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Nucleophosmin Is Essential for Stimulation of Rac1 Nuclear Accumulation by RNA‐binding Protein HuR in the Intestinal Epithelium
Author(s) -
Liu Lan,
Chung Hee Kyoung,
Kalakonda Sudhakar,
Yu Tingxi,
Xiao Lan,
Rao Jaladanki N.,
Wang JianYing
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.873.9
Subject(s) - nucleophosmin , intestinal epithelium , intestinal mucosa , microbiology and biotechnology , rac1 , biology , gene silencing , rna binding protein , epithelium , messenger rna , cancer research , signal transduction , biochemistry , gene , medicine , genetics , myeloid leukemia
The mammalian intestinal epithelium is a rapidly self‐renewing tissue in the body and its homeostasis is tightly regulated by numerous factors including the RNA‐binding protein HuR. HuR is highly expressed in the intestinal mucosa and acts as a key regulator of the epithelial homeostasis by regulating the stability and translation of target mRNAs. We have recently demonstrated that targeted deletion of HuR in intestinal epithelial cells (IECs) inhibits growth of the small intestinal mucosa and delays healing after injury in mice partially by altering subcellular distribution of a small GTP‐binding protein Rac1. HuR knockout in IECs increases the levels of cytoplasmic Rac1 in vivo as well as in vitro , but the exact mechanism underlying this process remains unknown. Nucleophosmin (NPM) is a phosphoprotein that binds to proteins containing nuclear localization signals for their import and functions as a molecular chaperone. In this study, we investigated the role of NPM in HuR‐dependent nuclear accumulation of Rac1 in the intestinal epithelium. Methods Studies in vivo were conducted in intestinal epithelial tissue‐specific HuR knockout (IE‐HuR −/− ) mice, and experiments in vitro were carried out in cultured IEC‐6 cells that were derived from rat small intestinal crypts. Functions of NPM and HuR in vitro were investigated via their gene silencing by using siRNAs specifically targeting NPM (siNPM) or HuR (siHuR) and their overexpression. Levels of NPM, Rac1, and HuR proteins were measured by Western blotting analysis, and the cellular distributions of Rac1 and NPM were examined by immunofluorescent staining. Results The levels of NPM protein in the intestinal mucosa decreased significantly in IE‐HuR −/− mice compared with control littermates. Decreased NPM was associated with elevated cytoplasmic Rac1 in the HuR‐deficient epithelium, along with a decrease in nuclear Rac1 abundance. In cultured IEC‐6 cells, HuR silencing by transient transfection with siHuR also decreased cellular NPM, but it increased the levels of cytoplasmic Rac1. NPM was found to physically interact with Rac1 and formed NPM/Rac1 complex in normal IEC‐6 cells. NPM silencing by transfection with siNPM increased cytoplasmic Rac1 levels and decreased nuclear Rac1 abundance. Furthermore, ectopically expressed NPM abolished the increased levels of cytoplasmic Rac1 in HuR‐silenced cells and restored the nuclear Rac1 levels to near normal. Conclusions These results indicate that 1) HuR regulates NPM expression in the intestinal epithelium; and 2) HuR regulates Rac1 subcellular trafficking by altering NPM. Support or Funding Information National Institutes of Health (JYW) and US Department of Veterans Affairs (JYW and JNR) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .