Insulin from the β-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor Neurogenin3 initiates the differentiation of the β-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurogenin3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate β-cells for patients with diabetes.

During pancreas development, both the exocrine and endocrine lineages differentiate from a common pool of progenitor cells with similarities to mature pancreatic duct cells. A small set of transcription factors, including Tcf2, Onecut1, and Foxa2, has been identified in these pancreatic progenitor cells. The Sry/HMG box transcription factor Sox9 is also expressed in the early pancreatic epithelium and is required for normal pancreatic exocrine and endocrine development in humans. In this study, we found Sox9 in mice specifically expressed with the other progenitor transcription factors in both pancreatic progenitor cells and duct cells in the adult pancreas. Sox9 directly bound to all three genes in vitro and in intact cells, and regulated their expression. In turn, both Foxa2 and Tcf2 regulated Sox9 expression, demonstrating feedback circuits between these genes. Furthermore, Sox9 activated the expression of the proendocrine factor Neurogenin3, which also depends on the other members of the progenitor transcription network. These studies indicate that Sox9 plays a dual role in pancreatic progenitor cells: both maintaining a stable transcriptional network and supporting the programs by which these cells differentiate into distinct lineages.

Neurogenin3 (ngn3), a basic helix-loop-helix (bHLH) transcription factor, functions as a pro-endocrine factor in the developing pancreas: by itself, it is sufficient to force undifferentiated pancreatic epithelial cells to become islet cells. Because ngn3 expression determines which precursor cells will differentiate into islet cells, the signals that regulate ngn3 expression control islet cell formation. To investigate the factors that control ngn3 gene expression, we mapped the human and mouse ngn3 promoters and delineated transcriptionally active sequences within the human promoter. Surprisingly, the human ngn3 promoter drives transcription in all cell lines tested, including fibroblast cell lines. In contrast, in transgenic animals the promoter drives expression specifically in regions of ngn3 expression in the developing pancreas and gut; and the addition of distal sequences greatly enhances transgene expression. Within the distal enhancer, binding sites for several pancreatic transcription factors, including hepatocyte nuclear factor (HNF)-1 and HNF-3, form a tight cluster. HES1, an inhibitory bHLH factor activated by Notch signaling, binds to the proximal promoter and specifically blocks promoter activity. Together with previous genetic data, these results suggest a model in which the ngn3 gene is activated by the coordinated activities of several pancreatic transcription factors and inhibited by Notch signaling through HES1. Diabetes 50:928 -936, 2001

The paired-homeodomain transcription factor PAX4 is expressed in the developing pancreas and along with PAX6 is required for normal development of the endocrine cells. In the absence of PAX4, the numbers of insulin-producing beta cells and somatostatin-producing delta cells are drastically reduced, while the numbers of glucagon-producing alpha cells are increased. To gain insight into PAX4 function, we cloned a full-length Pax4 cDNA from a beta-cell cDNA library and identified a bipartite consensus DNA binding sequence consisting of a homeodomain binding site separated from a paired domain binding site by 15 nucleotides. The paired half of this consensus sequence has similarities to the PAX6 paired domain consensus binding site, and the two proteins bind to common sequences in several islet genes, although with different relative affinities. When expressed in an alpha-cell line, PAX4 represses transcription through the glucagon or insulin promoters or through an isolated PAX4 binding site. This repression is not simply due to competition with the PAX6 transcriptional activator for the same binding site, since PAX4 fused to the unrelated yeast GAL4 DNA binding domain also represses transcription through the GAL4 binding site in the alpha-cell line and to a lesser degree in beta-cell lines and NIH 3T3 cells. Repressor activity maps to more than one domain within the molecule, although the homeodomain and carboxyl terminus give the strongest repression. PAX4 transcriptional regulation apparently plays a role only early in islet development, since Pax4 mRNA as determined by reverse transcriptase PCR peaks at embryonic day 13.5 in the fetal mouse pancreas and is undetectable in adult islets. In summary, PAX4 can function as a transcriptional repressor and is expressed early in pancreatic development, which may allow it to suppress alpha-cell differentiation and permit beta-cell differentiation.

Insulin upstream factor 1 (IUF1), a transcription factor present in pancreatic ␤-cells, binds to the sequence C(C/T)TAATG present at several sites within the human insulin promoter. Here we isolated and sequenced cDNA encoding human IUF1 and exploited it to identify the signal transduction pathway by which glucose triggers its activation. In human islets, or in the mouse ␤-cell line MIN6, high glucose induced the binding of IUF1 to DNA, an effect mimicked by serine/threonine phosphatase inhibitors, indicating that DNA binding was induced by a phosphorylation mechanism. The glucose-stimulated binding of IUF1 to DNA and IUF1-dependent gene transcription were both prevented by SB 203580, a specific inhibitor of stress-activated protein kinase 2 (SAPK2, also termed p38 mitogen-activated protein kinase, reactivating kinase, CSBP, and Mxi2) but not by several other protein kinase inhibitors. Consistent with this finding, high glucose activated mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase-2) (a downstream target of SAPK2) in MIN6 cells, an effect that was also blocked by SB 203580. Cellular stresses that trigger the activation of SAPK2 and MAP-KAP kinase-2 (arsenite, heat shock) also stimulated IUF1 binding to DNA and IUF1-dependent gene transcription, and these effects were also prevented by SB 203580.IUF1 expressed in Escherichia coli was unable to bind to DNA, but binding was induced by incubation with MgATP, SAPK2, and a MIN6 cell extract, which resulted in the conversion of IUF1 to a slower migrating form. SAPK2 could not be replaced by p42 MAP kinase, MAP-KAP kinase-2, or MAPKAP kinase-3. The glucose-stimulated activation of IUF1 DNA binding and MAPKAP kinase-2 (but not the arsenite-induced activation of these proteins) was prevented by wortmannin and LY 294002 at concentrations similar to those that inhibit phosphatidylinositide 3-kinase. Our results indicate that high glucose (a cellular stress) activates SAPK2 by a novel mechanism in which a wortmannin/LY 294002-sensitive component plays an essential role. SAPK2 then activates IUF1 indirectly by activating a novel IUF1-activating enzyme.