HNF-3 Beta Research Articles

Expression and differential posttranscriptional regulation of the elongation factor 1 alpha 1 gene in endometrial caruncle and intercaruncle of Japanese Black cattle at early and mid-gestation stages.

The elongation factor 1 alpha 1 (EEF1A1), an isoform of EEF1A, is one of the most abundant cytoplasmic proteins and an important component of the translational machinery. We investigated the relative expression, alternative polyadenylation (APA), and changes in poly(A) tail length of EEF1A1 mRNA in the endometrial caruncle (CAR) and intercaruncle (ICAR) at early and mid-gestation in Japanese Black cattle. The relative EEF1A1 mRNA expression levels in the CAR were the highest on Gestation day 20 and were significantly decreased at mid-gestation. The expression levels in the ICAR were significantly higher than those in the CAR, and the gestation stage had no significant impact. Four different EEF1A1 transcripts with distinct 3' untranslated regions (UTRs) (proximal and distal types) and poly(A) tails (medium and short types) of different lengths were identified. The EEF1A1 mRNAs with distal 3' UTR and medium-length poly(A) tails were specific from the CAR of uterus horn at early gestation. RNA-sequencing data analyses revealed that the HSF1, MZF1, E47, SRF, GATA2, GATA3, GATA6, HNF-3 beta (FOXA2), CPSF1, and Ataxin-2 genes might affect the EEF1A1 gene expression or poly(A) length.

SUN-LB033 New Onset Diabetes in a Post-Renal Transplant Pediatric Patient with a Mutation of the Hepatocyte Nuclear Factor-1β Gene

Background: Maturity onset diabetes of the young (MODY) 5 is a rare type of dominantly inherited diabetes mellitus (DM). It is caused by mutations of the hepatocyte nuclear factor-1beta (HNF-1 beta) gene. Most are missense mutations that produce truncated proteins resulting in a variable clinical spectrum that may include kidney, genital and pancreatic abnormalities. We describe a case of a young patient diagnosed with DM after renal transplant; also, his diagnosis disclosed extensive family history of DM. Case: Six-year-old African-American patient with history of end stage renal disease due to bilateral cystic dysplasia presented with significant hyperglycemia three days after renal transplant. Polycystic kidney disease observed in prenatal ultrasound led to genetic testing. He was found to have a 1.0-1.8Mb deletion on chromosome microarray at 17q12 that included TCF2/HNF1B genes. His HbA1C was 5.8% at time of endocrine evaluation. He received one time high dose steroids in operating room and was started on tacrolimus after transplant. Pancreatic ultrasound showed normal pancreatic head, body not visualized. He was sent home with intensive insulin management on day 20 after renal transplant. Patient’s family history revealed mother with diagnosis of type 2 diabetes mellitus (T2DM) at age 19, maternal grandmother and great-grandmother diagnosed at early age with T2DM as well. Discussion: HNF-1 beta gene encodes a transcription factor that regulates nephron and embryonic pancreas development. MODY 5 is clinically heterogeneous disorder and the degree of insulin deficiency is variable, some family members who carry the gene remain free of diabetes into later adult life. The mean age of diagnosis of DM is 26 years with a range of 10–61 years. Our patient is only six years old and he may have presented earlier due post-transplant status, high dose steroids and tacrolimus use, all factors known to be triggers for hyperglycemia but the persistence of insulin requirement and levels of HbA1C being on pre-diabetes range at time of evaluation might be a sign of early deterioration of pancreatic function. Diagnosis of MODY5 has important implications for treatment, prognosis, and genetic counseling. Diabetes in these patients requires early use of insulin. Patient’s family members were advised to undergo genetic testing and screening for renal abnormalities and DM. Further studies on the correlation of phenotypes and genotypes will help delineate the clinical course of MODY 5. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Alpha-fetoprotein-producing ovarian clear cell adenocarcinoma with fetal gut differentiation: a rare case report and literature review

BackgroundAlpha-fetoprotein (AFP) is a useful tumor marker for ovarian germ cell tumors, particularly yolk sac tumor (YST). It is valuable for both diagnosis and further follow-up. Epithelial ovarian carcinoma (EOC) rarely secretes AFP, especially for clear cell type and in the postmenopausal women. Based on the limited knowledge about AFP-producing clear cell type EOC, a case and literature review on this topic is extensively reviewed.Case presentationWe report a 55-year-old postmenopausal woman experienced vaginal spotting for one month, and serum level of AFP was 60,721 ng/ml initially. Histological examination was clear cell type EOC. Tumor cells revealed strong immunoreactivity for glypican-3 (GPC3) and AFP and weak for hepatocyte nuclear factor-1 beta (HNF-1 beta), but negative for CD30, making the diagnosis of AFP-producing clear cell type EOC with fetal gut differentiation in focal areas, FIGO (International Federation of Gynecology and Obstetrics) IIIc. Although the patient underwent an intensive treatment, including optimal debulking surgery and multi-agent chemotherapy, the patient died of disease. To provide a better understanding of clinical and molecular characteristics of the AFP-producing clear cell type EOC, we conducted a systematic literature review.ConclusionsA total of three papers described the AFP-producing clear cell type EOC are available. The overall survival rate of these cases, including the current case is 50%. Although immunohistochemical examination is not always needed in routine for the diagnosis of clear cell type EOC, to distinguish from other tumors, especially germ cell tumors, or to provide the better way to monitor therapeutic response or to evaluate the disease status, immunostaining, including GPC3, HNF-1 beta, CD30, cytokeratin 7 or 20, and AFP is taken into account. Due to rarity, the appropriate chemotherapy regimen and the biological behavior of AFP-producing clear cell type EOC are still unclear.

Generation of Mouse Notochord Cell Lines for the Study of Notochord Function during Intervertebral Disk Development

Introduction Degeneration of intervertebral disks (IVDs) is the most common cause of back pain. Herniation of disk material into vertebral column and the reduction of disk thickness are the two prominent clinical features. Our understanding of the mechanisms regulating the processes of IVD development, maintenance, and degeneration is still limited. Therefore, there is no effective biological treatment for IVD degeneration. Nucleus pulposus (NP) is the core of IVD and it is derived from the notochord. However, the biological properties of the NP cell and its precursor, the notochord (NC cell), are not well understood. Here, we aim to establish an in vitro culturing system for maintenance of NP and NC cells. This can help to identify the characteristics (individual growth factors, secrete molecules and matrix components that constitute the unique extracellular environment of the notochord and the IVD) of both cell types and develop cell-based therapy for IVD degeneration. Materials and Methods We have generated a mouse model ( Foxa2mNE-Cre/Z/EG double transgenic mouse) to tag the notochord cells and its descendants by GFP, by which notochord lineages can be isolated at different embryonic and postnatal stages in the animal by fluorescence-activated cell sorting (FACS). The enhancer element responsible for Foxa2 expression in the node and notochord has previously been localized to a 520 bp fragment, the minimal notochord element (mNE) (Sasaki and Hogan, 1996). We have utilized this mNE of Foxa2to generate transgenic mice expressing the Crerecombinase ( Cre) gene in the node and notochord. The notochord descendants were labeled after crossing Foxa2mNE-Cre mice with the reporter strain, Z/EG (Novak et al., 2000 ). Enhanced green fluorescent protein (EGFP) signal was detected in the node and the notochord from E8.0 and continued at 1 year of age. EGFP tagged notochord cells were isolated from (a) E8.5 and (b) E9.5 Foxa2mNE-Cre/Z/EG embryos by FACS. The sorted cells were immortalized by SV40 large T antigen. Results The established cell lines proliferate and expand in adherent culture. They maintain the in vivo notochord characteristics including the following: (1) Expression of notochord specific transcription factors (c) Foxa2 and (d) Brachyury. (2) Secretion of extracellular matrix component, (e) type II collagen. (3) Sonic hedgehog, (f) Shh, a crucial signaling molecule secreted by the notochord for patterning surrounding tissues is also detected in the cell line. Conclusion We have established an initial protocol for the isolation and immortalization of notochord cells. We aim to use these cell lines initially to determine the gene and protein expression profiles, then by induction and ablation of signals to study the impact on NCC fate and their differentiation. This study will contribute to our understanding of notochord biology and their potential for intervertebral disk repair. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Novak, A., Guo,C., Yang,W., Nagy,A., and Lobe,C.G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 2000;28, 147–155 Sasaki, H. and Hogan,B.L. Enhancer analysis of the mouse HNF-3 beta gene: regulatory elements for node/notochord and floor plate are independent and consist of multiple sub-elements. Genes Cells 1996;1:59–72

Growth hormone therapy-related hyperglycaemia in a boy with renal cystic hypodysplasia and a new mutation of the HNF1 gene

We provide a molecular and pathophysiological characterization of an 11-year-old male patient, with a diagnosis of renal hypodysplasia, cysts and chronic renal failure. Although previously normoglycaemic and with a negative familial history for diabetes mellitus, he developed fasting hyperglycaemia within 12 months of the start of treatment with recombinant human growth hormone (rhGH). Direct sequencing of the HNF1 beta gene revealed a de novo heterozygous mutation in exon 2, c.535delC [Pro118LeuX7]+[=]. The appearance of fasting hyperglycaemia following rhGH treatment in children with renal cystic hypodysplasia suggests that investigation of the HNF1 beta gene is warranted, even when familial history is negative for diabetes. This is particularly important in regard to genetic counselling.

Diabète MODY-5 et malformations génitales : prise en charge diagnostique. À propos d’un cas

With an incidence of 5% in the female population, genital malformations are a frequent clinical occurrence presented by Oppelt et al. (2007) [1]. First, we have to diagnostic them and search for extra genital malformation when it is necessary. Think about maturity-onset-diabetes-of-the young 5 (MODY-5) when there's a personnel or family story of diabetes. This is often within the competence of the diabetologist and our speciality can be put in contribution in the research for the associated genital malformation. We report on the case of a young woman age of 19 with diabetes diagnosed during her adolescence, a nephropathy with polycystic kidneys, biological cytolysis, a bicornuate unicervical uterus while this may be a clinical diabetes MODY-5 confirmed by the discovery of the complete deletion of the gene coding for HNF-1 beta in the heterozygous state.

High Levels of Protein C Are Determinated by PROCR H3 in a Dutch Family.

Abstract 2139Poster Board II-116The natural anticoagulant protein C (PC) circulates in blood at a concentration of about 60 nM. Inter-individual variations in the levels of PC are in part genetically determined, but which loci in the genome are involved is only partially known. In a recent study we identified a locus on chromosome 20 which was associated with high PC levels in a large pedigree from the GENES study (LOD score >5 at 55 cMorgan). Candidate genes related to the PC pathway under the LOD-1 region encoded FOXA2 (previously known as HNF3 beta, a nuclear factor regulating protein C gene transcription), thrombomodulin (THBD,which is key to activation of PC), and the endothelial protein C receptor (PROCR). Here we present data that pinpoint a SNP in PROCRas being responsible for the observed segregation of high PC levels. The pedigree has 218 members and was ascertained through a proband with a family history of venous thrombosis (VT). Classical genetic risk factors for thrombosis (i.e. PC-, PS-, antithrombin deficiency, factor V Leiden and prothrombin G20201A) were not present. Complete medical data, plasma measurements and DNA was available for 161 family members. The mean age was 47±15 (range 15-87) years. The mean PC plasma level was 116±25% (range 72-212). Four family members had experienced VT and 2 had had recurrence. These symptomatic members had normal to high PC levels (66, 82, 114 and 178%).Haplotypes (and genotypes) for PROCR were determined in the family members by TaqMan assay using tag SNPs (single nucleotide polymorphisms) and PROCR H3 was associated with the levels of PC in the family. Furthermore, the promoter, exons, and 3`UTR of the 3 candidate genes were sequenced in 13 individuals, 9 with high and 4 with normal plasma PC levels. Critical SNPs that were encountered during sequencing were genotyped in all family members, namely FOXA2 rs1055080 (3`UTR) and rs2277764 (promoter region). As those 2 SNPs were inherited together in the set of 13 patients and also in the LETS (data not shown), our further analysis used only rs1055080. Plasma soluble endothelial protein C receptor (sEPCR) and soluble thrombomodulin (sTM) levels were measured with an ELISA assay. PC and sEPCR and levels were compared between PROCR H3 and FOXA2 rs1055080 carriers and non carriers by Student's t-test. sTM was analyzed by Mann-Whitney test. Association between PC levels and sEPCR/sTM levels were evaluated using linear regression analysis. Afterwards associations were adjusted for the PROCR H3 and FOXA2 rs1055080 SNP separately to detect their possible confounding effect. DNA sequencing only yielded previously reported SNPs in FOXA2, THBD and PROCR. Only the above mentioned SNPs were associated with PC plasma levels. Linkage analysis for PC levels using the original markers (from Marshifield) and adding the new PROCR and FOXA2 SNPs did not change the LOD score. When the analysis was adjusted for the mentioned markers, the LOD score dropped below 2. sEPCR has a bimodal distribution; mean ± SD was 103±27 ng/ml for the first mode and 262±70 ng/ml for the second mode. Median (range) sTM was 1.2 ng/ml (0.1-4). Linkage analysis for sEPCR levels yielded a high LOD score (above 6) that was accentuated to above 8 when PROCR H3 was included as a marker. For sTM, the LOD score was low with every combination of markers. PC, sEPCR and sTM levels were compared between PROCR H3 carriers and non-carriers and both PC levels and sEPCR levels were influenced by this PROCR haplotype, but not sTM. In conclusion, chromosome 20 harbors a locus which influences PC levels and also the levels of sEPCR, but not the levels of sTM. A detailed analysis with SNPs in PROCR, THBD and FOXA2suggests that the so-called PROCR H3 is directly responsible for the increased PC and sEPCR levels in this family. PROCR H3 is known to represent a g.A6936G substitution leading to a p.Ser219Gly replacement in the transmembrane domain of EPCR. The Gly219 isoform is more sensitive to sheddases (such as the ADAM17 metalloprotease) and is associated with generation of truncated mRNA lacking the transmembrane domain. However, the exact mechanism by which EPCR and sEPCR levels influence the level of PC remains to be determined Disclosures:No relevant conflicts of interest to declare.

