Syk Regulates AP-1 Dependent Response to mmLDL Figure 2. MAP kinases and transcription factors phosphorylation in WT and Syk2/2 macrophages stimulated with mmLDL and KLA. (A) Upper blots: Ba/F3 cells stably expressing GFP-TLR4 and Flag-TLR4 were incubated with media or mmLDL (50 mg/ml) for 15 min. Cell lysates were immunoprecipitated with an anti-GFP antibody and immunoblotted with anti-Flag and anti-GFP antibodies. Lower blots: Ba/F3 cells were preincubated with 5 mg/ml of an anti-mouse TLR4/MD2 antibody (UT12) or an isotype IgG3k control for 1 hour and then stimulated with mmLDL (50 mg/ml) for 15 min. Cell lysates were immunoprecipitated with a different anti-mouse TLR4 antibody (UT18) and immunoblotted with anti-Syk and anti-Flag antibodies. (B) WT and Syk2/2 BMDM were incubated with media or mmLDL (50 mg/ml) for 15 min. Cell lysates were separated on SDS- PAGE and immunoblotted with antibodies against Syk, phospho-ERK1/2, phospho-c-Jun, phospho-JNK and GAPDH. (C) In vitro kinase assay in J774 macrophages. Cells were incubated with mmLDL (50 mg/ml) for indicated periods of time and then precipitated with anti-JNK, anti-IKKe and anti- ERK1/2 antibodies. Endogenous JNK and IKKe kinase activities were determined using GST-c-Jun (1–79 aa) as a substrate, and endogenous ERK1/2 kinase activity was determined using GST-c-Fos (300–380 aa) as a substrate. Protein levels of endogenous JNK, IKKe and ERK1/2 in the same cell lysates were determined by immunoblot. This experiment informed the optimal stimulation time periods for the experiment in panel D. (D) In vitro kinase assay in WT and Syk2/2 BMDM. Cells were treated with media or mmLDL (50 mg/ml) for 30 min (JNK), 5 min (IKKe) or 15 min (ERK1/2). Kinase activities were determined using the same method as in C. doi:10.1371/journal.pone.0032378.g002 chemiluminescent plate-based assay. For these experiments, we used cell lysates of Syk2/2 BMDM and of Syk knockdown J774 cells, with their respective controls. mmLDL induced c-Jun and c- Fos DNA binding in WT cells, and this effect was completely inhibited in Syk-deficient macrophages (Fig. 3). In gain-of-function experiments, CHO cells were transiently transfected with a Syk expression plasmid (or empty vector) and a AP-1-Luc reporter. Because CHO cells express endogenous TLR4/MD2 [25], mmLDL induced a 2-fold increase in AP-1 activity in empty vector-transfected cells. Overexpression of Syk potentiated mmLDL-induced AP-1 activity and increased it additional 3-fold (Fig. 4A). Furthermore, Syk expression induced phosphorylation of JNK and c-Jun in mmLDL-stimulated CHO cells (Fig. 4B). These results collectively suggest that Syk regulates a AP-1 transcription program in macrophages. Syk regulates expression of CXCL2 and IL-6 induced by mmLDL Among AP-1 dependent genes, our previous studies identified Cxcl2 (MIP-2) as one of the best responders to mmLDL activation in macrophages [4,5,26,27]. Transcription of IL-6 is also AP-1- dependent, and it has a strong NF-kB component as well [28]. In experiments shown in Fig. 5, mmLDL induced Cxcl2 and Il-6 mRNA expression both in WT BMDM and in WT J774 cells. The Syk deficiency in BMDM completely abolished mmLDL-induced Cxcl2 expression and reduced in half expression of Il-6. PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32378