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The Role of Lysosomal Acid Lipase in Regulation of the Atp

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The Role of Lysosomal Acid Lipase in Regulation of the Atp THE ROLE OF LYSOSOMAL ACID LIPASE IN REGULATION OF THE ATP- BINDING CASSETTE TRANSPORTER A1, HIGH DENSITY LIPOPROTEIN AND REVERSE CHOLESTEROL TRANSPORT by Kristin Louise Bowden B.Sc., Queen’s University, 2005 M.Sc., Dalhousie University, 2008. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2013 © Kristin Louise Bowden, 2013. Abstract The key regulator of initial HDL particle formation by cells is the ATP-binding cassette transporter A1 (ABCA1). ABCA1 expression is regulated primarily by oxysterol dependent activation of the liver X receptor (LXR). We investigated the role of lysosomal cholesterol on ABCA1 regulation by studying the lysosomal disorder Cholesteryl Ester Storage Disease (CESD). CESD is caused by genetic mutations in the LIP-A gene that result in only 5% of normal activity of lysosomal acid lipase (LAL), an enzyme that hydrolyzes cholesteryl esters (CE) and triglycerides on internalized lipoproteins specifically within the lysosome. We hypothesized that the flux of unesterified cholesterol out of the lysosomes from LAL- mediated hydrolysis of LDL cholesteryl esters is a key regulator of cellular ABCA1 expression, HDL formation and reverse cholesterol transport (RCT). We found that primary skin fibroblasts derived from individuals with CESD had impaired upregulation of ABCA1 in response to LDL loading, reduced phospholipid and cholesterol efflux to apoA-I, lower production of 27-hydroxycholesterol (27-OH) production in response to LDL loading and reduced α-HDL particle formation. This defect was recapitulated in normal fibroblasts following treatment with LAL inhibitors, whereas, treatment with conditioned medium from normal fibroblasts containing secreted LAL rescued ABCA1 expression, apoA-I-mediated cholesterol efflux, HDL particle formation and production of 27-OH by CESD cells. We further investigated the role of LAL in RCT from macrophages specifically using an immortalized macrophage cell line created from LAL-deficient mouse peritoneal macrophages (LAL-/-). LAL-/- macrophages exhibited reduced basal and cholesterol-stimulated ABCA1 expression in culture, and reduced ability to support RCT in LAL-/- mice compared to wild-type (LAL+/+) macrophages injected into LAL+/+ mice. ABCA1 protein expression was reduced in lal-/- mouse liver and mRNA ii expression of several LXR-dependent genes involved in reverse cholesterol transport (ABCG1, ABCG5, ABCG8, CYP7A1, SR-B1) were differentially modulated compared to LAL+/+ controls. These results indicate a critical role of LAL in promoting lysosomal flux of cholesterol for ABCA1 expression, cellular cholesterol efflux and RCT in vivo. iii Preface Chapter 2: LAL deficiency impairs regulation of ABCA1 and formation of HDL in CESD Kristin Bowden contributed 70% towards all aspects of this work. She contributed 60% towards experiments (Figures 2.3, 2.5, 2.6 and 2.7, 2.9, 2.10, 2.11 and 2.12), completed analysis of the data, and drafted the original version of this manuscript. Contribution of co-authors: Nicolas Bilbey, MSc performed 40% of experiments. This includes Figures 1, 2 and 4. Leanne Bilawchuk, BSc and Teddy Chan, MSc provided technical assistance and performed experiments for supplementary Figure 2.11. Emmanuel Boadu, PhD performed the 2-dimensional gel electrophoresis of medium samples (Figures 2.6 and 2.12). Roshni Sidhu, BSc, performed analysis of oxysterol mass by Gas Chromatography / Mass Spectroscopy of cell and medium samples (Figure 2.8) under supervision of a collaborator Daniel Ory, PhD. Hong Du, PhD, provided expert advice and assisted with revision of the manuscript. Gordon Francis, MD, supervised this project and assisted with editing and writing of the manuscript. All work was completed under approval of the UBC research ethics board (certificate #H07- 02720 – High Density Lipoprotein Formation). A version of this work is published: *Bowden KL, *Bilbey NJ, Bilawchuk LM, Boadu E, Sidhu R, Ory DS, Du H, Chan T, Francis GA. (2011) Lysosomal acid lipase deficiency impairs regulation of ABCA1 gene and formation of high density lipoproteins in cholesteryl ester storage disease. J Biol Chem. 286 (35): 30624-35. (*both authors contributed equally to this manuscript) iv Chapter 3: LAL promotes Reverse Cholesterol Transport from Macrophages Kristin Bowden contributed 95% towards all aspects of this work, including all experiments, analysis of data and writing of the original manuscript. Contribution of co-authors: Hejin (Julia) Kong and