Fgf10 gene expression is delayed in the embryonic lung mesenchyme in the adriamycin mouse model

The adriamycin mouse model is a well-established teratogenic model of esophageal atresia/tracheoesophageal fistula. Fibroblast growth factor 10 (Fgf10) plays a key role in branching of the lung buds during lung morphogenesis. Fgf10 knockout mice exhibit the absence of the lungs. Optical projection tomography (OPT) is a technique that allows three-dimensional (3D) imaging of gene expression in small tissue specimens in an anatomical context. The aim of this study was to investigate the temporo-spatial expression of Fgf10 during the critical period of separation of the trachea and esophagus in normal and adriamycin-treated embryos using OPT. Time-mated CBA/Ca mice received intraperitoneal injections of adriamycin (6 mg/kg) or saline on days 7 and 8 of gestation. Embryos were harvested on days 10-13, stained after whole mount in situ hybridization with labeled RNA probes to detect Fgf10 transcripts (n = 5 for each treatment/day of gestation). Immunolocalization with endoderm marker Hnf3 beta was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of gene expression domains. Computer reconstructed specimens allowed precise staging of developing embryos according to Theiler Staging (TS) criteria. OPT elegantly displayed Fgf10 gene expression in the pulmonary mesenchyme around the tip of the lung buds in both controls and treated embryos in the same spatial territory. Fgf10 gene expression was first detected in the control embryos at TS17. However, Fgf10 gene expression in adriamycin-treated embryos was first only observed at TS18 in 67% of the specimens. Delayed Fgf10 gene expression during the critical period of separation of the trachea and esophagus may affect lung bud formation in the adriamycin model leading to tracheoesophageal malformations.

Clinical spectrum associated with recurrent genomic rearrangements in chromosome 17q12

Deletions in chromosome 17q12 encompassing the HNF1 beta gene cause cystic renal disease and maturity onset diabetes of the young, and have been recently described as the first recurrent genomic deletion leading to diabetes. Earlier reports of patients with this microdeletion syndrome have suggested an absence of cognitive impairment, differentiating it from most other contiguous gene deletion syndromes. The reciprocal duplication of 17q12 is rare and has been hypothesized to be associated with an increased risk of epilepsy and mental retardation. We conducted a detailed clinical and molecular characterization of four patients with a deletion and five patients with a reciprocal duplication of this region. Our patients with deletion of 17q12 presented with cognitive impairment, cystic renal disease, seizures, and structural abnormalities of the brain. Patients with reciprocal duplications manifest with cognitive impairment and behavioral abnormalities, but not with seizures. Our findings expand the phenotypic spectrum associated with rearrangements of 17q12 and show that cognitive impairment is a part of the phenotype of individuals with deletions of 17q12.

