 weeks of atherogenic diet. Reduced calcification was correlated with decreases in macrophage accumulation and tumor necrosis factor alpha staining, suggesting that the reduction in calcification was in part due to decreased inflammation. The chondrogenic markers type II collagen, type X collagen, and Sox-9 were expressed within the mineralized foci. An in vitro assay performed with vascular smooth muscle cells revealed that DDR1 was required for cell-mediated calcification of the matrix, and Ddr1(+/+) smooth muscle cells expressed more alkaline phosphatase activity, whereas Ddr1(-/-) smooth muscle cells expressed elevated levels of mRNA for nucleotide pyrophosphatase phosphodiesterase 1, an inhibitor of tissue mineralization. Taken together, our results demonstrate that DDR1 mediates an important mechanism for atherosclerotic calcification.

  6. Id2 Complexes with the SNAG Domain of Snai1 Inhibiting Snai1-Mediated Repression of Integrin β4

    PubMed Central

    Chang, Cheng; Yang, Xiaofang; Pursell, Bryan

    2013-01-01

    The epithelial-mesenchymal transition (EMT) is a fundamental process that underlies development and cancer. Although the EMT involves alterations in the expression of specific integrins that mediate stable adhesion to the basement membrane, such as α6β4, the mechanisms involved are poorly understood. Here, we report that Snai1 inhibits β4 transcription by increasing repressive histone modification (trimethylation of histone H3 at K27 [H3K27Me3]). Surprisingly, Snai1 is expressed and localized in the nucleus in epithelial cells, but it does not repress β4. We resolved this paradox by discovering that Id2 complexes with the SNAG domain of Snai1 on the β4 promoter and constrains the repressive function of Snai1. Disruption of the complex by depleting Id2 resulted in Snai1-mediated β4 repression with a concomitant increase in H3K27Me3 modification on the β4 promoter. These findings establish a novel function for Id2 in regulating Snai1 that has significant implications for the regulation of epithelial gene expression. PMID:23878399

  7. Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

    PubMed

    Kim, Eugene Y; Sudini, Kuladeep; Singh, Anil K; Haque, Mahamudul; Leaman, Douglas; Khuder, Sadik; Ahmed, Salahuddin

    2018-05-25

    Rheumatoid arthritis (RA) is characterized by hyperplastic pannus formation mediated by activated synovial fibroblasts (RASFs) that cause joint destruction. We have shown earlier that RASFs exhibit resistance to apoptosis, primarily as a result of enhanced expression of myeloid cell leukemia-1 (Mcl-1). In this study, we discovered that ursolic acid (UA), a plant-derived pentacyclic triterpenoid, selectively induces B-cell lymphoma 2 homology 3-only protein Noxa in human RASFs. We observed that UA-induced Noxa expression was followed by a consequent decrease in Mcl-1 expression in a dose-dependent manner. Subsequent evaluation of the signaling pathways showed that UA-induced Noxa is primarily mediated by the JNK pathway in human RASFs. Chromatin immunoprecipitation (IP) studies into the promoter region of Noxa indicated the role of transcription factor specificity protein 1 in JNK-mediated Noxa expression. Furthermore, the results from IP studies and proximity ligation assays indicated that UA-induced Noxa colocalizes and associates with Mcl-1 to prime it for proteasomal degradation through K 48 -linked ubiquitination by the selective recruitment of Mcl-1 ubiquitin ligase E3, a homologous to E6-associated protein C terminus domain-containing E3 ubiquitin ligase. These findings unveil a novel mechanism of inducing apoptosis in RASFs and a potential adjunct therapeutic strategy of regulating synovial hyperplasia in RA.-Kim, E. Y., Sudini, K., Singh, A. K., Haque, M., Leaman, D., Khuder, S., Ahmed, S. Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

  8. Polycystin-1 promotes PKC{alpha}-mediated NF-{kappa}B activation in kidney cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Banzi, Manuela; Aguiari, Gianluca; Trimi, Viky

    2006-11-17

    Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-{kappa}B signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293{sup CTT}), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-{kappa}B nuclear levels and NF-{kappa}B-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-{kappa}B promoter activation was mediated by PKC{alpha}more » because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293{sup CTT} cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-{kappa}B inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKC{alpha}-mediated NF-{kappa}B signalling and cell survival.« less

  9. Alu-mediated recombination defect in IGF1R: haploinsufficiency in a patient with short stature.

    PubMed

    Harmel, Eva-Maria; Binder, Gerhard; Barnikol-Oettler, Anja; Caliebe, Janina; Kiess, Wieland; Losekoot, Monique; Ranke, Michael B; Rappold, Gudrun A; Schlicke, Marina; Stobbe, Heike; Wit, Jan M; Pfäffle, Roland; Klammt, Jürgen

    2013-01-01

    The insulin-like growth factor (IGF) receptor (IGF1R) is essential for normal development and growth. IGF1R mutations cause IGF-1 resistance resulting in intrauterine and postnatal growth failure. The phenotypic spectrum related to IGF1R mutations remains to be fully understood. Auxological and endocrinological data of a patient identified previously were assessed. The patient's fibroblasts were studied to characterize the IGF1R deletion, mRNA fate, protein expression and signalling capabilities. The boy, who carries a heterozygous IGF1R exon 6 deletion caused by Alu element-mediated recombination and a heterozygous SHOX variant (p.Met240Ile), was born appropriate for gestational age but developed proportionate short stature postnatally. IGF-1 levels were low-normal. None of the stigmata associated with SHOX deficiency or sporadically observed in IGF1R mutation carriers were present. Nonsense-mediated mRNA decay led to a substantial decline of IGF1R dosage and IGF-1-dependent receptor autophosphorylation but not impaired downstream signalling. We present the first detailed report of an intragenic IGF1R deletion identified in a patient who, apart from short stature, deviates from all established markers that qualify a growth-retarded child for IGF1R analysis. Although such children will usually escape routine clinical mutation screenings, they can contribute to the understanding of factors and mechanisms that cooperate with the IGF1R. © 2013 S. Karger AG, Basel.

  10. Inhibition of PTP1B Restores IRS1-Mediated Hepatic Insulin Signaling in IRS2-Deficient Mice

    PubMed Central

    González-Rodríguez, Águeda; Gutierrez, Jose A. Mas; Sanz-González, Silvia; Ros, Manuel; Burks, Deborah J.; Valverde, Ángela M.

    2010-01-01

    OBJECTIVE Mice with complete deletion of insulin receptor substrate 2 (IRS2) develop hyperglycemia, impaired hepatic insulin signaling, and elevated gluconeogenesis, whereas mice deficient for protein tyrosine phosphatase (PTP)1B display an opposing hepatic phenotype characterized by increased sensitivity to insulin. To define the relationship between these two signaling pathways in the regulation of liver metabolism, we used genetic and pharmacological approaches to study the effects of inhibiting PTP1B on hepatic insulin signaling and expression of gluconeogenic enzymes in IRS2−/− mice. RESEARCH DESIGN AND METHODS We analyzed glucose homeostasis and insulin signaling in liver and isolated hepatocytes from IRS2−/− and IRS2−/−/PTP1B−/− mice. Additionally, hepatic insulin signaling was assessed in control and IRS2−/− mice treated with resveratrol, an antioxidant present in red wine. RESULTS In livers of hyperglycemic IRS2−/− mice, the expression levels of PTP1B and its association with the insulin receptor (IR) were increased. The absence of PTP1B in the double-mutant mice restored hepatic IRS1-mediated phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 signaling. Moreover, resveratrol treatment of hyperglycemic IRS2−/− mice decreased hepatic PTP1B mRNA and inhibited PTP1B activity, thereby restoring IRS1-mediated PI 3-kinase/Akt/Foxo1 signaling and peripheral insulin sensitivity. CONCLUSIONS By regulating the phosphorylation state of IR, PTB1B determines sensitivity to insulin in liver and exerts a unique role in the interplay between IRS1 and IRS2 in the modulation of hepatic insulin action. PMID:20028942

  11. Silibinin induces apoptosis of HT29 colon carcinoma cells through early growth response-1 (EGR-1)-mediated non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) up-regulation.

    PubMed

    Woo, Seon Min; Min, Kyoung-Jin; Kim, Shin; Park, Jong-Wook; Kim, Dong Eun; Chun, Kyung-Soo; Kim, Young Ho; Lee, Tae-Jin; Kim, Sang Hyun; Choi, Yung Hyun; Chang, Jong-Soo; Kwon, Taeg Kyu

    2014-03-25

    Silibinin, an effective anti-cancer and chemopreventive agent, has been shown to exert multiple effects on cancer cells, including inhibition of both cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We observed that silibinin significantly induced the expression of the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in both p53 wild-type and p53-null cancer cell lines, suggesting that silibinin-induced NAG-1 up-regulation is p53-independent manner. Silibinin up-regulates early growth response-1 (EGR-1) expression. The ectopic expression of EGR-1 significantly increased NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner. Furthermore, down-regulation of EGR-1 expression using siRNA markedly reduced silibinin-mediated NAG-1 expression, suggesting that the expression of EGR-1 is critical for silibinin-induced NAG-1 expression. We also observed that reactive oxygen species (ROS) are generated by silibinin; however, ROS did not affect silibinin-induced NAG-1 expression and apoptosis. In addition, we demonstrated that the mitogen-activated protein kinase (MAP kinase) signal transduction pathway is involved in silibinin-induced NAG-1 expression. Inhibitors of p38 MAP kinase (SB203580) attenuated silibinin-induced NAG-1 expression. Furthermore, we found that siRNA-mediated knockdown of NAG-1 attenuated silibinin-induced apoptosis. Collectively, the results of this study demonstrate for the first time that up-regulation of NAG-1 contributes to silibinin-induced apoptosis in cancer cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

    PubMed

    Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

    2017-06-26

    The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

  13. Homo sapiens Systemic RNA Interference-defective-1 Transmembrane Family Member 1 (SIDT1) Protein Mediates Contact-dependent Small RNA Transfer and MicroRNA-21-driven Chemoresistance*

    PubMed Central

    Elhassan, Mohamed O.; Christie, Jennifer; Duxbury, Mark S.

    2012-01-01

    Locally initiated RNA interference (RNAi) has the potential for spatial propagation, inducing posttranscriptional gene silencing in distant cells. In Caenorhabditis elegans, systemic RNAi requires a phylogenetically conserved transmembrane channel, SID-1. Here, we show that a human SID-1 orthologue, SIDT1, facilitates rapid, contact-dependent, bidirectional small RNA transfer between human cells, resulting in target-specific non-cell-autonomous RNAi. Intercellular small RNA transfer can be both homotypic and heterotypic. We show SIDT1-mediated intercellular transfer of microRNA-21 to be a driver of resistance to the nucleoside analog gemcitabine in human adenocarcinoma cells. Documentation of a SIDT1-dependent small RNA transfer mechanism and the associated phenotypic effects on chemoresistance in human cancer cells raises the possibility that conserved systemic RNAi pathways contribute to the acquisition of drug resistance. Mediators of non-cell-autonomous RNAi may be tractable targets for novel therapies aimed at improving the efficacy of current cytotoxic agents. PMID:22174421

  14. Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

    PubMed Central

    Burch, R M; Connor, J R; Axelrod, J

    1988-01-01

    Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

  15. Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

    PubMed

    Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

    2014-02-15

    Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

  16. Cambrian origin of the CYP27C1-mediated vitamin A1-to-A2 switch, a key mechanism of vertebrate sensory plasticity

    USGS Publications Warehouse

    Morshedian, Ala; Toomery, Matthew B.; Pollock, Gabriel E.; Frederiksen, Rikard; Enright, Jennifer; McCormick, Stephen; Cornwall, M. Carter; Fain, Gordon L.; Corbo, Joseph C.

    2017-01-01

    The spectral composition of ambient light varies across both space and time. Many species of jawed vertebrates adapt to this variation by tuning the sensitivity of their photoreceptors via the expression of CYP27C1, an enzyme that converts vitamin A1 into vitamin A2, thereby shifting the ratio of vitamin A1-based rhodopsin to red-shifted vitamin A2-based porphyropsin in the eye. Here, we show that the sea lamprey (Petromyzon marinus), a jawless vertebrate that diverged from jawed vertebrates during the Cambrian period (approx. 500 Ma), dynamically shifts its photoreceptor spectral sensitivity via vitamin A1-to-A2 chromophore exchange as it transitions between photically divergent aquatic habitats. We further show that this shift correlates with high-level expression of the lamprey orthologue of CYP27C1, specifically in the retinal pigment epithelium as in jawed vertebrates. Our results suggest that the CYP27C1-mediated vitamin A1-to-A2 switch is an evolutionarily ancient mechanism of sensory plasticity that appeared not long after the origin of vertebrates.

