Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells

103

Apoptosis plays a critical role in the maintenance of gut mucosal homeostasis and is regulated by numerous factors including polyamines. Although the exact roles of polyamines in apoptotic pathway are still unclear, inhibition of polyamine synthesis promotes the resistance of intestinal epithelial cells to apoptosis. Akt is a serine-threonine kinase that has been established as an important intracellular signaling in regulating cell survival. The current studies test the hypothesis that polyamines are involved in the control of Akt activity in normal intestinal epithelial cells (IEC-6 line) and that activated Akt mediates suppression of apoptosis following polyamine depletion. Depletion of cellular polyamines by ␣-difluoromethylornithine induced levels of phosphorylated Akt and increased Akt kinase activity, although it had no effect on expression of total Akt, pERK, p38, and Bcl-2 proteins. This activated Akt was associated with both decreased levels of active caspase-3 and increased resistance to tumor necrosis factor-␣/cycloheximide-induced apoptosis. Inactivation of Akt by either treatment with LY294002 or ectopic expression of a dominant negative Akt mutant (DNMAkt) not only enhanced the caspase-3 activation in polyamine-deficient cells but also prevented the increased resistance to tumor necrosis factor-␣/cycloheximide-induced apoptosis. Phosphorylation of glycogen synthase kinase-3, a downstream target of Akt, was also increased in ␣-difluoromethylornithine-treated cells, which was prevented by inactivation of Akt by LY294002 or DNMAkt overexpression. These results indicate that polyamine depletion induces the Akt activation mediating suppression of apoptosis via inhibition of caspase-3 in normal intestinal epithelial cells.

Section: Effect Of Polyamine Depletion On Akt Phosphorylation and Itssupporting

confidence: 92%

“…High Performance Liquid Chromatography Analysis of Cellular Polyamines-The cellular polyamine content was determined as previously described (10,36). The results are expressed as nanomoles of polyamines per milligram of protein.…”

Section: Methodsmentioning

confidence: 99%

Akt Kinase Activation Blocks Apoptosis in Intestinal Epithelial Cells by Inhibiting Caspase-3 after Polyamine Depletion

Zhang

Rao

Guo

et al. 2004

103

“…ODC is the enzyme responsible for catalyzing the first rate-limiting step in polyamine biosynthesis. Inhibition of ODC activity by DFMO decreases mRNA levels for the c-fos, c-myc, and c-jun proto-oncogenes in IEC-6 cells (18). In the present report we investigated the potential role of the NF-B transcription factor family in the regulation of gene expression by polyamines.…”

Section: Discussionmentioning

confidence: 92%

Polyamine Depletion Induces Rapid NF-κB Activation in IEC-6 Cells

Pfeffer¹,

Yang²,

Murti³

et al. 2001

The proliferation of the rat intestinal mucosal IEC-6 cell line requires polyamines, whose synthesis is catalyzed by the enzyme ornithine decarboxylase (ODC). ODC inhibition leads to polyamine depletion, as well as inhibition of both cell proliferation and apoptosis by regulating gene expression. The NF-B transcription factor regulates genes involved in apoptotic, immune, and inflammatory responses. In the present study we tested the hypothesis that NF-B is activated following ODC inhibition. We found that the inhibition of ODC by ␣-difluoromethylornithine (DFMO) resulted in a ϳ50% decrease in intracellular putrescine levels within 1 h. NF-B is activated by DFMO through the degradation of the inhibitory protein IB␣ that sequesters NF-B in the cytoplasm. The DFMO-induced NF-B complexes contain the p65 and p50 members of the Rel protein family. DFMO-induced NF-B activation was accompanied by the translocation of p65 from the cytoplasm into the nucleus. DFMO selectively inhibited a gene reporter construct dependent on the B site present in the HLA-B7 gene. In contrast, DFMO had no effect on a gene reporter construct dependent on the B site present in the interleukin-8 gene. Thus, we report that ODC inhibition activates the NF-B transcription factor, which may mediate the altered physiological state of intestinal cells that occurs following polyamine depletion.

“…Although the molecular mechanisms whereby polyamines control the growth of intestinal mucosal cells have not been fully elucidated, they are known to be involved in the regulation of growth-related genes (8,9,13,32,33). In this regard, polyamines have been shown to suppress the expression of growthinhibiting genes such as p53 (10,12), NPM (14), p21 (34), TGF␤/TGF␤-Rs (TGF␤-receptors) (8,35), and junD (11,39), primarily by controlling mRNA stability.…”

Section: Discussionmentioning

confidence: 99%

Polyamine Depletion Increases Cytoplasmic Levels of RNA-binding Protein HuR Leading to Stabilization of Nucleophosmin and p53 mRNAs

Zou¹,

Mazan-Mamczarz

Rao³

et al. 2006

114

167

Polyamines are essential for maintaining normal intestinal epithelial integrity, an effect that relies, at least in part, on their ability to keep low levels of nucleophosmin (NPM) and p53 mRNAs. The RNA-binding protein HuR associates with the p53 mRNA, as reported previously, and with the NPM mRNA, computationally predicted to be a target of HuR. Here, we show that HuR binds the NPM and p53 3-untranslated regions and stabilizes these mRNAs in polyaminedepleted intestinal epithelial cells. Depletion of cellular polyamines by inhibiting ornithine decarboxylase with ␣-difluoromethylornithine dramatically enhanced the cytoplasmic abundance of HuR, whereas ectopic ornithine decarboxylase overexpression decreased cytoplasmic HuR; neither intervention changed whole-cell HuR levels. HuR was found to specifically bind the 3-untranslated regions of NPN and p53 mRNAs. HuR silencing rendered the NPM and p53 mRNAs unstable and prevented increases in NPM and p53 mRNA and protein in polyamine-deficient cells. These results indicate that polyamines modulate cytoplasmic HuR levels in intestinal epithelial cells, in turn controlling the stability of the NPM and p53 mRNAs and influencing NPM and p53 protein levels.