Search

ACAT catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acids. There are two known genes encoding the two ACAT enzymes, ACAT1 and ACAT2 (also known as Soat1 and Soat2). In adult humans, ACAT1 is present in most tissues, whereas ACAT2 is localized to enterocytes and hepatocytes. In this report, we elucidate the mechanisms that control the liver-specific expression of the human ACAT2 gene. We identified hepatic nuclear factor 1 (HNF1) as an important liver-specific trans-acting element for the human ACAT2 gene using the human hepatocellular carcinoma cell lines HuH7 and HepG2. Targeted deletion of the HNF1 binding site in the DNA sequence abolished not only the basal promoter function in HepG2 and HuH7 cells but also the induction of the ACAT2 promoter by HNF1. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the transcription factors HNF1alpha and HNF1beta interact with this region in the human ACAT2 gene in vitro and in vivo. These data indicate that a) the identified HNF1 binding site serves as a positive regulator sequence, b) the binding site is functionally active both in vivo and in vitro, and c) the transcription factors HNF1alpha and HNF1beta, which bind to this site, play an important part in the regulation of the human ACAT2 promoter.

Mutations in transcription factors hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta cause maturity-onset diabetes of the young (MODY) types 3 and 5, respectively. HNF-1alpha and HNF-1beta mutations are well studied in some tissues, but the mechanism by which HNF-1alpha and HNF-1beta mutations affect sucrase-isomaltase (SI) transcription in the small intestine is unclear. We studied the effects of 13 HNF-1alpha mutants and 2 HNF-1beta mutants on human SI gene transcription, which were identified in subjects with MODY3 and MODY5, respectively. Transactivation activity of 11 HNF-1alpha and 2 HNF-1beta mutants was significantly lower than that of wild (wt)-HNF-1alpha and wt-HNF-1beta. Furthermore, in co-expression studies with mutant (mu)-HNF-1alpha/ wt-HNF-1beta and wt-HNF-1alpha/mu-HNF-1beta, the combination of mu-HNF-1alpha (P379fsdelCT and T539fsdelC)/wt-HNF-1beta impaired SI transcription, but the others were not remarkably different from wt-HNF-1alpha/wt-HNF-1beta. Although wt-HNF-1beta inhibited the transactivation activity of wt-HNF-1alpha on SI transcription, the inhibitory effect was reduced by 2 HNF-1beta mutants. These results suggest that SI transcription might tend to be unchanged or lower in MODY3, while occurring more in MODY5.

Targeted disruption of the onecut transcription factor, hnf-6, alters mammalian biliary system development. We have identified a related zebrafish cDNA expressed in the developing liver that is a functional ortholog of mammalian hnf-6. Antisense-mediated knockdown of zebrafish hnf-6 perturbs development of the intrahepatic biliary system. Knockdown of zebrafish hnf-6 alters expression of vhnf1 and the zebrafish orthologs of other mammalian genes regulated by hnf-6. Coinjection of mRNA encoding zebrafish vhnf1 rescues the biliary phenotype of hnf-6 morphants. These experiments strongly suggest that hnf-6 and vhnf1 function within an evolutionarily conserved pathway that regulates biliary development. Forced expression of either hnf-6 or vhnf1 also produces biliary phenotypes. Altered bile duct development in both loss- and gain-of-function experiments suggests that zebrafish biliary cells are sensitive to the dosage of hnf-6-mediated gene transcription.

Retinoic acid (RA) plays important roles in cellular differentiation and proliferation in various tissues including the liver. To explore a possible role of RA in the postnatal development of hepatic function, we analyzed RA-generation enzyme activity and the RA-related hepatic gene expressions in the suckling and weaning rats. At 5 days after birth, retinal dehydrogenase (RALDH) activity in the liver was relatively high. Its activity decreased by 70% until day 17, and then it gradually increased to a high level by the completion of weaning period. Northern blot analysis showed that RALDH2 mRNA levels decreased in the suckling period, whereas RALDH1 mRNA levels increased in the weaning period. Retinoid X receptor alpha (RXRalpha) mRNA levels increased in the suckling period and attained to a higher level at 17 days after birth. Retinoic acid receptor alpha (RARalpha) mRNA level showed only a slight and temporary increase on day 13. The mRNA levels of hepatocyte nuclear factors (HNF-4 and HNF-1alpha) exhibited parallel increases around suckling-weaning period, and the transcript levels of albumin, a typical target gene of the hepatocyte nuclear factors, increased during the suckling-weaning transition period. Electrophoretic mobility shift assay using a putative nuclear receptor-binding element on rat HNF-1 alpha gene revealed that HNF-4 homodimer, but not RXRalpha homodimer, bound to this element. These results suggest that postnatal expressions of hepatocyte-specific genes might be up-regulated by retinoid receptors, which may be related with the alterations of RALDH expression during postnatal development in the liver.

To achieve the liver-directed expression in sufficient amounts of therapeutic genes for successful and safe gene therapy, natural liver-specific promoters can be used to direct the expression of therapeutic genes in the liver, whereas strong viral enhancers were used to obtain sufficient amounts of expressed therapeutic gene products. However, very often use of either the former or the latter does not guarantee both potent and liver-specific therapeutic gene expression. Here we conglomerate them and thus create a potent tissue-specific promoter by characterizing and using the liver-type pyruvate kinase proximal promoter (LPKPP) harboring its TATA box and a HNF-1alpha binding site. Alone it hardly activated its reporter gene expression in non-hepatocytes or hepatocytes. However, in the presence of the SV40 viral enhancer (SV40VE), which is active in multiple cell types, it was able to potently activate its reporter gene expression specifically in hepatocytes. The tissue-specific activation of the LPKPP by the viral enhancer was attributed to HNF-1alpha binding to the LPKPP. Taken together, these results support the idea that the constitutively active SV40VE could be used to activate the LPKPP in a tissue-specific manner in the presence of HNF-1alpha. To our knowledge, this is the first study to utilize HNF-1alpha and its binding site, in the context of the LPKPP, to generate a basal promoter that is transcriptionally activated potently in a tissue-specific manner by a viral enhancer that is active in multiple cell types.

