Role of TRAF interacting protein in TNF-R Signaling Pathway

다국어 초록 (Multilingual Abstract)

TNF-receptor-associated factor 2 (TRAF2) is a key adaptor molecule in TNF-R signaling complexes that promotes the activation of transcription factor such as NF-kappaB. Previous studies demonstrated that K63-ubiquitination of TRAF2 contributes to recruitment of TAB/TAK1 complex. It leads to activation of NF-kappaB. TRAF2-ubiquitination is dependent on the E3 ligase activity of TRAF2 RING domain. Recent study showed that sphingosine-1-phosphate (S1P), is phosphorylated by sphingosine kinase 1(SPHK1), is an essential cofactor for TRAF2 ubiquitin ligase activity. S1P is important for the activation of TRAF2- ubiquitin ligase, enabling it to mediate the activation of NF-kappaB in TNFR1 signaling. However, a regulator of the mechanism remains largely unknown.
One of the known binding partners of the TRAF2, TRAF-interacting protein (TRIP), contains a RING finger motif, rod-like coiled coil domain and leucine zipper domain in its amino-terminal region. In this study, I reported that TRIP reduces Ubc13-dependent K63-linked ubiquitination of TRAF2. Moreover, rod-like coiled coil domain of TRIP is essential for inhibition of TRAF2 ubiquitination and NF-kappaB activity. In addition, I identified that TRIP associates with TRAF2-SPHK1 complex and suppresses NF-kappaB activation induced by TRAF2-SPHK1 complex. TRIP also inhibits binding of S1P to TRAF2. Consistently, overexpression of TRIP suppresses production of TNF-alpha-induced pro-inflammatory cytokines. Indeed, knockdown of TRIP promotes TNF-alpha-induced pro-inflammatory cytokine. Recently, it has been found that osteoclast differentiation is induced by TNF-alpha. Here, I found that TRIP as a key regulator of osteoclastogenesis that plays a role in suppressing TNF-alpha-induced osteoclast differentiation. Furthermore, TRIP depletion promotes TNF-alpha-induced osteoclastogenesis. These results suggest that TRIP is critical for suppression of TNF-alpha-induced osteoclast formation. In conclusion, these findings demonstrate that TRIP plays a regulator in TNF-R signaling. Therefore, I suggest that TRIP is a potential therapeutic target of inflammatory responses.

국문 초록 (Abstract)

TNF-R 신호전달기작에서 중요한 adaptor로서 알려진 TRAF2 단백질은 NF-kappaB를 포함하는 전사인자의 활성을 조절한다. 기존의 논문에 의하면, TRAF2 단백질의 K63-ubiquitination은 TAB/TAK1 complex와의 상호결합을 유도하여 NF-kappaB 활성기작을 촉진시킨다. TRAF2 단백질의 ubiquitination은 TRAF2 단백질의 RING finger motif에 존재하는 E3 ubiquitin ligase 활성에 의해 영향을 받는 것으로 알려져 있다. 최근 연구결과를 통해, sphingosine-1-phosphate (S1P)가 TRAF2 단백질의 E3 ubiquitin ligase의 cofactor로 작용함이 밝혀졌다. S1P는 sphingosine kinase 1 (SPHK1)에 의해 인산화되며, TNF-R 신호전달기작내에 존재하는 NF-kappaB 활성기작 조절을 매개하는 TRAF2 단백질의 ubiquitin ligase의 활성을 조절하는데 중요한 영향을 미친다. 그러나 이러한 S1P를 통한 TRAF2 단백질의 ubiquitin ligase의 활성을 억제하는 단백질에 대해서는 아직 충분한 연구가 이루어지지 않았다.
TRAF2 단백질과 상호결합을 하는 것으로 밝혀진 TRIP 단백질은 amino-말단 부위에 RING finger motif, rod-like coiled coil domain, leucine zipper domain 으로 구성되어있다. 본 연구에서는 이러한 TRIP이 TRAF2 단백질의 K63-ubiquitination을 억제하는 조절자로서 작용한다는 사실을 밝혔다. 또한, TRAF2 단백질의 ubiquitination 을 억제하는 TRIP의 조절기작에서 rod-like coiled coil domain 부위가 중요하게 작용하는 것으로 보인다. 본 연구는 TRIP이 TRAF2-SPHK1 complex와 결합하여 TRAF2-SPHK1 complex에 의해 매개되는 NF-kappaB 활성을 억제하는 사실과 TRIP이 S1P와 TRAF2간의 상호결합을 억제함을 추가로 밝혀내었다. 이처럼, TRIP은 S1P와 TRAF2의 상호결합을 억제함으로써 TRAF2의 ubiquitination을 억제하고 TRAF2에 의해 유도되는 NF-kappaB 활성기작을 조절하는 조절자 역할을 하는 것으로 여겨진다.
본 연구를 통해, 세포수준에서 TRIP 단백질의 과발현이 TNF-alpha에 의해 유도되는 염증성 사이토카인 생성을 억제하며, TRIP의 결핍은 TNF-alpha에 의해 유도되는 염증성 사이토카인 생성을 촉진한다는 사실을 확인하였다. 세포 내에서 TRIP은 NF-kappaB 활성 기작을 조절하여 NF-kappaB에 의해 유도되는 염증성 사이토카인의 생성을 억제하는 것으로 보인다.
본？두번째？연구에서는？TRIP 단백질이 TNF-alpha에 의해 유도되는 파골 세포의 분화에 영향을 미친다는 사실을 밝혔다. 최근 연구에 따르면 TNF-alpha가？파골？세포의？분화에도？중요한 영향을？미친다는？사실이？보고되었다.
본？연구를？통해？TRIP이 과발현 되었을 때 TNF-alpha에 의한 파골 세포의 분화가 억제되는 사실과 TRIP이 결핍되었을 때는 TNF-alpha에 의한 파골 세포의 분화가 촉진되는 현상을 확인하였다.
본 연구를 통하여, TRIP 단백질이 새로운 TNF-R 염증성 신호전달기작의 조절자임을 규명하였으며 이러한 TRIP의 역할은 염증성 질환의 치료 개발까지 확대 적용할 수 있을 것으로 사료된다.

