Erschienen in:
01.03.2006 | Article
Downregulation of protein kinase B/Akt-1 mediates INS-1 insulinoma cell apoptosis induced by dominant-negative suppression of hepatocyte nuclear factor-1alpha function
verfasst von:
H. Wobser, C. Bonner, J. J. Nolan, M. M. Byrne, J. H. M. Prehn
Erschienen in:
Diabetologia
|
Ausgabe 3/2006
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Abstract
Aims/hypothesis
Inactivating mutations in Tcf1, which encodes the transcription factor hepatocyte nuclear factor (HNF)-1alpha, cause maturity-onset diabetes of the young-3. We have previously shown that a dominant-negative mutant (DN-HNF-1alpha) renders INS-1 insulinoma cells sensitive to the mitochondrial apoptosis pathway, but the underlying alterations in signal transduction remain unknown.
Materials and methods
Using a reverse tetracycline-dependent transactivator system, DN-HNF-1alpha-induced apoptosis was assessed by immunoblotting and caspase assays. Alterations in AKT1 kinase/protein kinase B (AKT1) survival signalling during DN-HNF-1alpha-induced apoptosis were investigated by phospho-specific immunodetection and transient transfection experiments.
Results
Induction of DN-HNF-1alpha caused significant changes in the activation-specific phosphorylation status of AKT1 that were preceded by a downregulation of Ins1 gene transcription. Phosphorylation of AKT1 at Ser473 was dramatically reduced after 36 to 48 h of DN-HNF-1alpha induction and coincided with maximal apoptosis activation. Overexpression of a constitutively active mutant of Akt1 rescued INS-1 cells from DN-HNF-1alpha-induced apoptosis, while ectopic expression of a dominant-negative mutant mimicked the effect of DN-HNF-1alpha on apoptosis activation. Pharmacological suppression of growth factor survival signalling through administration of the phosphatidylinositol-3 kinase (PI-3K) inhibitor wortmannin accelerated the induction of apoptosis by DN-HNF-1alpha.
Conclusions/interpretation
These data suggest that a decrease in PI-3K/AKT1 survival signalling mediates DN-HNF-1alpha-induced apoptosis in insulin-secreting cells.