Of all of the epithelial ovarian cancers, clear cell carcinoma (CCC) of the ovary has the worst prognosis. We applied the oligonucleotide array technique to identify genes generally involved in CCC. Of the approximately 12,600 genes that were analyzed, 28 were expressed significantly differently between four CCC and seven non-CCC cell lines. Among 16 up-regulated genes in CCC, we further investigated a transcription factor, hepatocyte nuclear factor-1 beta (HNF-1 beta). We validated up-regulation of HNF-1 beta in CCC in terms of both mRNA and protein level using real-time quantitative reverse transcriptase-polymerase chain reaction and immunoblotting. Immunohistochemical analysis of 83 surgically resected ovarian cancers showed that almost all CCC specimens (21 of 22 cases) had nuclear staining for HNF-1 beta, whereas most non-CCC specimens (60 of 61 cases) showed no immunostaining or only focal and faint staining in the nucleus. Furthermore, we investigated the significance of HNF-1 beta expression in CCC using RNA interference. The reduction of HNF-1 beta expression by RNA interference induced apoptotic cell death in ovarian CCC cells, which was confirmed by terminal dUTP nick-end labeling and fluorescence-activated cell-sorting analyses. Our results suggest that HNF-1 beta is not only an excellent CCC-specific molecular marker but also a molecular target for therapy of ovarian CCC.

The human AE2 gene (SLC4A2) is transcribed in a widespread fashion from the upstream promoter, the resultant full-length transcript AE2a being encountered in most tissues. Moreover, alternate promoter sequences within intron 2 may drive tissue-restricted expression of variants AE2b(1) and AE2b(2), mainly in liver and kidney. AE2b(2) proximal promoter sequences are highly active in transfected liver-derived HepG2 cells and contain an HNF1 motif. Mutation-disruption of this motif dramatically decreased alternate promoter activity in HepG2 cells but not in prostate-derived PC-3 cells. Electromobility shift and supershift assays indicated that HNF1alpha from HepG2 nuclear extracts binds the HNF1 sequence. Transactivation studies in PC-3 cells showed enhanced activity of the wild-type construct upon cotransfection with an HNF1alpha expression plasmid, while activity of the HNF1-mutated construct remained unaffected. Since liver AE2 is putatively involved in the biliary secretion of bicarbonate, HNF1alpha may have a role in increasing bicarbonate secretion in response to certain stimuli.

Background/aims: The portal tracts contain bile ducts associated with branches of the portal vein and of the hepatic artery. Hepatic artery malformations are found in diseases in which fetal biliary structures persist after birth (ductal plate malformations). Here we investigated how hepatic artery malformations relate to abnormal bile duct development., Methods: Hepatic artery and biliary development was analyzed in fetuses with Jeune syndrome or Meckel syndrome, which show ductal plate malformations. We also analyzed hepatic artery development in transgenic mice which exhibit biliary anomalies following inactivation of the genes for hepatocyte nuclear factor (HNF)-6 or HNF-1beta, two transcription factors expressed in biliary cells, but not in arteries., Results: We show that arterial anomalies occurred in fetuses with Jeune syndrome or Meckel syndrome. We provide the first description of hepatic artery branch development in the mouse and show that inactivation of the Hnf6 or Hnf1beta gene results in anomalies of the hepatic artery branches. In the transgenic mice and in the human syndromes, the biliary anomalies preceded the arterial anomalies., Conclusions: A primary defect in biliary epithelial cells is associated with hepatic artery malformations in mice. Our data provide a model to interpret and study hepatic artery anomalies in humans.

Background: Type 2 diabetes shows evidence of underlying heterogeneity. No studies have assessed whether different causes for diabetes change the response to oral hypoglycaemic therapy. In a few cases, patients with diabetes caused by mutations in the hepatocyte nuclear factor 1alpha (HNF-1alpha) gene have been described as sensitive to the hypoglycaemic effects of sulphonylureas. We aimed to see whether the glycaemic response to the sulphonylurea gliclazide and the biguanide metformin differed in HNF-1alpha diabetes and type 2 diabetes, and to investigate the mechanism for differences in sulphonylurea sensitivity., Methods: We did a randomised crossover trial of glicazide and metformin in 36 patients, either with diabetes caused by HNF-1alpha mutations or type 2 diabetes, who were matched for body-mass index and fasting plasma glucose. The primary outcome was reduction in fasting plasma glucose. Analysis was by intention to treat. We assessed possible mechanisms for sulphonylurea sensitivity through insulin sensitivity, insulin secretory response to glucose and tolbutamide, and tolbutamide clearance., Findings: Patients with HNF-1alpha diabetes had a 5.2-fold greater response to gliclazide than to metformin (fasting plasma glucose reduction 4.7 vs 0.9 mmol/L, p=0.0007) and 3.9-fold greater response to gliclazide than those with type 2 diabetes (p=0.002). Patients with HNF-1alpha diabetes had a strong insulin secretory response to intravenous tolbutamide despite a small response to intravenous glucose, and were more insulin sensitive than those with type 2 diabetes. Sulphonylurea metabolism was similar in both patient groups., Interpretation: The cause of hyperglycaemia changes the response to hypoglycaemic drugs; HNF-1alpha diabetes has marked sulphonylurea sensitivity. This pharmacogenetic effect is consistent with models of HNF-1alpha deficiency, which show that the beta-cell defect is upstream of the sulphonylurea receptor. Definition of the genetic basis of hyperglycaemia has implications for patient management.

