Gene inactivation studies have shown that members of the Gata family of transcription factors are critical for endoderm development throughout evolution. We show here that Gata-4 and/or Gata-6 are not only expressed in the adult exocrine pancreas but also in glucagonoma and insulinoma cell lines, whereas Gata-5 is restricted to the exocrine pancreas. During pancreas development, Gata-4 is expressed already at embryonic d 10.5 and colocalizes with early glucagon+ cells at embryonic d 12.5. Gata-4 was able to transactivate the glucagon gene both in heterologous BHK-21 (nonislet Syrian baby hamster kidney) and in glucagon-producing InR1G9 cells. Using gel-mobility shift assays, we identified a complex formed with nuclear extracts from InR1G9 cells on the G5 control element (-140 to -169) of the glucagon gene promoter as Gata-4. Mutation of the GATA binding site on G5 abrogated the transcriptional activation mediated by Gata-4 and reduced basal glucagon gene promoter activity in glucagon-producing cells by 55%. Furthermore, Gata-4 acted more than additively with Forkhead box A (hepatic nuclear factor-3) to trans-activate the glucagon gene promoter. We conclude that, besides its role in endoderm differentiation, Gata-4 might be implicated in the regulation of glucagon gene expression in the fetal pancreas and that Gata activity itself may be modulated by interactions with different cofactors.

Pax2 is a paired box transcription factor expressed in a spatially and temporally restricted manner and its absence results in major developmental defects of the central nervous system, eyes, ears and urogenital system. We recently reported that Pax2 is expressed in pancreatic endocrine cell lines and adult islets of Langerhans and activates glucagon gene expression. We have shown here that the Pax2 gene is expressed during pancreas development as early as embryonic day 10.5. Its absence, as assessed in Pax2(1Neu) mutant mice, results in a two- to threefold increase in the average pancreas volume occupied by the islets in both heterozygous and homozygous mutant mice with a gene-dependent dosage effect. This increase, which is due to a change in the number of islets per unit pancreas volume and in the size of individual islets, is not accompanied by significant modification in the insulin or glucagon content of the pancreas, indicating that the content of these hormones per cell is decreased. We have concluded that Pax2 may be implicated in the prenatal determination of the relative proportion of the endocrine and exocrine tissues of the pancreas.

Neonatal diabetes mellitus can be transient or permanent. The severe form of permanent neonatal diabetes mellitus can be associated with pancreas agenesis. Normal pancreas development is controlled by a cascade of transcription factors, where insulin promoter factor 1 (IPF1) plays a crucial role. Here, we describe two novel mutations in the IPF1 gene leading to pancreas agenesis. Direct sequence analysis of exons 1 and 2 of the IPF1 gene revealed two point mutations within the homeobox in exon 2. Genetic analysis of the parents showed that each mutation was inherited from one parent. Mutations localized in helices 1 and 2, respectively, of the homeodomain, decreased the protein half-life significantly, leading to intracellular IPF1 levels of 36% and 27% of wild-type levels. Both mutant forms of IPF1 were normally translocated to the nucleus, and their DNA binding activity on different known target promoters was similar to that of the wild-type protein. However, transcriptional activity of both mutant IPF1 proteins, alone or in combination with HNF3 beta/Foxa2, Pbx1, or the heterodimer E47-beta 2 was reduced, findings accounted for by decreased IPF1 steady state levels and not by impaired protein-protein interactions. We conclude that the IPF1 level is critical for human pancreas formation.

p8 is a nuclear DNA-binding protein, which was identified because its expression is strongly activated in response to several stresses. Biochemical and biophysical studies revealed that despite a weak sequence homology p8 is an HMG-I/Y-like protein, suggesting that p8 may be involved in transcription regulation. Results reported here strongly support this hypothesis. Using a pull-down approach, we found that p8 interacts with the general co-activator p300. We also found that, similar to the HMG proteins, p300 was able to acetylate recombinant p8in vitro, although the significance of such modification remains to be determined. Then a screening by the two-hybrid system, using p8 as bait, allowed us to identify the Pax2trans-activation domain-interacting protein (PTIP) as another partner of p8. Transient transfection studies revealed that PTIP is a strong inhibitor of the trans-activation activities of Pax2A and Pax2B on the glucagon gene promoter, which was chosen as a model because it is a target of the Pax2A and Pax2B transcription factors. This effect is completely abolished by co-transfection of p8 in glucagon-producing InRIG9 cells, indicating that p8 binding to PTIP prevents inhibition of the glucagon gene promoter. This was not observed in NIH3T3 fibroblasts that do not express glucagon. Finally, expression of p8 enhances the effect of p300 on Pax2A and Pax2B trans-activation of the glucagon gene promoter. These observations suggest that in glucagon-producing cells p8 is a positive cofactor of the activation of the glucagon gene promoter by Pax2A and Pax2B, both by recruiting the p300 cofactor to increase the Pax2A and Pax2B activities and by binding the Pax2-interacting protein PTIP to suppress its inhibition.

