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Vol. 18, Issue 11, 4579-4590, November 2007

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Polyamines Regulate the Stability of Activating Transcription Factor-2 mRNA through RNA-binding Protein HuR in Intestinal Epithelial Cells

Lan Xiao^*,, Jaladanki N. Rao^*,, Tongtong Zou^*,, Lan Liu^*,, Bernard S. Marasa^*,, Jie Chen^*,, Douglas J. Turner^*,, Huiping Zhou, Myriam Gorospe^||, and Jian-Ying Wang^*,,

*Cell Biology Group, Department of Surgery, and Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201; Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201; Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298; and ^||Laboratory of Cellular and Molecular Biology, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD 21224

Submitted July 17, 2007; Revised August 17, 2007; Accepted August 29, 2007
Monitoring Editor: William Tansey

Maintenance of intestinal mucosal epithelial integrity requires polyamines that modulate the expression of various genes involved in cell proliferation and apoptosis. Recently, polyamines were shown to regulate the subcellular localization of the RNA-binding protein HuR, which stabilizes its target transcripts such as nucleophosmin and p53 mRNAs. The activating transcription factor-2 (ATF-2) mRNA encodes a member of the ATF/CRE-binding protein family of transcription factors and was computationally predicted to be a target of HuR. Here, we show that polyamines negatively regulate ATF-2 expression posttranscriptionally and that polyamine depletion stabilizes ATF-2 mRNA by enhancing the interaction of the 3'-untranslated region (UTR) of ATF-2 with cytoplasmic HuR. Decreasing cellular polyamines by inhibiting ornithine decarboxylase (ODC) with -difluoromethylornithine increased the levels of ATF-2 mRNA and protein, whereas increasing polyamines by ectopic ODC overexpression repressed ATF-2 expression. Polyamine depletion did not alter transcription via the ATF-2 gene promoter but increased the stability of ATF-2 mRNA. Increased cytoplasmic HuR in polyamine-deficient cells formed ribonucleoprotein complexes with the endogenous ATF-2 mRNA and specifically bound to 3'-UTR of ATF-2 mRNA on multiple nonoverlapping 3'-UTR segments. Adenovirus-mediated HuR overexpression elevated ATF-2 mRNA and protein levels, whereas HuR silencing rendered the ATF-2 mRNA unstable and prevented increases in ATF-2 mRNA and protein. Furthermore, inhibition of ATF-2 expression prevented the increased resistance of polyamine-deficient cells to apoptosis induced by treatment with tumor necrosis factor- and cycloheximide. These results indicate that polyamines modulate the stability of ATF-2 mRNA by altering cytoplasmic HuR levels and that polyamine-modulated ATF-2 expression plays a critical role in regulating epithelial apoptosis.

Address correspondence to: Jian-Ying Wang (jwang{at}smail.umaryland.edu)

Abbreviations used: AREs, AU-rich elements; ATF-2, activating transcription factor-2; CHX, cycloheximide; Con, control; pfu, plaque-forming units; CR, coding region; CRE, cyclic AMP response element; CREB, CRE-binding protein; DFMO, DL--difluoromethylornithine; HEK, human embryonic kidney; IECs, intestinal epithelial cells; IP, immunoprecipitation; Luc, luciferase; NPM, nucleophosmin; ODC, ornithine decarboxylase; Put, putrescine; RBPs, RNA-binding proteins; RNP, ribonucleoprotein; si, small interfering; siATF-2, siRNA targeting ATF-2 mRNA; siHuR, siRNA targeting HuR mRNA; TNF-, tumor necrosis factor-; UTRs, untranslated regions.

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