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

在先天免疫系統(innate immune system)中，Toll-like receptors (TLRs)是胞漿內模式識別受體 (pattern recognition receptors, PRRs)家族成員中的一個主要份子，而它的活化反應對於免疫扮演著重要的角色。在免疫系統方面，雖然脾酪氨酸蛋白激酶（Syk）在含免疫酪氨酸受體激活基序 (ITAM)的免疫受體（ITAM-contained immunoreceptor）所引起的訊息傳遞過程中，是一個主要的調控分子，但是Syk在Toll-like receptor (TLR) 訊息傳遞路徑扮演的角色就較不清楚。在此，我們利用研究小鼠的巨噬細胞，在TLR主導的訊息傳導與基因調控的過程，探討Syk在其中扮演角色。不論由小鼠骨髓細胞分化而成的巨噬細胞(BMDM)或是小鼠巨噬細胞株(RAW 264.7 macrophages)，TLR的專一辨識配體 [poly(I:C)-TLR3、LPS-TLR4、CpG-TLR9]都可以活化IFNbeta、TNFalpha、MIP2、IL-6、IL-12beta、iNOS及COX-2 mRNA表現，且這些基因表現會受到專一性抑制Syk活性的小分子抑制劑(Syk inhibitor, SykI)及JNK抑制劑所抑制。另外，我們發現TLR3、TLR4、TLR9均有誘發Syk和JNK活化的能力，這是根據增加在Syk (Y519/520)位置自磷酸化、JNK被磷酸化及它們的in vitro激酶能力試驗證據顯示得知。在BMDM及RAW巨噬細胞中，我們發現由TLR所主導而活化的JNK會被SykI及siRNA所抑制，但IKK活化或IkappaBalpha降解過程則不受影響，顯示 Syk為JNK活化的正調控因子。在TLR訊息路徑中，MyD88、TRIF、TRAF3及TRAF6是上游必要的訊息銜接子(adaptor)，因此我們使用基因剔除chimera鼠的BMDM研究，Syk與四種分子的訊息傳遞網關係。結果顯示由TLR3/TRIF-, TLR4/MyD88-, TLR4/TRIF-及TLR9/MyD88所主導的JNK活化訊息傳遞路徑中是需要Syk參與在其中。此外TLR3及TLR4活化JNK路徑需要TRAF3及TRAF6，而 TLR9所引起JNK活化只需要TRAF6。為了進一步闡明受體、Syk和TRAFs蛋白之間的關連，我們建構不同區段的質體，包含(deletion of N-terminal SH2 domain, C-terminal SH2 domain, both SH2 domains, or kinase domain) 。在HEK 293T大量表現蛋白的系統下，我們發現Syk能夠與TLR3, TLR4, TLR9, TRAF3及 TRAF6結合，且Syk會利用其kinase domain與TRAF6結合。此外HeNC2巨噬細胞在沒有刺激狀態下，Syk已與TRAF6結合，但當有LPS刺激時，此Syk/TRAF6複合體會被徵召到TLR4受體。總括而言，目前結果顯示在TLR主導JNK活化訊息路徑中，Syk是一個必要分子，對於IKK活化路徑則非必要。這一個訊息任務依靠著Syk與TRAF6蛋白互相結合，且當LPS刺激情況下，Syk會被徵召到TLR4經過TLR4-MyD88或 TRIF-TRAF6複合體形成進而調控下游JNK活化的訊息傳遞。

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.