Spleen Tyrosine Kinase Regulates AP-1 Dependent Transcriptional Response to Minimally Oxidized LDL Soo-Ho Choi, Philipp Wiesner, Felicidad Almazan, Jungsu Kim, Yury I. Miller* Department of Medicine, University of California San Diego, La Jolla, California, United States of America Abstract Oxidative modification of low-density lipoprotein (LDL) turns it into an endogenous ligand recognized by pattern- recognition receptors. We have demonstrated that minimally oxidized LDL (mmLDL) binds to CD14 and mediates TLR4/MD- 2-dependent responses in macrophages, many of which are MyD88-independent. We have also demonstrated that the mmLDL activation leads to recruitment of spleen tyrosine kinase (Syk) to TLR4 and TLR4 and Syk phosphorylation. In this study, we produced a macrophage-specific Syk knockout mouse and used primary Syk2/2 macrophages in our studies. We demonstrated that Syk mediated phosphorylation of ERK1/2 and JNK, which in turn phosphorylated c-Fos and c-Jun, respectively, as assessed by an in vitro kinase assay. c-Jun phosphorylation was also mediated by IKKe. c-Jun and c-Fos bound to consensus DNA sites and thereby completed an AP-1 transcriptional complex and induced expression of CXCL2 and IL-6. These results suggest that Syk plays a key role in TLR4-mediated macrophage responses to host-generated ligands, like mmLDL, with subsequent activation of an AP-1 transcription program. Citation: Choi S-H, Wiesner P, Almazan F, Kim J, Miller YI (2012) Spleen Tyrosine Kinase Regulates AP-1 Dependent Transcriptional Response to Minimally Oxidized LDL. PLoS ONE 7(2): e32378. doi:10.1371/journal.pone.0032378 Editor: David M. Ojcius, University of California Merced, United States of America Received October 11, 2011; Accepted January 28, 2012; Published February 22, 2012 Copyright: 2012 Choi 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 study was supported by National Institutes of Health grants HL081862 and HL088093 as well as a grant from the Leducq Foundation. 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: yumiller@ucsd.edu Introduction Spleen tyrosine kinase (Syk) is best known as a tyrosine kinase involved in signaling initiated by B cell receptor, T cell receptor or Fc receptor. Ligand binding to these receptors leads to recruitment of signaling proteins with immunoreceptor tyrosine-based activa- tion motifs (ITAMs). Ensuing phosphorylation of tandem tyrosines in an ITAM leads to the ITAM binding with Syk via Syk’s tandem SH2 domains, with subsequent Syk activation. Activated Syk directly binds to Vav, PLCc, PI3K, and SLP76/SLP65 and engages a plethora of other signaling intermediates. Resulting cellular responses range from cytoskeletal changes and ROS production to cell differentiation, proliferation and survival [1]. In addition to the SH-2-mediated binding to ITAMs, Syk also binds to b-integrins via a site distinct from the site of phospho-tyrosine binding [2,3] and thus, coordinates integrin- and ITAM- dependent signaling cascades. We have recently demonstrated, using yeast-two-hybrid and immunoprecipitation assays, that Syk constitutively binds to the intracellular domain of toll-like receptor-4 (TLR4) [4,5]. This binding is further increased and both TLR4 and Syk become phosphorylated in macrophages stimulated with a host-derived TLR4 ligand, minimally oxidized low-density lipoprotein (mmLDL). Further downstream from Syk, mmLDL activates the Vav-Ras-Raf-MEK1-ERK1/2 pathway, small GTPases Cdc42, Ras and Rho, and phosphorylates paxillin, which collectively lead to actin polymerization and extensive membrane ruffling, culminating in robust macropinocytosis. This mmLDL-induced and TLR4/Syk-dependent macropinocytosis is suggested to constitute an important mechanism of excessive lipid accumula- tion in macrophages, resulting in formation of lipid-laden macrophage foam cells, a hallmark of atherosclerosis [4–8]. Interestingly, unlike LPS, the mmLDL activation of TLR4 does not engage MyD88-dependent pathways [4,5]. This makes the Syk- dependent signaling a central component of mmLDL-induced macrophage activation via TLR4. In the present work, we generated Sykflox/flox/LysM-Cre mice and performed the majority of experi- ments with primary macrophages derived from bone marrow in which Syk expression was reduced by 60–70%, to study the role of Syk in transcriptional regulation. We demonstrate that Syk regulates ERK1/2-dependent phosphorylation of c-Fos and JNK/IKKe- dependent phosphorylation of c-Jun, as well as c-Fos and c-Jun binding to DNA and AP-1-dependent expression of CXCL2 and IL- 6. These results suggest the role of Syk in TLR4-mediated chronic inflammation induced by modified host ligands, as it may relate to inflammation in atherosclerotic lesions induced by oxidized LDL. Materials and Methods Ethics Statement All animal experiments were performed according to the NIH guidelines and were approved by the Animal Subjects Committee of the UC San Diego (protocol S04155). Human plasma, used for LDL isolation, was obtained from normal volunteers according to a protocol approved by the UC San Diego Human Research Protection Program (project #071402). Animals Sykflox/flox mice were kindly provided by Alexander Tara- khovsky from the Rockfeller University [9]. LysM-Cre mice were PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32378