Akt kinase activation blocks apoptosis in intestinal epithelial cells by inhibiting caspase-3 after polyamine depletion.
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 alpha-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-alpha/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-alpha/cycloheximide-induced apoptosis. Phosphorylation of glycogen synthase kinase-3, a downstream target of Akt, was also increased in alpha-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.
Zhang
Huifang M
HM
Departments of Surgery and Pathology, University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201, USA.
Rao
Jaladanki N
JN
Guo
Xin
X
Liu
Lan
L
Zou
Tongtong
T
Turner
Douglas J
DJ
Wang
Jian-Ying
JY
D000818:Animals; D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid; D002867:Chromones / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D000518:Eflornithine / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000473:pathology*; D005799:Genes, Dominant; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D007422:Intestines / Q000166:cytology*; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D016366:Open Reading Frames; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011073:Polyamines / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D017304:Annexin A5; D061945:Caspase Inhibitors; D002867:Chromones; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D009025:Morpholines; D011073:Polyamines; D011518:Proto-Oncogene Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D011065:Poly(ADP-ribose) Polymerases; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D000518:Eflornithine