L’hétérogénéité d’expression phénotypique de la mutation du gène TCF2 codant pour HNF-1β illustrée par une famille

TCF2 gene's mutation of autosomal dominant inheritance, encoding for the HNF-1 beta transcription factor, is associated with monogenic Mody5 diabetes, renal structural and urogenital abnormalities, and hepatic cholestasis. We have identified a family with HNF-1 beta gene's mutation, and very different phenotypic expression: renal abnormalities with cysts, nephrocalcinosis, polyuropolydipsic syndrome, Mody5 diabetes, genital malformations. Molecular analysis identified a mutation of exon 4 of the TCF2 gene. The coexistence in the same family of pleiomorphic renal malformations (cysts, renal agenesia or hypoplasia, renal failure) with Mody-type diabetes, with an autosomal inheritance must lead to the search for a mutation of TCF2, one of the most frequent genetic renal diseases.

Heat-shock inducible Cre strains to study organogenesis in transgenic Xenopus laevis

The frog Xenopus is a well established vertebrate model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a whole series of methods. We have expanded these approaches by establishing two transgenic Xenopus strains that allow specific interference with the activity of defined genes using a heat-shock inducible Cre recombinase that can induce upon heat-shock expression of a reporter gene in crossings to a corresponding reporter strain. We have applied this binary technique of gene interference in Xenopus development to overexpress the mutated HNF1 beta transcription factor at distinct developmental stages. Induction of HNF1 beta P328L329del by heat-shock at the gastrula stage resulted in a dramatic phenotype including malformation of the pronephros, gut, stomach, abnormal tail development and massive edemas indicative for kidney dysfunction. Thus, we have established the first binary inducible gene expression system in Xenopus laevis that can be used to study organogenesis.

Global analysis of in vivo Foxa2-binding sites in mouse adult liver using massively parallel sequencing

Foxa2 (HNF3β) is a one of three, closely related transcription factors that are critical to the development and function of the mouse liver. We have used chromatin immunoprecipitation and massively parallel Illumina 1G sequencing (ChIP–Seq) to create a genome-wide profile of in vivo Foxa2-binding sites in the adult liver. More than 65% of the ∼11.5 k genomic sites associated with Foxa2 binding, mapped to extended gene regions of annotated genes, while more than 30% of intragenic sites were located within first introns. 20.5% of all sites were further than 50 kb from any annotated gene, suggesting an association with novel gene regions. QPCR analysis demonstrated a strong positive correlation between peak height and fold enrichment for Foxa2-binding sites. We measured the relationship between Foxa2 and liver gene expression by overlapping Foxa2-binding sites with a SAGE transcriptome profile, and found that 43.5% of genes expressed in the liver were also associated with Foxa2 binding. We also identified potential Foxa2-interacting transcription factors whose motifs were enriched near Foxa2-binding sites. Our comprehensive results for in vivo Foxa2-binding sites in the mouse liver will contribute to resolving transcriptional regulatory networks that are important for adult liver function.

Pancreatic Development and Disease

Pancreatic Development and Disease

In vitro and Pathological Investigations of MODY5 with the R276X-HNF1.BETA. (TCF2) Mutation

Maturity-onset diabetes of the young type 5 (MODY5) is caused by mutation of hepatocyte nuclear factor 1beta (HNF1 beta) (TCF2) gene, resulting in a wide range of phenotypes including diabetes and renal abnormalities, but little is known about the pathogenesis of the clinical spectrum. We describe a 27-year-old Japanese male with the MODY phenotype including an atrophic kidney and multiple renal cysts. Genetic analysis revealed the patient to be heterozygous for a nonsense mutation in codon 276 of the HNF1beta gene (CGA or Arginine to TGA or stop codon; R276X). To clarify the pathophysiological relevance of this mutation, we conducted an in vitro study monitoring human C-peptide secretion after transfecting both the HNF1beta mutant cDNA and preproinsulin cDNA into a murine beta cell line, MIN6. Functional studies of the transformed MIN6 cells indicated that expression of the R276X caused a significant decrease in glucose-stimulated insulin secretion but no change in either KCl-stimulated or basal insulin secretion. These results suggest that the R276X functions in a negative manner in regard to metabolic responses of insulin secretion in beta cells. Analysis with light and electron microscopy on biopsied kidney specimens suggested that the origin of the cysts might be glomeruli but the primary lesion could be tubules.

