Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371,[606][607][608][609]. In adults, IPF1 expression is restricted to the a-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of 8-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet a-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta2/NeuroD, which is expressed in both a-and P-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-8 islet cells, which suggests an important role of IPF1 in a-cell maturation.Insulin promoter factor 1 (IPF1) is expressed in precursor cells during pancreas ontogeny (1, 2), and expression is required for pancreas formation (3,4). During ontogeny, IPF1 expression becomes restricted to the nuclei of the insulin-producing pancreatic islet (3-cells, suggesting that maintenance of IPF1 expression is necessary for the differentiation islet p3-cells from an IPF1-positive precursor common to all islet cells (2, 5). This restricted expression profile within the islets is reflected in the transplantable rat pancreatic insulinoma (IN) and glucagonoma (AN), which show substantial similarity to the mature islet f3-and a-cells, respectively (6-8). Thus, the AN is lacking IPF1 expression, as is the normal a-cell, and was recently found to be similar to normal a-cells in its expression of glucokinase as well as of the glucose-regulated insulinotropic peptide and glucagon-like peptide 1 receptors (9, 10). In vitro IPF1 binds to multiple sites in the insulin promoter and activates insulin gene reporter constructs when cotransfected into cell lines (1,5,11,12). This activity is dependent on cooperation between IPF1 and insulin enhancer factor-1 (IEF-1; refs. 5 and 12), a heterodimer composed of Beta2/ NeuroD, which is present in both a-and (3-cells, and ubiquitous class A helix-loop-helix proteins, such as Betal/rat E-box binding protein (REB; ref. 13) and products of the E2A gene (E47, E12, and ITF-1; refs. 14-19). In addition to IPF1 and IEF-1 binding sites, transcriptional regulation of the insulin gene requires a number of other cis-elements to which factors not yet cloned are binding (20)(21)(22)(23). To address whether IPF1 could activate transcription of the otherwise silent insulin genes in islet cells lacking IPF1 but expressing at least a subset of the other insulin gene transcription factors, a cDNA encoding rat IPF1 (24) under transcriptional control of the cytomegalovirus promoter was stably tra...
The somatostatin upstream enhancer (SMS-UE) is a highly complex enhancer element. The distal A-element contains overlapping Pdx1 and Pbx binding sites. However, a point mutation in the A-element that abolishes both Pdx1 and Pbx binding does not impair promoter activity. In contrast, a point mutation that selectively eliminates Pdx1 binding to a proximal B-element reduces the promoter activity. The B-element completely overlaps with a Pax6 binding site, the C-element. A point mutation in the C-element demonstrates that Pax6 binding is essential for promoter activity. Interestingly, a block mutation in the A-element reduces both Pax6 binding and promoter activity. In heterologous cells, Pdx1 potentiated Pax6 mediated activation of a somatostatin reporter. We conclude that the L L/N N-cell-specific activity of the SMS-UE is achieved through simultaneous binding of Pdx1 and Pax6 to the B-and Celements, respectively. Furthermore, the A-element appears to stabilise Pax6 binding.z 1999 Federation of European Biochemical Societies.
Insulin promoter factor-1 (IPF1) (renamed to pancreatic-duodenal homeobox factor-1, PDX1) was originally cloned and characterized as an islet beta-cell specific insulin gene transcription factor (1) and later shown to be essential for the formation of the mature pancreas (2, 3). In the adult normal pancreas PDX1 is almost exclusively expressed in the beta-cell compartment and generally absent from the alpha-cell while it is widely expressed in the pancreatic epithelium during development. Using pluripotent rat islet tumor cultures and derived insulinomas and glucagonomas we have analyzed differential expression of a large number of genes including the transcription factors PDX1, Nkx6.1, Pax6, and NeuroD. While NeuroD and Pax6 expression was detectable among all phenotypes, PDX1 was expressed in the pluripotent culture and maintained in the insulinoma, while Nkx6.1 was selectively co-induced with insulin during insulinoma formation. Both factors were not detectable in the glucagonoma. Nkx6.1 proved to have a highly beta-cell restricted expression in the adult rat. Forced expression of recombinant PDX1 in the glucagonoma resulted in efficient transcriptional activation of the endogenous insulin and IAPP genes, but did not affect glucagon gene activity. In this hybrid alpha/beta-cell phenotype the endogenous Nkx6.1 gene remained silent. We conclude that PDX1 in synergy with NeuroD specifies part of the beta-cell phenotype including transcriptional activation of insulin and IAPP genes, but that other factors such as Nkx6.1 and Pax6 are required for additional features of the fully mature beta-cell phenotype.
K K-cell specific transcription of the glucagon gene is mainly conferred by the glucagon promoter G1-element, while additional elements G2, G3, and G4 have broad islet cell specificity. Transcription of the glucagon gene has been shown to be stimulated by Pax6 through binding to the glucagon gene promoter G3-element. In this report, we show that Pax6 additionally binds the glucagon gene promoter G1-element and forms a transcriptionally active complex with another homeodomain protein, Cdx2/3. Two distinct mutations in the G1-element, that both reduce promoter activity by 85^90%, is shown to eliminate binding of either Pax6 or Cdx2/3. Additionally, Pax6 enhanced Cdx2/3 mediated activation of a glucagon reporter in heterologous cells. We discuss how Pax6 may contribute to cell-type specific transcription in the pancreatic islets by complex formation with different transcription factors.z 1999 Federation of European Biochemical Societies.
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