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Jae-Seon So,
Kyu Yeon Hur,
Margarite Tarrio,
Vera Ruda, Maria Frank-Kamenetsky,
Kevin Fitzgerald,
Victor Koteliansky,
Andrew H Lichtman,
Takao Iwawaki,
Laurie H Glimcher,
Ann-Hwee Lee
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ABSTRACT: XBP1 is a key regulator of the unfolded protein response (UPR), which is involved in a wide range of physiological and pathological processes. XBP1 ablation in liver causes profound hypolipidemia in mice, highlighting its critical role in lipid metabolism. XBP1 deficiency triggers feedback activation of its upstream enzyme IRE1α, instigating regulated IRE1-dependent decay (RIDD) of cytosolic mRNAs. Here, we identify RIDD as a crucial control mechanism of lipid homeostasis. Suppression of RIDD by RNA interference or genetic ablation of IRE1α reversed hypolipidemia in XBP1-deficient mice. Comprehensive microarray analysis of XBP1 and/or IRE1α-deficient liver identified genes involved in lipogenesis and lipoprotein metabolism as RIDD substrates, which might contribute to the suppression of plasma lipid levels by activated IRE1α. Ablation of XBP1 ameliorated hepatosteatosis, liver damage, and hypercholesterolemia in dyslipidemic animal models, suggesting that direct targeting of either IRE1α or XBP1 might be a feasible strategy to treat dyslipidemias.
Cell metabolism 10/2012; 16(4):487-99. · 17.35 Impact Factor
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ABSTRACT: The mammalian stress sensor IRE1α plays a central role in the unfolded protein, or endoplasmic reticulum (ER), stress response by activating its downstream transcription factor XBP1 via an unconventional splicing mechanism. IRE1α can also induce the degradation of a subset of mRNAs in a process termed regulated IRE1-dependent decay (RIDD). Although diverse mRNA species can be degraded by IRE1α in vitro, the pathophysiological functions of RIDD are only beginning to be explored. Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in young adults in the United States and is primarily caused by CYP1A2-, CYP2E1-, and CYP3A4-driven conversion of APAP into hepatotoxic metabolites. We demonstrate here that genetic ablation of XBP1 results in constitutive IRE1α activation in the liver, leading to RIDD of Cyp1a2 and Cyp2e1 mRNAs, reduced JNK activation, and protection of mice from APAP-induced hepatotoxicity. A pharmacological ER stress inducer that activated IRE1α suppressed the expression of Cyp1a2 and Cyp2e1 in WT, but not IRE1α-deficient mouse liver, indicating the essential role of IRE1α in the down-regulation of these mRNAs upon ER stress. Our study reveals an unexpected function of RIDD in drug metabolism.
Journal of Experimental Medicine 02/2012; 209(2):307-18. · 13.85 Impact Factor
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ABSTRACT: Insulin resistance leads to hypertriglyceridemia and hepatic steatosis and is associated with increased SREBP-1c, a transcription factor that activates fatty acid synthesis. Here, we show that steatosis in insulin-resistant ob/ob mice was abolished by deletion of Scap, an escort protein necessary for generating nuclear isoforms of all three SREBPs. Scap deletion reduced lipid synthesis and prevented fatty livers despite persistent obesity, hyperinsulinemia, and hyperglycemia. Scap deficiency also prevented steatosis in mice fed high-fat diets. Steatosis was also prevented when siRNAs were used to silence Scap in livers of sucrose-fed hamsters, a model of diet-induced steatosis and hypertriglyceridemia. This silencing reduced all three nuclear SREBPs, decreasing lipid biosynthesis and abolishing sucrose-induced hypertriglyceridemia. These results demonstrate that SREBP activation is essential for development of diabetic hepatic steatosis and carbohydrate-induced hypertriglyceridemia, but not insulin resistance. Inhibition of SREBP activation has therapeutic potential for treatment of hypertriglyceridemia and fatty liver disease.
Cell metabolism 02/2012; 15(2):240-6. · 17.35 Impact Factor
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Kiran Musunuru,
Alanna Strong, Maria Frank-Kamenetsky,
Noemi E Lee,
Tim Ahfeldt,
Katherine V Sachs,
Xiaoyu Li,
Hui Li,
Nicolas Kuperwasser,
Vera M Ruda, [......],
Timothy Racie,
Kenechi G Ejebe,
Marju Orho-Melander,
Olle Melander,
Victor Koteliansky,
Kevin Fitzgerald,
Ronald M Krauss,
Chad A Cowan,
Sekar Kathiresan,
Daniel J Rader
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ABSTRACT: Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.
