The Journal of Immunology Liver X Receptors Inhibit Macrophage Proliferation through Downregulation of Cyclins D1 and B1 and Cyclin-Dependent Kinases 2 and 4 Mo ´nica Pascual-Garcı ´a,* Jose ´ M. Carbo ´,* Theresa Leo ´n,* Jonathan Matalonga,* Ruud Out, † Theo Van Berkel, † Maria-Rosa Sarrias, ‡ Francisco Lozano, x,{ Antonio Celada, ‖,# and Annabel F. Valledor* Macrophages serve essential functions as regulators of immunity and homeostasis, and their proliferation contributes to patho- genesis of certain disorders. In this report, we show that induction of macrophage proliferation by the growth factor M-CSF is negatively modulated by agonists that activate the nuclear receptor liver X receptor (LXR), both in vitro and in vivo. Both iso- forms LXR a and b are involved in the antiproliferative actions of LXR ligands in macrophages. In contrast, M-CSF does not exert negative effects on LXR-mediated gene expression. Treatment with LXR agonists results in the accumulation of macro- phages in the G 0 /G 1 phase of the cell cycle without affecting ERK-1/2 phosphorylation. The use of small interfering RNA or genetically modified mice revealed that, in contrast to other cellular models, functional expression of either the cyclin-dependent kinase inhibitor p27KIP1 or the cholesterol transporters ATP-binding cassette A1 or ATP-binding cassette G1 was not required for the antiproliferative effects of LXR agonists in macrophages. Western blot analysis revealed that protein expression of key molecules that regulate progression through the cell cycle, such as cyclins D1 and B1 and cyclin-dependent kinases 2 and 4, was downregulated upon LXR activation. These observations suggest a role for LXR agonists in limiting macrophage proliferative responses associated to pathogenic disorders. The Journal of Immunology, 2011, 186: 4656–4667. M acrophages serve essential functions as regulators of immunity and homeostasis (1). Macrophage activities include the production of cytokines and other signaling molecules that amplify acute inflammatory responses, Ag pre- sentation and modulation of T cell responses, and the involvement in cholesterol homeostasis (2). Regulation of macrophage pro- liferation, differentiation, and survival is critical to the overall control of the magnitude, duration, and characteristics of immune and homeostatic responses (3). Among the growth factors that influence myeloid differentiation, M-CSF acts specifically on bone marrow precursors committed to the monocytic/macrophagic lin- eage to promote their proliferation and differentiation (4). In ad- dition, M-CSF prolongs macrophage survival and enhances their functional activities in vitro (5, 6). Effects of M-CSF are mediated through a type III tyrosine kinase receptor, which is encoded by the proto-oncogene Fms. Binding of M-CSF induces dimerization of this receptor and autophosphorylation of its cytoplasmic tyro- sine residues (7, 8), which then associate with a large number of proteins containing Src homology 2 domains that consequently activate numerous signaling pathways. Among these pathways, activation of the MEK/ERK cascade is required for macrophage proliferation (9). At the level of transcription, M-CSF signaling induces the expression of genes associated with progression through the cell cycle, including immediate early genes such as Myc and delayed early genes such as D-type cyclins (10). D-type cyclins form complexes with cyclin-dependent kinases (CDK) 4 and 6, and their activity is crucial for the progression into the S phase of the cell cycle (11). The expression of cyclin E is also induced in response to M-CSF, although at later stages during G1. Cyclin E interacts with CDK2 and helps maintain the retino- blastoma protein in a hyperphosphorylated state during late G 1 - to-S transition. Nuclear receptors constitute a family of ligand-dependent transcription factors that regulate diverse aspects of development and homeostasis (12). Liver X receptors (LXRs) represent a subset of the nuclear receptor superfamily that are regulated by oxidized forms of cholesterol (oxysterols) and intermediate products of the cholesterol biosynthetic pathway (13). Two LXR isoforms have *Nuclear Receptor Group, Department of Physiology, School of Biology, University of Barcelona, 08028 Barcelona, Spain; † Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands; ‡ Health Sciences Research Institute, “Germans Trias i Pujol” Foundation, 08916 Badalona, Spain; x Servei d’Immunologia, Hospital Clı ´nic de Barcelona, Institut d’Investigacions Biome `diques August Pi i Sunyer, 08036 Barcelona, Spain; { De- partment of Cellular Biology, Immunology and Neurosciences, School of Medicine, University of Barcelona, 08036 Barcelona, Spain; ‖ Macrophage Biology Group, In- stitute for Research in Biomedicine, 08028 Barcelona, Spain; and # Department of Physiology, School of Biology, University of Barcelona, 08028 Barcelona, Spain Received for publication February 19, 2010. Accepted for publication February 13, 2011. This work was supported by grants from the Marie Curie International Reintegration Program (IRG-031137), the Spanish Ministry of Science and Innovation (SAF2007- 63543), and the Fundacio ´ Marato ´ de TV3 (080930) (to A.F.V.). M.P.-G. and J.M.C. are recipients of fellowships from the Spanish Ministry of Education (Formacio ´n del Profesorado Universitario and Beca de colaboracio ´n en departamentos, respectively). J.M. is the recipient of a Formacio ´n de Personal Investigador fellowship from the Spanish Ministry of Science and Innovation. M.-R.S. is supported by a grant from the Spanish Ministry of Science and Innovation (FISCP08/124). Address correspondence and reprint requests to Dr. Annabel F. Valledor, Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, planta 3, 08028 Barcelona, Spain. E-mail address: afernandezvalledor@ub.edu The online version of this article contains supplemental material. Abbreviations used in this article: ABCA1, ATP-binding cassette A1; ABCG1, ATP- binding cassette G1; AIM, apoptosis inhibitory factor secreted by macrophages; Apoc2, apolipoprotein CII; APOE, apolipoprotein E; BMDM, bone marrow-derived macro- phage; CCNB1, cyclin B1; CCND1, cyclin D1; CDK, cyclin-dependent kinase; L- cell cm, L-cell conditioned medium; LXR, liver X receptor; Pltp, phospholipid transfer protein; siRNA, small interfering RNA; SKP2, S phase kinase-associated protein 2; Srebp-1c, sterol response element binding protein-1c; wT, wild-type. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000585