Immunosuppressive effects of Sodium orthovanadate by interference with IKKβ dephosphorylation in RAW264.7 cells

다국어 초록 (Multilingual Abstract)

Sodium orthovanadate (SO, the complexes of vanadate with hydrogen peroxide) are two commonly used general protein-tyrosine phosphatase (PTP) inhibitors. These compounds also have insulin-mimetic properties, an observation that has generated a great deal of interest and study. Although inhibitory effects of Sodium orthovanadate have been reported in various types of cells, its immunosuppressive effect has not been elucidated. In this study, the immunesuppressive effect of Sodium orthovanadate was examined in lipopolysaccharide (LPS) treated macrophages (RAW 264.7 cells). The molecular mechanism of its anti-inflammatory activity was studied, employing immunoblotting analysis, nuclear fractionation, overexpression strategies, luciferase reporter gene assays, and kinase assays. Sodium orthovanadate was found to diminish the levels of nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-stimulated RAW264.7cells. In addition, mRNA expressions of tumor necrosis factor-a (TNF-a), cyclooxygenase-2 (COX-2), and inducible NO synthase (iNOS) were downregulated by Sodium orthovanadate. This also blocked the activation of NFκB, IκBα, but not IKKα/b, AKT, and upstream proteins in NF-κB signaling pathway, which indicate the key molecule to the anti-inflammatory effect of SO is IKKα/b proteins. According to previous reports, IKKα/b undergo progressive auto-phosphorylation at the C-terminal serine cluster, and become sensitized to deactivation. Therefore, this reference implicates that Sodium orthovanadate plays an important role in inhibition of Serine/Threonine Phosphatases PP1, holding IKKs auto-phosphorylated, allegedly in a low activity state. Taken together, immunosuppressive effects of Sodium orthovanadate can be attributed to its interference with PP1 to IKKβ dephosphorylation in NF-κB pathway. Further knowledge of these activities may be applied to prevent or suppress chronic inflammatory conditions such as diabetes, and atherosclerosis. In particular, the point that COX-2 could play a potential role in gastrointestinal tract malignancy, and Sodium orthvanadate, able to strongly suppress COX-2 expression, can be gastroprotective.

국문 초록 (Abstract)

Sodium orthovanadate (SO, vanadate 와 hydrogen peroxide 복합체)는 일반적으로 널리 사용되는 protein-tyrosine phosphatase (PTP) 억제제이다. 또한 이 화합물은 인슐린 모방 특성을 지니며 많은 관심과 연구를 이끌었다. 다양한 형태의 세포에서 Sodium orthovanadate 의 억제 효과가 보고되었지만 면역 억제 효과는 아직 밝혀지지 않았다. 본 연구에서는 LPS 처리된 대식세포 (RAW 264.7 cells)에서 Sodium orthovanadate 의 면역 억제 효과를 검증하였다. 항염증 활성의 분자 메커니즘을 연구를 위해 immunoblotting analysis을 비롯 nuclear fractionation, overexpression strategies, luciferase reporter gene assays, kinase assays 을 이용했다. Sodium orthovanadate는 LPS 자극 RAW 264.7 세포에서 nitric oxide (NO)와 prostaglandin E2 (PGE2) 생성을 감소시켰으며, tumor necrosis factor-α (TNF- α), cyclooxygenase-2 (COX-2) 및 inducible NO synthease (iNOS)의 mRNA 발현을 하향 조절하였다. 또한 NF-κB 신호 경로의 IκBα 를 제외한 IKKα/β, AKT를 비롯한 상류 단백질의 활성화를 차단하였다. 이는 IKKα/b가 SO의 항염증 효과에 중요한 요소임을 시사한다. 이전의 보고에 따르면, IKKα/b는 C-말단 serine cluster에서 자가인산화를 겪고 비활성화에 민감해진다. 따라서 SO는 Serine / Threonine Phosphatases PP1의 억제에 중요한 역할을 하며 IKKs의 자가인산화를 붙들어 낮은 활성 상태에 머문다. 종합적으로 말하자면, Sodium orthovanadate의 면역 억제 효과는 NF-κB 경로에서 IKKβ 탈 인산화 효소 PP1의 간섭에 기인한다. 이러한 활성에 대한 지식은 당뇨병 및 죽상경화증과 같은 만성적 염증 상태의 예방 또는 억제에 적용될 수 있으며, 특히 COX-2가 위장관 악성 종양에서 잠재력을 가진다는 점에서 COX-2 발현을 강력하게 억제하는 Sodium orthovanadate가 위장 보호 효과를 가질 수 있다.

목차 (Table of Contents)

• Ⅰ. Introduction
• 1. Immunity
• 1.1 Innate immunity
• 1.2 Adaptive immunity
• 1.3 Mechanisms of innate immune recognition
• 2. TLR
• 2.1 Receptors of the innate immune system
• 2.2 Signal transduction
• 3. AKTs
• 4. IKKs
• 4.1 The structure of IKKs
• 4.2 The negative regulation of IKKs
• 4.3 The three-step model of IKK activation
• 4.4 The role of protein phosphatase 1 on IKKb inactivation
• 5. Sodium orthovanadate
• Ⅱ. Materials and Methods
• Ⅲ. Results
• Ⅳ. Discussion
• Ⅴ. References