Gamma-secretase inhibitor suppressed Notch1 intracellular domain combination with p65 and resulted in the inhibition of the NF-κB signaling pathway induced by IL-1β and TNF-α in nucleus pulposus cells.
Gamma-secretase inhibitor suppressed Notch1 intracellular domain combination with p65 and resulted in the inhibition of the NF-κB signaling pathway induced by IL-1β and TNF-α in nucleus pulposus cells.
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DOI：
10.1002/jcb.27504
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发表时间：
2019-03
影响因子：
4
通讯作者：
Fan J
中科院分区：
文献类型：
--
作者：
Huang Y;Mei W;Chen J;Jiang T;Zhou Z;Yin G;Fan J
在本实验中，我们研究了notch与NF - κB信号通路之间的交叉对话，以揭示炎症细胞因子影响II型胶原（ColII）和聚集蛋白变性的减缓机制。采用免疫组织化学和苏木精-伊红染色+阿利新蓝染色分别观察ColII和aggrecan在椎间盘中的表达水平。Western blot检测各组人髓核细胞（hNPCs）细胞核中ColII、aggrecan、Runx2、NF‐κB的表达水平以及p65的磷酸化和乙酰化水平。用含有Notch1细胞内结构域过表达的相对结构域（NICD1）的质粒转染293T细胞，通过免疫沉淀（IP）观察NICD1与p65的结合情况。用缺乏NICD1 ANK区的慢病毒过表达转染HNPCs，并通过IP观察NICD1和p65的结合。当在椎间盘培养基中加入γ -分泌酶抑制剂N‐[N‐（3,5‐二氟苯乙酰基）‐1‐丙烯基]‐苯甘氨酸t‐丁基酯（DAPT）时，ColII和聚集蛋白在椎间盘培养物中的表达增加。Western blot结果显示，DAPT抑制p65磷酸化和乙酰化，细胞核内p65和p50水平下降。研究发现NICD1与p65结合，而hNPCs中ANK结构域缺失后的结果相反。在髓核细胞中，p65和NICD1的ANK结构域的结合是与IL - 1β和TNF - α诱导的NF - κB信号通路激活相关的变性的关键过程。
In this experiment, the cross‐talk betweenNotch and the NF‐κB signaling pathway was examined to reveal the mechanism of slowing down the type II collagen (ColII) and aggrecan degeneration affected by inflammatory cytokines. The expression levels of ColII and aggrecan in the intervertebral disc were observed through immunohistochemistry and hematoxylin‐eosin staining+alcian blue staining, respectively. The expression levels of ColII, aggrecan, Runx2, and NF‐κB in the nuclei of human nucleus pulposus cells (hNPCs) in each group, as well as the phosphorylation and acetylation levels of p65, were examined through Western blot analysis. The 293T cells were transfected with a plasmid containing the overexpressed relative domain of Notch1 intracellular domain (NICD1), and immunoprecipitation (IP) was performed to observe the combination of NICD1 and p65. HNPCs were transfected with a lentiviral‐contained overexpression lacking the ANK region of NICD1, and IP was performed to observe the combination of NICD1 and p65. The expression of ColII and aggrecan in the intervertebral disc culture increased when γ‐secretase inhibitor N‐[N‐(3,5‐difluorophenacetyl)‐1‐alanyl]‐Sphenylglycine t‐butyl ester (DAPT) was added to the disc culture medium. Western blot revealed that DAPT inhibited p65 phosphorylation and acetylation, and the p65 and p50 levels in the nucleus decreased. NICD1 was found to be combined with p65 in contrast to the reverse consequences after ANK domain deletion in hNPCs. In nucleus pulposus cells, the combination of p65 and the ANK domain of NICD1 is a critical procedure for the degeneration related to the NF‐κB signaling pathway activation induced by IL‐1β and TNF‐α.
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类风湿关节炎患者外周血T辅助细胞中Notch相关分子的表达分析
影响因子：
2.1
作者：
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通讯作者：
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影响因子：
20.3
作者：
Aurora, Aryn B.;Biyashev, Dauren;Volpert, Olga V.
通讯作者：
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影响因子：
3
作者：
Nasto LA;Seo HY;Robinson AR;Tilstra JS;Clauson CL;Sowa GA;Ngo K;Dong Q;Pola E;Lee JY;Niedernhofer LJ;Kang JD;Robbins PD;Vo NV
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Vo NV
影响因子：
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作者：
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通讯作者：
Im, Hee-Jeong
影响因子：
7.3
作者：
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通讯作者：
Hottiger MO