Maturity-onset diabetes of the young (MODY) is a monogenic subtype of Type 2 diabetes, defined as having an early age of onset, with a dominant inheritance pattern. Hepatocyte nuclear factor 1 (HNF1), which is encoded by the MODY3 gene, has been shown to bind the insulin promoter. Since the promoters of three pancreas-specific genes involved in glucose homeostasis-insulin, glucokinase, and amylin bind similar transcription factors, we were interested in whether HNF1 could also regulate amylin expression. In the present study, we used the electrophoretic mobility shift assay, to demonstrate that the HNF1 transcription factor can specifically bind to the amylin promoter. Moreover, co-transfection of an HNF1 expression vector with an amylin-CAT reporter plasmid decreased the activity of the amylin promoter by 85%. These data support the hypothesis that the amylin gene is regulated by HNF1 in a negative manner and may explain partially how HNF1 mutations result in diabetes.

Background: Mutations in hepatocyte nuclear factor-1beta (HNF-1beta) lead to a syndrome with diabetes and urogenital malformations [maturity onset of diabetes of the young, type 5 (MODY5)]. The aim of this study was to perform a clinicopathologic investigation of the renal disease in members of a Norwegian family with the HNF-1beta mutation R137-K161del., Methods: The study was based on long-term clinical observations of five mutation carriers, combined with renal biopsies from four of these. The biopsies were examined by light microscopy, immunohistochemistry, and transmission electron microscopy. The diameter of the glomerulus, proximal and distal tubules, in addition to thickness of the glomerular basement membrane (GBM), were measured in light microscopic slides and transmission electron micrographs. The results were compared with biopsies from adult patients with diabetic glomerulopathy, glomerulonephritis, and/or benign nephrosclerosis, and children with minimal-change glomerulopathy or glomerulonephritis, respectively., Results: Clinically, there was a wide intrafamilial variation from stable or slightly increasing serum creatinine to progressive renal failure and end-stage renal disease (ESRD). In all cases, the kidney disease was diagnosed prior to diabetes. Hypertrophy of the proximal and distal tubules as well as enlarged glomeruli were found in three of four mutation carriers. Essentially normal nephrons were found in the 10-year-old boy. The thickness of the GBM was considered near normal in all mutation carriers. Oligomeganephronia was found in one patient., Conclusion: Histopathologic and morphometric studies of kidney biopsies from four carriers of an HNF-1beta mutation revealed enlarged glomeruli and tubular structures. Long-term clinical follow-up demonstrated that the renal disease developed prior to and independently of diabetes. Finally, there is a wide phenotypic variation of the renal disease caused by HNF-1beta mutations.

Previous studies have shown that hST6Gal I mRNA is overexpressed in colorectal cancer tissues compared with non-malignant or benign tissue. Moreover, Form 1 (hepatic form) mRNA isoform had a marked tendency to accumulate in colon cancer [Int. J. Cancer 88 (2000) 58-65]. These findings suggest that the transcriptional regulation of Form 1 is altered during malignant transformation. We report here transcriptional regulation of the hST6Gal I gene in colon adenocarcinoma cell lines. We characterized P1 promoter region, which regulates Form 1 mRNA expression, using luciferase assays. The result indicates that the nt-156 to -1 region is important for transcriptional activity of hST6Gal I gene in colon adenocarcinoma cell lines. The nt-156 to -1 region contains HNF1 recognition element. Mutation of the HNF1 site reduced luciferase activity by about 80% compared with the wild-type construct, suggesting that HNF1 site is involved in the transcription of Form 1 mRNA in colon cancer cells.

Background: Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression., Objectives: The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance., Methods: We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs., Results: We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions., Conclusions: The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.

Type 2 diabetes (T2D) is a complex multifactorial disease. Genetic susceptibility is clearly needed, but in most cases, is not sufficient to induce T2D. Environmental factors are determinant in most patients to develop T2D. Monogenic forms of diabetes such as the maturity-onset diabetes of the young (MODY) represent a model to study the genetic basis of T2D. Studies in MODY individuals and families lead to a genetic definition of MODY subtypes with profound implications for our understanding of gene mutations involved in the development of T2D through our understanding on the major role of these genes and on their transcriptional activity on b-cell development and function; and on the regulation of glucose and insulin metabolism. Our current knowledge on the heterogeneity of MODY diabetes and contribution to our comprehension of the molecular mechanisms involved in T2D are described.

It is important to consider in clinical practice the diagnosis of MODY and to characterise the MODY sub-type. Indeed, evolution, clinical consequences and treatment of these forms of diabetes will be different according to the genetic origin of MODY. The main characteristics and consequences in clinical practice of these genetic abnormalities are briefly described, with a particular emphasis on the two most frequent sub-types, MODY-2 and MODY-3, caused by mutations of the enzyme glucokinase and of the transcription factor hepatocyte nuclear factor-1 alpha (HNF-1a) respectively.

Background: Familial juvenile hyperuricemic nephropathy (FJHN) is a dominantly inherited condition characterized by young-onset hyperuricemia, gout, and renal disease. The etiologic genes are unknown, although a locus on chromosome 16 has been identified in some kindreds. Mutations in the gene encoding hepatocyte nuclear factor (HNF)-1beta have been associated with dominant inheritance of a variety of disorders of renal development, particularly renal cystic disease and early onset diabetes; hyperuricemia has been reported in some kindreds., Methods: To assess a possible role for the HNF-1beta gene in some FJHN kindreds we sequenced the HNF-1beta gene in subjects from three unrelated FJHN families with atypical features of renal cysts or abnormalities of renal development. We also compared serum urate levels in subjects with HNF-1beta mutations with populations of controls, type 2 diabetic subjects, and subjects with mild chronic renal failure without HNF-1beta mutations., Results: A splice-site mutation in intron 2, designated IVS2+1G>T, showed complete co-segregation with FJHN in one family with diabetes. Serum urate levels were significantly higher in the HNF-1beta subjects compared with the normal control subjects (384 micromol/L vs. 264 micromol/L, P = 0.002) and the type 2 diabetic subjects (397 micromol/L vs. 271 micromol/L, P = 0.01). Comparison of serum urate levels in the HNF-1beta subjects with gender-matched subjects with renal impairment of other causes did not reach significance (402 micromol/L vs. 352 micromol/L, P = 0.2)., Conclusion: Hyperuricemia and young-onset gout are consistent features of the phenotype associated with HNF-1beta mutations, but the mechanism is uncertain. Families with HNF-1beta mutations may fit diagnostic criteria for FJHN. Identification of HNF-1beta patients by recognizing the features of diabetes and disorders of renal development is important in resolving the genetic heterogeneity in FJHN.

