The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.
The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.
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DOI：
10.1371/journal.pone.0059913
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Dimaline R
中科院分区：
文献类型：
--
作者：
O'Hara A;Howarth A;Varro A;Dimaline R
胃泌素在生理上调节胃酸分泌，并通过调节纤溶酶原激活物抑制剂2 （PAI-2）和再生蛋白1（Reg1）等基因的表达来维持胃上皮结构。在这里，我们研究了蛋白酶体亚基PSMB1在胃泌素对PAI-2和Reg1的转录调节中的作用，以及它在胃泌素刺激下的亚细胞分布。我们使用永久转染CCK2受体的胃癌细胞系AGS (AGS- gr)，研究了当PSMB1被siRNA敲除时，胃泌素刺激PAI-2和Reg1报告基因构建的表达。通过染色质免疫沉淀法（ChIP）评估PSMB1与PAI-2和Reg1启动子的结合。免疫细胞化学和Western Blot检测PSMB1的亚细胞分布。胃泌素在AGS-GR细胞中显著增加PAI-2和Reg1的表达，但当PSMB1被敲除时，反应显著降低。在ChIP实验中，用PSMB1抗体对染色质进行免疫沉淀后，与用对照IgG沉淀的染色质相比，PAI-2和Reg1启动子的胃泌素反应区域的DNA大量富集。在胃泌素刺激的AGS-GR细胞中，在ChIP试验中发现，在相同的时间尺度上，核细胞质PSMB1的比例与PSMB1向PAI-2和Reg1启动子募集的比例显著增加。我们得出结论，PSMB1是胃泌素刺激PAI-2和Reg1表达所需的转录机制的一部分，并且它在亚细胞分布中响应胃泌素的变化与这一作用一致。
The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and regenerating protein 1(Reg1). Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR) to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP) assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role.
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