Downregulation of protein kinase B/Akt-1 mediates INS-1 insulinoma cell apoptosis induced by dominant-negative suppression of hepatocyte nuclear factor-1alpha function.
OBJECTIVE
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.


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
These data suggest that a decrease in PI-3K/AKT1 survival signalling mediates DN-HNF-1alpha-induced apoptosis in insulin-secreting cells.
Wobser
H
H
Department of Physiology, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, Dublin 2, Ireland.
Bonner
C
C
Nolan
J J
JJ
Byrne
M M
MM
Prehn
J H M
JH
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000235:genetics / Q000378:metabolism*; D007340:Insulinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010768:Phosphoserine / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013997:Time Factors; D000077191:Wortmannin
D000730:Androstadienes; D051538:Hepatocyte Nuclear Factor 1-alpha; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010768:Phosphoserine; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin