Ectopic expression of the beta-cell specific transcription factor Pdx1 inhibits glucagon gene transcription.
OBJECTIVE
The transcription factor Pdx1 is required for the development and differentiation of all pancreatic cells. Beta-cell specific inactivation of Pdx1 in developing or adult mice leads to an increase in glucagon-expressing cells, suggesting that absence of Pdx1could favour glucagon gene expression by a default mechanism.


METHODS
We investigated the inhibitory role of Pdx1 on glucagon gene expression in vitro. The glucagonoma cell line InR1G9 was transduced with a Pdx1-encoding lentiviral vector and insulin and glucagon mRNA levels were analysed by northern blot and real-time PCR. To understand the mechanism by which Pdx1 inhibits glucagon gene expression, we studied its effect on glucagon promoter activity in non-islet cells using transient transfections and gel-shift analysis.


RESULTS
In glucagonoma cells transduced with a Pdx1-encoding lentiviral vector, insulin gene expression was induced while glucagon mRNA levels were reduced by 50 to 60%. In the heterologous cell line BHK-21, Pdx1 inhibited by 60 to 80% the activation of the alpha-cell specific element G1 conferred by Pax-6 and/or Cdx-2/3. Although Pdx1 could bind three AT-rich motifs within G1, two of which are binding sites for Pax-6 and Cdx-2/3, the affinity of Pdx1 for G1 was much lower as compared to Pax-6. In addition, Pdx1 inhibited Pax-6 mediated activation through G3, to which Pdx1 was unable to bind. Moreover, a mutation impairing DNA binding of Pdx1 had no effect on its inhibition on Cdx-2/3. Since Pdx1 interacts directly with Pax-6 and Cdx-2/3 forming heterodimers, we suggest that Pdx1 inhibits glucagon gene transcription through protein to protein interactions with Pax-6 and Cdx-2/3.


CONCLUSIONS
Cell-specific expression of the glucagon gene can only occur when Pdx1 expression extinguishes from the early alpha cell precursor.
Ritz-Laser
B
B
Diabetes Unit, University Hospital Geneva, 24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. Beate.Laser@medecine.unige.ch
Gauthier
B R
BR
Estreicher
A
A
Mamin
A
A
Brun
T
T
Ris
F
F
Salmon
P
P
Halban
P A
PA
Trono
D
D
Philippe
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D005822:Genetic Vectors; D005934:Glucagon / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000235:genetics; D007515:Islets of Langerhans / Q000502:physiology; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
D018398:Homeodomain Proteins; D007328:Insulin; D009838:Oligodeoxyribonucleotides; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D005934:Glucagon