목차 (Table of Contents)

• Chapter 1 Introduction and literature reviews 1
• 1. TNF signaling and TRAFs 3
• 2. NF-κB regulation by ubiquitination 8
• 3. TRAF interacting protein (TRIP) 14
• 4. Role of TNF-α in Inflammatory disease 17
• 5. Reference 20
• Chapter 2 TRAF-interacting protein (TRIP) down-regulates TNF-α-mediated NF-κB signaling by inhibition of TRAF2 ubiquitination 25
• 1. Introduction 27
• 2. Material and Methods 32
• 2.1 Cell culture, Mice and reagents 32
• 2.2 Plasmids 33
• 2.3 RNA interference (RNAi) synthesis 34
• 2.4 Transfection and in-vivo binding assay 34
• 2.5 In vivo Ubiquitination assay 35
• 2.6 Pull downs with lipid affinity matrices 36
• 2.7 Immunoblot analysis 37
• 2.8 NF-κB reporter assay 38
• 2.9 Retroviral infection of BMDM and IL-6 & IL-1β ELISA assay 38
• 2.10 Mouse cytokine expression array 39
• 2.11 Real-time RT-PCR analysis 40
• 3. Results 41
• 3.1 TRIP suppresses TNF-α induced NF-κB activation by inhibition of TRAF2 ubiquitination 41
• 3.2 Rod-like coiled coil and leucine zipper domain are essential for TRIP-TRAF2 interaction 47
• 3.3 Rod-like coiled coil domain of TRIP is required for inhibition of TRAF2-mediated NF-κB activity and TRAF2 ubiquitination 50
• 3.4 RING E3 ligase activity of TRIP is dispensable for regulation of TRAF2 ubiquitination and TRAF2-mediated NF-κB activity 54
• 3.5 TRIP does not affect in oligomerization and stability of TRAF2 57
• 3.6 TRIP down-regulates NF-kB activity induced by TRAF2-SPHK1 complex and binding affinity of S1P to TRAF2 59
• 3.7 TNF-α-induced pro-inflammatory cytokines is down-regulated by overexpression of TRIP 63
• 3.8 Knockdown of TRIP promotes expressions of TNF-α-induced pro-inflammatory cytokines 68
• 3.9 TRIP suppresses TNF-α-induced phosphorylation of p65, IkB-a and MAPKs 72
• 3.10 Overexpression of TRIP does not influence Macrophage differentiation 75
• 4. Discussion 80
• 5. Reference 83
• Chapter 3 TRAF-interacting protein (TRIP) inhibits TNF-α-induced osteoclastogenesis 87
• 1. Introduction 89
• 2. Materials and Methods 92
• 2.1 Cell culture, Mice and reagents 92
• 2.2 Plasmids 93
• 2.3 Retroviral infection of BMDM and Osteoclastogenesis 93
• 2.4 Analysis of Osteoclasts 94
• 2.5 Transfection and in-vivo binding assay 95
• 2.6 NF-κB reporter assay 95
• 3. Results 97
• 3.1 TRIP suppresses TNF-α-induced osteoclastogenesis 97
• 3.2 TRIP does not affect RANKL-induced osteoclastogenesis 100
• 3.3 TRIP knockdown promotes TNF-α-induced osteoclastogenesis 103
• 3.4 TRIP regulates TRAFs-mediated NF-κB signaling 106
• 4. Discussion 108
• 5. Reference 110
• Summary (in Korean) 113
• Acknowledgments 116