Abnormal splicing of hepatocyte nuclear factor-1 beta in the renal cysts and diabetes syndrome.

Mutations in the hepatocyte nuclear factor-1 beta ( HNF-1 beta) gene result in disorders of renal development, typically involving renal cysts and early-onset diabetes (the RCAD syndrome/ MODY5). Sixteen mutations have been reported, including three splicing mutations of the intron 2 splice donor site. Because tissues showing abundant expression (kidney, liver, pancreas, gut, lung and gonads) are not easily accessible for analysis in living subjects, it has previously proven difficult to determine the effect of HNF-1 beta mutations at the mRNA level. This is the aim of the present study. We have developed a nested RT-PCR assay that exploits the presence of ectopic HNF-1 beta transcripts in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines derived from subjects carrying HNF-1 beta splice site mutations. We report a fourth mutation of the intron 2 splice donor site, IVS2nt+2insT. Sequence analysis of ectopic HNF-1 beta transcripts showed that both IVS2nt+2insT and IVS2nt+1G>T result in the deletion of exon 2 and are predicted to result in premature termination of the HNF-1 beta protein. Mutant transcripts were less abundant than the normal transcripts but there was no evidence of nonsense-mediated decay. This is the first study to define the pathogenic consequences of mutations within the HNF-1 beta gene by mRNA analysis. This type of approach is a useful and important tool to define mutational mechanisms and determine pathogenicity.

Expression Profiling in Ovarian Clear Cell Carcinoma: Identification of Hepatocyte Nuclear Factor-1β as a Molecular Marker and a Possible Molecular Target for Therapy of Ovarian Clear Cell Carcinoma

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.

Type-II pneumocyte differentiation in pulmonary sclerosing hemangioma: ultrastructural differentiation and immunohistochemical distribution of lineage-specific transcription factors (TTF-1, HNF-3 alpha, and HNF-3 beta) and surfactant proteins

Sclerosing hemangioma (PSH) is a rare pulmonary tumor, in which two types of tumor cells could be histologically discerned--surface and stromal tumor cells. Nine tumor-tissue specimens from six female Japanese patients were studied, focusing on the distribution of several transcription factors related to lung epithelial development and surfactant proteins and comparing the ultrastructural features of the tumor cells. The immunohistochemical analysis revealed that the surfactant proteins of surfactant apoprotein A, surfactant protein B, and prosurfactant protein C were distributed in many of the surface-lining cells and in a small number of stromal-tumor cells. In addition, the nuclei of the tumor cells stained positive for thyroid transcription factor 1 (TTF)-1, hepatocyte nuclear factor (HNF)-3 alpha, and HNF-3 beta. In situ hybridization staining for TTF-1 showed similar positive signals. Ultrastructurally, two types of tumor cells showed similar features, but stromal tumor cells lost the definitive apico-lateral differentiation compared with the surface tumor cells and showed restricted surface differentiation between the adjacent tumor cells, forming small lumina accompanied by microvilli and occasional multi-vesicle or multi-lamellar bodies. Conclusively, the real tumoral population being undifferentiated stromal cells, the lining cells are fully differentiated type-II pneumonocytes. PSH is a proliferation of rather fetal type-II pneumonocytes (pneumocytoma or pneumoblastoma?).

The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes.

