The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6.
OBJECTIVE
The paired-homeobox genes pax-4 and pax-6 are crucial for islet development; whereas the null mutation of pax-6 results in the nearly absence of glucagon-producing alpha cells, pax-4 homozygous mutant mice lack insulin and somatostatin-producing beta and delta cells but contain an increased number of alpha cells suggesting that alpha cells could develop by a default mechanism.


METHODS
To investigate whether beta-cell specific factors act negatively on glucagon gene transcription, we ectopically expressed pax-4 in glucagon producing InR1G9 cells; Pax-4 inhibited basal transcription of the glucagon gene promoter by 60%. To assess the mechanism of this inhibition, we cotransfected the non-islet cell line BHK-21 with Pax-4 and various transcription factors present in alpha cells.


RESULTS
In addition to a general repressor activity on basal glucagon gene promoter activity of 30-50%, a specific 90% inhibition of Pax-6 mediated transactivation was observed. In contrast, Pax-4 had no effect on Cdx-2/3 or HNF3alpha mediated transcriptional activation. Pax-4 showed similar affinity to the Pax-6 binding sites on the glucagon gene promoter compared to Pax-6, but varying with KCl concentrations.


CONCLUSIONS
Pax-4 impairs glucagon gene transcription specifically through inhibition of Pax-6 mediated transactivation. Transcriptional inhibition seems to be mediated by direct DNA binding competition with Pax-6 and potentially additional mechanisms such as protein-protein interactions and a general repressor activity of Pax-4. Glucagon gene expression in alpha cells could thus result from both the presence of islet cell specific transcription factors and the absence of Pax-4.
Ritz-Laser
Beate
B
Diabetes Unit, Geneva University Hospital, 24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. Beate.Laser@medecine.unige.ch
Estreicher
A
A
Gauthier
B R
BR
Mamin
A
A
Edlund
H
H
Philippe
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D005136:Eye Proteins; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter; D005934:Glucagon / Q000235:genetics*; D018398:Homeodomain Proteins / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D007668:Kidney; D008969:Molecular Sequence Data; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D005934:Glucagon; D015500:Chloramphenicol O-Acetyltransferase