探討巨噬細胞Spleen tyrosine kinase在Toll-Like Receptors的訊息傳遞及誘發的發炎基因表現中所扮演的角色

Abstract

Toll-like receptor (TLR) is a major family of pattern recognition receptors (PRRs) and plays a crucial role in innate immune system. Even though non-receptor spleen tyrosine kinase (Syk) is a key signaling molecule of ITAM-contained immunoreceptors, its role in TLRs signaling is not clearly understood. Herein, we investigated the role of Syk in TLR-mediated signaling and gene regulation in murine macrophages. In both mouse bone marrow-derived macrophages (BMDM) and murine RAW 264.7 macrophages, poly (I:C), LPS and CpG, which are specific ligands for TLR3, TLR4 and TLR9 respectively, can increase the mRNA levels of several pro-inflammatory cytokines and mediators, including IFNbeta, TNFalpha, MIP2, IL-6 and IL-12beta, iNOS and COX-2. These transcriptional gene upregulation caused by TLR activation were inhibited by specific Syk inhibitor (SykI) and JNK inhibitor (SP600125). Accordingly we found the abilities of TLR3, TLR4 and TLR9 to induce Syk and JNK activation, as evidenced by the increase of Syk auto-phosphorylation on Y519/Y520, JNK phosphorylation as well as their in vitro kinase activities. We also found that TLRs-mediated activation of JNK, but not IKK or IkappaBalpha degradation in BMDM and RAW 264.7 macrophages was blocked by SykI and Syk siRNA, suggesting that Syk is a positive regulator of JNK. Since MyD88, TRIF, TRAF3 and TRAF6 are crucial upstream molecules for TLR signaling, we thus used BMDM isolated from genetic knockout chimera mice to study the coupling between Syk and these adaptors. Results revealed that Syk is involved in TLR3/TRIF-, TLR4/MyD88-, TLR4/TRIF- and TLR9/MyD88-mediated JNK signaling. In addition, both TRAF3 and TRAF6 contribute to JNK activation elicited by TLR3 and TLR4, while only TRAF6 is required for JNK activation by TLR9. To further dissect the protein interactions among receptors, Syk and TRAFs, we constructed Syk plasmids with different domains (deletion of N-terminal SH2 domain, C-terminal SH2 domain, both SH2 domains, or kinase domain. We found that in expression cell system Syk is able to associate with TLR3, TLR4, TLR9, TRAF3 and TRAF6. Moreover, kinase domain of Syk is required for its association with TRAF6. In resting HeNC2 macrophages, Syk can associate with TRAF6, and upon LPS stimulation this Syk/TRAF6 complex is recruited to TLR4. All together, our data highlight the role of Syk in coupling TLR signaling to JNK but not to IKK pathway. This signaling role relies on the interaction of Syk to TRAF6, and upon LPS stimulation, Syk can be recruited to TLR4 through the TLR4-adaptors (MyD88 or TRIF)-TRAF6 complex formation, and then regulates downstream JNK signal.