Growth hormone induces insulin-like growth factor-I gene transcription by a synergistic action of STAT5 and HNF-1alpha.
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Salmon insulin-like growth factor-I (sIGF-I) expression is, as in mammals, induced by growth hormone (GH). To elucidate the mechanism by which GH stimulates the transcription of the IGF-I gene, we transiently transfected Hep3B cells expressing the rat GH receptor with a sIGF-I promoter-luciferase reporter construct. Activation of the construct by GH added to the medium of the transfected cells was observed when two specific transcription factors, STAT5 and HNF-1alpha, were simultaneously overexpressed in these cells. This finding demonstrates for the first time a GH-dependent activation of an IGF-I promoter construct in an immortalized laboratory cell line. (+info)
Regulation of alpha1-antitrypsin gene expression in human intestinal epithelial cell line caco-2 by HNF-1alpha and HNF-4.
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There is still relatively limited information about mechanisms of gene expression in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial cell line Caco-2, which spontaneously differentiates from a cryptlike to a villouslike enterocyte, we have previously shown that there is a marked increase in transcription of the well-characterized alpha1-antitrypsin (alpha1-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements and trans-acting DNA-binding proteins responsible for expression of the alpha1-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-1alpha (HNF-1alpha), HNF-1beta, and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the alpha1-AT gene. Cotransfection studies showed that HNF-1alpha and HNF-4 had a synergistic effect on alpha1-AT gene expression. RNA blot analysis showed that HNF-1alpha and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1alpha binding activity increase coordinately with alpha1-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1alpha in Caco-2 cells resulted in a selective decrease in endogenous alpha1-AT gene expression. Together, these results provide evidence that HNF-1alpha and HNF-4 play a role in the mechanism by which the alpha1-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system. (+info)
Functional study of the E276Q mutant hepatocyte nuclear factor-4alpha found in type 1 maturity-onset diabetes of the young: impaired synergy with chicken ovalbumin upstream promoter transcription factor II on the hepatocyte nuclear factor-1 promoter.
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Seven mutations in the hepatocyte nuclear factor (HNF)-4alpha gene have been shown to correlate with type 1 maturity-onset diabetes of the young (MODY 1), a monogenic form of type 2 diabetes. Up to now, only the functional properties of two MODY 1 HNF-4alpha mutants, Q268X and V393I, have been investigated to address how the mutations in the HNF-4alpha gene, found by genetic studies, can give rise to impaired activities of mutated HNF-4alpha proteins and can cause this disease. The E276Q mutation results in a nonconservative substitution occurring in the HNF-4alpha E domain, which is involved in dimerization and transactivation activities as well as in protein-protein interactions with other transcription factors or coactivators. Using the mutated human HNF-4alpha2, we have found that, in the absence of chicken ovalbumin upstream promoter transcription factor II (COUP TFII), the E276Q substitution does not significantly affect the dimerization and transactivating activities of HNF-4alpha, at least on the promoters studied herein. On the other hand, in the presence of COUP TFII, the substitution impairs the enhancement of HNF-4-mediated activation of HNF-1 promoter. The impaired synergy between COUP TFII and HNF-4 on the HNF-1 promoter results from an alteration of their interaction. HNF-1 expression plays a crucial role in transactivation of insulin promoter and of numerous genes coding for enzymes involved in glucose homeostasis. Therefore, its downregulation resulting from the E276Q mutation in HNF-4alpha gene most probably impairs the function of pancreatic beta-cells. (+info)
Phenotypic characteristics of early-onset autosomal-dominant type 2 diabetes unlinked to known maturity-onset diabetes of the young (MODY) genes.
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OBJECTIVE: To investigate whether there are forms of early-onset autosomal-dominant type 2 diabetes that are distinct from typical maturity-onset diabetes of the young (MODY) and to characterize their phenotypic characteristics. RESEARCH DESIGN AND METHODS: The study included 220 affected subjects from 29 families in which early-onset type 2 diabetes occurred in multiple generations and was not linked to known MODY genes (MODY gene-negative families). All individuals underwent an oral glucose tolerance test and other clinical measurements aimed at investigating the underlying metabolic defect and the presence of diabetic complications. For comparison, 79 affected carriers of MODY3 (hepatocyte nuclear factor [HNF]-1 alpha) mutations were similarly examined. RESULTS: Subjects from MODY gene-negative pedigrees were diagnosed with diabetes at an older age (36 +/- 17 vs. 21 +/- 10 years, P = 0.0001) and were more frequently obese (52 vs. 18%, P = 0.0001) than MODY3 individuals. MODY gene-negative patients who were insulin treated required more exogenous insulin than did MODY3 subjects (0.7 +/- 0.4 vs. 0.45 +/- 0.2 U.kg-1.day-1, P = 0.04), despite similar C-peptide levels. Among subjects not treated with insulin, MODY gene-negative subjects had significantly higher serum insulin levels, both fasting (16.5 +/- 15 vs. 6.5 +/- 5 microU/ml, P = 0.027) and 2 h after a glucose load (53 +/- 44 vs. 11 +/- 10, P = 0.002). They also had higher serum triglycerides (P = 0.02), higher cholesterol levels (P = 0.02), more hypertension (P = 0.0001), and more nephropathy (P = 0.001). Differences persisted when families were matched for age at diagnosis. CONCLUSIONS: Our findings indicate the existence of forms of early-onset autosomal-dominant type 2 diabetes that are distinct from MODY and are frequently characterized by insulin resistance, similar to later-onset type 2 diabetes. Because of the Mendelian pattern of inheritance, the goal of identifying the genes involved in these forms of diabetes appears to be particularly feasible. (+info)
Activation of the mouse TATA-less and human TATA-containing UDP-glucuronosyltransferase 1A1 promoters by hepatocyte nuclear factor 1.
