title: Increased expression and activation of c-Jun contributes to human amylin-induced apoptosis in pancreatic islet beta-cells.
abstract: The role of c-Jun in apoptosis evoked by human amylin was investigated using human and rat insulinoma beta-cell lines. Two transient increases in the levels of c-jun mRNA were detected at 30 minutes and eight hours after treatment with human amylin. The level of c-Jun protein was also up-regulated in a time-dependent manner, reaching maximal levels after eight hours of exposure. However, no c-Jun induction was detected in cells treated with vehicle only or with rat amylin, indicating that the amyloidogenic feature of the human peptide may be important for c-Jun induction. We found that c-Jun was activated by phosphorylation specifically at Ser63 at four hours, but not at Ser73, after treatment with human amylin, preceding increased c-Jun protein. Furthermore, expression of an antisense c-jun (AS-c-jun), which suppressed protein levels of both c-Jun and phosphorylated-c-Jun, caused a marked reduction in apoptotic cell death, whereas the corresponding sense c-jun (S-c-jun) had no effect on changes of either c-Jun production or apoptosis. This indicated that increased expression and activation of c-Jun is required for human amylin-induced apoptosis. Immunocytochemical studies showed a significant increase in nuclear staining for c-Jun, phosphorylated-c-Jun (Ser63) and phosphorylated-JNK, suggesting that c-Jun may be activated through activation of JNK. In addition, electrophoretic mobility-shift assays showed that the increase in expression and phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays demonstrated that c-Jun, c-Fos and ATF-2 are part of the AP-1 complex, indicating that activated c-Jun is dimerized with c-Fos or ATF-2 for control of its target gene expression. Finally, we showed that human amylin triggers AP-1-mediated transcriptional activation. Our results suggest strongly that human amylin induces apoptosis through stimulation of expression and activation of c-Jun, and that co-expression and dimerization of c-Jun and c-fos or ATF-2 may be important for activation of the downstream apoptotic process.
authors.affiliation: School of Biological Sciences, Faculty of Science, University of Auckland, Level 4 3A Symonds Street, Auckland, New Zealand.
authors.forename: Shaoping
authors.initials: S
authors.lastname: Zhang
authors.forename: Junxi
authors.initials: J
authors.lastname: Liu
authors.forename: Geraldine
authors.initials: G
authors.lastname: MacGibbon
authors.forename: Michael
authors.initials: M
authors.lastname: Dragunow
authors.forename: Garth J S
authors.initials: GJ
authors.lastname: Cooper
mesh_terms: D000682:Amyloid / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015152:Blotting, Northern; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D017931:DNA Primers / Q000737:chemistry; D016373:DNA, Antisense / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007124:Immunoenzyme Techniques; D007340:Insulinoma / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D058228:Islet Amyloid Polypeptide; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D016755:Proto-Oncogene Proteins c-jun / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000682:Amyloid; D017931:DNA Primers; D016373:DNA, Antisense; D058228:Islet Amyloid Polypeptide; D020319:Oligodeoxyribonucleotides, Antisense; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D008156:Luciferases