Post-transcriptional regulation of MEK-1 by polyamines through the RNA-binding protein HuR modulating intestinal epithelial apoptosis.

NIH Public Access Author Manuscript Biochem J. Author manuscript; available in PMC 2011 January 16. NIH-PA Author Manuscript Published in final edited form as: Biochem J. ; 426(3): 293–306. doi:10.1042/BJ20091459. Posttranscriptional Regulation of MEK-1 by Polyamines through the RNA–binding Protein HuR Modulating Intestinal Epithelial Apoptosis Peng-Yuan Wang*,†,§, Jaladanki N. Rao*,†, Tongtong Zou*,†, Lan Liu*,†, Lan Xiao*,‡, TingXi Yu*,†, Douglas J. Turner*,†, Myriam Gorospe¶, and Jian-Ying Wang*,†,‡,# *Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201 ‡Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland 21201 †Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201 NIH-PA Author Manuscript ¶Laboratory of Cellular and Molecular Biology, National Institute on Aging-IRP, NIH, Baltimore, Maryland 21224 Abstract NIH-PA Author Manuscript Mitogen-activated protein kinase kinase 1 (MEK-1) is an important signal transducing enzyme that is implicated in many aspects of cellular functions. Here, we report that cellular polyamines regulate MEK-1 expression at the posttranscriptional level through the RNA-binding protein HuR in intestinal epithelial cells (IECs). Decreasing the levels of cellular polyamines by inhibiting ornithine decarboxylase (ODC) stabilized MEK-1 mRNA and promoted its translation through enhancement of HuR interaction with the 3′-untranslated region of MEK-1 mRNA, whereas increasing polyamine levels by ectopic ODC overexpression destabilized the MEK-1 transcript and repressed its translation by reducing [HuR/MEK-1 mRNA] complex; neither intervention changed MEK-1 gene transcription via its promoter. HuR silencing rendered the MEK-1 mRNA unstable and inhibited its translation, thus preventing increases in MEK-1 mRNA and protein in polyamine-deficient cells. Conversely, HuR overexpression induced MEK-1 mRNA stability and promoted its translation. Inhibition of MEK-1 expression by MEK-1 silencing or HuR silencing prevented the increased resistance of polyamine-deficient cells to apoptosis. Moreover, HuR overexpression did not protect against apoptosis if MEK-1 expression was silenced. These results indicate that polyamines destabilize the MEK-1 mRNA and repress its translation by inhibiting HuR association with MEK-1 transcript. Our findings indicate that MEK-1 is a key effector of the HuR-elicited antiapoptotic program in intestinal epithelial cells. Keywords 3′-untranslated region; mRNA stability; translational regulation; ribonucleoprotein; ornithine decarboxylase # Corresponding author: Dr. Jian-Ying Wang, Baltimore Veterans Affairs Medical Center (112), 10 North Greene Street, Baltimore, MD 21201; Phone: 410-605-7000 ×5678; Fax: 410-605-7919; . §Current Address is: Peking University First Hospital, Peking University, Beijing 100034, CHINA. Supplemental Data: Supplemental data include three Figures (Supplemental Figures A1-A3). Wang et al. Page 2 INTRODUCTION NIH-PA Author Manuscript NIH-PA Author Manuscript The epithelium of mammalian intestinal mucosa is a rapidly self-renewing tissue in the body, and maintenance of its integrity depends on a dynamic balance between cell proliferation and apoptosis [1,2]. In response to stress, rapid changes in gene expression patterns in intestinal epithelial cells (IECs) control cell division and survival, thereby preserving the epithelial homeostasis [3]. Although gene expression is critically regulated at the transcription level in IECs, the essential contribution of posttranscriptional events, particularly altered mRNA turnover and translation, is becoming increasingly recognized [2,4-6]. The posttranscriptional fate of a given mRNA is primarily controlled by the interaction of specific mRNA sequences (cis-elements) with specific trans-acting factors such as RNA-binding proteins (RBPs) and noncoding regulatory RNAs (such as microRNAs) [7-10]. The most common cis-elements responsible for rapid regulation of mRNA decay and translation in mammalian cells are U- and UA-rich elements (AREs) located in the 3′-untranslated regions (3′-UTRs) of many mRNAs [11-14]. Ribonucleoprotein (RNP) associations either increase or decrease mRNA stability or/and translation depending on the particular mRNA sequence, cellular growth conditions, and the stimulus type [5,15,17]. Among the RBPs that regulate specific subsets of mRNAs are several RBPs that modulate mRNA turnover (HuR, NF90, AUF1, BRF1, TTP, KSRP) and RBPs that modulate translation (HuR, TIAR, NF90, TIA-1), collectively known as translation and turnover-regulatory (TTR)-RBPs [16-18]. NIH-PA Author Manuscript The Hu antigen R (HuR) protein is the ubiquitously expressed member of the ELAV-like family of TTR-RBPs. HuR has two N-terminal RNA-recognition motifs (RRMs), followed by a nucleocytoplasmic shuttling sequence, and a C-terminal RRM [19-22]. HuR is predominantly located in the nucleus in unstimulated cells, but it rapidly translocates to the cytoplasm, where it directly interacts with and regulates target mRNA stability and/or translation in response to specific stimuli [20,23]. Recently, HuR was shown to play an important role in the regulation of intestinal epithelial homeostasis by modulating IEC proliferation and apoptosis [6,24-27]. The subcellular localization of HuR and its binding affinity for specific target transcripts in IECs are tightly regulated by numerous factors, including cellular polyamines [6,26]. The natural polyamines spermidine, spermine, and their precursor putrescine are ubiquitous, small basic molecules that are intimately involved in the control of epithelial homeostasis [27-29]. Normal IEC proliferation in the mucosa depends on the supply of polyamines to the dividing cells in the crypts [2,4,5,30]; polyamines also regulate IEC apoptosis [31,32]. Decreasing cellular polyamines by inhibiting ornithine decarboxylase (ODC, the first rate-limiting step for polyamine biosynthesis) is found to increase cytoplsmic levels of HuR-stabilizing p53 and nucleophosmin (NPM) mRNAs, thus contributing to the inhibition of IEC proliferation [6,26]. Polyamines are also necessary for HuR phosphorylation in IECs, and polyamine depletion represses c-Myc translation by reducing [HuR/c-Myc mRNA] complex through inhibition of Chk2-dependent HuR phosphorylation [25]. Indeed, growing evidence shows that HuR is emerging as a pivotal posttranscriptional regulator essential for maintaining the intestinal epithelial integrity. Mitogen-activated protein kinase (MAPK) kinase-1 (MEK-1) is a dual-specificity kinase that plays a role in the regulation of various cellular functions including proliferation, development, differentiation, migration, and apoptosis by activating MAPK/ERK signals [33-35]. While a single MEK gene is present in C. elegans, Drosophila and Xenopus, there are two MEK homologs, MEK-1 and MEK-2, in mammalian systems [33]. Studies anal (...truncated)