TIRAP Functions as an Adaptor Protein for RAGE Figure 3. Specific activation of endogenous RAGE by AGE induced cytokines in HUVECs. (A and B) Activation of downstream signaling molecules by specific activation of endogenous RAGE. Experiments were performed under the conditions similar to those described in Fig. 2A except for down regulation of RAGE with siRNA, immunoprecipitation using an antibody against RAGE, and type of cells used. The results were confirmed by an independent and two related experiments (A) or by an independent and three related experiments (B). (C) Induction of IL-6 by AGE (100 mg/ml for 12 h) demonstrated by Northern blot analysis in HUVECs in serum- and supplement-free conditions. Cell-permeable inhibitory peptides (100 mM) were added 12 h prior to the addition of AGE. The results were confirmed by two independent and one related experiments. (D) Induction of IL-6 in HUVECs but not in HASMCs by treatment with AGE. HUVECs co-cultured with HASMCs pre-infected with an adenovirus carrying GFP were treated under conditions similar to those described in (C). Fixed cells were immunostained with anti-IL-6 antibody. Bars, 20 mm. The results were confirmed by three independent experiments. (E) Detection of cytokines secreted into the medium by HUVECs treated with AGE using an antibody array [Human cytokine Antibody Array VI & 6.1 (60), RayBiotech, Norcross GA]. Dotted squares indicate antibodies against corresponding cytokines. Cells were treated under conditions similar to those described in (C). The results were confirmed by an independent experiment. doi:10.1371/journal.pone.0023132.g003 [23], which was accompanied by activation of caspase-8 (Figure S3C). Notably, this effect was almost completely inhibited by siRNA for PKCf and the inhibitor peptides for TIRAP and MyD88 (Figure S3I), indicating the critical role of these proteins in RAGE-mediated apoptotic cell death. Hence, diabetes- associated vascular damages through AGE-RAGE interaction are probably mediated by TIRAP and MyD88. It remains to be clarified how ligand-activated RAGE led to activation of PKCf PKCf requires activated Ras, a small GTPase, for its own kinase activity [29]. Lander et al. [9] reported that AGE-albumin activated p21 (ras) in rat pulmonary artery smooth muscle cells that express RAGE. Hudson et al. [18] showed by immunoprecipitation that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1 and that down-regulation of Dia-1 blocks RAGE- mediated activation of Rac-1 and Cdc42 and RAGE ligand- stimulated cellular migration. We could not detect endogenous Dia-1 protein in immunoprecipitates prepared from HEK293 cells using an antibody against tagged RAGE under the conditions in which endogenous TIRAP and MyD88 were co- precipitated with overexpressed RAGE. In accordance with the results by Hudson et al., however, overexpressed FH1 domain of Dia-1 was co-precipitated with the cytoplasmic domain of RAGE (data not shown). In addition, our preliminary data suggest that TIRAP and the FH1 domain of Dia-1 compete for binding to the cytoplasmic domain of RAGE. Precise interac- tion among the three proteins remains to be elucidated. Binding of TIRAP but not MyD88 to the cytoplasmic domain of RAGE was confirmed to be direct by an in vitro protein interaction assay using recombinant proteins (data not shown). Phosphorylation of RAGE at Ser391 greatly enhanced affinity to TIRAP (Figure 2), indicating that more negative charge at the residue 391 is important for efficient binding with TIRAP. When the residue is phosphorylated, Q(390)SEE becomes equivalent to QEEE. Intriguingly, the motif of Q followed by 3 successive negatively charged residues is found at another site of RAGE (QEEE stating at 379) and in the cytoplasmic domain of TLR4 (QDED starting at 683) and in TLR2 (QELE starting at 661) but not of TLR1 and TLR3. The motif might play a role in interaction of intracellular domains of receptors and TIRAP. PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23132