Teddy Chan, MSc provided technical assistance with some aspects of animal experiments. You-Hai Xu, PhD, performed breeding and husbandry of the LAL knockout mice under supervision of our collaborator Gregory Grabowski, MD. Hong Du, PhD provided immortalized peritoneal macrophage cell lines used for experiments in Figures 3.1-3.4 and provided scientific advice. Gordon Francis, MD, supervised this work and provided assistance with editing of the manuscript. Animal work was performed under approval by the UBC Animal Care Committee (Certificate #A12-0067). v Table of contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of contents .......................................................................................................................... vi List of tables.................................................................................................................................. xi List of figures ............................................................................................................................... xii List of abbreviations .................................................................................................................. xiv Acknowledgements .................................................................................................................... xvi Dedication .................................................................................................................................. xvii Chapter 1: Introduction ...............................................................................................................1 1.1 Overview ........................................................................................................................ 1 1.2 Regulation of cellular cholesterol metabolism ........................................................... 2 1.2.1 SREBPs in the regulation of cholesterol homeostasis ................................................ 3 1.2.2 LDL-receptor mediated uptake of exogenous cholesterol .......................................... 5 1.2.3 Uptake of modified LDL by scavenger receptors ....................................................... 8 1.3 Reverse cholesterol transport .................................................................................... 10 1.3.1 Step 1: Cellular cholesterol efflux and HDL formation............................................ 12 1.3.2 Step 2: HDL cholesterol esterification ...................................................................... 12 1.3.3 Step 3: Uptake of HDL cholesterol and cholesteryl esters by the liver .................... 13 1.3.3.1 HDL remodeling in the plasma ......................................................................... 14 1.3.4 Step 4: Hepatobiliary excretion of cholesterol and bile acids ................................... 15 1.3.5 The role of the intestine in RCT ............................................................................... 15 1.3.6 Macrophage-specific reverse cholesterol transport .................................................. 16 vi 1.3.7 Regulation of reverse cholesterol transport genes by the liver X receptor ............... 18 1.3.7.1 Enzymatic and non-enzymatic synthesis of oxysterols ...................................... 19 1.3.7.2 The role of oxysterols in regulation of cholesterol homeostasis....................... 20 1.4 ABCA1 and ABCG1 function and regulation .......................................................... 21 1.4.1 ABCA1 transcriptional and post-transcriptional regulation ..................................... 23 1.4.2 The ABCG1 and its role in RCT............................................................................... 24 1.5 Regulation of cholesterol metabolism in Niemann Pick disease type C (NPC) ..... 25 1.6 Lysosomal acid lipase (LAL)...................................................................................... 26 1.6.1 The history of discovery of LAL .............................................................................. 26 1.6.2 The molecular biology of LAL ................................................................................. 27 1.6.2.1 Gene regulation of LIPA ................................................................................... 28 1.6.3 LAL secretion and uptake ......................................................................................... 28 1.6.4 LAL enzyme properties ............................................................................................ 29 1.6.4.1 How LAL activity is measured .......................................................................... 30 1.6.4.2
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