Smad7和Tollip的功能研究
文献类型：学位论文
| 作者 | 朱路 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-11 |
| 授予单位 | 中国科学院上海生命科学研究院营养科学研究所 |
| 授予地点 | 中国科学院上海生命科学研究院 |
| 导师 | 陈雁 |
| 关键词 | TGF-beta Smad7 酒精性肝损伤 Tollip 细胞内体 |
| 其他题名 | The functional investigation of Smad7 and Tollip |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 转化生长因子transforming growth factor-β (TGF-β) 在众多肝脏疾病中扮演着非常重要的角色。这些疾病包括肝脏纤维化和酒精性脂肪肝等。Smad7是TGF-β信号通路在细胞内最重要的负调控因子之一，但目前内源性的Smad7对于肝脏功能是否具有调节功能尚不清楚。近年来我国由于过度饮酒引起的酒精性脂肪肝和肝损伤的发生率呈现明显上升趋势，已经成为威胁人民身体健康的重要因素。TGF-β信号通路与酒精性肝损伤的关系却罕有报道。首先，我们利用Cre/loxP同源重组系统建立了Smad7肝脏特异性敲除的小鼠模型，发现Smad7的敲除特异地增强了肝脏细胞中的TGF-β信号通路，部分小鼠表现出自发性的肝功能损伤。然后，通过对小鼠进行为期6周的酒精饮食诱导，发现与对照组相比，Smad7基因敲除的小鼠表现出更为严重的肝功能损伤和酒精性脂肪肝。进一步的研究发现，在酒精饮食的刺激下Smad7基因敲除小鼠肝脏中酒精代谢的关键酶乙醇脱氢酶 ADH-1的表达量较对照组显著降低，而脂肪合成的相关基因以及多种炎性因子的表达却显著增加。这些结果表明Smad7基因的缺失可以改变酒精在体内的代谢途径，并影响了脂代谢和炎性反应。因此，这一部分工作中我们运用动物模型直接揭示了Smad7在肝脏中的功能，首次发现并阐释了Smad7在酒精导致的肝功能损害中的重要作用，也为以后寻找有效干预酒精导致的肝脏损伤的措施奠定了理论基础。 在配体的刺激下，激活的转化生长因子受体TGF receptors (TβRs) 会被泛素化，该过程主要由Smad7招募E3泛素酶体至受体复合物来完成。然而，泛素化的转化生长因子受体在细胞内被转运从而进入降解途径的过程尚不十分清楚。我们的研究发现了一个新的Smad7结合蛋白Tollip (Toll interacting protein), 并发现它能有效地抑制TGF-beta信号通路。Tollip是新近发现的一种接头蛋白，它广泛表达于体内各组织。早期的发现提示Tollip对Toll样受体TLRs/IL-1R信号通路具有负调控作用，并参与IL-1R在细胞中的转运及泛素化降解过程。我们的研究证实Tollip同样与TGF-β的I型受体 (TβRI) 相互作用，而且这种相互作用和受体的泛素化相关。Smad7能有效地促进Tollip和TβRI之间的相互作用并增加他们在细胞中的共定位。另外，Tollip可以加速活化的TβRI的降解，这可能与Tollip可以改变活化的TβRI在细胞中的定位以及影响其在细胞内体中的转运有关。因此，我们这一部分的研究表明Tollip和Smad7协同参与了TβRI的细胞内转运和降解过程，从而负调控TGF-β信号通路。 |
| 索取号 | D2012-143 |
| 英文摘要 | TGF-β has been known to play an important role in various liver diseases including fibrosis and alcohol-induced fatty liver. Smad7 is an intracellular negative regulator of TGF-β signaling. It is currently unclear whether endogenous Smad7 has an effect on liver function and alcoholic liver damage. We used Cre/loxP system by crossing Alb-Cre mice with Smad7(loxP/loxP) mice to generate liver-specific deletion of Smad7 with loss of the indispensable MH2 domain. Alcoholic liver injury was achieved by feeding mice with a liquid diet containing 5% ethanol for 6 weeks, followed by a single dose of ethanol gavage. Deletion of Smad7 in the liver was associated with increased Smad2/3 phosphorylation in the liver or upon TGF-β treatment in primary hepatocytes. The majority of mice with liver specific deletion of Smad7 (Smad7(liver-KO)) were viable and phenotypically normal, accompanied by only slight or no reduction of Smad7 expression in the liver. However, about 30% of Smad7(liver-KO) mice with high efficiency of Smad7 deletion had spontaneous liver dysfunction, demonstrated as low body weight, overall deterioration, and increased serum levels of AST and ALT. In addition, alcohol-induced liver injury and steatosis were profoundly aggravated in Smad7 deficient mice, associated with upregulation of critical genes involved in lipogenesis and inflammation. Furthermore, alcohol-induced ADH1 expression was significantly abrogated by Smad7 deletion in hepatocytes. In this study, we provided in vivo evidence revealing that endogenous Smad7 plays an important role in liver function and alcohol-induced liver injury. Upon activation, TGF-β type I receptor (TβRI) undergoes active ubiquitination via recruitment of E3 ligases to the receptor complex by Smad7. However, how ubiquitination of TβRI is coupled to intracellular trafficking and protein degradation remains unclear. We report here that Tollip, an adaptor protein that contains both ubiquitin associated domains and endosome-targeting domain, plays an important role in modulating trafficking and degradation of TβRI. Tollip was previously demonstrated to possess a functional role in modulating the signaling of interleukin-1 and Toll-like receptors. We identify here that Tollip interacts with Smad7, a major modulatory protein involved in the negative regulation of TGF-β signaling. Overexpression of Tollip antagonizes TGF-β-stimulated transcriptional response, Smad2 phosphorylation and epithelial–mesenchymal transition. Tollip also interacts with ubiquitinated TβRI and such interaction requires ubiquitin-associated domains of Tollip. The interaction and intracellular colocalization of Tollip with TβRI is enhanced by Smad7. Overexpression of Tollip accelerates protein degradation of activated TβRI. In addition, Tollip alters subcellular compartmentalization and endosomal trafficking of activated TβRI. Thus, our studies reveal that Tollip cooperates with Smad7 to modulate intracellular trafficking and degradation of ubiquitinated TβRI, whereby negatively regulates TGF-β signaling pathway. |
| 语种 | 中文 |
| 源URL | [http://202.127.25.144/handle/331004/109]  |
| 专题 | 中国科学院上海生命科学研究院营养科学研究所_信号转导与营养相关疾病研究组 |
| 推荐引用方式 GB/T 7714 | 朱路. Smad7和Tollip的功能研究[D]. 中国科学院上海生命科学研究院. 中国科学院上海生命科学研究院营养科学研究所. 2012. |
入库方式： OAI收割
来源：上海营养与健康研究所
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