  17. Promiscuous dimerization of the growth hormone secretagogue receptor (GHS-R1a) attenuates ghrelin-mediated signaling.

    PubMed

    Schellekens, Harriët; van Oeffelen, Wesley E P A; Dinan, Timothy G; Cryan, John F

    2013-01-04

    G protein-coupled receptors (GPCRs), such as the ghrelin receptor (GHS-R1a), the melanocortin 3 receptor (MC(3)), and the serotonin 2C receptor (5-HT(2C)), are well known for their key role in the homeostatic control of food intake and energy balance. Ghrelin is the only known gut peptide exerting an orexigenic effect and has thus received much attention as an anti-obesity drug target. In addition, recent data have revealed a critical role for ghrelin in dopaminergic mesolimbic circuits involved in food reward signaling. This study investigates the downstream signaling consequences and ligand-mediated co-internalization following heterodimerization of the GHS-R1a receptor with the dopamine 1 receptor, as well as that of the GHS-R1a-MC(3) heterodimer. In addition, a novel heterodimer between the GHS-R1a receptor and the 5-HT(2C) receptor was identified. Interestingly, dimerization of the GHS-R1a receptor with the unedited 5-HT(2C)-INI receptor, but not with the partially edited 5-HT(2C)-VSV isoform, significantly reduced GHS-R1a agonist-mediated calcium influx, which was completely restored following pharmacological blockade of the 5-HT(2C) receptor. These results combined suggest a potential novel mechanism for fine-tuning GHS-R1a receptor-mediated activity via promiscuous dimerization of the GHS-R1a receptor with other G protein-coupled receptors involved in appetite regulation and food reward. These findings may uncover novel mechanisms of significant relevance for the future pharmacological targeting of the GHS-R1a receptor in the homeostatic regulation of energy balance and in hedonic appetite signaling, both of which play a significant role in the development of obesity.

  18. Activation of TNFR2 sensitizes macrophages for TNFR1-mediated necroptosis

    PubMed Central

    Siegmund, Daniela; Kums, Juliane; Ehrenschwender, Martin; Wajant, Harald

    2016-01-01

    Macrophages express TNFR1 as well as TNFR2 and are also major producers of tumor necrosis factor (TNF), especially upon contact with pathogen-associated molecular patterns. Consequently, TNF not only acts as a macrophage-derived effector molecule but also regulates the activity and viability of macrophages. Here, we investigated the individual contribution of TNFR1 and TNFR2 to TNF-induced cell death in macrophages. Exclusive stimulation of TNFR1 showed no cytotoxic effect whereas selective stimulation of TNFR2 displayed mild cytotoxicity. Intriguingly, the latter was strongly enhanced by the caspase inhibitor zVAD-fmk. The strong cytotoxic activity of TNFR2 in the presence of zVAD-fmk was reversed by necrostatin-1, indicating necroptotic cell death. TNFR1- and TNF-deficient macrophages turned out to be resistant against TNFR2-induced cell death. In addition, the cIAP-depleting SMAC mimetic BV6 also enforced TNF/TNFR1-mediated necroptotic cell death in the presence of zVAD-fmk. In sum, our data suggest a model in which TNFR2 sensitizes macrophages for endogenous TNF-induced TNFR1-mediated necroptosis by the known ability of TNFR2 to interfere with the survival activity of TRAF2-cIAP1/2 complexes. PMID:27899821

  19. Activation of TNFR2 sensitizes macrophages for TNFR1-mediated necroptosis.

    PubMed

    Siegmund, Daniela; Kums, Juliane; Ehrenschwender, Martin; Wajant, Harald

    2016-09-22

    Macrophages express TNFR1 as well as TNFR2 and are also major producers of tumor necrosis factor (TNF), especially upon contact with pathogen-associated molecular patterns. Consequently, TNF not only acts as a macrophage-derived effector molecule but also regulates the activity and viability of macrophages. Here, we investigated the individual contribution of TNFR1 and TNFR2 to TNF-induced cell death in macrophages. Exclusive stimulation of TNFR1 showed no cytotoxic effect whereas selective stimulation of TNFR2 displayed mild cytotoxicity. Intriguingly, the latter was strongly enhanced by the caspase inhibitor zVAD-fmk. The strong cytotoxic activity of TNFR2 in the presence of zVAD-fmk was reversed by necrostatin-1, indicating necroptotic cell death. TNFR1- and TNF-deficient macrophages turned out to be resistant against TNFR2-induced cell death. In addition, the cIAP-depleting SMAC mimetic BV6 also enforced TNF/TNFR1-mediated necroptotic cell death in the presence of zVAD-fmk. In sum, our data suggest a model in which TNFR2 sensitizes macrophages for endogenous TNF-induced TNFR1-mediated necroptosis by the known ability of TNFR2 to interfere with the survival activity of TRAF2-cIAP1/2 complexes.

  20. A herbal formula comprising Rosae Multiflorae Fructus and Lonicerae Japonicae Flos inhibits the production of inflammatory mediators and the IRAK-1/TAK1 and TBK1/IRF3 pathways in RAW 264.7 and THP-1 cells.

    PubMed

    Cheng, Brian Chi Yan; Yu, Hua; Su, Tao; Fu, Xiu-Qiong; Guo, Hui; Li, Ting; Cao, Hui-Hui; Tse, Anfernee Kai-Wing; Kwan, Hiu-Yee; Yu, Zhi-Ling

    2015-11-04

    As documented in the Chinese Materia Medica Grand Dictionary (), a herbal formula (RL) consisting of Rosae Multiflorae Fructus (multiflora rose hips) and Lonicerae Japonicae Flos (Japanese honeysuckle flowers) has traditionally been used in treating inflammatory disorders. RL was previously reported to inhibit the expression of various inflammatory mediators regulated by NF-κB and MAPKs that are components of the TLR4 signalling pathways. This study aims to provide further justification for clinical application of RL in treating inflammatory disorders by further delineating the involvement of the TLR4 signalling cascades in the effects of RL on inflammatory mediators. RL consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos (in 5:3 ratio) was extracted using absolute ethanol. We investigated the effect of RL on the production of cytokines and chemokines that are regulated by three key transcription factors of the TLR4 signalling pathways AP-1, NF-κB and IRF3 in LPS-stimulated RAW264.7 cells using the multiplex biometric immunoassay. Phosphorylation of AP-1, NF-κB, IRF3, IκB-α, IKKα/β, Akt, TAK1, TBK1, IRAK-1 and IRAK-4 were examined in LPS-stimulated RAW264.7 cells and THP-1 cells using Western blotting. Nuclear localizations of AP-1, NF-κB and IRF3 were also examined using Western blotting. RL reduced the secretion of various pro-inflammatory cytokines and chemokines regulated by transcription factors AP-1, NF-κB and IRF3. Phosphorylation and nuclear protein levels of these transcription factors were decreased by RL treatment. Moreover, RL inhibited the activation/phosphorylation of IκB-α, IKKα/β, TAK1, TBK1 and IRAK-1. Suppression of the IRAK-1/TAK1 and TBK1/IRF3 signalling pathways was associated with the effect of RL on inflammatory mediators in LPS-stimulated RAW264.7 and THP-1 cells. This provides further pharmacological basis for the clinical application of RL in the treatment of inflammatory disorders. Copyright © 2015 Elsevier

  1. Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression

    PubMed Central

    Zhang, Yaochen; Kim, Don-Kyu; Lee, Ji-Min; Park, Seung Bum; Jeong, Won-IL; Kim, Seong Heon; Lee, In-Kyu; Lee, Chul-Ho; Chiang, John Y.L.; Choi, Hueng-Sik

    2017-01-01

    Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ -binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism. PMID:26348907

  2. Degradation of Mcl-1 by granzyme B: implications for Bim-mediated mitochondrial apoptotic events.

    PubMed

    Han, Jie; Goldstein, Leslie A; Gastman, Brian R; Froelich, Christopher J; Yin, Xiao-Ming; Rabinowich, Hannah

    2004-05-21

    Recent studies have suggested that in the absence of Bid, granzyme B (GrB) can utilize an unknown alternative pathway to mediate mitochondrial apoptotic events. The current study has elucidated just such a pathway for GrB-mediated mitochondrial apoptotic alterations. Two Bcl-2 family members have been identified as interactive players in this newly discovered mitochondrial response to GrB: the pro-survival protein Mcl-1L and the pro-apoptotic protein, Bim. Expression of Mcl-1L, which localizes mainly to the outer mitochondrial membrane, decreases significantly in cells subjected to CTL-free cytotoxicity mediated by a combination of GrB and replication-deficient adenovirus. The data suggest that Mcl-1L is a substrate for GrB and for caspase-3, but the two enzymes appear to target different cleavage sites. The cleavage pattern of endogenous Mcl-1L resembles that of in vitro translated Mcl-1L subjected to similar proteolytic activity. Co-immunoprecipitation experiments performed with endogenous as well as with in vitro translated proteins suggest that Mcl-1L is a high affinity binding partner of the three isoforms of Bim (extra-long, long, and short). Bim, a BH3-only protein, is capable of mediating the release of mitochondrial cytochrome c, and this activity is inhibited by the presence of exogenous Mcl-1L. The findings presented herein imply that Mcl-1L degradation by either GrB or caspase-3 interferes with Bim sequestration by Mcl-1L.

  3. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong

    2014-10-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 andmore » CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO

  4. An LXR agonist promotes GBM cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway

    PubMed Central

    Guo, Deliang; Reinitz, Felicia; Youssef, Mary; Hong, Cynthia; Nathanson, David; Akhavan, David; Kuga, Daisuke; Amzajerdi, Ali Nael; Soto, Horacio; Zhu, Shaojun; Babic, Ivan; Tanaka, Kazuhiro; Dang, Julie; Iwanami, Akio; Gini, Beatrice; DeJesus, Jason; Lisiero, Dominique D.; Huang, Tiffany T.; Prins, Robert M.; Wen, Patrick Y.; Robins, H. Ian; Prados, Michael D.; DeAngelis, Lisa M.; Mellinghoff, Ingo K.; Mehta, Minesh P.; James, C. David; Chakravarti, Arnab; Cloughesy, Timothy F.; Tontonoz, Peter; Mischel, Paul S.

    2011-01-01

    Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. EGFR mutations (EGFRvIII) and PI3K hyperactivation are common in GBM, promoting tumor growth and survival, including through SREBP-1-dependent-lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. Here, studies in GBM cell lines, xenograft models and GBM clinical samples, including from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the LDL receptor. Targeting LDLR with the Liver X Receptor (LXR) agonist GW3965 caused IDOL (Inducible Degrader Of LDLR)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results demonstrate that EGFRvIII can promote tumor survival through PI3K-SREBP-1 dependent up-regulation of LDLR, and suggest a role for LXR agonists in the treatment of GBM patients. PMID:22059152

  5. Eleostearic acid induces RIP1-mediated atypical apoptosis in a kinase-independent manner via ERK phosphorylation, ROS generation and mitochondrial dysfunction

    PubMed Central

    Obitsu, S; Sakata, K; Teshima, R; Kondo, K

    2013-01-01

    RIP1 is a serine/threonine kinase, which is involved in apoptosis and necroptosis. In apoptosis, caspase-8 and FADD have an important role. On the other hand, RIP3 is a key molecule in necroptosis. Recently, we reported that eleostearic acid (ESA) elicits caspase-3- and PARP-1-independent cell death, although ESA-treated cells mediate typical apoptotic morphology such as chromatin condensation, plasma membrane blebbing and apoptotic body formation. The activation of caspases, Bax and PARP-1, the cleavage of AIF and the phosphorylation of histone H2AX, all of which are characteristics of typical apoptosis, do not occur in ESA-treated cells. However, the underlying mechanism remains unclear. To clarify the signaling pathways in ESA-mediated apoptosis, we investigated the functions of RIP1, MEK, ERK, as well as AIF. Using an extensive study based on molecular biology, we identified the alternative role of RIP1 in ESA-mediated apoptosis. ESA mediates RIP1-dependent apoptosis in a kinase independent manner. ESA activates serine/threonine phosphatases such as calcineurin, which induces RIP1 dephosphorylation, thereby ERK pathway is activated. Consequently, localization of AIF and ERK in the nucleus, ROS generation and ATP reduction in mitochondria are induced to disrupt mitochondrial cristae, which leads to cell death. Necrostatin (Nec)-1 blocked MEK/ERK phosphorylation and ESA-mediated apoptosis. Nec-1 inactive form (Nec1i) also impaired ESA-mediated apoptosis. Nec1 blocked the interaction of MEK with ERK upon ESA stimulation. Together, these findings provide a new finding that ERK and kinase-independent RIP1 proteins are implicated in atypical ESA-mediated apoptosis. PMID:23788031

  6. Direct involvement of sigma-1 receptors in the dopamine D1 receptor-mediated effects of cocaine.

    PubMed

    Navarro, Gemma; Moreno, Estefanía; Aymerich, Marisol; Marcellino, Daniel; McCormick, Peter J; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Canela, Enric I; Ortiz, Jordi; Fuxe, Kjell; Lluís, Carmen; Ferré, Sergi; Franco, Rafael

    2010-10-26

    It is well known that cocaine blocks the dopamine transporter. This mechanism should lead to a general increase in dopaminergic neurotransmission, and yet dopamine D(1) receptors (D(1)Rs) play a more significant role in the behavioral effects of cocaine than the other dopamine receptor subtypes. Cocaine also binds to σ-1 receptors, the physiological role of which is largely unknown. In the present study, D(1)R and σ(1)R were found to heteromerize in transfected cells, where cocaine robustly potentiated D(1)R-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK activation induced by D(1)R stimulation in a dopamine transporter-independent and σ(1)R-dependent manner. Some of these effects were also demonstrated in murine striatal slices and were absent in σ(1)R KO mice, providing evidence for the existence of σ(1)R-D(1)R heteromers in the brain. Therefore, these results provide a molecular explanation for which D(1)R plays a more significant role in the behavioral effects of cocaine, through σ(1)R-D(1)R heteromerization, and provide a unique perspective toward understanding the molecular basis of cocaine addiction.

  7. A slow excitatory postsynaptic current mediated by a novel metabotropic glutamate receptor in CA1 pyramidal neurons.

    PubMed

    Sheng, Nengyin; Yang, Jing; Silm, Katlin; Edwards, Robert H; Nicoll, Roger A

    2017-03-15

    Slow excitatory postsynaptic currents (EPSCs) mediated by metabotropic glutamate receptors (mGlu receptors) have been reported in several neuronal subtypes, but their presence in hippocampal pyramidal neurons remains elusive. Here we find that in CA1 pyramidal neurons a slow EPSC is induced by repetitive stimulation while ionotropic glutamate receptors and glutamate-uptake are blocked whereas it is absent in the VGLUT1 knockout mouse in which presynaptic glutamate is lost, suggesting the slow EPSC is mediated by glutamate activating mGlu receptors. However, it is not inhibited by known mGlu receptor antagonists. These findings suggest that this slow EPSC is mediated by a novel mGlu receptor, and that it may be involved in neurological diseases associated with abnormal high-concentration of extracellular glutamate. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis.

    PubMed

    Chae, Wook-Jin; Park, Jong-Hyun; Henegariu, Octavian; Yilmaz, Saliha; Hao, Liming; Bothwell, Alfred L M

    2017-10-01

    Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3 + regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4 + T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3 + Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3 + Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3 + Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

  9. Ideal crop plant architecture is mediated by tassels replace upper ears1, a BTB/POZ ankyrin repeat gene directly targeted by TEOSINTE BRANCHED1.

    PubMed

    Dong, Zhaobin; Li, Wei; Unger-Wallace, Erica; Yang, Jinliang; Vollbrecht, Erik; Chuck, George

    2017-10-10

    Axillary branch suppression is a favorable trait bred into many domesticated crop plants including maize compared with its highly branched wild ancestor teosinte. Branch suppression in maize was achieved through selection of a gain of function allele of the teosinte branched1 (tb1) transcription factor that acts as a repressor of axillary bud growth. Previous work indicated that other loci may function epistatically with tb1 and may be responsible for some of its phenotypic effects. Here, we show that tb1 mediates axillary branch suppression through direct activation of the tassels replace upper ears1 ( tru1 ) gene that encodes an ankyrin repeat domain protein containing a BTB/POZ motif necessary for protein-protein interactions. The expression of TRU1 and TB1 overlap in axillary buds, and TB1 binds to two locations in the tru1 gene as shown by chromatin immunoprecipitation and gel shifts. In addition, nucleotide diversity surveys indicate that tru1 , like tb1 , was a target of selection. In modern maize, TRU1 is highly expressed in the leaf trace vasculature of axillary internodes, while in teosinte, this expression is highly reduced or absent. This increase in TRU1 expression levels in modern maize is supported by comparisons of relative protein levels with teosinte as well as by quantitative measurements of mRNA levels. Hence, a major innovation in creating ideal maize plant architecture originated from ectopic overexpression of tru1 in axillary branches, a critical step in mediating the effects of domestication by tb1.

  10. Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model.

    PubMed

    Pawlinski, Rafal; Pedersen, Brian; Kehrle, Bettina; Aird, William C; Frank, Rolf D; Guha, Mausumee; Mackman, Nigel

    2003-05-15

    In septic shock, tissue factor (TF) activates blood coagulation, and cytokines and chemokines orchestrate an inflammatory response. In this study, the role of Egr-1 in lipopolysaccharide (LPS) induction of TF and inflammatory mediators in vivo was evaluated using Egr-1(+/+) and Egr-1(-/-) mice. Administration of LPS transiently increased the steady-state levels of Egr-1 mRNA in the kidneys and lungs of Egr-1(+/+) mice with maximal induction at one hour. Egr-1 was expressed in epithelial cells in the kidneys and lungs in untreated and LPS-treated mice. LPS induction of monocyte chemoattractant protein mRNA in the kidneys and lungs of Egr-1(-/-) mice was not affected at 3 hours, but its expression was significantly reduced at 8 hours compared with the expression observed in Egr-1(+/+) mice. Similarly, LPS induction of TF mRNA expression in the kidneys and lungs at 8 hours was reduced in Egr-1(-/-) mice. However, Egr-1 deficiency did not affect plasma levels of tumor necrosis factor alpha in endotoxemic mice. Moreover, Egr-1(+/+) and Egr-1(-/-) mice exhibited similar survival times in a model of acute endotoxemia. These data indicate that Egr-1 does not contribute to the early inflammatory response in the kidneys and lungs or the early systemic inflammatory response in endotoxemic mice. However, Egr-1 does contribute to the sustained expression of inflammatory mediators and to the maximal expression of TF at 8 hours in the kidneys and lungs.

  11. Wt1a, Foxc1a, and the Notch mediator Rbpj Physically Interact and Regulate the Formation of Podocytes in Zebrafish

    PubMed Central

    O’Brien, Lori L.; Grimaldi, Michael; Kostun, Zachary; Wingert, Rebecca A.; Selleck, Rori; Davidson, Alan J.

    2011-01-01

    Podocytes help form the glomerular blood filtration barrier in the kidney and their injury or loss leads to renal disease. The Wilms’ tumor suppressor-1 (Wt1) and the FoxC1/2 transcription factors, as well as Notch signaling, have been implicated as important regulators of podocyte fate. It is not known whether these factors work in parallel or sequentially on different gene targets, or as higher-order transcriptional complexes on common genes. Here, we use the zebrafish to demonstrate that embryos treated with morpholinos against wt1a, foxc1a, or the Notch transcriptional mediator rbpj develop fewer podocytes, as determined by wt1b, hey1 and nephrin expression, while embryos deficient in any two of these factors completely lack podocytes. From GST-pull-downs and co-immunoprecipitation experiments we show that Wt1a, Foxc1a, and Rbpj can physically interact with each other, whereas only Rbpj binds to the Notch intracellular domain (NICD). In transactivation assays, combinations of Wt1, FoxC1/2, and NICD synergistically induce the Hey1 promoter, and have additive or repressive effects on the Podocalyxin promoter, depending on dosage. Taken together, these data suggest that Wt1, FoxC1/2, and Notch signaling converge on common target genes where they physically interact to regulate a podocyte-specific gene program. These findings further our understanding of the transcriptional circuitry responsible for podocyte formation and differentiation during kidney development. PMID:21871448

  12. Immune Tolerance to Apoptotic Self Is Mediated Primarily by Regulatory B1a Cells.

    PubMed

    Miles, Katherine; Simpson, Joanne; Brown, Sheila; Cowan, Graeme; Gray, David; Gray, Mohini

    2017-01-01

    The chronic autoimmune inflammatory diseases, systemic lupus erythematosus and Sjogren's syndrome, develop when tolerance to apoptotic cells (ACs) is lost. We have previously reported that this tolerance is maintained by innate-like, IL-10 secreting regulatory B cells. Two questions remained. First, do these regulatory B cells belong predominantly to a single subset of steady-state B cells and second, what is their specificity? We report here that innate-like B cells with markers characteristic for B1a cells (CD43 +ve CD19 hi CD5 +ve IgM hi IgD lo ) constitute 80% of splenic and 96% of peritoneal B cells that respond to ACs by secreting IL-10. AC responsive B1a cells secrete self-reactive natural antibodies (NAbs) and IL-10, which is augmented by toll-like receptor (TLR) 7 or TLR9 stimulation. In so doing, they both accelerate the clearance of dying cells by macrophages and inhibit their potential to mount proinflammatory immune responses. While B1a cells make prolonged contact with ACs, they do not require TIM1 or complement to mediate their regulatory function. In an animal model of neural inflammation (experimental autoimmune encephalomyelitis), just 10 5 activated B1a B cells was sufficient to restrain inflammation. Activated B1a B cells also induced antigen-specific T cells to secrete IL-10. Hence, regulatory B1a cells specifically recognize and augment tolerance to apoptotic self via IL-10 and NAbs; but once activated, can also prevent autoimmune mediated inflammation.

  13. FAX1, a Novel Membrane Protein Mediating Plastid Fatty Acid Export

    PubMed Central

    Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin

    2015-01-01

    Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734

  14. Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

    PubMed Central

    Savory, Joanne G. A.; Hsu, Brian; Laquian, Ian R.; Giffin, Ward; Reich, Terry; Haché, Robert J. G.; Lefebvre, Yvonne A.

    1999-01-01

    Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1− GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export. PMID:9891038

  15. Hindbrain GLP-1 receptor mediation of cisplatin-induced anorexia and nausea.

    PubMed

    De Jonghe, Bart C; Holland, Ruby A; Olivos, Diana R; Rupprecht, Laura E; Kanoski, Scott E; Hayes, Matthew R

    2016-01-01

    While chemotherapy-induced nausea and vomiting are clinically controlled in the acute (<24 h) phase following treatment, the anorexia, nausea, fatigue, and other illness-type behaviors during the delayed phase (>24 h) of chemotherapy are largely uncontrolled. As the hindbrain glucagon-like peptide-1 (GLP-1) system contributes to energy balance and mediates aversive and stressful stimuli, here we examine the hypothesis that hindbrain GLP-1 signaling mediates aspects of chemotherapy-induced nausea and reductions in feeding behavior in rats. Specifically, hindbrain GLP-1 receptor (GLP-1R) blockade, via 4th intracerebroventricular (ICV) exendin-(9-39) injections, attenuates the anorexia, body weight reduction, and pica (nausea-induced ingestion of kaolin clay) elicited by cisplatin chemotherapy during the delayed phase (48 h) of chemotherapy-induced nausea. Additionally, the present data provide evidence that the central GLP-1-producing preproglucagon neurons in the nucleus tractus solitarius (NTS) of the caudal brainstem are activated by cisplatin during the delayed phase of chemotherapy-induced nausea, as cisplatin led to a significant increase in c-Fos immunoreactivity in NTS GLP-1-immunoreactive neurons. These data support a growing body of literature suggesting that the central GLP-1 system may be a potential pharmaceutical target for adjunct anti-emetics used to treat the delayed-phase of nausea and emesis, anorexia, and body weight loss that accompany chemotherapy treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Lack of mutational hot spots during decitabine-mediated HIV-1 mutagenesis.

