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The Regulation of Cholesterol Absorption: Nuclear Hormone Receptors and Niemann-Pick C1 Like 1

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THE REGULATION OF CHOLESTEROL ABSORPTION: NUCLEAR HORMONE RECEPTORS AND NIEMANN-PICK C1 LIKE 1 APPROVED BY SUPERVISORY COMMITTEE Dr. Lourdes Malú Tansey, Ph.D. Dr. Richard G. W. Anderson, Ph.D. Dr. Kristen W. Lynch, Ph.D. Dr. Joyce J. Repa, Ph.D. Dr. David J. Mangelsdorf, Ph.D. For Mandy THE REGULATION OF CHOLESTEROL ABSORPTION: NUCLEAR HORMONE RECEPTORS AND NIEMANN-PICK C1 LIKE 1 by MARK ANDREW VALASEK DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas August, 2006 ACKNOWLEDGEMENTS Many thanks go to all those who encouraged me to pursue research, especially Mike Brown, M.D. and Masashi Yanagisawa, M.D., Ph.D. I’m most thankful to my mentor, Joyce Repa, Ph.D. for adopting me into the lab and motivating me to do better science through her leadership by example. Many thanks go to our collaborators, John Dietschy, M.D., Stephen Turley, Ph.D., Richard Anderson, Ph.D, and Malu Tansey, Ph.D. who have kindly allowed me to pursue and participate in various projects and have shared their priceless experience and expertise in addition to material resources. I’m grateful for the Committee’s helpful comments and encouragement. I also would like to thank the Medical Scientist Training Program, Dennis McKearin, Ph.D., Rodney Ulane, Ph.D., Robin Downing, and Stephanie Robertson, and the NIH Pharmacology Training Grant Program, David J. Mangelsdorf, Ph.D., Alfred G. Gilman, M.D., Ph.D., Rama Ranganathan, M.D., Ph.D. and Carla Childers, who have provided both funding and counseling for this work, but also have broadened my scientific experience through insightful discussion. I greatly appreciate family and friends who have supported me throughout. My deepest gratitude is extended to my wife, Mandy, without whose dedication, wisdom, and persevering love, this work would not have been as enjoyable or even possible, and to my children, Ruby and Andrew, may they always seek to understand. THE REGULATION OF CHOLESTEROL ABSORPTION: NUCLEAR HORMONE RECEPTORS AND NIEMANN-PICK C1 LIKE 1 Publication No. Mark Andrew Valasek, Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2006 Supervising Professor: Joyce J. Repa, Ph.D. Cholesterol plays fundamental roles in cellular physiology, but it is also involved in many pathophysiological processes including atherosclerosis, cholelithiasis, and some forms of neurodegenerative disease. The ability of mammals to selectively absorb cholesterol from the diet while largely excluding plant sterols has been known for more than 75 years, but the precise repertoire of molecular events necessary for this process are just beginning to be elucidated. Recently, several candidates have been put forth as putative intestinal cholesterol “permeases” responsible for cholesterol transport across the intestinal brush border. In addition, members of the nuclear receptor superfamily of v ligand-activated transcription factors are known to modulate expression of genes involved in cholesterol and bile acid homeostasis, and cholesterol absorption. Therefore, we wanted not only to clarify the essential molecular mechanisms by which cholesterol was absorbed, but also to investigate the potential role of nuclear receptors in regulating essential steps in this process. Here, we show that a candidate component of the cholesterol transport machinery, caveolin-1 (CAV1), is neither required for intestinal cholesterol transport or sensitivity to the novel cholesterol absorption blocking agent, ezetimibe. This rules out a critical role for caveolin-1 and lends further support to the contention that Niemann-Pick C1 like 1 (NPC1L1) is the bona fide intestinal cholesterol permease. Therefore to better understand new ways in which nuclear receptors could regulate cholesterol absorption, we studied nuclear receptor regulation of NPC1L1 and determined that several nuclear receptors could modulate its expression in small intestine, including peroxisome proliferator-activated receptor alpha (PPARα) and retinoid X receptor (RXR). Thus, cholesterol absorption can be regulated by nuclear receptor modulation of NPC1L1 expression. vi TABLE OF CONTENTS Title...................................................................................................................................... i Dedication........................................................................................................................... ii Acknowledgements............................................................................................................ iv Abstract............................................................................................................................... v Table of Contents.............................................................................................................. vii Prior Publications............................................................................................................. xiv List of Figures................................................................................................................... xv List of Tables .................................................................................................................. xvii List of Abbreviations .....................................................................................................xviii CHAPTER 1: Introduction.............................................................................................. 1 1.1 The Cholesterol Molecule......................................................................................... 