The promoter element G1, critical for a-cell-specific expression of the glucagon gene, contains two AT-rich sequences important for transcriptional activity. Pax-6, a paired homeodomain protein previously shown to be required for normal a-cell development and to interact with the enhancer element G3 of the glucagon gene, binds as a monomer to the distal AT-rich site of G1. However, although the paired domain of Pax-6 is sufficient for interaction with the G3 element, the paired domain and the homeodomain are required for high affinity binding to G1. In addition to monomer formation, Pax-6 interacts with Cdx-2/3, a caudal-related homeodomain protein binding to the proximal AT-rich site, to form a heterodimer on G1. Both proteins are capable of directly interacting in the absence of DNA. In BHK-21 cells, Pax-6 activates glucagon gene transcription both through G3 and G1, and heterodimerization with Cdx-2/3 on G1 leads to more than additive transcriptional activation. In glucagon-producing cells, both G1 and G3 are critical for basal transcription, and the Pax-6 and Cdx-2/3 binding sites are required for activation. We conclude that Pax-6 is not only critical for a-cell development but also for glucagon gene transcription by its independent interaction with the two DNA control elements, G1 and G3.

The promoter element G(1), critical for alpha-cell-specific expression of the glucagon gene, contains two AT-rich sequences important for transcriptional activity. Pax-6, a paired homeodomain protein previously shown to be required for normal alpha-cell development and to interact with the enhancer element G(3) of the glucagon gene, binds as a monomer to the distal AT-rich site of G(1), However, although the paired domain of Pax-6 is sufficient for interaction with the G(3) element, the paired domain and the homeodomain are required for high affinity binding to G(1), In addition to monomer formation, Pax-6 interacts with Cdx-2/3, a caudal-related homeodomain protein binding to the proximal AT-rich site, to form a heterodimer on G(1), Both proteins are capable of directly interacting in the absence of DNA. In BHK-21 cells, Pax-6 activates glucagon gene transcription both through G(1) and G(3), and heterodimerization with Cdx-2/3 on G(1) leads to more than additive transcriptional activation, In glucagon-producing cells, both G(3) and G(1) are critical for basal transcription, and the Pax-6 and Cdx-2/3 binding sites are required for activation. We conclude that Pax-6 is not only critical for cu-cell development but also for glucagon gene transcription by its independent interaction with the two DNA control elements, G(1) and G(3).

The factors that regulate glucagon biosynthesis and proglucagon gene expression are poorly defined. We previously reported that insulin inhibits proglucagon gene expression in vitro. In vivo, however, the effects of insulin on the regulation of the proglucagon gene have been controversial. Furthermore, whether glucose plays any role alone or in conjunction with insulin on proglucagon gene expression is unknown. We investigated the consequences of insulinopenic diabetes on glucagon gene expression in the endocrine pancreas and intestine and whether insulin and/or glucose could correct the observed abnormalities. We show here that in the first 3 days after induction of hyperglycemia by streptozotocin, rats have levels of plasma glucagon and proglucagon messenger RNA comparable to those of normoglycemic controls despite hyperglycemia. With more prolonged diabetes, plasma glucagon and proglucagon messenger RNA levels increase; this increase is corrected by insulin treatment, but not by phloridzin despite normalization of the glycemia by both treatments. Proglucagon gene expression exhibits the same regulatory response to glucose and insulin in both pancreas and ileum. We conclude that insulin tonically inhibits proglucagon gene expression in the pancreas and ileum and that glucose plays a minor, if any, role in this regulation.