The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages Emmanuelle Girard1,2, Jean Louis Paul1,2, Natalie Fournier1,2, Philippe Beaune1, Ludger Johannes3,4, Christophe Lamaze3,4*., Benoıˆt Ve´ die1. 1 AP-HP (Assistance Publique – H oˆpitaux de Paris), H oˆpital Europe´en Georges Pompidou, Service de Biochimie, Paris, France, 2 Universite´ Paris-Sud, EA 4529, UFR de Pharmacie, Chaˆtenay-Malabry, France, 3 CNRS UMR144, Paris, France, 4 Institut Curie, Centre de Recherche, Laboratoire Trafic, Signalisation et Ciblage Intracellulaires, Paris, France Abstract Intracellular transport of cholesterol contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we characterized the impact of dynasore, a recently described drug that specifically inhibits the enzymatic activity of dynamin, a GTPase regulating receptor endocytosis and cholesterol trafficking. Dynasore strongly inhibited the uptake of low-density lipoprotein (LDL) in HeLa cells, and to a lower extent in human macrophages. In both cell types, dynasore treatment led to the abnormal accumulation of LDL and free cholesterol (FC) within the endolysosomal network. The measure of cholesterol esters (CE) further showed that the delivery of regulatory cholesterol to the endoplasmic reticulum (ER) was deficient. This resulted in the inhibition of the transcriptional control of the three major sterol-sensitive genes, sterol-regulatory element binding protein 2 (SREBP-2), 3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR), and low-density lipoprotein receptor (LDLR). The sequestration of cholesterol in the endolysosomal compartment impaired both the active and passive cholesterol efflux in HMDM. Our data further illustrate the importance of membrane trafficking in cholesterol homeostasis and validate dynasore as a new pharmacological tool to study the intracellular transport of cholesterol. Citation: Girard E, Paul JL, Fournier N, Beaune P, Johannes L, et al. (2011) The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol- Sensitive Genes Transcription in Human HeLa Cells and Macrophages. PLoS ONE 6(12): e29042. doi:10.1371/journal.pone.0029042 Editor: Wanjin Hong, Institute of Molecular and Cell Biology, Singapore Received July 20, 2011; Accepted November 19, 2011; Published December 19, 2011 Copyright: 2011 Girard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a grant from Agence Nationale de la Recherche (ANR- 08-GENO-002-01). E.G. is supported by a doctoral fellowship from Ministe`re de la Recherche. C.L. is the recipient of a Contrat Hospitalier de Recherche Translationnelle (CHRT) with Institut National de la Sante´ et de la Recherche Me´dicale (INSERM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: christophe.lamaze@curie.fr . These authors contributed equally to this work. Introduction Membrane trafficking contributes to cell cholesterol homeostasis through the control of intracellular cholesterol levels and compartmentalization. Little is known about the transport pathways involved in cholesterol trafficking and the associated molecular machinery [1]. The level of cellular cholesterol results from the tight control of both cholesterol neosynthesis and low- density lipoprotein (LDL)-derived cholesterol uptake. The uptake of LDL-derived cholesterol at the plasma membrane occurs primarily through clathrin-dependent endocytosis of the low- density lipoprotein receptor (LDLR), as shown in the seminal studies by Brown and Goldstein [2]. This process delivers lipoprotein-associated cholesterol esters (CE) first to the early endosome (EE) and then to the late endosomal/lysosomal network. At this stage, the acid hydrolases de-esterify CE into free cholesterol (FC), which will leave the endolysosomal network to reach the endoplasmic reticulum (ER) either directly or indirectly after transiting through the Golgi apparatus and the plasma membrane [3]. ER membranes, which are characterized by a poor content in cholesterol, are home to the principal effectors of cellular cholesterol homeostasis. Key of this regulation is a family of membrane-bound transcription factors, sterol regulatory element binding proteins (SREBPs). Under low sterol concentration, SREBP is transported to the Golgi complex where it is activated by proteolytic processing. This cleavage releases the active form of SREBP, which is translocated to the nucleus where it regulates the transcription of sterol responsive genes involved in cholesterol synthesis (3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR)) or uptake (LDLR) [4]. Cholesterol transport to the ER is therefore a key element of the cholesterol homeostasis machinery. The levels of cholesterol are constantly monitored by the two chaperone proteins SCAP (SREBP cleavage activating protein) and INSIG (insulin-induced gene protein). SCAP is a SREBP Golgi escort protein while INSIG is an ER anchored protein. Binding of cholesterol or oxysterols to SCAP or to INSIG, respectively, promotes the interaction between SCAP and INSIG. The formation of this complex masks the binding site on SCAP that is recognized by the vesicular coat transport complex COPII, thereby blocking SREBP transport from the ER to the Golgi apparatus and the release of the cleaved SREBP active form [5,6]. Another important actor of this tight regulation is the acyl PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29042