We have used antisense morpholino oligos to establish the developmental roles of three Xsox17 proteins in Xenopus development (Xsox17alpha(1), alpha(2) and beta). We show that their synthesis can be inhibited with modest amounts of oligo. The inhibition of each individually produces defects in late midgut development. Loss of activity of the Xsox17alpha proteins additionally inhibits hindgut formation, and inhibiting Xsox17alpha(1) disrupts foregut development with variable penetrance. When all Xsox17 activity is inhibited cell movements are halted during late gastrulation and the transcription of several endodermally expressed genes is reduced. Thus the Xsox17 proteins have redundant roles in early development of the endoderm and partly distinct roles during later organogenesis.

Human epidermal melanocytes hold the full capacity for autocrine de novo synthesis/regulation/recycling of the essential cofactor 6-tetrahydrobiopterin (6BH(4)) for conversion of L-phenylalanine via phenylalanine hydroxylase to L-tyrosine and for production of L-Dopa via tyrosine hydroxylase to initiate both pigmentation and catecholamine synthesis in these neural crest-derived cells. Earlier we have demonstrated pterin-4a-carbinolamine dehydratase (PCD) mRNA and enzyme activities in epidermal melanocytes and keratinocytes. This protein dimerises also the transcription factor hepatocyte nuclear factor 1 (HNF-1), leading to activation of multiple genes. This study demonstrates for the first time DCoH/HNF-1 alpha expression and transcriptional activity in human epidermal melanocytes in vitro and in situ and identified tyrosinase, the key enzyme for pigmentation, as a new transcriptional target. Specific binding of DCoH/HNF-1 complex to the human tyrosinase promoter was confirmed by gel shift analysis. These results provide a novel mechanism in the regulation of skin pigmentation.

Connexin32 (Cx32) encodes the predominant gap junction protein expressed by hepatocytes. We investigated the transcriptional control of Cx32 in expressing and nonexpressing rat liver cell lines and hypothesized that a putative hepatocyte nuclear factor-1 (HNF-1) binding site (centered at mp -187) in the liver-active, P1 promoter is essential for transcription of Cx32. HNF-1alpha was expressed by Cx32-expressing rat liver cell lines and bound the promoter at the -187 site, but was not expressed by non-Cx32-expressing hepatic lines. Stable transfection of non-Cx32-expressing WB-F344 rat liver epithelial cells with HNF-1alpha stimulated a transfected Cx32 promoter element (mp -244 to -33), binding of HNF-1alpha to the -187 site, and expression of endogenous Cx32. Site-directed mutagenesis of this HNF-1 binding site abolished HNF-1alpha binding and proximal promoter activity. Hepatic Cx32 expression was also significantly decreased in HNF-1alpha(-/-) mice. These data indicate that HNF-1alpha is a positive regulator of Cx32 expression in hepatic cells.

Hepatocyte nuclear factor 1 alpha (HNF1alpha) is a transcription factor that is expressed in liver, pancreas, kidney and intestine. Mice lacking HNF1alpha are born normally but suffer from several defects including hyperphenylalaninemia, defective bile acid and cholesterol metabolism, an insulin secretion defect and renal Fanconi syndrome. The renal phenotype involves a defect in renal proximal tubule reabsorption, leading to polyuria, glucosuria, aminoaciduria and phosphaturia. We investigated the expression of genes encoding members of the sodium/phosphate cotransporter (Na(+)/Pi) family (namely Npt1, Npt2, Npt4 and Ram1). We show that Npt1 and Npt4 genes were expressed at reduced levels in the kidneys of HNF1alpha -/- mice, whereas the expression of Npt2, the major renal phosphate transporter, was not affected. Analysis of the Npt1 genomic sequence revealed the existence of several alternative promoters activated in liver and/or in kidney. All of these were down-regulated in the kidneys of HNF1alpha -/- animals. Several HNF1alpha binding sites (BS) play an important role in the transcriptional control of this locus, including low-affinity HNF1 BSs localised in a DNase I hypersensitivity site (HSS3). Transient transfection experiments confirmed that HNF1alpha directly transactivates the Npt1 promoter and that the HSS3 region contributes to this activation.

We applied phage display technology to DNA-protein interaction studies. A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line. Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence. One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated. Specific binding of the selected protein domain was confirmed in a phage-independent context. By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified. The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.

Recently, we found a region A (a hepatocyte nuclear factor (HNF)-4-binding site from nucleotides -701 to -684) and a region B (an HNF-1-binding site from nucleotides -682 to -666) as cis-acting elements necessary for the transcriptional activation of the human dihydrodiol dehydrogenase (DD)4 gene in human hepatoblastoma HepG2 cells, which express DD4 mRNA. Thus, to investigate the mechanism(s) responsible for the cell-type-specific expression of DD4 mRNA, we constructed a reporter plasmid, pDD4 Foot A+B: -95/+28, in which regions A and B were linked to the human DD4 minimal promoter (-95 to +28) fused to the luciferase gene. The luciferase activity was detectable in HepG2 cells but not in human renal adenocarcinoma ACHN cells transfected with the pDD4 Foot A+B: -95/+28, which do not express DD4 mRNA. A supershift assay using antibodies to HNF-4 alpha, -4 gamma, -1 alpha, or valiant HNF (vHNF)-1 revealed that HNF-4 alpha, -4 gamma, and -1 alpha recognized regions A and B in HepG2 cells and that only vHNF-1 bound to regions A and B in ACHN cells. Semiquantitative reverse-transcribed polymerase chain reaction showed that a large amount of vHNF-1-C, which lacked transcriptional activation domain, was expressed in ACHN cells. Transfection of ACHN cells with an expression plasmid of vHNF-1-C did not activate the pDD4 Foot A+B: -95/+28 reporter gene. Taken together, we conclude that the cell-type-specific expression of DD4 mRNA is regulated by vHNF-1-C.