1 1.1.1 Structure of Cholesterol ..................................................................................... 2 1.1.2 Biosynthesis of Cholesterol ............................................................................... 2 1.1.3 Regulation of Cholesterol Biosynthesis............................................................. 4 1.1.4 Endogenously Synthesized Derivatives of Cholesterol ..................................... 6 1.2 Introduction to Cholesterol Absorption .................................................................... 9 1.2.1 A Brief History of Cholesterol Absorption........................................................ 9 1.2.2 Cholesterol Absorption and Transport and the Enterohepatic Circulation of Bile Acids.................................................................................................................. 10 1.2.3 Phases and Molecular Mechanisms of Intestinal Cholesterol Absorption....... 13 1.2.3.1 Intraluminal Phase .................................................................................... 13 1.2.3.2 Transmembrane Phase (Uptake and Efflux)............................................. 15 vii 1.2.3.3 Intracellular Phase..................................................................................... 17 1.2.4 Nuclear Receptor Regulation of Cholesterol Absorption ................................ 20 1.2.4.1 Classification of Nuclear Hormone Receptors ......................................... 20 1.2.4.2 Nuclear Receptors Important in Cholesterol Absorption.......................... 21 Liver X Receptors (LXRs)................................................................................ 21 Farnesoid X Receptor (FXR)............................................................................ 24 Peroxisome Proliferator-activated Receptors (PPARs) .................................... 25 1.2.5 Candidates for the Intestinal Cholesterol Transporter ..................................... 26 1.2.5.1 Evidence for an Intestinal Cholesterol Permease...................................... 26 1.2.5.2 Cholesterol Effluxers ................................................................................ 29 ATP-binding cassette transporter A1 (ABCA1)............................................... 30 ATP-binding cassette transporters G5 and G8 (ABCG5/8).............................. 31 1.2.5.3 Cholesterol Importers................................................................................ 33 Scavenger receptor BI (SR-BI)......................................................................... 33 Aminopeptidase N (APN)................................................................................. 35 Caveolin 1 (CAV1) and Annexin 2 (ANXA2) Complex.................................. 36 Niemann-Pick C1 Like 1 (NPC1L1)................................................................. 37 1.3 Rationale ................................................................................................................. 45 CHAPTER 2: Caveolin-1 is not Required for Murine Intestinal Cholesterol Transport......................................................................................................................... 46 2.1 Abstract................................................................................................................... 46 2.2 Introduction............................................................................................................. 47 2.3 Experimental Procedures ........................................................................................ 48 2.3.1 Animal studies ................................................................................................. 49 2.3.2 Plasma Lipid Analyses....................................................................................
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    Gain-of-Function Effects of Mutant p53 Explored Using a Three- Dimensional Culture Model of Breast Cancer William A. Freed-Pastor Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2012 © 2011 William A. Freed-Pastor All Rights Reserved ABSTRACT Gain-of-Function Effects of Mutant p53 Explored Using a Three-Dimensional Culture Model of Breast Cancer William A. Freed-Pastor p53 is the most frequent target for mutation in human tumors and mutation at this locus is a common and early event in breast carcinogenesis. Breast tumors with mutated p53 often contain abundant levels of this mutant protein, which has been postulated to actively contribute to tumorigenesis by acquiring pro-oncogenic (“gain- of-function”) properties. To elucidate how mutant p53 might contribute to mammary carcinogenesis, we employed a three-dimensional (3D) culture model of breast cancer. When placed in a laminin-rich extracellular matrix, non-malignant mammary epithelial cells form structures highly reminiscent for many aspects of acinar structures found in vivo. On the other hand, breast cancer cells, when placed in the same environment, form highly disorganized and sometimes invasive structures. Modulation of critical oncogenic signaling pathways has been shown to phenotypically revert breast cancer cells to a more acinar-like morphology. We examined the role of mutant p53 in this context by generating stable, regulatable p53 shRNA derivatives of mammary carcinoma cell lines to deplete endogenous mutant p53. We demonstrated that, depending on the cellular context, mutant p53 depletion is sufficient to significantly reduce invasion or in some cases actually induce a phenotypic reversion to more acinar-like structures in breast cancer cells grown in 3D culture.