SARM (sterile a-and armadillo-motif-containing protein), the fifth identified TIR (Tollinterleukin 1 receptor (IL-1R)) domain-containing adaptors in humans, downregulates NF-jB and IRF3 (interferon-regulatory factor 3)-mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR-domain-containing adaptor protein inducing IFN-b)-dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF-and MyD88-mediated, as well as basal MAPK activity, were repressed, indicating that SARM-mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP-1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N-terminal and sterile alpha motif domains in the autoregulation of SARM activity.Key words: Activator protein 1 inhibition . MAPK . N-terminal polybasic and glycine-rich region motif . Sterile a-and armadillo-motif-containing protein .
TLR Supporting Information available online
IntroductionThe transmembrane TLR play a vital role in initiating innate immunity against pathogens [1]. To date, 13 members of the TLR family have been identified in mammals [2], all of which contain an intracellular TIR (Toll-interleukin 1 receptor (IL-1R)) domain [3]. TLR are a family of PRR which recognize PAMP. Different TLR recognize different PAMP, such as LPS (a ligand for TLR4) or double-stranded viral RNA (a ligand for TLR3). After activation by PAMP, TLR transduce specific immune-related signals to the nucleus via transcription factors such as NF-kB, interferonregulatory factor 3 (IRF3) and activator protein 1 (AP-1), which in turn induces pro-inflammatory mediators, including type I interferons, chemokines and cytokines [4].TLR exert their functions via a family of five TIR domaincontaining adaptor proteins: MyD88 (myeloid differentiation à These authors have contributed equally to this work. primary-response gene 88), Mal (MyD88-adaptor-like protein), TRIF (TIR-domain-containing adaptor protein inducing IFN-b), TRAM (TRIF-related adaptor molecule) and SARM (sterile a-and armadillo-motif-containing protein). MyD88, Mal, TRIF and TRAM all activate the TLR signaling pathways. All TLR except TLR3 recruit MyD88 to their cytoplasmic TIR domain to mediate innate immune signaling [5,6]. Mal is required by TLR2 and TLR4 to bridge the recruitment of MyD88 [7][8][9][10]. TRIF mediates TLR3 signaling and TLR4-induced MyD88-independent pathway, such as delayed NF-kB activation [11][12][13]. The interaction between TRIF and TLR4 is mediated by TRAM [14][15][16].As a newly discovered member of the TLR-adaptor family, the function of S...