The functional study of GSK3β in TLR3 signaling

Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is critical for the production of inflammatory cytokines in various Toll-like receptor (TLR)-mediated signaling pathways. However, it is poorly understood how TRAF6 regulates TLR3 responses. Here, I demonstrate that GSK3β interacts with TRAF6 and positively regulates the TLR3-mediated signaling. Suppression of GSK3β expression or its kinase activity drastically reduced the production of inflammatory cytokines and the induction of c-Fos by decreasing extracellular signal-regulated kinase (ERK) and p38 phosphorylation. GSK3β physically associated with TRAF6 in a poly I:C-dependent manner. TRAF6 was determined to be a direct E3 ligase for GSK3β and TRAF6-mediated GSK3β ubiquitination was essential for poly I:C-dependent cytokine production by promoting the TRIF-assembled signaling complex.;Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) 는 다양한 Toll-like receptor (TLR) 과 관련된 신호 전달 과정에서 inflammatory cytokine을 생성하는 데에 있어 매우 중요한 adaptor 기능을 하는 것으로 알려져 있다. 그러나 TRAF6가 어떻게 TLR3 반응을 조절하는지에 대해서는 거의 알려진 바가 없다. 이 논문에서 우리는 GSK3β 가 TRAF6와 interaction하며 TLR3 관련 신호 전달 체계를 양성적으로 조절함을 보였다. GSK3β 의 발현이나 kinase 활성을 억제시켰을때, extracelluar signal-regulated kinase (ERK)와 p38의 phosphorylation을 감소시킴으로써 inflammatory cytokine의 생성과 c-Fos induction양을 극적으로 감소시켰다. 또한GSK3β는poly I:C 에 의해 TRAF6와 생리적으로 interaction을 하였다. TRAF6는 GSK3β의직접적인 E3 ubiquitin ligase로 보여지며, TRAF6에 의해 유도된 GSK3β의ubiquitination은 TRIF에 결합하는 신호 전달 complex를 증진시킴으로써 poly I:C에 의한 cytokine 생성에 필수적으로 작용하고 있다.INTRODUCTION 1 MATERIALS AND METHODS 7 Cell culture and transfection 7 Reagents and antibodies 8 Plasmid constructs 9 Immunoprecipitation (IP) and Western blot analysis 9 Enzyme-linked immunosorbent assay (ELISA) 10 Real-time PCR analysis 10 siRNA transfection 13 GST pull-down assay 14 Native PAGE assay 14 Mapping of ubiquitination sites on GSK3β 14 Statistical analysis 15 RESULTS 16 GSK3β positively regulates TLR3-mediated inflammatory cytokine production and type I IFN-β induction 16 GSK3β regulates TLR3-mediated ERK and p38 activation 27 GSK3β regulates expression of c-Fos through ERK and p38 in TLR3 signaling 31 GSK3β is required for the recruitment of TRAF6-TAK1-TAB1-TAB2 complex to TLR3 36 GSK3β interacts with TRAF6 and TAK1 39 TRAF6-mediated GSK3β ubiquitination at lysine 183 is essential for TLR3 signaling 45 Ubiquitination of GSK3β is required for interaction with TRAF6 58 GSK3β is required for TRAF6 phosphorylation in TLR3 signaling 62 GSK3β is required for BTK activation in TLR3 signaling 64 DISCUSSION 67 REFERENCES 74 국문초록 8

파골세포 발생과 TLR 신호전달에 미치는 GSK3β의 역할

Glycogen synthase kinase 3 (GSK3) is a ubiquitous serine/threonine kinase and regulates multiple cellular functions. A delicate balance between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs) is critical for the maintenance of bone homeostasis. A lot of studies demonstrated roles for GSK3β in bone remodeling by regulation of OBs, but roles by which GSK3β regulates OCs fate remains unclear. The skeletal and the immune systems share a variety of regulatory mechanisms. Toll-like receptors (TLRs) are important that contribute innate immunity and adaptive immunity and GSK3 has also been identified as a regulator in TLR signaling. Here, I developed a transgenic mice, which express kinase-dead GSK3β, and cell-permeable peptide, which specifically inhibits activity of GSK3β. By using these tools, I found that GSK3β negatively regulated RANKL-induced OC differentiation and TLR-mediated proinflammatory cytokines production. In addition, administration of the GSK3β inhibitor peptide protected mice against LPS-induced lethality. These findings demonstrate a regulatory function of GSK3β in modulating the bone remodeling and the inflammatory response, and may be useful in the development of selective therapeutic agents for the treatment of septic shock and other inflammatory diseases.;GSK3 (Glycogen synthase kinase 3) 는 대부분의 세포에 존재하는 Serine/Threonine 인산화 효소이며 다양한 세포 기능에 관여한다. 조골세포 (Osteoblasts) 와 파골세포 (Osteoclasts) 의 정교한 균형은 뼈의 재구성 (Remodeling) 과 항상성 (Homeostasis) 에 매우 중요하다. 조골세포 조절에 있어서의 GSK3의 역할에 대해서는 많이 알려진 반면, 파골세포를 통한 GSK3의 조절 기작은 거의 밝혀지지 않은 상태이다. 골격계 (Skeletal systems) 와 면역계 (Immune systems) 는 다양한 조절 기작을 공유한다. TLRs (Toll-like receptors) 는 선천성 면역체계 (Innate Immune system) 와 후천성 면역체계 (Adaptive immune system) 모두에서 중요하게 작용하며 최근 GSK3가 TLR 신호전달 (TLR signaling) 에서 중요한 조절자 (Regulator) 로서 작용한다는 사실이 밝혀지고 있다 인산화 활성이 억제된 GSK3β를 과발현 (Overexpression) 시킨 형질전환 마우스 (Transgenic mice) 와 GSK3β활성을 억제하는 세포 투과적 펩타이드 (Cell-permeable peptide) 를 사용하여 GSK3β가 RANKL (Receptor activator of nuclear factor кB ligand) 에 의한 파골세포 분화를 음성적으로 조절하며 TLR에 의한 cytokine 생성을 조절함을 확인하였다. 또한 이 펩타이드가 투여된 마우스는 LPS에 의해 유도된 패혈증 (Sepsis) 으로부터 보호되는 것을 확인하였다. 이러한 결과들은 GSK3β가 뼈의 재구성과 면역 반응에서 중요하게 작용하고 있으며, 패혈증을 비롯한 여러 면역 질병 치료제로서의 가능성 또한 시사한다.Ⅰ. INTRODUCTION = 1 Ⅱ. EXPERIMENTAL PROCEDURES = 6 1. Cell culture = 6 2. Reagents and antibodies = 7 3. Retrovirus preparation = 8 4. In vitro assay for osteoclasts formation = 8 5. Construction of transgene = 9 6. Production of transgenic mice = 10 7. Genotyping of transgenic mice = 11 8. Peptide synthesis = 12 9. Western blot analysis = 12 10. Measurement of cytokines = 13 11. Endotoxin shock model = 14 Ⅲ. RESULTS = 15 1. GSK3β inhibits RANKL-induced osteoclast differentiation = 15 2. Establishment of transgenic mice expressing kinase-dead human GSK3β = 24 3. The kinase-dead GSK3β transgene augments osteoclast differentiation ex vivo = 29 4. The GSK3β inhibitor peptide can regulate osteoclastogenesis = 33 5. The GSK3β inhibitor peptide can regulate TLR-mediated pro-inflammatory cytokine production = 36 6. Inhibition of GSK3β using the GSK3β inhibitor peptide regulates activation of MAPKs in TLR2 and TLR4 = 39 7. The GSK3β inhibition peptide protects mice from endotoxin shock = 43 Ⅳ. DISCUSSION = 46 Ⅵ. REFERENCES = 50 Ⅶ. 논문개요 = 5

Glycogen Synthase Kinase 3 beta Regulates Antiviral Responses of TLR3 via TRAF2-Src Axis

The protein tyrosine kinase Src regulates the synthesis of TLR3-mediated IFN-beta via the TBK1-IFN regulatory factor 3 axis. However, the molecular mechanisms regulating Src activity in TLR3 signaling remain unclear. In this study, we report that GSK3 beta regulates Src phosphorylation via TNFR-associated factor 2 (TRAF2)-mediated Src ubiquitination. GSK3 beta deficiency in mouse embryonic fibroblasts significantly reduces polyinosinic:polycytidylic acid-induced IFN-beta and IFN-stimulated gene expression, which is caused by diminished phosphorylation of Src at tyrosine 416. Src undergoes polyinosinic:polycytidylic acid-dependent lysine 63 chain ubiquitination, and TRAF2 is a direct E3 ligase for Src. Our study reveals novel mechanisms underlying TLR3-mediated antiviral responses mediated via the GSK3 beta-TRAF2-Src axis