SIRT1在活化的内皮细胞中抑制NFAT依赖的炎症基因的表达

发布时间：2018-05-19 05:50

本文选题：SIRT1 + NFAT　；参考：《北京协和医学院》2010年博士论文

【摘要】：目的：研究Ⅲ类组蛋白去乙酰化酶SIRTI对转录因子NFAT(Nuclear factor of activated T cells)的作用及下游炎症基因的表达,探讨其对内皮细胞炎症作用及其机制。背景：SIRTI是酵母染色质沉默因子(Silent information regulator 2,Sir2)的哺乳动物同源体,属于Ⅲ类组蛋白去乙酰化酶。Sir2家族的蛋白从细菌到人类都具有高度保守性,人Sirtuins是与Sir2具有同源性的一个家族,其中SIRTI是与Sir2同源性最高的一个成员,在许多生命过程,如细胞凋亡、细胞周期、DNA损伤修复、重组、长寿和基因沉默中都发挥了重要的作用。近年来的研究表明,SIRT1不仅可以去乙酰化组蛋白如组蛋白H3、H4等,而且与很多转录因子和转录辅因子相互作用,调节它们的转录活性。这些转录因子包括：p53、FOXO家族、NF-kB(P65)、MyoD等；转录辅因子包括：NcoR、p300、PGC-1a等。最近的研究认为SIRT1是血管内皮稳态的关键的调节者,它控制着血管生成,血管的收缩和内皮功能紊乱等,尤其可能具有抗炎的作用。但在血管内皮细胞中SIRT1确切的抗炎作用以及机制还不清楚。转录因子NFAT家族目前共发现有5个成员,分别是NFATcl, NFATc2, NFATc3, NFATc4和NFAT5,其中包括NFATc3前四个成员都依赖于钙信号通路调节。NFAT对细胞因子、生长因子等生理或病理信号发生应答,调节基因的转录,参与细胞的增殖、分化等过程。NFAT在血管系统中主要发挥功促进血管生成和炎症的功能。在内皮细胞中,NFAT下游炎症分子例如：COX-2, ICAM-1等在炎症中发挥了重要的作用。但在血管内皮细胞中NFAT参与炎症反应的调控机制还不清楚。材料和方法：本实验首先用Real-time PCR实验检测了PMA/Ionomycin(Io)在内皮细胞中是否诱导炎症基因的表达。为了证明NFATc3是否在内皮细胞中被PMA/Io诱导入核和激活,用免疫荧光的方法检测了PMA/Io处理内皮细胞后NFATc3核转位的改变。为了进一步证明NFAT是否介导了由PMA/Io诱导的炎症基因的表达,用NFAT的抑制剂CsA预处理内皮细胞,再用PMA/Io处理内皮细胞,Real-time PCR实验检测了炎症基因表达的改变。为了证明SIRT1对PMA/Io诱导的内皮细胞NFAT下游炎症基因表达的影响,用Real-time PCR和Western blotting (WB)的实验,在PMA/Io处理的内皮细胞中,过表达SIRTI或干扰SIRTI的表达,检测SIRTI对NFAT下游炎症分子表达的影响。为了进一步证明SIRT1对PMA/Io诱导的内皮细胞NFAT下游炎症基因的表达的影响,用SIRT1的激活剂resveratrol和抑制剂Sirtinol处理内皮细胞,Real-time PCR实验检测了炎症基因表达的改变。通过免疫共沉淀的方法,检测SIRT1与NFATc3之间的相互作用。为了证明SIRT1是否抑制了NFATc3的转录活性,在HEK293细胞中通过荧光素酶报告基因实验方法检测了SIRT1在基础水平和PMA/Io处理下对NFATc3的转录活性的影响。为了证明SIRT1是否通过去乙酰化NFATc3而抑制其转录活性,通过免疫共沉淀的方法,检测了SIRT1对NFATc3乙酰化水平的改变。为了进一步证明SIRT1对NFATc3的转录抑制是否影响了NFATc3与AP-1的相互作用,通过免疫共沉淀的方法,检测了SIRT1对NFATc3募集AP-1的能力的影响。结果：在内皮细胞中,PMA/Io诱导了炎症基因的表达,而且NFATc3介导PMA/Io诱导的炎症基因的表达；腺病毒介导的SIRT1高表达抑制了PMA/Io诱导的NFAT下游炎症基因的表达,并且SIRT1的激活剂resveratrol也能够抑制PMA/Io诱导的NFAT下游炎症基因的表达,并且SIRT1的抑制剂Sirtinol上调了NFAT下游炎症分子的表达。NFATc3可以和SIRT1相互作用,并且NFATc3 NHR和RHR结构域介导了与SIRT1之间的相互作用。进一步的研究发现SIRT1可以通过去乙酰化抑制NFATc3的转录活性。SIRT1不影响NFATc3募集AP-1的能力；最后研究还发现PMA/lo可以诱导内皮细胞内SIRT1的表达,这可能是一种细胞的负反馈保护机制。结论：我们首次发现NFAT可能是SIRT1的一个新的靶分子。SIRT1可以通过去乙酰化NFATc3,从而抑制其转录活性。在内皮细胞内,SIRT1可能通过抑制PMA/Io诱导的NFATc3活性,降低了下游炎症基因的表达及活性,从而抑制了炎症反应。因此,提高SIRT1的表达和活性可以作为一个调节炎症反应,保护机体的新手段。
[Abstract]:Objective: To study the effect of class III histone deacetylase (SIRTI) on the transcription factor NFAT (Nuclear factor of activated T cells) and the expression of the downstream inflammatory genes, and to explore the inflammatory effect and mechanism of it on endothelial cells.
Background: SIRTI is a mammalian homologous body of yeast chromatin silencing factor (Silent information regulator 2, Sir2). The proteins belonging to class III histone deacetylase.Sir2 family are highly conserved from bacteria to humans. Human Sirtuins is a family of homology with Sir2, and SIRTI is the highest homology with Sir2. Members have played an important role in many life processes, such as apoptosis, cell cycle, DNA damage repair, recombination, longevity and gene silencing. Recent studies have shown that SIRT1 can not only deacetylate histone, such as histone H3, H4, but also interact with many transcription factors and transcription cofactors to regulate their transcriptional transcription. Activity. These transcription factors include: p53, FOXO family, NF-kB (P65), MyoD, etc.; transcriptional cofactors include NcoR, P300, PGC-1a and so on. Recent studies suggest that SIRT1 is a key regulator of vascular endothelial homeostasis, which controls angiogenesis, vascular contractions and endothelial dysfunction, and is especially likely to have anti-inflammatory effects. The exact anti-inflammatory effect and mechanism of SIRT1 in skin cells are unclear.
