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

Abstract

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 alpha-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-alpha 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.

Sphingosine kinase 1 overexpression stimulates intestinal epithelial cell proliferation through increased c-Myc translation

“…Figure 6 demonstrates the effects of silencing HuR or Chk2, the abolition of the increased c-Myc in the SphK1 clones, and the loss of the enhanced proliferation seen in these cells. As reported previously, there are several potential triggers that can affect HuR binding, including modifications of the transcript itself (43,47) and the influence of microRNAs that interfere with HuR target binding (5,15), and current studies in our laboratory are focused on these interactions. In summary, these results indicate that SphK1 overexpression in intestinal epithelial cells regulates cell proliferation through c-Myc.…”

“…The synthesis of biotinylated transcripts and analysis of RBPs bound to biotinylated RNA were performed as described previously (43). Immunoprecipitation (IP) of endogenous RNA-protein complexes was performed as described previously (22).…”

Polyamines regulate E-cadherin transcription through c-Myc modulating intestinal epithelial barrier function

“…Equal amounts of total RNA (2 g) were transcribed to synthesize single-stranded cDNA with an RT-PCR kit (Invitrogen). Real-time quantitative PCR (Q-PCR) was performed using Applied Biosystems Instruments (Foster City, CA) specific primers, probes, and software as described in our previous publications (56,58). The levels of E-cadherin mRNA were quantified by Q-PCR analysis and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels.…”

ATF2 – at the crossroad of nuclear and cytosolic functions

“…Binding of the cytoplasmic RNA-binding protein HuR to the 39-untranslated region (UTR) stabilizes ATF2 mRNA, whereas intracellular polyamines destabilize ATF2 mRNA (Xiao et al, 2007). ATF2 transcript levels are also negatively regulated by the microRNA miR-26b in lung cancer cells, and this suppression is relieved by ionizing radiation (Arora et al, 2011).…”