Background/aims: Hepatobiliary transporters are down-regulated in cholestasis, but their expression in acute, non-cholestatic, cytokine-mediated liver injury is unknown. Thus we studied the molecular mechanisms, by which sodium taurocholate cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, multidrug-resistance protein 2 (Mrp2) and bile salt export pump (Bsep) are regulated in liver injury induced by carbon tetrachloride (CCl(4))., Methods: mRNA and protein levels were determined in rats 24 and 72h after CCl(4) injection. Transporter gene transcription and binding activities of Ntcp and Mrp2 transactivators were assessed by nuclear runoff and electrophoretic mobility shift assays., Results: mRNA levels significantly declined to 41+/-44% for Ntcp, 65+/-41% for Oatp1 and 64+/-28% for Oatp2, but remained unchanged for Oatp4, canalicular Mrp2 and Bsep. Protein levels declined only for Oatp4 (-50+/-17%) and Ntcp (-23+/-13%) at 24h. Reduced mRNA levels (Ntcp, Oatp1, Oatp2) were associated with decreased transcriptional activities. Binding activity of Ntcp transactivators (hepatocyte nuclear factor 1 alpha (HNF1alpha) and CAAT enhancer binding protein alpha (C/EBPalpha) were reduced by 24h, whereas retinoid X receptor alpha (RXRalpha):retinoid acid receptor alpha (RARalpha) as transactivator of both Ntcp and Mrp2 remained unaltered. Recovery of acute hepatitis and changes in gene expression occurred after 72h., Conclusions: Acute liver injury results in down-regulation of basolateral organic anion transporters similar to liver regeneration after partial hepatectomy, but in contrast to endotoxin-induced cholestasis. Maintained binding activity of RXRalpha:RARalpha may explain differences in Mrp2 expression.

Background: Renal tract malformations are, on occasion, associated with uterine malformations. The transcription factor hepatocyte nuclear factor (HNF)-1beta is expressed from the earliest stages of development of the Wolffian duct, the mesonephros and metanephros, and the Müllerian ducts in the mouse. In adult mice HNF-1beta is expressed in the kidney tubules, collecting ducts, and in the oviducts and uterus in the female (Müllerian duct derivatives) and in the epididymis, vas deferens and seminal vesicles (Wolffian duct derivatives) in the male. HNF-1beta mutations have been reported in two families where affected members have renal abnormalities, female genital tract malformations and early-onset diabetes. Renal and uterine abnormalities have not been described in families without early-onset diabetes., Methods: We sequenced the HNF-1beta gene in nine subjects with renal abnormalities and a personal or family history of female genital tract malformations, but no history of diabetes., Results: Two families were identified with novel HNF-1beta mutations: a missense mutation in exon 2 with conversion of serine to proline at codon 151 (S151P) and a frameshift mutation in exon 3 with a 1 base pair deletion at codon 243 (Q243fsdelC). The S151P mutation proband has cystic kidneys and uterus didelphys. Her affected second son has renal cysts and hypospadias. The Q243fsdelC proband has a single functioning kidney and her two children have renal dysplasia. Histology in one child shows cystic dysplasia with a lack of glomeruli. The proband's sister is a mutation carrier and has a bicornuate uterus. Diabetes is not a feature in either family., Conclusions: This study confirms an association between HNF-1beta mutations and renal and Müllerian anomalies. The hypospadias may be coincidental. This study describes the first HNF-1beta mutations that are associated with a single functioning kidney and the absence of diabetes. This study further reinforces the variability of the renal and non-renal phenotypes associated with HNF-1beta mutations.

Hepatocyte nuclear factors (HNFs) are upstream regulators of many liver-specific genes and are involved in many cellular functions in the body, but their existence, expression, and function in gonads are still poorly understood. Here we report on the first cloning of partial cDNAs of HNF-1alpha and -1beta and full HNF-3beta cDNA from a tilapia (Oreochromis mossambicus) liver cDNA library. The deduced amino acid sequence of tilapia HNF-3beta has a 90 to 96% identity with those of other fishes (dwarf gourami, medaka, and zebrafish), 74% with mammals (human, rat, and mouse), and 82% with Xenopus. RT-PCR detected IGF-I and -II and HNF-1alpha, -1beta, and -3beta in both liver and gonads and the identity of the PCR fragments was confirmed by PCR hybridization. Immunoprecipitation and Western blotting also detected all three HNF proteins in both liver and gonads. Expression of HNFs in the gonads of the tilapia suggests that multi-HNFs may form a cascade to regulate gonadal physiology in the bony fish., (Copyright 2001 Academic Press.)

Expression of the rodent phenylalanine hydroxylase (PAH) gene is dependent upon hormones. Induction by glucocorticoids and cAMP occurs slowly and maximal stimulation is obtained by a synergistic effect of the two compounds. Hormone responsiveness is conferred by the tissue-specific HSIII enhancer and involves (i) protein kinase A mediating the cAMP response, even though a consensus sequence for binding of the cAMP response element binding protein is not present; (ii) other serine/threonine kinases as deduced from inhibitor studies; (iii) glucocorticoid receptor protein bound to glucocorticoid response element half sites; and (iv) binding of the liver-enriched transcription factor hepatocyte nuclear factor 1 (HNF1) to sites in the enhancer. Glucocorticoid receptor and HNF1, bound to their cognate sites, cooperatively increase the glucocorticoid response of the PAH gene, this response being synergistically enhanced by cAMP after long-term treatment., (Copyright 2001 Academic Press.)

