Foxa2 (HNF3β) Controls Multiple Genes Implicated in Metabolism-Secretion Coupling of Glucose-induced Insulin Release

Abstract

The forkhead/winged-helix Foxa family of transcription factors, encoded by three genes Foxa1 (Hnf3␣), Foxa2 (Hnf3␤), and Foxa3 (Hnf3␥), regulate hepatic and/or pancreatic gene expression (1-9). Foxa1 and Foxa3 are required for maintaining glucose homeostasis by activation, respectively, of pancreatic glucagon and hepatic gluconeogenic enzymes (2, 3, 5). Targeted disruption of Foxa2 resulted in embryonic lethality with defective development of the foregut endoderm, from which the liver and pancreas arise (10). Foxa2, which is expressed in islets, has been suggested as the upstream transactivator of Hnf4␣, Hnf1␣, Pdx1, and Hnf1␤ in the transcriptional hierarchy (1, 9, 11). Mutations in the genes encoding these pancreatic transcription factors are linked to four monogenic forms of MODY 1 (maturity-onset diabetes of the young): MODY1/HNF4␣, MODY3/HNF1␣, MODY4/IPF1(PDX1), and MODY5/HNF1␤ (12, 13). However, the search for the association of FOXA2 mutations with MODY patients has not been successful (14, 15). Most recently, Sund et al. (16) have suggested that FOXA2 rather might be a candidate gene for familial hyperinsulinism. Pancreatic ␤-cell-specific deletion of Foxa2 resulted in postnatal death due to severe hyperinsulinemic hypoglycemia, and the down-regulation of ATP-sensitive K ϩ (K ATP ) channel subunits Sur1 and Kir6.2 has been demonstrated in these mutant mice (16).To assess whether Foxa2 indeed controls the expression of the transcription factors associated with MODY, we have established INS-1-derived stable cell lines, which allow conditional expression of the wild type Foxa2 or its dominant-negative mutant DN-Foxa2 under tight control of the reverse tetracycline-dependent transactivator (17). DN-Foxa2 is a Myctagged truncated Foxa2 mutant protein that possesses the intact DNA-binding domain but lacks the transactivation domain (7). DN-Foxa2 exerts its dominant-negative function by competing with the endogenous Foxa2 for cognate DNA binding (7). The impact of altered Foxa2 function on glucose metabolism and insulin secretion was assessed in these stable clones. The gene expression profile before and after induction of the Foxa2 or DN-Foxa2 was quantified. EXPERIMENTAL PROCEDURESEstablishment of Stable Cell Lines-Rat insulinoma INS-1 cell linederived stable clones were cultured in RPMI 1640 in 11.2 mM glucose (18), unless otherwise indicated. The first step stable clone INSr␣␤, which expresses the reverse tetracycline-dependent transactivator, was described previously (17,18). Plasmids used in the secondary stable transfection were constructed by subcloning the cDNAs encoding the mouse Foxa2 (kindly supplied by Prof. G. Schü tz) and its dominantnegative mutant (DN-Foxa2) into the expression vector PUHD10 -3 (a kind gift from Prof. H. Bujard). DN-Foxa2 (truncated mutation lacking the transactivation domain but containing the intact DNA-binding domain) (7) was PCR-amplified from Foxa2 cDNA using the following primers: 5Ј-gcaggatccgtaatggtgctcgggcttcaggtg-3Ј and 5Ј-gcaggatccggcgccatggcgggcatgagcggctca3-Ј. The ...

Myostatin Induces DNA Damage in Skeletal Muscle of Streptozotocin-induced Type 1 Diabetic Mice

“…Previous studies demonstrated that in the absence of insulin, Foxa2 activity and level was enhanced in the liver. Foxa2 has been reported to regulate genes implicated in glucose (26) and lipid metabolism in the liver (28). Our results indicate that in the muscle from STZ-treated WT mice, the Foxa2 expression and protein was up-regulated ( Fig Previously it has been reported that insulin signaling inhibits Foxa2 activity by phosphorylation mediated by Akt (28,30).…”

“…The sequence analysis identified a putative Foxa2 binding site (5Ј-CAAATATTTGTT-3Ј) within the 1.7 kb sequence of the mouse Mstn promoter. Foxa2 has been shown to regulate glucose homeostasis and glucose-induced insulin release (26). RTqPCR and Western blot analysis of Foxa2 indicated that Foxa2 mRNA expression and protein level ( Fig.…”

MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

“…Moreover, Foxa2 plays a central role in insulin release via the regulation of genes coding for both Kir6.2 (inward rectifier potassium channel member 6.2) and Sur1 (sulfonylurea receptor 1) genes, the two subunits of the ATP-dependent K ϩ (K ATP ) channel. In brief, it appears that Foxa2 plays a pivotal role in key ␤-cell functions and physiology (22)(23)(24)(25).…”

Does chasing selected ‘Fox’ to the nucleus prevent diabetes?

“…Human loss-of-function mutations in these three genes are linked to persistent hyperinsulinemic hypoglycemia of infancy [25,26]. Conversely, the gain of function following Foxa2 overexpression in rat insulinoma INS-1 cells suppresses insulin secretion [27]. This is in line with the requirement for Foxa2 in the regulation of glucagon gene expression and pancreatic a-cell differentiation [15].…”