cytoplasmic domain of RAGE finally led to the identification of TIRAP as an adaptor protein for RAGE (Figure S2A). TIRAP and MyD88, but not TRAM, were co-precipitated with overexpressed RAGE and RAGE was phosphorylated in HEK293 cells predominantly when the cells were treated with RAGE ligands, S100A11, S100A12, HMGB1 and AGE but not with a TLR4 ligand (LPS) and TLR2 ligands (Figure 2A). TLR2/4 blocker mixture did not affect ligand-induced activation of RAGE and its downstream signaling. The amount of TIRAP and MyD88 co-precipitated with the cytoplasmic domain of RAGE was shown to depend on phosphorylation status of Ser391 as demonstrated using phosphorylation-mimic and non-phosphorylatable variants (Figure S2B). In HEK293 cells, endogenous TIRAP, MyD88, and IRAK4 were co-precipitated with overexpressed wild-type RAGE but not with a non-phosphorylatable variant when the transfected cells were treated with AGE (Figure 2B). In accordance with this, downstream signal tansducers of TIRAP including Akt, p38, IKKa, NFkB, and caspase 8 were activated, as demonstrated by elevated phosphorylation levels for Akt, p38 and IKKa, increase in the amount of the truncated form for caspase 8, and increase in binding to NFkB-responsible element for NFkB (Figure 2C). Mutation in a protein-protein interaction domain of TIRAP resulted in loss of not only its binding to RAGE but also that of wild-type MyD88 (Figure S2C), indicating that MyD88 binds to RAGE only in the presence of TIRAP. Binding of TIRAP to the cytoplasmic domain of RAGE was confirmed to be direct using both recombinant proteins (data not shown). This mode of interaction is similar to that known for TLR2/4, TIRAP, and MyD88 [21]. Essential nature of TIRAP and MyD88 for RAGE signaling was demonstrated using TIRAP-down regulated NIH3T3 cells and MyD88-/- mouse fibroblasts (Figures S2D, 2D and 2E). Overexpression of TIRAP or MyD88 rescued the defective protein interaction and signal transduction in corre- sponding cells. These results indicate that TIRAP and MyD88 function as essential adaptor proteins for RAGE, binding to ligand-activated phosphorylated RAGE and transducing a signal from it. Signal transduction from activated endogenous RAGE and its biological consequence in human umbilical vein endothelial cells (HUVECs) We next examined activation and downstream signaling of endogenous RAGE using HUVECs. RAGE was expressed in HUVECs (Figure S3A). When HUVECs were exposed to AGE, the downstream signaling molecules from RAGE were activated in dose- and time-dependent manners (Figures S3B and S3C). Nuclear translocation of NFkB induced by AGE was abrogated by treatment with inhibitor peptides for MyD88 and TIRAP (Figure S3D). Application to HUVECs with RAGE ligands such as S100A11, S100A12, HMGB1, and AGE but not LPS and TLR2 ligands induced phosphorylation of endogenous RAGE, interac- tion with TIRAP, MyD88, and IRAK4, and transduction of signal to the downstream molecules (Figures 3A and 3B). A mixture of TLR2/4 blockers showed no effect on these processes. Activation of the molecules downstream from RAGE was abrogated by treatment of HUVECs with inhibitor peptides for MyD88 and TIRAP and siRNA for PKCf (Figures S3F and S3G). On the other hand, LPS did not induce phosphorylation of RAGE and its interaction with the adaptor proteins (Figures 3A and S3F) but activated the downstream molecules from TIRAP since TLR4 shares the adaptor protein with RAGE (Figures 3B and S3G). Endogenous PKCf was activated by RAGE ligands and the activation was canceled by down regulation of RAGE using siRNA in HUVECs (Figure S3H). TIRAP Functions as an Adaptor Protein for RAGE It is well known that AGE induces IL-6 in endothelial cells through binding to RAGE [22]. Application of AGE to HUVECs resulted in induction of IL-6 mRNA, which was abrogated by pretreatment of the cells with inhibitory peptides for TIRAP and MyD88 (Figure 3C). When AGE was applied to a co-culture of HUVECs and human arterial sooth muscle cells (HASMCs), AGE induced IL-6 selectively in HUVECs (Figure 3D). HASMCs did not express RAGE at any appreciable levels (Figure S3A). A wider screening using an antibody array showed that angiogenin, BDNF, EGF, IL-1a, IL-1b, MIP3a, PDGF-BB, and RANTES in addition to IL-6 were induced by AGE in HUVECs and that the induction was abrogated by an inhibitor peptide for MyD88 (Figure 3E). Another well-known effect of AGE on endothelial cells is induction of apoptosis [23]. Application of AGE to HUVECs resulted in increase in annexin V-positive cell rate, which was abrogated by inhibitor peptides for TIRAP and MyD88 and by siRNA for PKCf (Figure S3I). These results indicate that the signal transduction pathway we found in the present study, i.e., phosphorylation of RAGE by PKCf and function of TIRAP and MyD88 as adaptor proteins, endogenously works in HUVECs, which are one of the most critical target cells of RAGE activation in physiological and pathological contexts. Effect of differential stimulation of RAGE and TLR4 Since TIRAP and MyD88 function as adaptor proteins for TLR2/4, we next examined the mode of signal transduction when RAGE and TLR4 were differentially stimulated. AGE and LPS pre-incubated with LBP for efficient transfer of LPS to the receptor [24] were used to selectively stimulate RAGE and TLR4, respectively, and a mixture of LPS and HMGB1 [25] or S100A8 and S100A9 (S100A8/A9) [17] were used to simultaneously stimulate both receptors. Since CD14 and MD2 have been shown to be involved in activation of TLR4 by ligands [26], we used HEK293 cells constitutively expressing both genes. Immunoprecipitation using antibodies against respective tags of TLR4 and RAGE followed by Western blot analysis showed that RAGE was co-precipitated with TIRAP, MyD88 and IRAK-4, while TLR4 was co-precipitated with TRAM in addition to the three proteins only when the receptors were activated by the ligands (Figure 4A and S5B). In accordance with the binding mode of adaptor proteins to respective receptors, downstream signaling molecules were activated (Figure S4A and S5A). Mutations in a protein-protein interaction domain, Toll/IL-1R intracellular domain (TIR), of RAGE and TLR4 (Figures S4B, S4C, and S5C) resulted in abrogation of binding to the adaptor proteins and downstream signal transduction (Figures 4B, 4C, S5C, and S5D). Activation of Rac1 and NFkB and phosphorylation of Akt, p38, JNK, and IKKa/b were observed when either RAGE or TLR4 was stimulated (Figures 4C, S4A, S4D, S5A, and S5D). Phosphorylation of IRF-3 was enhanced by stimulation of TLR4 but not by stimulation of RAGE (Figures 4C, S4A, S4D, S5A, and S5D). This is conceivable because IRF-3 is a downstream effector of TRAM and all other signaling molecules examined are downstream to TIRAP/MyD88 [27,28]. Addition of polymixin B blocked effect of LPS but not that of AGE (Figure S4D). IL-6, IL-8 and TNFa were induced by a ligand that can bind to either RAGE or TLR4, but interferon b (IFNb) was induced only by a TLR-ligand, HMGB1/LPS (Figure 4D). It is notable that the extent of activation of the signaling molecules and induction level of the target genes were higher when both receptors were simultaneously stimulated than that observed with stimulation of an individual receptor. These results indicate that activation of TIRAP/MyD88 triggers similar downstream signaling pathways irrespective of their binding partners. PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23132