Intracellular mechanisms mediating the anti-apoptotic action of gastrin.
We previously reported that gastrin (G17) inhibits apoptosis of AR4-2J pancreatic adenocarcinoma cells, through the activation of Akt. We dissected the mechanisms responsible for this effect. D2, a CCKB receptor antagonist, inhibited G17 induction of Akt phosphorylation, measured by Western blots with anti-phospho-Akt antibodies. The intracellular calcium chelator BAPTA-AM, but not the PKC inhibitor GF109203X, blocked G17 induction of Akt. G17 stimulated BAD phosphorylation, measured by both Western blots with anti-phospho-BAD antibodies and by in vitro Akt kinase assays using recombinant BAD as substrate. G17 also induced FOXO3 phosphorylation assessed by Western blots with anti-phospho-FOXO3 antibodies, and BAPTA-AM inhibited this effect. Gastrin inhibited luciferase activity in cells transfected with FOXO1 together with a vector containing insulin-responsive sequences upstream of the luciferase reporter gene. In conclusion, G17 induces Akt through activation of CCKB receptors and of intracellular calcium-dependent, PKC-independent, pathways. This effect leads to BAD phosphorylation and to forkhead transcription factors inactivation.
Ramamoorthy
Saravanan
S
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI, USA.
Stepan
Vinzenz
V
Todisco
Andrea
A
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051858:Forkhead Transcription Factors; D005755:Gastrins / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology*; D017930:Genes, Reporter; D005822:Genetic Vectors; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D051025:bcl-Associated Death Protein; D000071161:Forkhead Box Protein O1
D002118:Calcium; D002352:Carrier Proteins; D002614:Chelating Agents; D004268:DNA-Binding Proteins; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D051858:Forkhead Transcription Factors; D005755:Gastrins; D007211:Indoles; D008156:Luciferases; D008301:Maleimides; D009419:Nerve Tissue Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D014157:Transcription Factors; D051025:bcl-Associated Death Protein; C494821:Bad protein, rat; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C074639:Foxo1 protein, rat; D000071161:Forkhead Box Protein O1; C494920:Akt1 protein, rat; C070515:bisindolylmaleimide I