    PubMed

    Rawson, Jonathan M O; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Patterson, Steven E; Mansky, Louis M

    2015-11-01

    Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Involvement of IGF-1 and MEOX2 in PI3K/Akt1/2 and ERK1/2 pathways mediated proliferation and differentiation of perivascular adipocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Ping, E-mail: lping@sdu.edu.cn; Kong, Feng; Wang, Jue

    Perivascular adipocyte (PVAC) proliferation and differentiation were closely involved in cardiovascular disease. We aimed to investigate whether phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways enhance PVAC functions activated by insulin-like growth factor 1(IGF-1) and suppressed by mesenchyme homeobox 2 (MEOX2). In this study, PVACs from primary culture were cultured and induced to differentiate. Cell viability assays demonstrated that IGF-1 promoted PVAC proliferation and differentiation. However MEOX2 counteracted these IGF-1-mediated actions. Flow Cytometry revealed that IGF-1 increased S phase cells and decreased apoptosis; however, MEOX2 decreased S phase cells, increased G0–G1 phase cells, and promoted apoptosis. During PVACmore » proliferation and differentiation, IGF-1 activated PI3K/Akt1/2 and ERK1/2 signaling pathways, upregulated the expression of these signaling proteins and FAS, and increased PVAC lipid content. In contrast, MEOX2 constrained the phosphorylation of ERK1/2 and Akt1/2 protein, down-regulated these signaling molecules and FAS, and decreased PVAC lipid content. Instead, MEOX2 knockdown enhanced the ERK1/2 and Akt1/2 phosphorylation, augmented the expression of these signaling molecules and FAS, and increased PVAC lipid content. Our findings suggested that PI3K/Akt1/2 and ERK1/2 activation mediated by IGF-1 is essential for PVAC proliferation and differentiation, and MEOX2 is a promising therapeutic gene to intervene in the signaling pathways and inhibit PVAC functions. - Highlights: • IGF-1 activated PI3K/Akt2 and ERK1/2 pathways to mediate PVAC proliferation and differentiation. • The expression of ERK1, ERK 2, PI3K, Akt1 and Akt2 showed different change trends between PVAC proliferation and differentiation. • MEOX2 effectively expressed in PVAC, increased early and late cellular apoptosis, and inhibited its proliferation. • MEOX2 depressed PVAC differentiation and FAS

  18. Neuroprotective effects of curcumin on endothelin-1 mediated cell death in hippocampal neurons.

    PubMed

    Stankowska, Dorota L; Krishnamoorthy, Vignesh R; Ellis, Dorette Z; Krishnamoorthy, Raghu R

    2017-06-01

    Alzheimer's disease is a progressive neurodegenerative disease characterized by loss of hippocampal neurons leading to memory deficits and cognitive decline. Studies suggest that levels of the vasoactive peptide endothelin-1 (ET-1) are increased in the brain tissue of Alzheimer's patients. Curcumin, the main ingredient of the spice turmeric, has been shown to have anti-inflammatory, anti-cancer, and neuroprotective effects. However, the mechanisms underlying some of these beneficial effects are not completely understood. The objective of this study was to determine if curcumin could protect hippocampal neurons from ET-1 mediated cell death and examine the involvement of c-Jun in this pathway. Primary hippocampal neurons from rat pups were isolated using a previously published protocol. Viability of the cells was measured by the live/dead assay. Immunoblot and immunohistochemical analyses were performed to analyze c-Jun levels in hippocampal neurons treated with either ET-1 or a combination of ET-1 and curcumin. Apoptotic changes were evaluated by immunoblot detection of cleaved caspase-3, cleaved fodrin, and a caspase 3/7 activation assay. ET-1 treatment produced a 2-fold increase in the levels of c-Jun as determined by an immunoblot analysis in hippocampal neurons. Co-treatment with curcumin significantly attenuated the ET-1 mediated increase in c-Jun levels. ET-1 caused increased neuronal cell death of hippocampal neurons indicated by elevation of cleaved caspase-3, cleaved fodrin and an increased activity of caspases 3 and 7 which was attenuated by co-treatment with curcumin. Blockade of JNK, an upstream effector of c-Jun by specific inhibitor SP600125 did not fully protect from ET-1 mediated activation of pro-apoptotic enzymes in primary hippocampal cells. Our data suggests that one mechanism by which curcumin protects against ET-1-mediated cell death is through blocking an increase in c-Jun levels. Other possible mechanisms include decreasing pro

  19. HTLV-1 Tax plugs and freezes UPF1 helicase leading to nonsense-mediated mRNA decay inhibition.

    PubMed

    Fiorini, Francesca; Robin, Jean-Philippe; Kanaan, Joanne; Borowiak, Malgorzata; Croquette, Vincent; Le Hir, Hervé; Jalinot, Pierre; Mocquet, Vincent

    2018-01-30

    Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.

  20. HOPM1 mediated disease resistance to Pseudomonas syringae in Arabidopsis

    DOEpatents

    He, Sheng Yang [Okemos, MI; Nomura, Kinya [East Lansing, MI

    2011-11-15

    The present invention relates to compositions and methods for enhancing plant defenses against pathogens. More particularly, the invention relates to enhancing plant immunity against bacterial pathogens, wherein HopM1.sub.1-300 mediated protection is enhanced, such as increased protection to Pseudomonas syringae pv. tomato DC3000 HopM1 and/or there is an increase in activity of an ATMIN associated plant protection protein, such as ATMIN7. Reagents of the present invention further provide a means of studying cellular trafficking while formulations of the present inventions provide increased pathogen resistance in plants.

  1. The Mas receptor mediates modulation of insulin signaling by angiotensin-(1-7).

    PubMed

    Muñoz, Marina C; Giani, Jorge F; Burghi, Valeria; Mayer, Marcos A; Carranza, Andrea; Taira, Carlos A; Dominici, Fernando P

    2012-08-20

    Angiotensin (Ang)-(1-7) stimulates proteins belonging to the insulin signaling pathway and ameliorates the Ang II negative effects at this level. However, up to date, receptors involved and mechanisms behind these observations remain unknown. Accordingly, in the present study, we explored the in vivo effects of antagonism of the Ang-(1-7) specific Mas receptor on insulin signal transduction in rat insulin-target tissues. We evaluated the acute modulation of insulin-stimulated phosphorylation of Akt, GSK-3β (Glycogen synthase kinase-3β) and AS160 (Akt substrate of 160kDa) by Ang-(1-7) and/or Ang II in the presence and absence of the selective Mas receptor antagonist A-779 in insulin-target tissues of normal rats. Also using A-779, we determined whether the Mas receptor mediates the improvement of insulin sensitivity exerted by chronic Ang-(1-7) treatment in fructose-fed rats (FFR), a model of insulin resistance, dyslipidemia and mild hypertension. The two major findings of the present work are as follows; 1) Ang-(1-7) attenuates acute Ang II-mediated inhibition of insulin signaling components in normal rats via a Mas receptor-dependent mechanism; and 2). The Mas receptor appears to be involved in beneficial effects of Ang-(1-7) on the phosphorylation of crucial insulin signaling mediators (Akt, GSK-3β and AS160), in liver, skeletal muscle and adipose tissue of FFR. These results shed light into the mechanism by which Ang-(1-7) exerts its positive physiological modulation of insulin actions in classical metabolic tissues and reinforces the central role of Akt in these effects. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Proprotein convertase 1 mediated proneuropeptide proteolytic processing in ischemic neuron injury.

    PubMed

    Tang, S S; Liang, Z Y; Guo, L R; Zhang, J H; Zhou, D

    2017-01-01

    Pro-protein processing mechanism plays an important role in neuron injury. To study the protein convertase 1 (PC1) mediated processing mechanism, the ischemic cellular or tissue proPC1/PC1 or proCgA/CgA (pro-chromogranin A) was analyzed. NS20Y differentiated cells were stressed by 0-6 h of oxygen and glucose deprivation (OGD) in glucose-free DMEM and an anaerobic jar environment. Ischemic C57BL/J mouse model was established by performing 60-min of middle cerebral artery occlusion (MCAO) operation and subsequent 4 or 24-h reperfusion. The TUNEL, immunochemistry, and Western blot methods were used to detect protein expression in ischemic cells or tissues. The OGD or MCAO stress caused substantial cell death in a dose-dependent manner (p < 0.05 or 0.01). With the increasing OGD dose, proPC1 and PC1 proteins gradually increased (p < 0.05 or 0.01) whereas proCgA and CgA proteins decreased (p < 0.05). In vivo the proPC1 and PC1 expressions presented with a peak at 4-h and then decreased at 24-h reperfusion (p < 0.05 or 0.01). The tissue proCgA and CgA proteins decreased with the increasing reperfusion time (p < 0.05). The results suggest that the increasing PC1 expression promoted the transformation of proCgA into CgA or smaller peptides, i.e. Pancreastatin or Secretoneurin, and the PC1 mediated processing plays a critical role (Fig. 4, Ref. 15).

  3. The Chromatin Remodeler SPLAYED Negatively Regulates SNC1-Mediated Immunity.

    PubMed

    Johnson, Kaeli C M; Xia, Shitou; Feng, Xiaoqi; Li, Xin

    2015-08-01

    SNC1 (SUPPRESSOR OF NPR1, CONSTITUTIVE 1) is one of a suite of intracellular Arabidopsis NOD-like receptor (NLR) proteins which, upon activation, result in the induction of defense responses. However, the molecular mechanisms underlying NLR activation and the subsequent provocation of immune responses are only partially characterized. To identify negative regulators of NLR-mediated immunity, a forward genetic screen was undertaken to search for enhancers of the dwarf, autoimmune gain-of-function snc1 mutant. To avoid lethality resulting from severe dwarfism, the screen was conducted using mos4 (modifier of snc1, 4) snc1 plants, which display wild-type-like morphology and resistance. M2 progeny were screened for mutant, snc1-enhancing (muse) mutants displaying a reversion to snc1-like phenotypes. The muse9 mos4 snc1 triple mutant was found to exhibit dwarf morphology, elevated expression of the pPR2-GUS defense marker reporter gene and enhanced resistance to the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Via map-based cloning and Illumina sequencing, it was determined that the muse9 mutation is in the gene encoding the SWI/SNF chromatin remodeler SYD (SPLAYED), and was thus renamed syd-10. The syd-10 single mutant has no observable alteration from wild-type-like resistance, although the syd-4 T-DNA insertion allele displays enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Transcription of SNC1 is increased in both syd-4 and syd-10. These data suggest that SYD plays a subtle, specific role in the regulation of SNC1 expression and SNC1-mediated immunity. SYD may work with other proteins at the chromatin level to repress SNC1 transcription; such regulation is important for fine-tuning the expression of NLR-encoding genes to prevent unpropitious autoimmunity. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please

  4. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.

    Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from themore » carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are

  5. Transcription factor YY1 can control AID-mediated mutagenesis in mice.

    PubMed

    Zaprazna, Kristina; Basu, Arindam; Tom, Nikola; Jha, Vibha; Hodawadekar, Suchita; Radova, Lenka; Malcikova, Jitka; Tichy, Boris; Pospisilova, Sarka; Atchison, Michael L

    2018-02-01

    Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1 f/f ) or that express normal AID levels (yy1 f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sμ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Contributions of the three CYP1 monooxygenases to pro-inflammatory and inflammation-resolution lipid mediator pathways.

    PubMed

    Divanovic, Senad; Dalli, Jesmond; Jorge-Nebert, Lucia F; Flick, Leah M; Gálvez-Peralta, Marina; Boespflug, Nicholas D; Stankiewicz, Traci E; Fitzgerald, Jonathan M; Somarathna, Maheshika; Karp, Christopher L; Serhan, Charles N; Nebert, Daniel W

    2013-09-15

    All three cytochrome P450 1 (CYP1) monooxygenases are believed to participate in lipid mediator biosynthesis and/or their local inactivation; however, distinct metabolic steps are unknown. We used multiple-reaction monitoring and liquid chromatography-UV coupled with tandem mass spectrometry-based lipid-mediator metabololipidomics to identify and quantify three lipid-mediator metabolomes in basal peritoneal and zymosan-stimulated inflammatory exudates, comparing Cyp1a1/1a2/1b1(⁻/⁻) C57BL/6J-background triple-knockout mice with C57BL/6J wild-type mice. Significant differences between untreated triple-knockout and wild-type mice were not found for peritoneal cell number or type or for basal CYP1 activities involving 11 identified metabolic steps. Following zymosan-initiated inflammation, 18 lipid mediators were identified, including members of the eicosanoids and specialized proresolving mediators (i.e., resolvins and protectins). Compared with wild-type mice, Cyp1 triple-knockout mice exhibited increased neutrophil recruitment in zymosan-treated peritoneal exudates. Zymosan stimulation was associated with eight statistically significantly altered metabolic steps: increased arachidonic acid-derived leukotriene B₄ (LTB₄) and decreased 5S-hydroxyeicosatetraenoic acid; decreased docosahexaenoic acid-derived neuroprotectin D1/protectin D1, 17S-hydroxydocosahexaenoic acid, and 14S-hydroxydocosahexaenoic acid; and decreased eicosapentaenoic acid-derived 18R-hydroxyeicosapentaenoic acid (HEPE), 15S-HEPE, and 12S-HEPE. In neutrophils analyzed ex vivo, elevated LTB₄ levels were shown to parallel increased neutrophil numbers, and 20-hydroxy-LTB₄ formation was found to be deficient in Cyp1 triple-knockout mice. Together, these results demonstrate novel contributions of CYP1 enzymes to the local metabolite profile of lipid mediators that regulate neutrophilic inflammation.

