胆汁酸受体Gpbar1（TGR5）抑制炎症的分子机制研究

Elucidating the mechanisms for suppressing chronic inflammation could lead to life-saving therapy for a large number of patients with inflammation-related diseases such as diabetes and cancer. Our recent publications have shown that bile acid membrane receptor Gpbar1 (G-protein-coupled bile acid receptor, TGR5) is a key negative regulator of inflammation. We found that TGR5 negatively regulates hepatic inflammatory response through antagonizing NF-κB signaling pathway. TGR5 activation suppresses the phosphorylation of IκBα, the translocation of p65, NF-κB DNA-binding activity, and its transcription activity. Furthermore, we found that TGR5 activation enhances the interaction of IκBα and β-arrestin2. Suppression of NF-κB transcription activity and its target gene expression by TGR5 agonist are specifically abolished by the expression of anti-β-arrestin2 small interfering RNA. These results show that TGR5 suppresses the NF-κB pathway by mediation of the interaction between IκBα and β-arrestin2. In Specific Aim 1 of this proposal, we will determine the molecular mechanisms by which TGR5 activation enhances the interaction of IκBα and β-arrestin2. In our preliminary study, we found that TGR5 activation inhibited phosphorylation of β-arrestin2. In this aim, we propose to test the hypothesis: TGR5 enhances the β-arrestin2-IκBα interaction through promoting dephosphorylation of β-arrestin2, which contributes to suppressing NF-κB signaling. In Specific Aim 2, we will focus on the molecular mechanisms of TGR5 antagonizing NF-κB DNA-binding activity. We will investigate the effects of TGR5 activation on a competition between cAMP response element-binding (CREB) and p65 for limiting amounts of CREB-binding protein (CBP), which may cause the inhibition of NF-κB DNA binding activity. These results from this proposal will help us determine the molecular mechanisms by which TGR5 suppress hepatic inflammation, and explore whether TGR5 is a potential therapeutic candidate for prevention and treatment of inflammation-related diseases.

阐明抑制炎症的机制有助于新药开发，挽救糖尿病和癌症等炎症相关疾病患者的生命。我们已发表的工作表明，胆汁酸膜受体Gpbar1（TGR5）是炎症的关键负调节子，它通过拮抗NF-κB信号通路抑制炎症。我们发现TGR5抑制炎症是依赖于IκBα和多重信号调控子β-arrestin2形成的蛋白质复合体，TGR5激活能抑制NF-κB的DNA结合活性。本课题中我们将鉴定TGR5调控IκBα和β-arrestin2之间蛋白质相互作用的分子机制，TGR5对于β-arrestin2去磷酸化的作用将被考察。此外，在本课题中我们将鉴定TGR5对NF-κB的DNA结合活性的抑制机理，cAMP反应元件结合蛋白（CREB）磷酸化后与p65竞争结合CBP的作用将被考察。该项目内容均属首次阐述，所得结果将揭示TGR5抑制炎症的分子机制，有助于鉴定TGR5是否是潜在的治疗炎症相关疾病的药物靶向，并为其提供分子水平的理论基础。

阐明抑制炎症的机制有助于新药开发，挽救糖尿病和癌症等炎症相关疾病患者的生命。我们已发表的工作表明，胆汁酸膜受体Gpbar1（TGR5）是炎症的关键负调节子，它通过拮抗NF-κB信号通路抑制肝炎。我们发现TGR5抑制炎症是依赖于IκBα和多重信号调控子β-arrestin2形成的蛋白质复合体，TGR5激活能抑制NF-κB的DNA结合活性。本课题中我们鉴定了TGR5调控IκBα和β-arrestin2之间蛋白质相互作用的分子机制，考察了TGR5对于β-arrestin2去磷酸化的作用。此外，在本课题中我们鉴定了TGR5对NF-κB的DNA结合活性的抑制机理，考察了cAMP反应元件结合蛋白（CREB）敲低后对于TGR5抑制炎症效果的影响。我们发现TGR5能够调控β-arrestin2的磷酸化作用，增强其去磷酸化从而调控β-arrestin2和IκBα之间的相互作用；β-arrestin2的基因沉默可以消除TGR5对于NF-κB下游特定基因的抑制影响；初步确定了β-arrestin2和IκBα复合体中还存在其它蛋白因子；其次，在肿瘤细胞和免疫细胞中可能存在不同的调控机理。进一步，我们鉴定了TGR5激活后诱导了CREB磷酸化，磷酸化的CREB进而干扰了p65与DAN的结合，从而抑制了NF-κB的转录活性。本项目鉴定出抑制炎症的新机制，即TGR5抑制NF-κB信号通路分子机制的鉴定。所得结果不仅揭示出TGR5抑制炎症的分子机制，并有可能发现新的炎症相关的辅助因子。这些结果能用于指导开发治疗和预防慢性炎症的药物和新疗法，具有现实意义，对众多炎症相关疾病的治疗具有潜在指导意义。另一方面，本课题鉴定出抑制炎症新的靶向因子，即胆汁酸膜受体TGR5作为抑制炎症的药物靶向的鉴定。胆汁酸膜受体TGR5是G蛋白偶联受体，适于作为药物设计的靶向。TGR5通过感受内源胆汁酸各成分的变化而被动态激活，调控其下游路径。TGR5的调控失衡可能是多种炎症相关疾病的主要发病原因之一。该项目所得结果将在分子水平提供TGR5作为治疗炎症相关疾病药物靶向的理论基础，有助于推动以其为药物靶向的新药和新疗法的开发。