Maturity-onset diabetes of the young is a heterogeneous group of autosomal dominantly inherited, young-onset beta-cell disorders. At least two consecutive generations are affected with a family member diagnosed before 25 years of age. Diabetes is caused either by mutations in the glucokinase gene (glucokinase MODY) or by mutations in transcription factors (transcription factor MODY). Glucokinase maturity-onset diabetes of the young is a mild, non-progressive hyperglycaemia caused by a resetting of the pancreatic glucose sensor. It is treated with diet, and complications are rare. Pregnancies affected by glucokinase mutations have specific management strategies and prognosis. Transcription factor maturity-onset diabetes of the young, caused by mutations in the hepatocyte nuclear factor genes HNF-1alpha, HNF-4alpha and HNF-1beta, and in insulin promoter factor-1 results in a progressive beta-cell defect with increasing treatment requirements and diabetic complications. Cystic renal disease is a prominent feature of HNF-1beta mutations. Further maturity-onset diabetes of the young genes remain to be identified. MODY is part of the differential diagnosis of diabetes presenting in the first to third decades of life. Diagnostic molecular genetic testing is available for the more common genes involved., (Copyright 2001 Harcourt Publishers Ltd.)

In hepatoma Huh-7 cells, inhibition of sphingosine kinase (SphK) activity by N,N-dimethylsphingosine (DMS) resulted in up-regulated production of liver-specific serum proteins including albumin and alpha-fetoprotein (AFP). The changes in these protein levels coincided well with those of two liver-enriched transcription factors, hepatocyte nuclear factor (HNF)-1 and -4, which regulate a number of liver-specific genes at the transcriptional level. Moreover, DMS induced the expression of retinoic acid receptor-alpha and retinoid X receptor-alpha. In DMS-treated cells, 9-cis retinoic acid (RA) further enhanced HNF-4alpha and albumin expression but it inhibited AFP accumulation. These results suggest that activation of SphK disengages cells from their liver-specific phenotype, and that 9-cis RA further induces differentiation of hepatoma cells when SphK activity is inhibited., (Copyright 2001 Academic Press.)

Maturity-onset diabetes mellitus of the young (MODY) is a human genetic syndrome most commonly due to mutations in hepatocyte nuclear factor-1alpha (HNF-1alpha). Here, we describe the crystal structure of the HNF-1alpha dimerization domain at 1.7 A resolution and assess its structural plasticity. The crystal's low solvent content (23%, v/v) leads to tight packing of peptides in the lattice. Two independent dimers, similar in structure, are formed in the unit cell by a 2-fold crystallographic symmetry axis. The dimers define a novel intertwined four-helix bundle (4HB). Each protomer contains two alpha-helices separated by a sharp non-canonical turn. Dimer-related alpha-helices form anti-parallel coiled-coils, including an N-terminal "mini-zipper" complementary in structure, symmetry and surface characteristics to transcriptional coactivator dimerization cofactor of HNF-1 (DCoH). A confluence of ten leucine side-chains (five per protomer) forms a hydrophobic core. Isotope-assisted NMR studies demonstrate that a similar intertwined dimer exists in solution. Comparison of structures obtained in multiple independent crystal forms indicates that the mini-zipper is a stable structural element, whereas the C-terminal alpha-helix can adopt a broad range of orientations. Segmental alignment of the mini-zipper (mean pairwise root-mean-square difference (rmsd) in C(alpha) coordinates of 0.29 A) is associated with a 2.1 A mean C(alpha) rmsd displacement of the C-terminal coiled-coil. The greatest C-terminal structural variation (4.1 A C(alpha) rmsd displacement) is observed in the DCoH-bound peptide. Diabetes-associated mutations perturb distinct structural features of the HNF-1alpha domain. One mutation (L12H) destabilizes the domain but preserves structural specificity. Adjoining H12 side-chains in a native-like dimer are predicted to alter the functional surface of the mini-zipper involved in DCoH recognition. The other mutation (G20R), by contrast, leads to a dimeric molten globule, as indicated by its 1H-NMR features and fluorescent binding of 1-anilino-8-naphthalene sulfonate. We propose that a glycine-specific turn configuration enables specific interactions between the mini-zipper and the C-terminal coiled-coil., (Copyright 2001 Academic Press.)

Variant Hepatocyte Nuclear Factor 1 (vHNF1/HNF1beta) is a homeodomain-containing transcription factor first expressed in the primitive endoderm and its derivatives, the visceral and parietal endoderm. It is subsequently expressed in epithelial cells of different organs, including the primitive gut and derivatives (liver, pancreas, lung), the kidney, and transiently, in the neural tube. We report here new data concerning vHnf1 expression in the mouse genital tract, using both RNA analyses and our vHnf1 heterozygous mutant mouse line, in which the first coding exon of the vHnf1 gene is replaced by the NLSLacZ reporter gene. Both beta-galactosidase activity and vHnf1 transcripts are detected in epididymus, vas deferens, seminal vesicle, prostate, uterus and oviduct. RNA analysis and in situ hybridization studies demonstrate that vHnf1 transcripts are restricted to the germinal cells of the testis. Unexpectedly, no beta-galactosidase activity is detected in the testis. We further show that, in addition to the somatic transcript, two more abundant vHnf1 transcript variants, which lack exons 1-4, appear in this organ after meiosis.

Maturity-onset diabetes of the young (MODY) is an autosomal dominant form of diabetes characterized by early onset of pancreatic dysfunction. MODY type 3 is caused by mutations in the hepatocyte nuclear factor (HNF)-1alpha. During a screening of Norwegian patients with suspected MODY we identified two novel HNF-1alpha mutations, P112L and Q466X. The molecular mechanisms underlying the disease were studied by analyzing the DNA binding properties, transcriptional activation, and subcellular localization of HNF-1alpha P112L and Q466X compared to wild type HNF-1alpha. P112L had reduced ability to bind an HNF1 consensus sequence and to activate transcription. Q466X did not differ from wild type HNF-1alpha in DNA binding activity. Transactivation, however, was markedly reduced. When both mutants were coexpressed with wild type HNF-1alpha in HeLa cells, transcriptional activity appeared unaffected, suggesting that a dominant-negative mechanism was not present. Immunolocalization experiments showed that P112L HNF-1alpha was correctly targeted to nuclei in HeLa cells. In contrast, some Q466X HNF-1alpha protein was retained in the cytoplasm, which indicated that the mechanism for nuclear localization was disturbed. Thus, the HNF-1alpha mutations P112L and Q466X both seem to impair pancreatic beta-cell function by loss-of-function mechanisms; P112L by reduced DNA binding and reduced ability to transactivate, and Q466X by reduced transactivation and incomplete nuclear targeting.

Hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF4alpha are liver-selective transcription factors and are essential for hepatocyte differentiation. This study demonstrates that HNF1alpha as well as HNF4alpha genes contain a direct repeat with a space of one nucleotide (DR1)-retinoic acid (RA) response element that can be bound and regulated by RA and retinoid x receptor alpha (RXRalpha) complex. Transient transfection experiments showed that RA increased the promoter activity of the HNF1alpha and HNF4alpha genes in Hep3B cells. Overexpression of RXRalpha further enhanced the activities of both genes. Two putative RXRalpha binding sites on the HNF1alpha (-295 to -276) and HNF4alpha (-418 to -399) genes have been characterized. By transient transfection, both sites positively responded to RA, and overexpression of RXRalpha in Hep3B cells increased the regulatory effect. Gel mobility shift assay demonstrated that these two DR-1 sites could be bound by RXRalpha specifically. These data suggest that the differentiation effect of RA on hepatocyte may be due to direct interaction of RXRalpha with the RA-responsive elements on the HNF1alpha and HNF4alpha genes., (Copyright 2000 Academic Press.)

Caco-2 cells undergo differentiation to an enterocytic-like cell when maintained in a post-confluent state for 1-2 weeks. During this period Caco-2 cells begin to express high levels brush border membrane associated enzymes such as dipeptidyl peptidase IV. Using the dipeptidyl peptidase IV gene promoter in electrophoretic mobility shift assays, we have shown for the first time that levels of hepatocyte nuclear factor 1alpha increase three- to fourfold during Caco-2 cell differentiation. Transient cotransfection experiments with 3T3 cells using dipeptidyl peptidase IV promoter constructs and expression vectors containing hepatocyte nuclear factor 1alpha and beta show that the ratio of alpha and beta modulates reporter gene expression. These results suggest that the increase in levels of hepatocyte nuclear factor 1alpha that occur during intestinal cell differentiation, are important for expression of dipeptidyl peptidase IV and other intestinal proteins., (Copyright 2000 Academic Press.)

Background: The transcription factor hepatocyte nuclear factor (HNF)-1 beta functions as a homodimer or as a heterodimer with the structurally related protein HNF-1 alpha. Both are expressed sequentially in rat kidney development, with HNF-1 beta being detected from the earliest inductory phases. HNF-1 beta gene mutations are associated with a unique disorder characterized by maturity-onset diabetes of the young (MODY) and early-onset and progressive nondiabetic renal dysfunction, which may lead to chronic renal failure., Methods: The HNF-1 beta gene was screened for mutations in six subjects with early-onset diabetes and a history of renal dysfunction in the subjects or their families., Results: A novel frameshift mutation in exon 4 of the HNF-1 beta gene and a deletion of CCTCT at codons 328 to 329 were detected in one subject. She was diagnosed as diabetic at the age of 21 in her second pregnancy. Glucose tolerance rapidly deteriorated over 18 months as a result of beta-cell dysfunction. The HNF-1 beta mutation arose de novo on a paternal chromosome and cosegregated with renal abnormalities in her family. The proband had bilateral small cysts in normal-sized kidneys and a reduced creatinine clearance of 66 mL/min (NR 80-120). Her first pregnancy was terminated at 17 weeks following an ultrasound diagnosis of bilateral, nonfunctioning cystic kidneys. Her first-born child had a small multicystic, dysplastic right kidney and a dysplastic left kidney with a reduced creatinine clearance (40 mL/min per 1.73 m2). Histologic examination of the large (5.8 vs. 1.4 g), polycystic fetal kidneys showed no normal nephrogenesis., Conclusions: These studies indicate that HNF-1 beta plays a central role in normal kidney development and pancreatic beta-cell function, and suggest that one mechanism by which HNF-1 beta gene mutations may cause renal dysfunction are by their effects on nephron development.

DCoH, the dimerization cofactor of the HNF-1 homeodomain proteins (hepatocyte nuclear factor-1alpha and beta), is involved in gene expression by associating with these transcription factors. The protein also called PCD for pterin-4alpha-carbinolamine dehydratase is a bifunctional factor as it catalyzes also the regeneration of tetrahydrobiopterin. This coenzyme is used by the enzyme phenylalanine hydroxylase, which generates tyrosine, the precursor of catecholamines and melanin. DCoH/PCD presumably cooperates with other partners, because it is expressed earlier than HNF1 and phenylalanine hydroxylase (PAH) in early vertebrate development. It is also found in cells lacking HNF1 and PAH like skin, brain and the pigmented epithelium of the eye suggesting a yet unknown function. We show that the overexpression of DCoH/PCD in Xenopus induces the formation of ectopic pigment cells in the epidermis, that are visible earlier than the endogenous pigmentation and broader distributed. This ectopic pigmentation is accompanied by an increase in tyrosinase activity and the amount of melanin. Overexpression of DCoH/PCD induces the appearance of pigment cells also in animal cap explants, that normally differentiate into atypical epidermis. DCoH/PCD mutants with impaired carbinolamine dehydratase activity retain the potential to induce pigmentation and we propose therefore that DCoH/PCD is not simply an essential enzyme for melanin biosynthesis, but also a regulator for the differentiation of pigment producing cells.