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UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1alpha and HNF1beta in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human. (+info)
The HNF-4/HNF-1alpha transactivation cascade regulates gene activity and chromatin structure of the human serine protease inhibitor gene cluster at 14q32.1.
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Hepatocyte-specific expression of the alpha1-antitrypsin (alpha1AT) gene requires the activities of two liver-enriched transactivators, hepatocyte nuclear factors 1alpha and 4 (HNF-1alpha and HNF-4). The alpha1AT gene maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene encoding corticosteroid-binding globulin (CBG), and the chromatin organization of this approximately 130-kb region, as defined by DNase I-hypersensitive sites, has been described. Microcell transfer of human chromosome 14 from fibroblasts to rat hepatoma cells results in activation of alpha1AT and CBG transcription and chromatin reorganization of the entire locus. To assess the roles of HNF-1alpha and HNF-4 in gene activation and chromatin remodeling, we transferred human chromosome 14 from fibroblasts to rat hepatoma cell variants that are deficient in expression of HNF-1alpha and HNF-4. The variant cells failed to activate either alpha1AT or CBG transcription, and chromatin remodeling failed to occur. However, alpha1AT and CBG transcription could be rescued by transfecting the cells with expression plasmids encoding HNF-1alpha or HNF-4. In these transfectants, the chromatin structure of the entire alpha1AT/CBG locus was reorganized to an expressing cell-typical state. Thus, HNF-1alpha and HNF-4 control both chromatin structure and gene activity of two cell-specific genes within the serpin gene cluster at 14q32.1. (+info)
Non-penetrance in a MODY 3 family with a mutation in the hepatic nuclear factor 1alpha gene: implications for predictive testing.
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The most common cause of maturity-onset diabetes of the young (MODY) is a mutation in the hepatic nuclear factor 1alpha (HNF1alpha) gene (MODY3). We describe a family in which a missense mutation causing a Thr-Ile substitution at codon 620 has been found in all affected members. The mutation is not fully penetrant as two family members aged 87 and 46 have the mutation but do not have diabetes. The severity and age of diagnosis of diabetes varies widely within the family, and most presented over the age of 25. HNF1alpha mutation screening should be considered in any family with autosomal dominant inheritance of diabetes where one member has presented with diabetes before the age of 25. Predictive testing is now possible within the majority of MODY families, and is of clinical benefit, but the possibility of non-penetrance should be addressed during counselling and interpretation of results. (+info)
Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation.
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vHNF1/HNF1beta, a member of the divergent HNF1/vHNF1 homeoprotein family, is expressed in polarized epithelia of several adult organs and may participate in controlling the transcription of specific genes. In addition to this late requirement, vHNF1 may play earlier roles during development, as it is first expressed in the visceral endoderm at the onset of gastrulation. In order to shed light on its function during embryogenesis, we have inactivated the murine gene by homologous recombination. The homozygous mutation results in embryonic lethality by day 7.5 of development and vHNF1(-)(/)(-) embryos display a disorganized visceral endoderm and a significantly reduced size. Studies of ES cell differentiation and aggregation with tetraploid morulae establish that vHNF1 expression is essential for visceral endoderm differentiation, both in vitro and in vivo. Analysis of differentiation markers confirms that vHNF1 is part of a genetic network that directs the expression of HNF4 and downstream endodermal genes. Furthermore, the complementation of the mutant embryos with wild-type visceral endoderm rescues the day 7.5 lethality and reveals an additional phenotype linked to vHNF1 later expression. The examination of chimeric embryos suggests that vHNF1 expression might be cell-autonomously required in the gut for the proper morphogenesis of the embryo. (+info)