TIRAP Functions as an Adaptor Protein for RAGE performed under conditions similar to those described in (A). The results were confirmed by two independent and two related experiments. (C) Downstream signal transduction from RAGE and TLR4. Experiments were performed under conditions similar to those described in (B). The results were confirmed by two independent and two related experiments. (D) Induction of cytokines by differential stimulation of RAGE and TLR4 as assayed by Northern blot analysis. Experiments were performed under conditions similar to those described in (B) except for treatment time (12 h). The results were confirmed by an independent and four related experiments. doi:10.1371/journal.pone.0023132.g004 Sharing of the adaptor proteins between RAGE and TLR2/4 is unexpected but not inconceivable. RAGE and TLR2/4 are categorized as pattern-recognizing receptors, and they use NFkB as a major intracellular signaling molecule and are involved in diverse cellular functions, inflammation in particular. Lin indicated the possibility that a TIR-containing protein may function as an adaptor protein for RAGE [20]. TLR4 has a signal transduction pathway of TRAM-TRIF in addition to TIRAP-MyD88 [30,31]. It is not yet clear whether Dia-1 is another arm for RAGE. Both RAGE and TLR2/4 have common and distinct ligands. Our findings indicate that RAGE and TLR2/ 4 partly share an intracellular signaling pathway. It is intriguing that different RAGE ligands often show different effects in given types of cells. One reason may come from simultaneous binding of RAGE ligands to TLR2/4 as described Figure 5. Schematic diagram of signal transduction from RAGE and TLR4. Both receptors partially share ligands and adaptor proteins. TLR4 transduces a signal to NFkB via TIRAP, MyD88 and IRAK4 and to IRF3 via TRAM and TRIF. RAGE phosphorylated by PKCf upon ligand binding also uses TIRAP, MyD88 and IRAK4 for signal transduction to NFkB. Presence of yet unknown signaling pathway from RAGE is still possible. Simultaneous activation of both receptors resulted in enhanced activation of NFkB. The signaling machinery is also linked to Akt and caspase-8, and possibly Rac1. doi:10.1371/journal.pone.0023132.g005 above. A complex of S100A8 and S00A9 (S100A8/A9) also binds both RAGE and TLR4 [5,16,32,33]. S100A8/A9 induced phosphorylation of RAGE (Figures S1C and S1H) and led to activation of diverse signal effectors such as Rac1, Akt, p38, JNK, IKK, and NFkB (Figure S5A and S5D). On the other hand, recruitment of the adaptor proteins triggered by S100A8/A9 was limited (Figure S5B and S5C). In addition, activation of p38, JNK, IKK, and NFkB was not completely inhibited by co-expression of RAGE- and TLR4-dominant negative forms, contrary to the complete inhibition observed when treated with HMGB1-LPS (Figure S5D). This may be explained by presence of other unidentified receptor(s) for S100A8/A9 in addition to RAGE and TLR4. Apparently, the system involving ligands-receptors-adaptor proteins for RAGE and TLR2/4 is very complicated. Further extensive studies are needed to clarify the functional interaction of proteins in the system and biological outcomes of such interactions. Studies in this direction will ultimately lead to better understanding of and development of therapeutic measures for many serious human diseases including diabetes mellitus and cancer [2–5,11,34–36]. Materials and Methods Cells and Chemicals Cell lines of HEK293, NIH3T3 (ATCC, Manassas, VA) and 293-hMD2-CD14 stably expressing human MD2 and CD14 genes (InvivoGen, San Diego, CA) and primary culture of lung fibroblasts prepared from MyD88-/- mice (Oriental Yeast, Tokyo, Japan) were cultivated in D/F medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS. HUVECs (KURABO, Osaka, Japan) were grown in Humedia-EG (KURABO) medium containing 2% FBS, FGF-B (5 ng/ml), EGF (10 ng/ml), hydro- cortisone (1 mg/ml), and heparin (10 mg/ml). HASMCs (KUR- ABO) were grown in Humedia-SG2 (KURABO) medium containing 5% FBS, FGF-B (2 ng/ml), EGF (0.5 ng/ml), and insulin (5.0 mg/ml). Cells at 3,6 passages were used. When necessary, cells were labeled with 32P- orthophosphate (ICN, Costa Mesa, CA) at a dose of 100 mCi/ml for 4 h. Sources of the materials are as follows: Lipopolysaccharide from E. coli 0111:B4 (LPS, Sigma-Aldrich), lipoteichoic acid from Staphylococcus aureus (LTA-SA), synthetic bacterial lipoprotein (Pam3CSK4), peptidoglycan from E. coli 0111:B4 (PGN-EC) and lipomannan from Mycobacterium smegmatis (LM-MS) (InvivoGen), polymixin B sulphate (PMB; Indofine Chemical Company, Hillsborough, NJ), recombinant human TLR2 extracellular domain (rexTLR2), TLR4 extracellular domain/MD-2 (rexTLR4/MD-2), LPS-binding protein (LBP) and HMGB1 (R&D Systems), AGE (made of BSA; Calbiochem, La Jolla, CA), cell-permeable peptide set comprising peptides that inhibit human TIRAP and MyD88 functions (IMGENEX, San Diego, CA), active types of recombinant MEK1, JNK1, p38a, Akt1, PKCa, PKCe, PKCd, PKCf, and PKCm (MILLIPORE, Gemini Crescent Dundee, UK), recombinant IRAK4 (BPS Bioscience, San Diego, CA), and annexin V-FITC (MBL). A TLR2 ligand mixture of LTA-SA, Pam3CSK4, PGN-EC and LM-MS at 10 ng/ml each was used to activate TLR2 (TLR2Ls). A mixture of PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23132