Androgens and mesenchymal factors are essential extracellular signals for the development as well as the functional activity of the prostate epithelium. Little is known of the intraepithelial determinants that are involved in prostatic differentiation. Here we found that hepatocyte nuclear factor-3 alpha (HNF-3 alpha), an endoderm developmental factor, is essential for androgen receptor (AR)-mediated prostatic gene activation. Two HNF-3 cis-regulatory elements were identified in the rat probasin (PB) gene promoter, each immediately adjacent to an androgen response element. Remarkably, similar organization of HNF-3 and AR binding sites was observed in the prostate-specific antigen (PSA) gene core enhancer, suggesting a common functional mechanism. Mutations that disrupt these HNF-3 motifs significantly abolished the maximal androgen induction of PB and PSA activities. Overexpressing a mutant HNF-3 alpha deleted in the C-terminal region inhibited the androgen-induced promoter activity in LNCaP cells where endogenous HNF-3 alpha is expressed. Chromatin immunoprecipitation revealed in vivo that the occupancy of HNF-3 alpha on PSA enhancer can occur in an androgen-depleted condition, and before the recruitment of ligand-bound AR. A physical interaction of HNF-3 alpha and AR was detected through immunoprecipitation and confirmed by glutathione-S-transferase pull-down. This interaction is directly mediated through the DNA-binding domain/hinge region of AR and the forkhead domain of HNF-3 alpha. In addition, strong HNF-3 alpha expression, but not HNF-3 beta or HNF-3 gamma, is detected in both human and mouse prostatic epithelial cells where markers (PSA and PB) of differentiation are expressed. Taken together, these data support a model in which regulatory cues from the cell lineage and the extracellular environment coordinately establish the prostatic differentiated response.

Nonsense and missense mutations in the human hepatocyte nuclear factor-1 beta gene (TCF2) and their relation to type 2 diabetes in Japanese.

Mutations in transcription factors expressed in the pancreatic beta-cell are a major cause of maturity-onset diabetes of the young (MODY). They have also been found in patients diagnosed with type 1 and type 2 diabetes mellitus, which may highlight the difficulty in diagnosing these forms of diabetes or perhaps indicate a direct role in the development of multiple forms of diabetes. We have screened the hepatocyte nuclear factor-1 beta (HNF-1 beta/MODY5) gene for mutations in a group of 126 unrelated Japanese patients with type 2 diabetes and a family history of at least one first degree relative with diabetes. We identified one patient with a nonsense mutation (R276X) and another with a missense mutation (S465R). These mutations were present in the heterozygous state and were not found in 132 nondiabetic subjects (264 normal alleles). We identified a second patient with the S465R mutation on screening a second group of 272 randomly selected type 2 diabetic patients but not in another 122 nondiabetic subjects. Functional studies indicated that R276X-HNF-1 beta was inactive and S465R-HNF-1 beta exhibited a 22% reduction in activity compared with the wild-type protein. The S465R mutation may function in a dominant-negative manner. The subject with the R276X mutation had MODY5 misdiagnosed as common type 2 diabetes. He was diagnosed with diabetes at 13 yr of age and also had small kidneys with multiple bilateral renal cysts and decreased urinary concentrating ability. The two subjects with the S465R mutation had typical late-onset type 2 diabetes and no evidence of kidney disease. We have identified two novel mutations in human HNF-1 beta gene. The prevalence of MODY5 among our population of Japanese diabetes patients with a strong positive family of disease is 0.8%. The S465R mutation was found in 0.5% of our patients with common type 2 diabetes and thus may be a rare genetic risk factor contributing to the development of type 2 diabetes rather than MODY5.

Mutations and intronic variants in the HNF-1beta gene in a group of German and Czech Caucasians with type 2 diabetes mellitus and progressive diabetic nephropathy

Mutations in the hepatocyte nuclear factor - 1 beta (HNF-1 beta) gene cause maturity onset diabetes of the young type 5 (MODY 5). A clinical feature of the resulting phenotype besides impaired glucose tolerance is a variety of renal abnormalities, ranging from renal cysts to end-stage renal failure. Using a candidate gene approach we investigated the prevalence of mutations in the HNF-1 beta gene in a group of 63 patients from two different European populations (33 Germans, 30 Czechs) with type 2 diabetes mellitus and diabetic nephropathy diagnosed by increased albuminuria (39 patients) or end-stage renal failure (24 patients). No mutations were found in any of the 9 exons or in a minimal promoter region. Three intronic variants (single nucleotide polymorphisms - SNPs) were detected. The frequencies of these variants showed no difference between the two studied populations and were comparable to data reported from healthy subjects. No association between SNPs or formed haplotypes and any clinical parameters (like age of disease onset, BMI and severity of renal failure) was found. The results confirm that the genetic variations in the HNF-1 beta gene would be a very uncommon cause of progressive nephropathy in patients with type 2 diabetes mellitus.