  7. Genome of the Netherlands population-specific imputations identify an ABCA6 variant associated with cholesterol levels

    PubMed Central

    van Leeuwen, Elisabeth M.; Karssen, Lennart C.; Deelen, Joris; Isaacs, Aaron; Medina-Gomez, Carolina; Mbarek, Hamdi; Kanterakis, Alexandros; Trompet, Stella; Postmus, Iris; Verweij, Niek; van Enckevort, David J.; Huffman, Jennifer E.; White, Charles C.; Feitosa, Mary F.; Bartz, Traci M.; Manichaikul, Ani; Joshi, Peter K.; Peloso, Gina M.; Deelen, Patrick; van Dijk, Freerk; Willemsen, Gonneke; de Geus, Eco J.; Milaneschi, Yuri; Penninx, Brenda W.J.H.; Francioli, Laurent C.; Menelaou, Androniki; Pulit, Sara L.; Rivadeneira, Fernando; Hofman, Albert; Oostra, Ben A.; Franco, Oscar H.; Leach, Irene Mateo; Beekman, Marian; de Craen, Anton J.M.; Uh, Hae-Won; Trochet, Holly; Hocking, Lynne J.; Porteous, David J.; Sattar, Naveed; Packard, Chris J.; Buckley, Brendan M.; Brody, Jennifer A.; Bis, Joshua C.; Rotter, Jerome I.; Mychaleckyj, Josyf C.; Campbell, Harry; Duan, Qing; Lange, Leslie A.; Wilson, James F.; Hayward, Caroline; Polasek, Ozren; Vitart, Veronique; Rudan, Igor; Wright, Alan F.; Rich, Stephen S.; Psaty, Bruce M.; Borecki, Ingrid B.; Kearney, Patricia M.; Stott, David J.; Adrienne Cupples, L.; Neerincx, Pieter B.T.; Elbers, Clara C.; Francesco Palamara, Pier; Pe'er, Itsik; Abdellaoui, Abdel; Kloosterman, Wigard P.; van Oven, Mannis; Vermaat, Martijn; Li, Mingkun; Laros, Jeroen F.J.; Stoneking, Mark; de Knijff, Peter; Kayser, Manfred; Veldink, Jan H.; van den Berg, Leonard H.; Byelas, Heorhiy; den Dunnen, Johan T.; Dijkstra, Martijn; Amin, Najaf; Joeri van der Velde, K.; van Setten, Jessica; Kattenberg, Mathijs; van Schaik, Barbera D.C.; Bot, Jan; Nijman, Isaäc J.; Mei, Hailiang; Koval, Vyacheslav; Ye, Kai; Lameijer, Eric-Wubbo; Moed, Matthijs H.; Hehir-Kwa, Jayne Y.; Handsaker, Robert E.; Sunyaev, Shamil R.; Sohail, Mashaal; Hormozdiari, Fereydoun; Marschall, Tobias; Schönhuth, Alexander; Guryev, Victor; Suchiman, H. Eka D.; Wolffenbuttel, Bruce H.; Platteel, Mathieu; Pitts, Steven J.; Potluri, Shobha; Cox, David R.; Li, Qibin; Li, Yingrui; Du, Yuanping; Chen, Ruoyan; Cao, Hongzhi; Li, Ning; Cao, Sujie; Wang, Jun; Bovenberg, Jasper A.; Jukema, J. Wouter; van der Harst, Pim; Sijbrands, Eric J.; Hottenga, Jouke-Jan; Uitterlinden, Andre G.; Swertz, Morris A.; van Ommen, Gert-Jan B.; de Bakker, Paul I.W.; Eline Slagboom, P.; Boomsma, Dorret I.; Wijmenga, Cisca; van Duijn, Cornelia M.

    2015-01-01

    Variants associated with blood lipid levels may be population-specific. To identify low-frequency variants associated with this phenotype, population-specific reference panels may be used. Here we impute nine large Dutch biobanks (~35,000 samples) with the population-specific reference panel created by the Genome of the Netherlands Project and perform association testing with blood lipid levels. We report the discovery of five novel associations at four loci (P value <6.61 × 10−4), including a rare missense variant in ABCA6 (rs77542162, p.Cys1359Arg, frequency 0.034), which is predicted to be deleterious. The frequency of this ABCA6 variant is 3.65-fold increased in the Dutch and its effect (βLDL-C=0.135, βTC=0.140) is estimated to be very similar to those observed for single variants in well-known lipid genes, such as LDLR. PMID:25751400

  8. Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation

    PubMed Central

    Ovejero, Sara; Ayala, Patricia; Bueno, Avelino; Sacristán, María P.

    2012-01-01

    The activity of Cdk1–cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation. The Cdk1-dependent phosphorylation of Wee1 primes phosphorylation by additional kinases such as Plk1, triggering Wee1 degradation at the onset of mitosis. Here we report that Cdc14A plays an important role in the regulation of Wee1 stability. Depletion of Cdc14A results in a significant reduction in Wee1 protein levels. Cdc14A binds to Wee1 at its amino-terminal domain and reverses CDK-mediated Wee1 phosphorylation. In particular, we found that Cdc14A inhibits Wee1 degradation through the dephosphorylation of Ser-123 and Ser-139 residues. Thus the lack of phosphorylation of these two residues prevents the interaction with Plk1 and the consequent efficient Wee1 degradation at the onset of mitosis. These data support the hypothesis that Cdc14A counteracts Cdk1–cyclin B1 activity through Wee1 dephosphorylation. PMID:23051732

  9. Rat organic solute carrier protein 1 (rOscp1) mediated the transport of organic solutes in Xenopus laevis oocytes: isolation and pharmacological characterization of rOscp1.

    PubMed

    Izuno, Hisanori; Kobayashi, Yasuna; Sanada, Yutaka; Nihei, Daisuke; Suzuki, Masako; Kohyama, Noriko; Ohbayashi, Masayuki; Yamamoto, Toshinori

    2007-09-22

    Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a Km value of 15.7+/-1.9 microM, and rOscp1-mediated organic solutes were exhibited in time- and Na+-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239-1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1.

  10. ERK1/2 mediates glucose-regulated POMC gene expression in hypothalamic neurons.

    PubMed

    Zhang, Juan; Zhou, Yunting; Chen, Cheng; Yu, Feiyuan; Wang, Yun; Gu, Jiang; Ma, Lian; Ho, Guyu

    2015-04-01

    Hypothalamic glucose-sensing neurons regulate the expression of genes encoding feeding-related neuropetides POMC, AgRP, and NPY - the key components governing metabolic homeostasis. AMP-activated protein kinase (AMPK) is postulated to be the molecular mediator relaying glucose signals to regulate the expression of these neuropeptides. Whether other signaling mediator(s) plays a role is not clear. In this study, we investigated the role of ERK1/2 using primary hypothalamic neurons as the model system. The primary neurons were differentiated from hypothalamic progenitor cells. The differentiated neurons possessed the characteristic neuronal cell morphology and expressed neuronal post-mitotic markers as well as leptin-regulated orexigenic POMC and anorexigenic AgRP/NPY genes. Treatment of cells with glucose dose-dependently increased POMC and decreased AgRP/NPY expression with a concurrent suppression of AMPK phosphorylation. In addition, glucose treatment dose-dependently increased the ERK1/2 phosphorylation. Blockade of ERK1/2 activity with its specific inhibitor PD98059 partially (approximately 50%) abolished glucose-induced POMC expression, but had little effect on AgRP/NPY expression. Conversely, blockade of AMPK activity with its specific inhibitor produced a partial (approximately 50%) reversion of low-glucose-suppressed POMC expression, but almost completely blunted the low-glucose-induced AgRP/NPY expression. The results indicate that ERK1/2 mediated POMC but not AgRP/NPY expression. Confirming the in vitro findings, i.c.v. administration of PD98059 in rats similarly attenuated glucose-induced POMC expression in the hypothalamus, but again had little effect on AgRP/NPY expression. The results are indicative of a novel role of ERK1/2 in glucose-regulated POMC expression and offer new mechanistic insights into hypothalamic glucose sensing. © 2015 Society for Endocrinology.

  11. IQGAP1 mediates the disruption of adherens junctions to promote Escherichia coli K1 invasion of brain endothelial cells.

    PubMed

    Krishnan, Subramanian; Fernandez, G Esteban; Sacks, David B; Prasadarao, Nemani V

    2012-09-01

    The transcellular entry of Escherichia coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain oedema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with its receptor, Ecgp96, to induce PKC-α phosphorylation, adherens junction (AJ) disassembly (by dislodging β-catenin from VE-cadherin), and remodelling of actin in HBMEC. We report here that IQGAP1 mediates β-catenin dissociation from AJs to promote actin polymerization required for E. coli K1 invasion of HBMEC. Overexpression of C-terminal truncated IQGAP1 (IQΔC) that cannot bind β-catenin prevents both AJ disruption and E. coli K1 entry. Of note, phospho-PKC-α interacts with the C-terminal portion of Ecgp96 as well as with VE-cadherin after IQGAP1-mediated AJ disassembly. HBMEC overexpressing either C-terminal truncated Ecgp96 (Ecgp96Δ200) or IQΔC upon infection with E. coli showed no interaction of phospho-PKC-α with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC-α phosphorylation and association of phospho-PKC-α with Ecgp96, and then signals IQGAP1 to detach β-catenin from AJs. Subsequently, IQGAP1/β-catenin bound actin translocates to the site of E. coli K1 attachment to promote invasion. © 2012 Blackwell Publishing Ltd.

  12. IQGAP1 mediates the disruption of adherens junctions to promote Escherichia coli K1 invasion of brain endothelial cells

    PubMed Central

    Krishnan, Subramanian; Fernandez, G. Esteban; Sacks, David B.; Prasadarao, Nemani V.

    2012-01-01

    The transcellular entry of E. coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain edema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with its receptor, Ecgp96 to induce PKC-α phosphorylation, adherens junction (AJ) disassembly (by dislodging β-catenin from VE-cadherin), and remodeling of actin in HBMEC. We report here that IQGAP1 mediates β-catenin dissociation from AJs to promote actin polymerization required for E. coli K1 invasion of HBMEC. Overexpression of C-terminal truncated IQGAP1 (IQΔC) that cannot bind β-catenin prevents both AJ disruption and E. coli K1 entry. Of note, phospho-PKC-α interacts with the C-terminal portion of Ecgp96 as well as with VE-cadherin after IQGAP1 mediated AJ disassembly. HBMEC overexpressing either C-terminal truncated Ecgp96 (Ecgp96Δ200) or IQΔC upon infection with E. coli showed no interaction of phospho-PKC-α with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC-α phosphorylation and association of phospho-PKC-α with Ecgp96, and then signals IQGAP1 to detach β-catenin from AJs. Subsequently, IQGAP1/β-catenin bound actin translocates to the site of E. coli K1 attachment to promote invasion. PMID:22519731

  13. Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-α induction

    PubMed Central

    Uematsu, Satoshi; Sato, Shintaro; Yamamoto, Masahiro; Hirotani, Tomonori; Kato, Hiroki; Takeshita, Fumihiko; Matsuda, Michiyuki; Coban, Cevayir; Ishii, Ken J.; Kawai, Taro; Takeuchi, Osamu; Akira, Shizuo

    2005-01-01

    Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-α in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7 signaling pathway. IRAK-1 directly bound and phosphorylated IRF7 in vitro. The kinase activity of IRAK-1 was necessary for transcriptional activation of IRF7. TLR7- and TLR9-mediated IFN-α production was abolished in Irak-1–deficient mice, whereas inflammatory cytokine production was not impaired. Despite normal activation of NF-κB and mitogen-activated protein kinases, IRF7 was not activated by a TLR9 ligand in Irak-1–deficient pDCs. These results indicated that IRAK-1 is a specific regulator for TLR7- and TLR9-mediated IFN-α induction in pDCs. PMID:15767370

  14. Intrarenal Mas and AT1 receptors play a role in mediating the excretory actions of renal interstitial angiotensin-(1-7) infusion in anaesthetized rats.

    PubMed

    O'Neill, Julie; Healy, Vincent; Johns, Edward J

    2017-12-01

    What is the central question of this study? Dietary sodium manipulation alters the magnitude of angiotensin-(1-7) [Ang-(1-7)]-induced natriuresis. The present study sought to determine whether this was related to relative changes in the activity of intrarenal Mas and/or AT 1 receptors. What is the main finding and its importance? Angiotensin-(1-7)-induced diuresis and natriuresis is mediated by intrarenal Mas receptors. However, intrarenal AT 1 receptor blockade also had an inhibitory effect on Ang-(1-7)-induced natriuresis and diuresis. Thus, Ang-(1-7)-induced increases in sodium and water excretion are dependent upon functional Mas and AT 1 receptors. We investigated whether angiotensin-(1-7) [Ang-(1-7)]-induced renal haemodynamic and excretory actions were solely dependent upon intrarenal Mas receptor activation or required functional angiotensin II type 1 (AT 1 ) receptors. The renin-angiotensin system was enhanced in anaesthetized rats by prior manipulation of dietary sodium intake. Angiotensin-(1-7) and AT 1 and Mas receptor antagonists were infused into the kidney at the corticomedullary border. Mas receptor expression was measured in the kidney. Mean arterial pressure, urine flow and fractional sodium excretion were 93 ± 4 mmHg, 46.1 ± 15.7 μl min -1  kg -1 and 1.4 ± 0.3%, respectively, in the normal-sodium group and 91 ± 2 mmHg, 19.1 ± 3.3 μl min -1  kg -1 and 0.7 ± 0.2%, respectively, in the low-sodium group. Angiotensin-(1-7) infusion had no effect on mean arterial pressure in rats receiving a normal-sodium diet but decreased it by 4 ± 5% in rats receiving a low-sodium diet (P < 0.05). Interstitial Ang-(1-7) infusion increased urine flow twofold and fractional sodium excretion threefold (P < 0.05) in rats receiving a normal-sodium diet and to a greater extent, approximately three- and fourfold, respectively, in rats receiving the low-sodium diet (both P < 0.05). Angiotensin-(1-7)-induced increases in urine flow and

  15. DNA methylation as a mediator of HLA-DRB1*15:01 and a protective variant in multiple sclerosis.

    PubMed

    Kular, Lara; Liu, Yun; Ruhrmann, Sabrina; Zheleznyakova, Galina; Marabita, Francesco; Gomez-Cabrero, David; James, Tojo; Ewing, Ewoud; Lindén, Magdalena; Górnikiewicz, Bartosz; Aeinehband, Shahin; Stridh, Pernilla; Link, Jenny; Andlauer, Till F M; Gasperi, Christiane; Wiendl, Heinz; Zipp, Frauke; Gold, Ralf; Tackenberg, Björn; Weber, Frank; Hemmer, Bernhard; Strauch, Konstantin; Heilmann-Heimbach, Stefanie; Rawal, Rajesh; Schminke, Ulf; Schmidt, Carsten O; Kacprowski, Tim; Franke, Andre; Laudes, Matthias; Dilthey, Alexander T; Celius, Elisabeth G; Søndergaard, Helle B; Tegnér, Jesper; Harbo, Hanne F; Oturai, Annette B; Olafsson, Sigurgeir; Eggertsson, Hannes P; Halldorsson, Bjarni V; Hjaltason, Haukur; Olafsson, Elias; Jonsdottir, Ingileif; Stefansson, Kari; Olsson, Tomas; Piehl, Fredrik; Ekström, Tomas J; Kockum, Ingrid; Feinberg, Andrew P; Jagodic, Maja

    2018-06-19

    The human leukocyte antigen (HLA) haplotype DRB1*15:01 is the major risk factor for multiple sclerosis (MS). Here, we find that DRB1*15:01 is hypomethylated and predominantly expressed in monocytes among carriers of DRB1*15:01. A differentially methylated region (DMR) encompassing HLA-DRB1 exon 2 is particularly affected and displays methylation-sensitive regulatory properties in vitro. Causal inference and Mendelian randomization provide evidence that HLA variants mediate risk for MS via changes in the HLA-DRB1 DMR that modify HLA-DRB1 expression. Meta-analysis of 14,259 cases and 171,347 controls confirms that these variants confer risk from DRB1*15:01 and also identifies a protective variant (rs9267649, p < 3.32 × 10 -8 , odds ratio = 0.86) after conditioning for all MS-associated variants in the region. rs9267649 is associated with increased DNA methylation at the HLA-DRB1 DMR and reduced expression of HLA-DRB1, suggesting a modulation of the DRB1*15:01 effect. Our integrative approach provides insights into the molecular mechanisms of MS susceptibility and suggests putative therapeutic strategies targeting a methylation-mediated regulation of the major risk gene.

  16. Multidrug Resistance Protein 1 (MRP1, ABCC1), a “Multitasking” ATP-binding Cassette (ABC) Transporter*

    PubMed Central

    Cole, Susan P. C.

    2014-01-01

    The multidrug resistance protein 1 (MRP1) encoded by ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. However, it is now clear that MRP1 serves a broader role than simply mediating the ATP-dependent efflux of drugs from cells. The antioxidant GSH and the pro-inflammatory cysteinyl leukotriene C4 have been identified as key physiological organic anions effluxed by MRP1, and an ever growing body of evidence indicates that additional lipid-derived mediators are also substrates of this transporter. As such, MRP1 is a multitasking transporter that likely influences the etiology and progression of a host of human diseases. PMID:25281745

  17. HIV-1 Nef in Macrophage-Mediated Disease Pathogenesis

    PubMed Central

    Lamers, Susanna L.; Fogel, Gary B.; Singer, Elyse J.; Salemi, Marco; Nolan, David J.; Huysentruyt, Leanne C.; McGrath, Michael S.

    2013-01-01

    Combined anti-retroviral therapy (cART) has significantly reduced the number of AIDS-associated illnesses and changed the course of HIV-1 disease in developed countries. Despite the ability of cART to maintain high CD4+ T-cell counts, a number of macrophage-mediated diseases can still occur in HIV-infected subjects. These diseases include lymphoma, metabolic diseases, and HIV-associated neurological disorders. Within macrophages, the HIV-1 regulatory protein “Nef” can modulate surface receptors, interact with signaling pathways, and promote specific environments that contribute to each of these pathologies. Moreover, genetic variation in Nef may also guide the macrophage response. Herein, we review findings relating to the Nef–macrophage interaction and how this relationship contributes to disease pathogenesis. PMID:23215766

  18. Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration

    PubMed Central

    Wang, Shujie; Watanabe, Takashi; Matsuzawa, Kenji; Katsumi, Akira; Kakeno, Mai; Matsui, Toshinori; Ye, Feng; Sato, Kazuhide; Murase, Kiyoko; Sugiyama, Ikuko; Kimura, Kazushi; Mizoguchi, Akira; Ginsberg, Mark H.; Collard, John G.

    2012-01-01

    Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration. PMID:23071154

  19. Structural Insights into the Niemann-Pick C1 (NPC1)-Mediated Cholesterol Transfer and Ebola Infection.

    PubMed

    Gong, Xin; Qian, Hongwu; Zhou, Xinhui; Wu, Jianping; Wan, Tao; Cao, Pingping; Huang, Weiyun; Zhao, Xin; Wang, Xudong; Wang, Peiyi; Shi, Yi; Gao, George F; Zhou, Qiang; Yan, Nieng

    2016-06-02

    Niemann-Pick disease type C (NPC) is associated with mutations in NPC1 and NPC2, whose gene products are key players in the endosomal/lysosomal egress of low-density lipoprotein-derived cholesterol. NPC1 is also the intracellular receptor for Ebola virus (EBOV). Here, we present a 4.4 Å structure of full-length human NPC1 and a low-resolution reconstruction of NPC1 in complex with the cleaved glycoprotein (GPcl) of EBOV, both determined by single-particle electron cryomicroscopy. NPC1 contains 13 transmembrane segments (TMs) and three distinct lumenal domains A (also designated NTD), C, and I. TMs 2-13 exhibit a typical resistance-nodulation-cell division fold, among which TMs 3-7 constitute the sterol-sensing domain conserved in several proteins involved in cholesterol metabolism and signaling. A trimeric EBOV-GPcl binds to one NPC1 monomer through the domain C. Our structural and biochemical characterizations provide an important framework for mechanistic understanding of NPC1-mediated intracellular cholesterol trafficking and Ebola virus infection. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. PTEN-mediated ERK1/2 inhibition and paradoxical cellular proliferation following Pnck overexpression

    PubMed Central

    Deb, Tushar B; Barndt, Robert J; Zuo, Annie H; Sengupta, Surojeet; Coticchia, Christine M; Johnson, Michael D

    2014-01-01

    Pregnancy upregulated non-ubiquitous calmodulin kinase (Pnck), a novel calmodulin kinase, is significantly overexpressed in breast and renal cancers. We present evidence that at high cell density, overexpression of Pnck in HEK 293 cells inhibits serum-induced extracellular signal-regulated kinase (ERK1/ERK2) activation. ERK1/2 inhibition is calcium-dependent and Pnck kinase activity is required for ERK1/2 inhibition, since expression of a kinase-dead (K44A) and a catalytic loop phosphorylation mutant (T171A) Pnck protein is unable to inhibit ERK 1/2 activity. Ras is constitutively active at high cell density, and Pnck does not alter Ras activation, suggesting that Pnck inhibition of ERK1/2 activity is independent of Ras activity. Pnck inhibition of serum-induced ERK1/2 activity is lost in cells in which phosphatase and tensin homolog (PTEN) is suppressed, suggesting that Pnck inhibition of ERK1/2 activity is mediated by PTEN. Overexpression of protein phosphatase-active but lipid phosphatase-dead PTEN protein inhibits ERK1/2 activity in control cells and enhances Pnck-mediated ERK1/2 inhibition, suggesting that Pnck increases availability of protein phosphatase active PTEN for ERK1/2 inhibition. Pnck is a stress-responsive kinase; however, serum-induced p38 MAP kinase activity is also downregulated by Pnck in a Pnck kinase- and PTEN-dependent manner, similar to ERK1/2 inhibition. Pnck overexpression increases proliferation, which is inhibited by PTEN knockdown, implying that PTEN acts as a paradoxical promoter of proliferation in ERK1/2 and p38 MAP kinase phosphorylation-inhibited, Pnck-overexpressing cells. Overall, these data reveal a novel function of Pnck in the regulation of ERK1/2 and p38 MAP kinase activity and cell proliferation, which is mediated by paradoxical PTEN functions. The possible biological implications of these data are discussed. PMID:24552815

  1. β-Adrenergic Receptor Stimulated Ncx1 Upregulation is Mediated via a CaMKII/AP-1 Signaling Pathway in Adult Cardiomyocytes

    PubMed Central

    Mani, Santhosh K.; Egan, Erin A.; Addy, Benjamin K.; Grimm, Michael; Kasiganesan, Harinath; Thiyagarajan, Thirumagal; Renaud, Ludivine; Brown, Joan Heller; Kern, Christine B.; Menick, Donald R.

    2013-01-01

    The Na+-Ca2+ exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. β-adrenergic receptor (β-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte but chronic activation in periods of cardiac stress contribute to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKIIδc) null mouse, we demonstrate that β-AR-stimulated Ncx1 upregulation is dependent on CaMKII. β-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that β-AR stimulation activates the ordered recruitment of JunB homodimers which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically β-AR-stimulated heart. PMID:19945464

  2. A zebrafish transgenic model of Ewing's sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis.

    PubMed

    Leacock, Stefanie W; Basse, Audrey N; Chandler, Garvin L; Kirk, Anne M; Rakheja, Dinesh; Amatruda, James F

    2012-01-01

    Ewing's sarcoma, a malignant bone tumor of children and young adults, is a member of the small-round-blue-cell tumor family. Ewing's sarcoma family tumors (ESFTs), which include peripheral primitive neuroectodermal tumors (PNETs), are characterized by chromosomal translocations that generate fusions between the EWS gene and ETS-family transcription factors, most commonly FLI1. The EWS-FLI1 fusion oncoprotein represents an attractive therapeutic target for treatment of Ewing's sarcoma. The cell of origin of ESFT and the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown, and few animal models of Ewing's sarcoma exist. Here, we report the use of zebrafish as a vertebrate model of EWS-FLI1 function and tumorigenesis. Mosaic expression of the human EWS-FLI1 fusion protein in zebrafish caused the development of tumors with histology strongly resembling that of human Ewing's sarcoma. The incidence of tumors increased in a p53 mutant background, suggesting that the p53 pathway suppresses EWS-FLI1-driven tumorigenesis. Gene expression profiling of the zebrafish tumors defined a set of genes that might be regulated by EWS-FLI1, including the zebrafish ortholog of a crucial EWS-FLI1 target gene in humans. Stable zebrafish transgenic lines expressing EWS-FLI1 under the control of the heat-shock promoter exhibit altered embryonic development and defective convergence and extension, suggesting that EWS-FLI1 interacts with conserved developmental pathways. These results indicate that functional targets of EWS-FLI1 that mediate tumorigenesis are conserved from zebrafish to human and provide a novel context in which to study the function of this fusion oncogene.