There are 5 members of the transcription factor NFAT family, which are NFATcl, NFATc2, NFATc3, NFATc4 and NFAT5, including the first four members of the NFATc3, which are dependent on the calcium signaling pathway to regulate the response of.NFAT to cytokines, growth factors and other physiological or pathological signals, regulate gene transcription, participate in cell proliferation, differentiation and so on. In the vascular system, it is important to play the role of promoting angiogenesis and inflammation. In endothelial cells, the downstream inflammatory molecules of NFAT, such as COX-2, ICAM-1, play an important role in the inflammation, but the regulatory mechanism of NFAT in the inflammatory response is not clear in vascular endothelial cells.
Materials and methods: in this experiment, Real-time PCR test was first used to detect whether PMA/Ionomycin (Io) induced the expression of inflammatory genes in endothelial cells. In order to prove whether NFATc3 was induced and activated by PMA/Io in endothelial cells, the change of NFATc3 nuclear transposition in PMA/Io cells was detected by immunofluorescence. It was further demonstrated whether NFAT mediated the expression of inflammatory genes induced by PMA/Io, pretreated endothelial cells with NFAT inhibitor CsA, and then treated endothelial cells with PMA/Io, and Real-time PCR test detected the changes in the expression of inflammatory genes. In order to prove the effect of SIRT1 on the expression of inflammatory genes downstream of PMA/Io induced endothelial cell NFAT, Real-t, using Real-t. IME PCR and Western blotting (WB) experiments, in PMA/Io treated endothelial cells, overexpress SIRTI or interfere with the expression of SIRTI to detect the effect of SIRTI on the expression of inflammatory molecules downstream of NFAT. The agent Sirtinol treated the endothelial cells, and the Real-time PCR test detected the changes in the expression of the inflammatory genes. The interaction between SIRT1 and NFATc3 was detected by immunoprecipitation. In order to prove whether SIRT1 inhibited the transcriptional activity of NFATc3, the fluorescein reporter gene test method was used to detect SIRT1 in the basis of HEK293 cells. The effect of level and PMA/Io on the transcriptional activity of NFATc3. In order to prove whether SIRT1 inhibits its transcriptional activity through deacetylation of NFATc3, the changes in the level of NFATc3 acetylation by SIRT1 are detected by immunoprecipitation. To further prove whether the SIRT1's transcript inhibition of NFATc3 affects the interaction between NFATc3 and AP-1. The effect of SIRT1 on the ability of NFATc3 to collect AP-1 was detected by CO immunoprecipitation.
Results: in endothelial cells, PMA/Io induces the expression of inflammatory genes, and NFATc3 mediates the expression of PMA/Io induced inflammatory genes, and the high expression of adenovirus mediated SIRT1 inhibits the expression of PMA/Io induced downstream inflammatory genes in NFAT, and SIRT1 activator resveratrol also inhibits the PMA/Io induced downstream inflammatory genes of NFAT. The expression of SIRT1 inhibitor Sirtinol up-regulated the expression of inflammatory molecules in the downstream NFAT,.NFATc3 can interact with SIRT1, and the NFATc3 NHR and RHR domains mediate the interaction with SIRT1. Further studies have found that SIRT1 can inhibit NFATc3 transcriptional activity by deacetylation. Finally, we also found that PMA/lo could induce the expression of SIRT1 in endothelial cells, which may be a negative feedback protection mechanism of cells.
Conclusion: we first found that NFAT may be a new target molecule of SIRT1,.SIRT1, which can deacetylate NFATc3 and inhibit its transcriptional activity. In endothelial cells, SIRT1 may inhibit the expression and activity of the downstream inflammatory genes by inhibiting the NFATc3 activity induced by PMA/Io and thus inhibiting the inflammatory response. Thus, SIRT1 is enhanced. Expression and activity can serve as a new way to regulate inflammation and protect organism.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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