The gene encoding the tissue specific transcription factor HNF1alpha is expressed in vertebrates in tissues of endodermal origin such as the liver and the gut as well as in the kidney, a mesoderm derived organ. Using a 6 kb HNF1alpha promoter fragment linked to GFP we observed green fluorescence in transgenic embryos restricted to the liver and gut as well as to the pronephros, the embryonic kidney. By deletion and mutation analysis of the HNF1alpha promoter we succeeded in dissecting the HNF1alpha promoter into two entities that are either active in the endoderm or the mesoderm. In conclusion, our data establish that the generation of transgenic Xenopus allows the functional dissection of promoters in the context of the entire organism.

Mutations in the transcription factor hepatocyte nuclear factor-1alpha (HNF-1alpha) cause maturity-onset diabetes of the young type 3 (MODY3), a form of diabetes mellitus characterized by autosomal dominant inheritance, early onset, and pancreatic beta-cell dysfunction. We have examined the effects of five diabetes-associated mutations (L12H, G191D, R263C, P379fsdelCT, and L584S585fsinsTC) on HNF-1alpha function including DNA binding ability, intracellular localization, and transactivation activity. L12H, P379fsdelCT, and L584S585fsinsTC mutations were found in patients with a clinical diagnosis of MODY, while G191D and R263C mutations were identified in patients diagnosed with type 2 diabetes. These mutations had diverse effects on the functional properties of HNF-1alpha. Comparison of the functional data with clinical information suggested that transactivation activity of mutant HNF-1alpha in beta cells like MIN6 may be the primary determinants of the phenotypic differences observed among diabetic patients with HNF-1alpha mutations., (Copyright 1999 Academic Press.)

Formation of tubular structures from an epithelial tissue is a process common to many morphogenetic events during organogenesis. We report here new data concerning the expression pattern of the vHNF1/HNF1beta gene during this process in the mouse. vHNF1 (variant Hepatocyte Nuclear Factor 1) is a member of the HNF1 homeoprotein family. Its expression domain includes organs such as the liver, the kidney, the lung and the pancreas, but is restricted to the epithelial cells of these organs. To follow vHNF1 expression during organogenesis, we have introduced a NLS-lacZ gene under the control of vHNF1 regulatory regions by homologous recombination. Detection of the beta-galactosidase activity in heterozygous mice demonstrates that this gene is expressed in numerous tubular epitheliums as soon as they appear and all along development.

Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance, early-onset, and impaired insulin secretion. The type 3 and type 5 forms of MODY result from mutations in the genes encoding the transcription factor, hepatocyte nuclear factor (HNF)-1alpha and HNF-1beta, respectively. The mechanism by which mutations in one allele of the HNF-1 gene impair pancreatic beta cell function is unclear. We studied the effects of wild-type and four mutant (L12H, R263C, P379fsdelCT, and L584S585fsinsTC) HNF-1alpha, which were identified in Japanese subjects with MODY3 on human insulin gene transcription. Both wild-type (WT) HNF-1alpha and HNF-1beta bound to the oligonucleotide containing the A3 element sequence in the human insulin promoter and transactivated the insulin-luciferase reporter gene by 30- and 31-fold, respectively. In contrast, binding of L12H, R263C and L584S585fsinsTC-HNF-1alpha to the probe was impaired. Transactivation activity by the four mutant HNF-1alpha was reduced (4.3 to 43.3% of WT). These data suggest that the insulin gene is a candidate target gene of HNF-1alpha/HNF-1beta and the impairment of insulin gene transcription by mutations in the HNF-1 gene might be involved in the pathogenesis of MODY., (Copyright 1999 Academic Press.)

Cholesterol 7alpha-hydroxylase (CYP7A1), a liver-specific enzyme, catalyzes the rate-limiting step in the degradation pathway of cholesterol to bile acids, and thus plays a key role in cholesterol homeostasis. To elucidate the mechanisms that control hepatic expression of the human CYP7A1 gene, we are studying the promoter region. Initially, we observed that up to 40% of the overall transcriptional activity of the promoter in HepG2 cells was associated with DNA sequences from -65 to -1 of the human gene. Within this region, a binding site for the liver-enriched transcription factor HNF-1 (-56 to -49) has been identified. Binding of HNF-1 to this site leads to transcriptional activation of the human promoter. The corresponding segment from the rat CYP7A1 gene does not bind HNF-1; instead, it is bound by the orphan receptors ARP-1 (COUP-TFII) and LXRalpha, that are implicated in dietary regulation., (Copyright 1999 Academic Press.)

d-Glucose and non-metabolisable analogues of D-glucose regulate the expression of intestinal SGLT1 at both transcriptional and post-transcriptional levels. In order to investigate the molecular mechanisms involved in the transcriptional regulation of the ovine intestinal SGLT1 gene, we have isolated an upstream element of about 1 kb in size. This DNA fragment contains a TATA box motif, 48 bp upstream of the transcriptional start site and includes transcription factor binding sites for HNF-1 and AP-2. We have shown that the ovine SGLT1 promoter fragment can drive the transcription of a reporter gene when transfected into the epithelial cell lines STC-1 and LLC-PK1, which endogenously express SGLT1. Deletion analyses of the promoter indicate that -66/+21 bp proximal sequence directs the highest level of reporter gene activity. There are one and possibly two sites of transcriptional suppression., (Copyright 1999 Academic Press.)

The retinoblastoma (Rb) gene is a key factor involved in cell cycle regulation. In the present study, alterations in cell growth and expression of the alpha-fetoprotein and albumin genes by the stable transfection of the Rb gene were analyzed in HuH-7 human hepatoma cells. Cell growth was almost similar in the Rb gene-transfected and nontransfected cells at the sparse cell density. However, under a confluent condition, contact inhibition of cell growth was stimulated by the Rb gene transfection. By Northern blotting, the levels of the alpha-fetoprotein and albumin mRNA as well as the transcript of hepatocyte nuclear factor-1 (HNF-1), a liver-specific transcriptional factor regulating both genes, were apparently elevated in the Rb gene-transfected cells, compared with the nontransfected cells. These results suggest that the Rb gene transduction promotes contact growth inhibition in conjunction with the enhanced HNF-1-mediated liver-specific gene transcription in human hepatoma cells., (Copyright 1998 Academic Press.)