Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland; Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland.
Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland; Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland; Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland.
Cell Mol Gastroenterol Hepatol. 2019;8(3):475-486. doi: 10.1016/j.jcmgh.2019.06.002. Epub 2019 Jun 10.
BACKGROUND & AIMS: The mammalian intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated via well-controlled mechanisms. The RNA-binding protein HuR is essential for maintaining gut epithelial integrity, and targeted deletion of HuR in intestinal epithelial cells (IECs) disrupts mucosal regeneration and delays repair after injury. Here, we defined the role of HuR in regulating subcellular distribution of small guanosine triphosphatase Rac1 and investigated the implication of nucleophosmin (NPM) as a molecular chaperone in this process.
Studies were conducted in intestinal epithelial tissue-specific HuR knockout (IE-HuR) mice and cultured IEC-6 cells, derived from rat small intestinal crypts. Functions of HuR and NPM in vitro were investigated via their gene silencing and overexpression.
The abundance of cytoplasmic Rac1 in the small intestinal mucosa increased significantly in IE-HuR mice, although HuR deletion did not alter total Rac1 levels. HuR silencing in cultured IECs also increased the cytoplasmic Rac1 levels, without an effect on whole-cell Rac1 content. In addition, HuR deficiency in the intestinal epithelium decreased the levels of NPM in IE-HuR mice and cultured IECs. NPM physically interacted with Rac1 and formed the NPM/Rac1 complex. NPM silencing decreased the NPM/Rac1 association and inhibited nuclear accumulation of Rac1, along with an increase in cytoplasmic abundances of Rac1. In contrast, ectopically expressed NPM enhanced Rac1 nuclear translocation and restored Rac1 subcellular localization to near normal in HuR-deficient cells.
These results indicate that HuR regulates Rac1 nucleocytoplasmic shuttling in the intestinal epithelium by altering NPM expression.
哺乳动物的肠道上皮组织是体内具有快速自我更新能力的组织，其通过严格控制的机制实现稳态平衡。RNA 结合蛋白 HuR 对维持肠道上皮组织的完整性至关重要，靶向敲除肠道上皮细胞（IEC）中的 HuR 会破坏黏膜再生，并延迟损伤后的修复。在此，我们定义了 HuR 在调节小 GTP 酶 Rac1 亚细胞分布中的作用，并研究了核磷蛋白（NPM）作为分子伴侣在这一过程中的作用。
在肠道上皮组织特异性 HuR 敲除（IE-HuR）小鼠和源自大鼠小肠隐窝的 IEC-6 细胞中进行了研究。通过基因沉默和过表达研究 HuR 和 NPM 在体外的功能。
IE-HuR 小鼠的小肠黏膜中细胞质 Rac1 的丰度显著增加，尽管 HuR 缺失并未改变 Rac1 的总水平。在培养的 IEC 中沉默 HuR 也会增加细胞质 Rac1 的水平，而对全细胞 Rac1 含量没有影响。此外，肠道上皮组织中 HuR 的缺失降低了 IE-HuR 小鼠和培养的 IEC 中的 NPM 水平。NPM 与 Rac1 物理相互作用并形成 NPM/Rac1 复合物。NPM 沉默减少了 NPM/Rac1 的结合，并抑制了 Rac1 的核内积累，同时细胞质 Rac1 的含量增加。相比之下，过表达的 NPM 增强了 Rac1 的核内易位，并在 HuR 缺陷细胞中恢复了 Rac1 的亚细胞定位接近正常。
这些结果表明 HuR 通过改变 NPM 的表达来调节肠道上皮组织中 Rac1 的核质穿梭。
Zotero 插件