  3. Transient Receptor Potential Vanilloid-1 (TRPV1) Is a Mediator of Lung Toxicity for Coal Fly Ash Particulate Material

    PubMed Central

    Deering-Rice, Cassandra E.; Johansen, Mark E.; Roberts, Jessica K.; Thomas, Karen C.; Romero, Erin G.; Lee, Jeewoo; Yost, Garold S.; Veranth, John M.

    2012-01-01

    Environmental particulate matter (PM) pollutants adversely affect human health, but the molecular basis is poorly understood. The ion channel transient receptor potential vanilloid-1 (TRPV1) has been implicated as a sensor for environmental PM and a mediator of adverse events in the respiratory tract. The objectives of this study were to determine whether TRPV1 can distinguish chemically and physically unique PM that represents important sources of air pollution; to elucidate the molecular basis of TRPV1 activation by PM; and to ascertain the contributions of TRPV1 to human lung cell and mouse lung tissue responses exposed to an insoluble PM agonist, coal fly ash (CFA1). The major findings of this study are that TRPV1 is activated by some, but not all of the prototype PM materials evaluated, with rank-ordered responses of CFA1 > diesel exhaust PM > crystalline silica; TRP melastatin-8 is also robustly activated by CFA1, whereas other TRP channels expressed by airway sensory neurons and lung epithelial cells that may also be activated by CFA1, including TRPs ankyrin 1 (A1), canonical 4α (C4α), M2, V2, V3, and V4, were either slightly (TRPA1) or not activated by CFA1; activation of TRPV1 by CFA1 occurs via cell surface interactions between the solid components of CFA1 and specific amino acid residues of TRPV1 that are localized in the putative pore-loop region; and activation of TRPV1 by CFA1 is not exclusive in mouse lungs but represents a pathway by which CFA1 affects the expression of selected genes in lung epithelial cells and airway tissue. PMID:22155782

  4. Selective Small Molecule Compounds Increase BMP-2 Responsiveness by Inhibiting Smurf1-mediated Smad1/5 Degradation

    PubMed Central

    Cao, Yu; Wang, Cheng; Zhang, Xueli; Xing, Guichun; Lu, Kefeng; Gu, Yongqing; He, Fuchu; Zhang, Lingqiang

    2014-01-01

    The ubiquitin ligase Smad ubiquitination regulatory factor-1 (Smurf1) negatively regulates bone morphogenetic protein (BMP) pathway by ubiquitinating certain signal components for degradation. Thus, it can be an eligible pharmacological target for increasing BMP signal responsiveness. We established a strategy to discover small molecule compounds that block the WW1 domain of Smurf1 from interacting with Smad1/5 by structure based virtual screening, molecular experimental examination and cytological efficacy evaluation. Our selected hits could reserve the protein level of Smad1/5 from degradation by interrupting Smurf1-Smad1/5 interaction and inhibiting Smurf1 mediated ubiquitination of Smad1/5. Further, these compounds increased BMP-2 signal responsiveness and the expression of certain downstream genes, enhanced the osteoblastic activity of myoblasts and osteoblasts. Our work indicates targeting Smurf1 for inhibition could be an accessible strategy to discover BMP-sensitizers that might be applied in future clinical treatments of bone disorders such as osteopenia. PMID:24828823

  5. Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis

    PubMed Central

    Marques Howarth, Michelle; Simpson, David; Ngok, Siu P.; Nieves, Bethsaida; Chen, Ron; Siprashvili, Zurab; Vaka, Dedeepya; Breese, Marcus R.; Crompton, Brian D.; Alexe, Gabriela; Hawkins, Doug S.; Jacobson, Damon; Brunner, Alayne L.; West, Robert; Mora, Jaume; Stegmaier, Kimberly; Khavari, Paul; Sweet-Cordero, E. Alejandro

    2014-01-01

    Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma–associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes. PMID:25401475

  6. Pro-resolving lipid mediator Resolvin D1 serves as a marker of lung disease in cystic fibrosis.

    PubMed

    Eickmeier, Olaf; Fussbroich, Daniela; Mueller, Klaus; Serve, Friederike; Smaczny, Christina; Zielen, Stefan; Schubert, Ralf

    2017-01-01

    Cystic fibrosis (CF) is an autosomal recessive genetic disorder that affects multiple organs, including the lungs, pancreas, liver and intestine. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) locus lead to defective proteins and reduced Cl- secretion and Na+ hyperabsorption in the affected organs. In addition, patients suffering from CF display chronic inflammation that contributes to the pathogenesis of CF. Recent work suggests that CF patients have a reduced capacity to biosynthesize specialized pro-resolving lipid mediators (SPMs), which contributes to the development and duration of the unwanted inflammation. Alterations in the metabolism of arachidonic acid (AA) and docosahexaenoic acid (DHA) to specialized pro-resolving mediators (SPMs), like lipoxins (LXs), maresins (MaRs), protectins (PDs) and resolvins (Rvs), may play a major role on clinical impact of airway inflammation in CF. In this study, our aims were to detect and quantitate Resolvin D1 (RvD1) in sputum and plasma from patients with CF and compare levels of RvD1 with biomarkers of inflammation and lung function. We studied 27 CF patients aged 6 to 55 years (median 16 years) in a prospective approach. DHA can be found in the plasma of our CF patients in the milligram range and is decreased in comparison to a healthy control group. The DHA-derived pro-resolving mediator Resolvin D1 (RvD1) was also present in the plasma (286.4 ± 50 pg/ mL, mean ± SEM) and sputum (30.0 ± 2.6 pg/ mL, mean ± SEM) samples from our patients with CF and showed a positive correlation with sputum inflammatory markers. The plasma concentrations of RvD1 were ten times higher than sputum concentrations. Interestingly, sputum RvD1/ IL-8 levels showed a positive correlation with FEV1 (rs = 0.3962, p< 0.05). SPMs, like RvD1, are well known to down-regulate inflammatory pathways. Our study shows that the bioactive lipid mediator RvD1, derived from DHA, was present in sputum and plasma of CF patients

  7. A dynamic ribosomal biogenesis response is not required for IGF-1-mediated hypertrophy of human primary myotubes.

    PubMed

    Crossland, Hannah; Timmons, James A; Atherton, Philip J

    2017-12-01

    Increased ribosomal DNA transcription has been proposed to limit muscle protein synthesis, making ribosome biogenesis central to skeletal muscle hypertrophy. We examined the relationship between ribosomal RNA (rRNA) production and IGF-1-mediated myotube hypertrophy in vitro Primary skeletal myotubes were treated with IGF-1 (50 ng/ml) with or without 0.5 µM CX-5461 (CX), an inhibitor of RNA polymerase I. Myotube diameter, total protein, and RNA and DNA levels were measured along with markers of RNA polymerase I regulatory factors and regulators of protein synthesis. CX treatment reduced 45S pre-rRNA expression (-64 ± 5% vs. IGF-1; P < 0.001) and total RNA content (-16 ± 2% vs. IGF-1; P < 0.001) in IGF-1-treated myotubes. IGF-1-mediated increases in myotube diameter (1.27 ± 0.09-fold, P < 0.05 vs. control) and total protein (+20 ± 2%; P < 0.001 vs. control) were not prevented by CX treatment. Suppression of rRNA synthesis during IGF-1 treatment did not prevent early increases in AKT (+203 ± 39% vs. CX; P < 0.001) and p70 S6K1 (269 ± 41% vs. CX; P < 0.001) phosphorylation. Despite robust inhibition of the dynamic ribosomal biogenesis response to IGF-1, myotube diameter and protein accretion were sustained. Thus, while ribosome biogenesis represents a potential site for the regulation of skeletal muscle protein synthesis and muscle mass, it does not appear to be a prerequisite for IGF-1-induced myotube hypertrophy in vitro. -Crossland, H., Timmons, J. A., Atherton, P. J. A dynamic ribosomal biogenesis response is not required for IGF-1-mediated hypertrophy of human primary myotubes. © The Author(s).

  8. Regression of atherosclerosis with apple procyanidins by activating the ATP-binding cassette subfamily A member 1 in a rabbit model.

    PubMed

    Wang, Liang; Fumoto, Toshio; Masumoto, Saeko; Shoji, Toshihiko; Miura, Tomisato; Naraoka, Masato; Matsuda, Naoya; Imaizumi, Tadaatsu; Ohkuma, Hiroki

    2017-03-01

    Apple polyphenol contains abundant procyanidins, which have been associated with an anti-atherosclerosis and cholesterol-lowering effect. The aim of this study was to investigate whether apple procyanidins (APCs) feature therapeutic efficacy in terms of regressing atherosclerosis and whether this efficacy is due to mechanisms other than a cholesterol-lowering effect. After eight weeks on an atherogenic diet, rabbits were given a normal diet for another eight weeks to normalize the increased serum lipids level. The rabbits in the baseline group were sacrificed at this stage. The control group was subsequently fed a normal diet for eight weeks, while the APCs group was administrated 50 mg/kg/day of APCs in addition to the normal diet. Serum lipids and aortic intimal-medial thickness (IMT) were serially examined, and the resected aorta was examined histologically and through molecular biology. Aortic IMT on ultrasonography and the lipid accumulation area examined using Sudan IV staining were significantly reduced in the APCs group as compared to the control group. Serum lipid profiles were not different between the groups. Immunohistochemistry showed significantly decreased staining of an oxidative stress marker and significantly increased staining of ATP-binding cassette subfamily A member 1 (ABCA1) in the APCs group. Western blotting and RT-PCR also showed increased expression of ABCA1 mRNA and its protein in the APCs group. This study revealed that APCs administration causes a regression of atherosclerosis. APCs might hold promise as an anti-atherosclerotic agent. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Role of NF-Kappa B Signaling in X-Box Binding Protein 1 (XBP1)-Mediated Antiestrogen Resistance in Breast Cancer

    DTIC Science & Technology

    2011-10-01

    cells. In this study, we aim to investigate the mechanism of XBP1-mediated antiestorgen resistance, specifically the involvement of NFkappaB ...signaling. We found that XBP1 regulates NFkappaB signaling at least at two levels. One, XBP1-S regulates RelA expression at the mRNA level; Second, XBP1...regulates NFkappaB transcriptional activity through ERalpha signaling. Furthermore, inhibition of NFkappaB with either Parthenolide (small molecule

  10. Regulation of androgen receptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation.

    PubMed

    Gaughan, Luke; Logan, Ian R; Neal, David E; Robson, Craig N

    2005-01-01

    The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors and plays a critical role in regulating the expression of genes involved in androgen-dependent and -independent tumour formation. Regulation of the AR is achieved by alternate binding of either histone acetyltransferase (HAT)-containing co-activator proteins, or histone deacetylase 1 (HDAC1). Factors that control AR stability may also constitute an important regulatory mechanism, a notion that has been confirmed with the finding that the AR is a direct target for Mdm2-mediated ubiquitylation and proteolysis. Using chromatin immunoprecipitation (ChIP) and re-ChIP analyses, we show that Mdm2 associates with AR and HDAC1 at the active androgen-responsive PSA promoter in LNCaP prostate cancer cells. Furthermore, we demonstrate that Mdm2-mediated modification of AR and HDAC1 catalyses protein destabilization and attenuates AR sactivity, suggesting that ubiquitylation of the AR and HDAC1 may constitute an additional mechanism for regulating AR function. We also show that HDAC1 and Mdm2 function co-operatively to reduce AR-mediated transcription that is attenuated by the HAT activity of the AR co-activator Tip60, suggesting interplay between acetylation status and receptor ubiquitylation in AR regulation. In all, our data indicates a novel role for Mdm2 in regulating components of the AR transcriptosome.

  11. Predicting Progression of ABCA4-Associated Retinal Degenerations Based on Longitudinal Measurements of the Leading Disease Front

    PubMed Central

    Cideciyan, Artur V.; Swider, Malgorzata; Schwartz, Sharon B.; Stone, Edwin M.; Jacobson, Samuel G.

