Transcriptional regulation of the distal promoter of the rat pyruvate carboxylase gene by hepatocyte nuclear factor 3β/Foxa2 and upstream stimulatory factors in insulinoma cells

Abstract

PC (pyruvate carboxylase) plays a crucial role in intermediary metabolism including glucose-induced insulin secretion in pancreatic islets. In the present study, we identified two regions of the 1.2 kb distal promoter, the -803/-795 site and the -408/-403 E-box upstream of the transcription start site, as the important cis-acting elements for transcriptional activation of the luciferase reporter gene. Site-directed mutagenesis of either one of these sites in the context of this 1.2 kb promoter fragment, followed by transient transfections in the insulinoma cell line, INS-1, abolished reporter activity by approx. 50%. However, disruption of either the -803/-795 or the -408/-403 site did not affect reporter gene activity in NIH 3T3 cells, suggesting that this promoter fragment is subjected to cell-specific regulation. The nuclear proteins that bound to these -803/-795 and -408/-403 sites were identified by gel retardation assays as HNF3beta (hepatocyte nuclear factor 3beta)/Foxa2 (forkhead/winged helix transcription factor box2) and USFs (upstream stimulatory factors), USF1 and USF2, respectively. Chromatin immunoprecipitation assays using antisera against HNF3beta/Foxa2, USF1 and USF2 demonstrated that endogenous HNF3beta/Foxa2 binds to the -803/-795 Foxa2 site, and USF1 and USF2 bind to the -408/-403 E-box respectively in vivo, consistent with the gel retardation assay results. Although there are weak binding sites located at regions -904 and -572 for PDX1 (pancreatic duodenal homeobox-1), a transcription factor that controls expression of beta-cell-specific genes, it did not appear to regulate PC expression in INS-1 cells in the context of the 1.2 kb promoter fragment. The results presented here show that Foxa2 and USFs regulate the distal promoter of the rat PC gene in a cell-specific manner.

Metabolic properties of chicken embryonic stem cells

“…However, undifferentiated chES cells completely block the gluconeogenic pathway, and thus pyruvate in these cells cannot be converted to sugar phosphates under the low glycolytic rate. This block occurs because some hormones, such as insulin, glucagon and glucocorticoid, and their receptors, which transcriptionally regulate gluconeogenic gene (Pcx and Pck2) expression [58][59][60][61] are not expressed until the gastrulation stage in the chicken [62,63]. We observed that yolk granules were either close to or directly associated with mitochondria.…”

Differential configurations involving binding of USF transcription factors and Twist1 regulateAlx3promoter activity in mesenchymal and pancreatic cells

“…USF1 and USF2 have been found to be important for the regulation of different pancreatic islet genes involved in the control of glucose metabolism [53,54], as well as in that of Pdx1 (pancreatic and duodenal homeobox 1) [29], a key regulator of pancreatic development and function whose defect is associated with maturity onset diabetes of the young [55]. Indeed, the USF1 locus in humans has been found to be associated with increased risk to develop Type 2 diabetes, although it cannot be ruled out that this association may be more related to lipid than to glucose metabolic defects [56].…”

Multiple E-Boxes in the Distal Promoter of the Rat Pyruvate Carboxylase Gene Function as a Glucose-Responsive Element

“…This promoter fragment has previously been characterized to contain full basal and tissue-specific cis -acting elements [21]. The transfected cells were maintained in the medium containing 5.5 mM or 25 mM glucose for 24 h. As shown in Figure 2, the transfected cells maintained in medium containing 25 mM glucose possessed luciferase activity approximately 2-fold higher than those maintained at 5.5 mM glucose, suggesting that glucose exerts its stimulatory effect on PC expression via GRE located within the 1.2 kb distal promoter of PC gene.…”

“…The luciferase and β-galactosidase assays were performed as described previously [21]. The luciferase activity was normalized with the β-galactosidase activity and presented as the relative luciferase activity.…”

“…Characterization of the distal promoter of rat PC gene revealed that a member of the forkhead box protein 3β (FoxA2) regulates PC expression in a β-cell specific manner [21]. Although previous work has shown that PC expression is inducible by exogenous glucose, it remains largely unknown how glucose increases PC expression [8], [17].…”