The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6.

Abstract

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. 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. 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. 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.