    2015-01-01

    Purpose To evaluate the progression of the earliest stage of disease in ABCA4-associated retinal degenerations (RDs). Methods Near-infrared excited reduced-illuminance autofluorescence imaging was acquired across the retina up to 80 degrees eccentricity in 44 patients with two ABCA4 alleles. The eccentricity of the leading disease front (LDF) corresponding to the earliest stage of disease was measured along the four meridians. A mathematical model describing the expansion of the LDF was developed based on 6 years of longitudinal follow-up. Results The extent of LDF along the superior, inferior, and temporal meridians showed a wide spectrum from 3.5 to 70 degrees. In patients with longitudinal data, the average centrifugal expansion rate was 2 degrees per year. The nasal extent of LDF between the fovea and ONH ranged from 4.3 to 16.5 degrees and expanded at 0.35 degrees per year. The extent of LDF beyond ONH ranged from 19 to 75 degrees and expanded on average at 2 degrees per year. A mathematical model fit well to the longitudinal data describing the expansion of the LDF. Conclusions The eccentricity of the LDF in ABCA4-RD shows a continuum from parafovea to far periphery along all four meridians consistent with a wide spectrum of severity observed clinically. The model of progression may provide a quantitative prediction of the LDF expansion based on the age and eccentricity of the LDF at a baseline visit, and thus contribute significantly to the enrollment of candidates appropriate for clinical trials planning specific interventions, efficacy outcomes, and durations. PMID:26377081

  12. Angeli's Salt, a nitroxyl anion donor, reverses endothelin-1 mediated vascular dysfunction in murine aorta.

    PubMed

    Wynne, Brandi M; Labazi, Hicham; Carneiro, Zidonia N; Tostes, Rita C; Webb, R Clinton

    2017-11-05

    Nitroglycerin (Gtn) is a treatment for cardiovascular patients due to its vasodilatory actions, but induces tolerance when given chronically. A proposed mechanism is the superoxide (O 2 - )-oxidative stress hypothesis, which suggests that Gtn increases O 2 - production. Nitric oxide (NO) exists in three different redox states; the protonated, reduced state, nitroxyl anion (HNO) is an emerging candidate in vascular regulation. HNO is resistant to scavenging and of particular interest in conditions where high levels of reactive oxygen species (ROS) exist. We hypothesize that treatment with Gtn will exacerbate endothelin 1 (ET-1) induced vascular dysfunction via an increase in ROS, while treatment with Angeli's Salt (AS), an HNO donor, will not. Aorta from mice were isolated and divided into four groups: vehicle, ET-1 [0.1μM, 1μM], ET-1+Gtn [Gtn 1μM] and ET-1+AS [AS 1μM]. Concentration response curves (CRCs) to acetylcholine (ACh) and phenylephrine (Phe) were performed. Aorta incubated with ET-1 (for 20-22h) exhibited a decreased relaxation response to ACh and an increase in Phe-mediated contraction. Aorta incubated with AS exhibited a reversal in ET-1 induced vascular and endothelial dysfunction. ET-1 increased ROS in aortic vascular smooth muscle cells (VSMCs), visualized by dihydroethidium (DHE) staining. AS incubated reduced this ROS generation, yet maintained with Gtn treatment. These data suggest that aorta incubated with the HNO donor, AS, can reverse ET-1 mediated vascular dysfunction, which may be through a decrease or prevention of ROS generation. We propose that HNO may be vasoprotective and that HNO donors studied as a therapeutic option where other organic nitrates are contraindicative. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Halvorsen, Bente, E-mail: Bente.Halvorsen@rr-research.no; Institute of Clinical Medicine, University of Oslo, Oslo; K.G. Jebsen Inflammation Research Center, University of Oslo, Oslo

    Highlights: • IL-10 promotes reverse cholesterol efflux from lipid loaded macrophages. • IL-10 increases the expression of ABCA-1 and ABCG-1. • IL-10 exhibits cross-talk with the nuclear receptor LXRα. - Abstract: Interleukin (IL)-10 is a prototypical anti-inflammatory cytokine that has been shown to attenuate atherosclerosis development. In addition to its anti-inflammatory properties, the anti-atherogenic effect of IL-10 has recently also been suggested to reflect a complex effect of IL-10 on lipid metabolism in macrophages. In the present study we examined the effects of IL-10 on cholesterol efflux mechanism in lipid-loaded THP-1 macrophages. Our main findings were: (i) IL-10 significantly enhancedmore » cholesterol efflux induced by fetal-calf serum, high-density lipoprotein (HDL){sub 2} and apolipoprotein A-1. (ii) The IL-10-mediated effects on cholesterol efflux were accompanied by an increased IL-10-mediated expression of the ATP-binding cassette transporters ABCA1 and ABCG1, that was further enhanced when the cells were co-activated with the liver X receptor (LXR)α agonist (22R)-hydroxycholesterol. (iii) The effect of LXRα activation on the IL-10-mediated effects on the ATP-binding cassette transporters seems to include enhancing effects on the IL-10 receptor 1 (IL10R1) expression and interaction with STAT-3 signaling. (iv) These enhancing effects on ABCA1 and ABCG1 was not seen when the cells were stimulated with the IL-10 family members IL-22 and IL-24. This study suggests that the anti-atherogenic properties of IL-10 may include enhancing effects on cholesterol efflux mechanism that involves cross-talk with LXRα activation.« less

  14. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermalmore » growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and

  15. Piwil1 mediates meiosis during spermatogenesis in chicken.

    PubMed

    Xu, Lu; Chang, Guobin; Ma, Teng; Wang, Hongzhi; Chen, Jing; Li, Zhiteng; Guo, Xiaomin; Wan, Fang; Ren, Lichen; Lu, Wei; Chen, Guohong

    2016-03-01

    Piwil1 mediates spermatogenesis and ensures stable cell division rates in germline cells in mammals. However, the involvement of Piwil1 in poultry spermatogenesis and meiosis is poorly understood. In the present study, we used TaqMan RT-qPCR to characterize Piwil1 mRNA expression in different types of spermatogenic cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia cells (Sa), tetraploid cells (Tp), round sperm cells (Rs), mature sperm, and in PGCs treated with retinoic acid. Our results revealed that Piwil1 is differentially expressed during spermatogenesis in chicken. Compared to PGCs, SSCs, Tp, and Sa, Rs cells presented the highest Piwil1 mRNA expression levels. Retinoic acid significantly upregulated Piwil1 and Stra8 mRNA expression as well as Piwil1 levels in chicken PGCs. In addition, retinoic acid induced PGCs to progress through all the meiotic stages, eventually leading to haploid cell formation, which was determined using flow cytometry and western blot analysis. Taken together, our results showed that during spermatogenesis, Piwil1 was first expressed at low levels in germ stem cells, PGCs, and SSCs. Its expression levels increased during later meiosis stages. Finally, no expression was detected in mature sperm after meiosis. Treatment of PGCs with retinoic acid further demonstrated that Piwil1 plays a key role in meiosis during chicken spermatogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Sulforaphane Reduces HMGB1-Mediated Septic Responses and Improves Survival Rate in Septic Mice.

    PubMed

    Lee, In-Chul; Kim, Dae Yong; Bae, Jong-Sup

    2017-01-01

    Sulforaphane (SFN), a natural isothiocyanate present in cruciferous vegetables such as broccoli and cabbage, is effective in preventing carcinogenesis, diabetes, and inflammatory responses. Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity is emerging as an attractive therapeutic strategy in the management of severe sepsis or septic shock. In this study, we examined the effects of SFN on HMGB1-mediated septic responses and survival rate in a mouse sepsis model. The anti-inflammatory activities of SFN were monitored based on its effects on lipopolysaccharide (LPS)- or cecal ligation and puncture (CLP)-mediated release of HMGB1. The antiseptic activities of SFN were determined by measuring permeability, leukocyte adhesion and migration, and the activation of pro-inflammatory proteins in HMGB1-activated human umbilical vein endothelial cells (HUVECs) and mice. SFN inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. SFN also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with SFN reduced CLP-induced release of HMGB1 and sepsis-related mortality and pulmonary injury in vivo. Our results indicate that SFN is a possible therapeutic agent that can be used to treat various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway.

  17. Mediating factors of coping process in parents of children with type 1 diabetes.

    PubMed

    Oskouie, Fatemeh; Mehrdad, Neda; Ebrahimi, Hossein

    2013-05-14

    Type 1 diabetes is a lifelong condition for children and their parents, the management for which imposes a vast responsibility. This study explores the mediating factors that affect Iranian parents' coping processes with their children's type 1 diabetes. Research was conducted using the grounded theory method. Participants were selected purposefully, and we continued with theoretical sampling. Constant comparative analysis was used to analyze the data. The mediating factors of the parental coping process with their child's diabetes consist of the child's cooperation, crises and experiences, economic challenges, and parental participation in care. Findings highlight the necessity of well-informed nurses with insightful understanding of the mediating factors in parental coping with juvenile diabetes in order to meet the particular needs of this group.

  18. Regulation of Neurospora crassa cell wall remodeling via the cot-1 pathway is mediated by gul-1.

    PubMed

    Herold, Inbal; Yarden, Oded

    2017-02-01

    Impairment of the Neurospora crassa Nuclear DBF2-related kinase-encoding gene cot-1 results in pleiotropic effects, including abnormally thick hyphal cell walls and septa. An increase in the transcript abundance of genes encoding chitin and glucan synthases and the chitinase gh18-5, but not the cell wall integrity pathway transcription factor rlm-1, accompany the phenotypic changes observed. Deletion of chs-5 or chs-7 in a cot-1 background results in a reduction of hyperbranching frequency characteristic of the cot-1 parent. gul-1 (a homologue of the yeast SSD1 gene) encodes a translational regulator and has been shown to partially suppress cot-1. We demonstrate that the high expression levels of the cell wall remodeling genes analyzed is curbed, and reaches near wild type levels, when gul-1 is inactivated. This is accompanied by morphological changes that include reduced cell wall thickness and restoration of normal chitin levels. We conclude that gul-1 is a mediator of cell wall remodeling within the cot-1 pathway.

  19. Didehydro-Cortistatin A inhibits HIV-1 Tat mediated neuroinflammation and prevents potentiation of cocaine reward in Tat transgenic mice

    PubMed Central

    Mediouni, Sonia; Jablonski, Joseph; Paris, Jason J.; Clementz, Mark A.; Thenin-Houssier, Suzie; McLaughlin, Jay P.; Valente, Susana T.

    2015-01-01

    HIV-1 Tat protein has been shown to have a crucial role in HIV-1-associated neurocognitive disorders (HAND), which includes a group of syndromes ranging from undetectable neurocognitive impairment to dementia. The abuse of psychostimulants, such as cocaine, by HIV infected individuals, may accelerate and intensify neurological damage. On the other hand, exposure to Tat potentiates cocaine-mediated reward mechanisms, which further promotes HAND. Here, we show that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid, crosses the blood-brain barrier, cross-neutralizes Tat activity from several HIV-1 clades and decreases Tat uptake by glial cell lines. In addition, dCA potently inhibits Tat mediated dysregulation of IL-1β, TNF-α and MCP-1, key neuroinflammatory signaling proteins. Importantly, using a mouse model where doxycycline induces Tat expression, we demonstrate that dCA reverses the potentiation of cocaine-mediated reward. Our results suggest that adding a Tat inhibitor, such as dCA, to current antiretroviral therapy may reduce HIV-1-related neuropathogenesis. PMID:25613133

  20. Gold nanoparticles functionalized with a fragment of the neural cell adhesion molecule L1 stimulate L1-mediated functions

    NASA Astrophysics Data System (ADS)

    Schulz, Florian; Lutz, David; Rusche, Norman; Bastús, Neus G.; Stieben, Martin; Höltig, Michael; Grüner, Florian; Weller, Horst; Schachner, Melitta; Vossmeyer, Tobias; Loers, Gabriele

    2013-10-01

    The neural cell adhesion molecule L1 is involved in nervous system development and promotes regeneration in animal models of acute and chronic injury of the adult nervous system. To translate these conducive functions into therapeutic approaches, a 22-mer peptide that encompasses a minimal and functional L1 sequence of the third fibronectin type III domain of murine L1 was identified and conjugated to gold nanoparticles (AuNPs) to obtain constructs that interact homophilically with the extracellular domain of L1 and trigger the cognate beneficial L1-mediated functions. Covalent conjugation was achieved by reacting mixtures of two cysteine-terminated forms of this L1 peptide and thiolated poly(ethylene) glycol (PEG) ligands (~2.1 kDa) with citrate stabilized AuNPs of two different sizes (~14 and 40 nm in diameter). By varying the ratio of the L1 peptide-PEG mixtures, an optimized layer composition was achieved that resulted in the expected homophilic interaction of the AuNPs. These AuNPs were stable as tested over a time period of 30 days in artificial cerebrospinal fluid and interacted with the extracellular domain of L1 on neurons and Schwann cells, as could be shown by using cells from wild-type and L1-deficient mice. In vitro, the L1-derivatized particles promoted neurite outgrowth and survival of neurons from the central and peripheral nervous system and stimulated Schwann cell process formation and proliferation. These observations raise the hope that, in combination with other therapeutic approaches, L1 peptide-functionalized AuNPs may become a useful tool to ameliorate the deficits resulting from acute and chronic injuries of the mammalian nervous system.The neural cell adhesion molecule L1 is involved in nervous system development and promotes regeneration in animal models of acute and chronic injury of the adult nervous system. To translate these conducive functions into therapeutic approaches, a 22-mer peptide that encompasses a minimal and functional L1