Studies On The Mechanism Of BCL10 In The LPS/TLR4 Signal Pathway

The innate immune response in vertebrates is the first line of defense against invading microorganisms. In innate immunity, different phagocytes such as neutrophils, macrophages and dendritic cells play crucial roles in discrimination between pathogens and self by utilizing signals from the Toll-like receptors(TLR). The most significant step in the process of initiating innate immunity to resist pathogens is recognition, the components, exist in pathogens instead of in cells, which is called pathogen-associated molecular pattern(PAMP), can be recognized by the receptors of killing cells. Different PAMPs are identified by different TLRs, for example, TLR4 can respond to LPS of Gram-negative bacteria. Herein, we demonstrated that BCL10, a critical molecule signaling between the TCR and IκB kinase complexes, was implicated in the innate immunity system and required for appropriate TLR4 pathway and NF-κB activation.As a traditionally accepted signaling protein in adaptive immunity, BCL10 has been found mediating signal transduction in TCR pathway and its mechanism has been well elucidated. Our previous studies reveal that besides its role in adaptive immunity, BCL10 also plays a dramatic part in signaling in innate immunity, since it can mediate nuclear factor-κB (NF-κB) activation in lipopolysaccharide (LPS) / Toll-like receptors-4 (TLR4) signaling. In this chapter we show that interleukin-1 receptor-associated kinase 1(IRAK1) acts as the pivotal upstream adaptor that recruits BCL10 to TLR4 signaling complex when stimulated by LPS, but RNAi of IRAK1 renders RAW264.7 cells failed to recruit BCL10 into the receptor complex. IRAK1 mainly act as a promixal adaptor in BCL10-mediated signal transduction independent on its kinase activity. When recruited into the receptor complex, BCL10 transduces signals directly downstream of IRAK1, leading to the activation of NF-kB. Studies have revealed that IRAK1 undergoes dimerization after LPS stimulation, which has been the major cause of its activation, here we proved that the IRAK1 oligomerization is an optimal factor for BCL10 oligomerization and activation. We also map the domains of IRAK1 required for the interaction of BCL10 and vice versa, finding that the axon 12(514-533 amino acid) of IRAK1 and the MALT1 binding domain and C-terminal Ser/Thr rich domain of BCL10 are in charge of their interaction.Pellino proteins play important roles in establishing and maintaining TLR signal subways as a newly identified signaling molecule in this process. Recent studies reveal that ectopic expression of a mouse Pellino2 antisense construct can inhibit LPS induced activation of NF-κB. In macrophages stimulated by LPS, the interaction in vivo between BCL10 and Pellino2 was demonstrated we designed a SiRNA construct pSUPER-Pellino2 to found a Pellino2 deficient cell line, in which the NF-κB activation was partly inhibited in response to LPS stimulation or BCL10 over-expression, suggests that Pellino2 have an important role in LPS pathway or BCL10-dependent signaling, we transfected cells with SiRNA construct pSUPER-BCL10 to form bcl10 gene silent cell line, we found that deficiency in BCL10 expression caused moderate reduction of NF-κB activation in response to LPS stimulation, whereas NF-κB activation triggered by TNF-αtreatment was similar in both wild type and BCL10-deficient cells, which directly give rise to a possibility that BCL10 might specifically signal downstream of TLR4. In subsequent research, after LPS stimulation, endogenous TLR4 co-precipitated with BCL10, indicating that BCL10 may be recruited to the TLR4 signal and be recruited to TLR4 signal complex. However, the RAW264.7 cells defect in Pellino2 expression had no effect on the recruitment of BCL10, which confirms that Pellino2 is an adaptor downstream of BCL10 in LPS signaling. SOCS3 blocks TLR4 and TCR signaling through its targeting and interacting with BCL10. It has been reported that SOCS3 is a negative regulator in LPS/TLR4 pathway and TCR pathway. To confirm the exact role SOCS3 plays in LPS pathway and whether BCL10 is the target of SOCS3, we carried out co-immunoprecipitation to study the binding activity of BCL10 and SOCS3 in vivo. we demonstrated that over-expressed SOCS3 could associated with BCL10 in cells stimulated with LPS or untreat. To get further proof, a cell line stably expressing SOCS3 was constructed and forced expression of SOCS3 was found to result in attenuation of BCL10-induce NF-κB activation indicating that BCL10 may be the targeted molecule of SOCS3 for negative regulation in LPS signaling. While in the same cell line, when SOCS3 is forced expressed, the association between BCL10 and Pellino2 was severely impaired, whereas BCL10 interact with more SOCS3 proteins compared to that in the wild type cells. However, neither Pellino2 was detected in SOCS3-precipitated complex, nor was SOCS3 in the Pellino2-precipitated complex, demonstrating that there is no association between SOCS3 and Pellino2. Together, SOCS3 may negatively regulate BCL10 function by declining its ability to interact with Pellino2.In TCR pathway MALT1 is a critical signaling protein directly associated with BCL10, transducing signals down to TRAF6 and leading to the activation of NF-kB. Recent studied have found that a 16-peptide sequence in BCL10 are responsible for its association with MALT1. We stably transfected RAW264.7 cells with RNAi vector for MALT1, find out that LPS induced NF-kB activation was obviously attenuated in the cells, suggesting MALT1 is required for LPS/TLR4 signaling. MALT1 interacts with BCL10 and TRAF6 in cytosol, and facilitating TRAF6 self- ubiquitination. Although we show the interaction between MALT1, BCL10 and TRAF6, the former can only be detected in cytosol complex, while the other two also present in membrane-bound complex, since a previous study has verified there are no direct interactions between BCL10 and TRAF6, so how does TRAF6 leave phosphorylated IRAK1 along with BCL10? Recently a signaling protein Pellino2 that identified mediating BCL10 signal in LPS pathway by our group plays a part in it. Our data indicate Pellino2 not only fulfill the role for bridging BCL10-TRAF6 interactions after disassociation from IRAK1, but also may facilitate the disassociation of BCL10 from IRAK1.