Gene Network Analysis of Bone Marrow Mononuclear Cells Reveals Activation of Multiple Kinase Pathways in Human Systemic Lupus Erythematosus Magdalene Nakou 1. , George Bertsias 1,2. , Ilias Stagakis 1,2 , Michael Centola 3 , Ioannis Tassiulas 1,2 , Maria Hatziapostolou 4,5 , Iraklis Kritikos 1{ , George Goulielmos 1 , Dimitrios T. Boumpas 1,2. , Dimitrios Iliopoulos 4,5 * . 1 Division of Rheumatology, Clinical Immunology and Allergy, University of Crete Medical School, Heraklion, Greece, 2 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Greece, 3 Microarray Research Facility, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America, 4 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America, 5 Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America Abstract Background: Gene profiling studies provide important information for key molecules relevant to a disease but are less informative of protein-protein interactions, post-translational modifications and regulation by targeted subcellular localization. Integration of genomic data and construction of functional gene networks may provide additional insights into complex diseases such as systemic lupus erythematosus (SLE). Methodology/Principal Findings: We analyzed gene expression microarray data of bone marrow mononuclear cells (BMMCs) from 20 SLE patients (11 with active disease) and 10 controls. Gene networks were constructed using the bioinformatic tool Ingenuity Gene Network Analysis. In SLE patients, comparative analysis of BMMCs genes revealed a network with 19 central nodes as major gene regulators including ERK, JNK, and p38 MAP kinases, insulin, Ca 2+ and STAT3. Comparison between active versus inactive SLE identified 30 central nodes associated with immune response, protein synthesis, and post-transcriptional modification. A high degree of identity between networks in active SLE and non- Hodgkin’s lymphoma (NHL) patients was found, with overlapping central nodes including kinases (MAPK, ERK, JNK, PKC), transcription factors (NF-kappaB, STAT3), and insulin. In validation studies, western blot analysis in splenic B cells from 5- month-old NZB/NZW F1 lupus mice showed activation of STAT3, ITGB2, HSPB1, ERK, JNK, p38, and p32 kinases, and downregulation of FOXO3 and VDR compared to normal C57Bl/6 mice. Conclusions/Significance: Gene network analysis of lupus BMMCs identified central gene regulators implicated in disease pathogenesis which could represent targets of novel therapies in human SLE. The high similarity between active SLE and NHL networks provides a molecular basis for the reported association of the former with lymphoid malignancies. Citation: Nakou M, Bertsias G, Stagakis I, Centola M, Tassiulas I, et al. (2010) Gene Network Analysis of Bone Marrow Mononuclear Cells Reveals Activation of Multiple Kinase Pathways in Human Systemic Lupus Erythematosus. PLoS ONE 5(10): e13351. doi:10.1371/journal.pone.0013351 Editor: Jacques Zimmer, Centre de Recherche Public de la Sante ´, Luxembourg Received February 13, 2010; Accepted September 20, 2010; Published October 14, 2010 Copyright: ß 2010 Nakou 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 has been supported by Hellenic Rheumatology Society grants. 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: dimitrios_iliopoulos@dfci.harvard.edu . These authors contributed equally to this work. { Deceased. Introduction Although gene profiling studies provide important information for key molecules relevant to a disease, they are less informative of protein-protein interactions, post-translational modifications and regulation by targeted subcellular localization. In several diseases, important proteins such as MAP kinases, are activated by phosphorylation while their mRNA and protein levels remain constant. To identify the molecular mechanisms by which these genes lead to complex disease phenotypes, such as obesity, diabetes, osteoarthritis, multiple sclerosis, juvenile rheumatoid arthritis and other autoimmunse diseases [1–4], it is essential to integrate genomic data and construct functional gene networks that will be predictive of these diseases. By the use of microarrays in the peripheral blood or bone marrow, we and others have previously shown specific gene signatures that are involved in SLE and correlate with disease activity [5–8]. Compared to peripheral blood, bone marrow may be more informative in accessing immune reactions in SLE patients [5]. In our study, BM gene analysis differentiated SLE patients based on their disease activity and identified genes involved in apoptosis [5]. Accelerated apoptosis and impaired clearance of apoptotic cells due to the decreased phagocytic ability of macrophages, monocytes and neutrophils have a pathogenic role in SLE [9–11]. PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13351