Nature 08/2010; 466(7307):714-9. · 36.28 Impact Factor
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Akin Akinc,
William Querbes,
Soma De,
June Qin, Maria Frank-Kamenetsky,
K Narayanannair Jayaprakash,
Muthusamy Jayaraman,
Kallanthottathil G Rajeev,
William L Cantley,
J Robert Dorkin, [......],
Anja Zeigerer,
Michael J Hope,
Marino Zerial,
Dinah W Y Sah,
Kevin Fitzgerald,
Mark A Tracy,
Muthiah Manoharan,
Victor Koteliansky,
Antonin de Fougerolles,
Martin A Maier
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ABSTRACT: Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.
Molecular Therapy 05/2010; 18(7):1357-64. · 6.87 Impact Factor
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Kevin T Love,
Kerry P Mahon,
Christopher G Levins,
Kathryn A Whitehead,
William Querbes,
J Robert Dorkin,
June Qin,
William Cantley,
Liu Liang Qin,
Timothy Racie, Maria Frank-Kamenetsky,
Ka Ning Yip,
Rene Alvarez,
Dinah W Y Sah,
Antonin de Fougerolles,
Kevin Fitzgerald,
Victor Koteliansky,
Akin Akinc,
Robert Langer,
Daniel G Anderson
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ABSTRACT: Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.
Proceedings of the National Academy of Sciences 02/2010; 107(5):1864-9. · 9.68 Impact Factor
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Maria Frank-Kamenetsky,
Aldo Grefhorst,
Norma N Anderson,
Timothy S Racie,
Birgit Bramlage,
Akin Akinc,
David Butler,
Klaus Charisse,
Robert Dorkin,
Yupeng Fan, [......],
Gang Wang,
Tracy Zimmermann,
Antonin de Fougerolles,
Hans-Peter Vornlocher,
Robert Langer,
Daniel G Anderson,
Muthiah Manoharan,
Victor Koteliansky,
Jay D Horton,
Kevin Fitzgerald
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ABSTRACT: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5'-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.
Proceedings of the National Academy of Sciences 08/2008; 105(33):11915-20. · 9.68 Impact Factor
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ABSTRACT: Recently, chemically synthesized short interfering RNA (siRNA) duplexes have been used with success for gene silencing. Chemical modification is desired for therapeutic applications to improve biostability and pharmacokinetic properties; chemical modification may also provide insight into the mechanism of silencing. siRNA duplexes containing the 2,4-difluorotoluyl ribonucleoside (rF) were synthesized to evaluate the effect of noncanonical nucleoside mimetics on RNA interference. 5'-Modification of the guide strand with rF did not alter silencing relative to unmodified control. Internal uridine to rF substitutions were well-tolerated. Thermal melting analysis showed that the base pair between rF and adenosine (A) was destabilizing relative to a uridine-adenosine pair, although it was slightly less destabilizing than other mismatches. The crystal structure of a duplex containing rFoA pairs showed local structural variations relative to a canonical RNA helix. As the fluorine atoms cannot act as hydrogen bond acceptors and are more hydrophobic than uridine, there was an absence of a well-ordered water structure around the rF residues in both grooves. siRNAs with the rF modification effectively silenced gene expression and offered improved nuclease resistance in serum; therefore, evaluation of this modification in therapeutic siRNAs is warranted.
ACS Chemical Biology 05/2006; 1(3):176-83. · 6.45 Impact Factor
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Maria Frank-Kamenetsky,
Xiaoyan M Zhang,
Steve Bottega,
Oivin Guicherit,
Hynek Wichterle,
Henryk Dudek,
David Bumcrot,
Frank Y Wang,
Simon Jones,
Janine Shulok,
Lee L Rubin,
Jeffery A Porter
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ABSTRACT: The Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults, Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover, stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy. The underlying mechanisms of Hh signal transduction remain obscure, however: little is known about the communication between the pathway suppressor Patched (Ptc), a multipass transmembrane protein that directly binds Hh, and the pathway activator Smoothened (Smo), a protein that is related to G-protein-coupled receptors and is capable of constitutive activation in the absence of Ptc.
We have identified and characterized a synthetic non-peptidyl small molecule, Hh-Ag, that acts as an agonist of the Hh pathway. This Hh agonist promotes cell-type-specific proliferation and concentration-dependent differentiation in vitro, while in utero it rescues aspects of the Hh-signaling defect in Sonic hedgehog-null, but not Smo-null, mouse embryos. Biochemical studies with Hh-Ag, the Hh-signaling antagonist cyclopamine, and a novel Hh-signaling inhibitor Cur61414, reveal that the action of all these compounds is independent of Hh-protein ligand and of the Hh receptor Ptc, as each binds directly to Smo.
Smo can have its activity modulated directly by synthetic small molecules. These studies raise the possibility that Hh signaling may be regulated by endogenous small molecules in vivo and provide potent compounds with which to test the therapeutic value of activating the Hh-signaling pathway in the treatment of traumatic and chronic degenerative conditions.
Journal of Biology 12/2002; 1(2):10.
