Gerhart U. Ryffel

HNF4alpha reduces proliferation of kidney cells and affects genes deregulated in renal cell carcinoma

Hepatocyte nuclear factor 4α (HNF4α) is a tissue-specific transcription factor known to regulate a large number of genes in hepatocytes and pancreatic β cells. Although HNF4α is highly expressed in some sections of the kidney, little is known about its role in this organ and about HNF4α-regulated genes in the kidney cells. The abundance and activity of HNF4α are frequently reduced in renal cell carcinoma (RCC) indicating some tumor suppressing function of HNF4α in renal cells. To determine the potential role of HNF4α in RCC, we used Flp recombinase-mediated gene integration to generate human embryonic kidney cells (HEK293) that conditionally express wild-type or mutated HNF4α. Expression of wild-type HNF4α but not of the mutants led to reduction of proliferation and alterations of cell morphology. These effects were reversible and induced at physiological concentrations of HNF4α. Using gene expression profiling by microarrays, we determined genes regulated by HNF4α. Interestingly, many of the genes regulated by HNF4α have been shown to be deregulated in RCC microarray studies. These genes (ACY1, WT1, SELENBP1, COBL, EFHD1, AGXT2L1, ALDH5A1, THEM2, ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) are good candidates for genes whose activity is changed upon the decrease of HNF4α in RCC.

Distinct Molecular and Morphogenetic Properties of Mutations in the Human HNF1β Gene That Lead to Defective Kidney Development

The homeobox transcription factor hepatocyte nuclear factor 1beta (HNF1beta) is a tissue-specific regulator that plays an essential role in early vertebrate development. In humans, heterozygous mutations in the HNF1beta gene are associated with young-onset diabetes as well as a variety of disorders of renal development with cysts as the most consistent feature. This report compares and classifies nine different HNF1beta mutations that lead in humans to distinct renal diseases, including solitary functioning kidney, renal dysplasia, glomerulocystic kidney disease, and oligomeganephronia. Analysis of these mutants in vitro identifies mutants that either retain or lack DNA binding. Investigation of the transactivation potential in transfected cell lines reveals a strict correlation between DNA binding and transactivation. Introduction of these mutants into developing Xenopus embryos shows that these mutants interfere with pronephros development, the first kidney form in amphibian. Whereas three mutants lead in Xenopus to a reduction or agenesis of the pronephric tubules and the anterior part of the duct, six mutants generate an enlargement of the pronephric structures. The differential morphogenetic potential in the developing embryo does not strictly correlate with the properties observed in vitro or in transfected cell lines. This suggests that the functional test in the developing embryo defines features of the HNF1beta protein that cannot be assessed in cell cultures. The distinct properties observed in the various HNF1beta mutants may guide the classification of the phenotypes observed in patients with a mutated HNF1beta gene.

HNF4α orchestrates a set of 14 genes to down-regulate cell proliferation in kidney cells

Abstract Few genes are known to be involved in renal cell carcinoma (RCC) development and progression. The cell-specific transcription factor hepatocyte nuclear factor 4α (HNF4α) is down-regulated in RCC and we have shown that HNF4α inhibits cell proliferation in the embryonic kidney cell line HEK293. To clarify the possible tumor suppressor activity of HNF4α we analyzed the whole human expression profile in HEK293 cells upon HNF4α induction. By comparing induced and uninduced cells, we identified 1411 differentially expressed genes. Using RNA interference, we screened 56 HNF4α-regulated genes for their possible role in mediating inhibition of cell proliferation triggered by HNF4α. We demonstrate that 14 of these regulated genes are able to contribute to the inhibitory effect of HNF4α on cell proliferation, including well-known cancer genes, such as CDKN1A (p21), TGFA, MME (NEP) and ADAMTS1. In addition, the genes SEPP1, THEM2, BPHL, DSC2, ANK3, ALDH6A1, EPHX2, NELL2, EFHD1 and PROS1 are also part of the network of HNF4α target genes that regulate proliferation in HEK293 cells. Therefore, we postulate that HNF4α orchestrates, at least, these 14 genes to regulate cell proliferation in HEK293 cells and that down-regulation of HNF4α could contribute to the progression of kidney cancer.

A Systematic Analysis of the 3′UTR of HNF4A mRNA Reveals an Interplay of Regulatory Elements Including miRNA Target Sites

Dysfunction of hepatocyte nuclear factor 4α (HNF4α) has been linked to maturity onset diabetes of the young (MODY1), diabetes type II and possibly to renal cell carcinoma (RCC). Whereas diabetes causing mutations are well known, there are no HNF4A mutations found in RCC. Since so far analyses have been constricted to the promoter and open reading frame of HNF4A, we performed a systematic analysis of the human HNF4A 3′UTR. We identified a short (1724 nt) and long (3180 nt) 3′UTR that are much longer than the open reading frame and conferred a repressive effect in luciferase reporter assays in HEK293 and INS-1 cells. By dissecting the 3′UTR into several pieces, we located two distinct elements of about 400 nt conferring a highly repressive effect. These negative elements A and B are counteracted by a balancer element of 39 nt located within the 5′ end of the HNF4A 3′UTR. Dicer knock-down experiments implied that the HNF4A 3′UTR is regulated by miRNAs. More detailed analysis showed that miR-34a and miR-21 both overexpressed in RCC cooperate in downregulation of the HNF4A mRNA. One of the identified miR-34a binding sites is destroyed by SNP rs11574744. The identification of several regulatory elements within the HNF4A 3′UTR justifies the analysis of the 3′UTR sequence to explore the dysfunction of HNF4α in diabetes and RCC.

Primary structure, chromosomal mapping, expression and transcriptional activity of murine hepatocyte nuclear factor 4γ.

We demonstrate the presence of a new member of the orphan nuclear receptor hepatocyte nuclear factor 4 (HNF4) subfamily in mouse which is genetically distinct from the previously characterized mouse HNF4alpha gene. The new member of the HNF4 subfamily shows highest amino acid identity, similar tissue distribution and syntenous chromosomal localization to the recently described human HNF4gamma (NR2A2), we therefore classify it as mouse HNF4gamma (mHNF4gamma). A combination of RT-PCR and immunohistochemical analysis showed expression of mHNF4gamma mRNA and protein in the endocrine pancreas, testes, kidney and gut. By co-transfection experiments, we show that mHNF4gamma is able to activate transcription, acting through binding sites that have been previously characterized as HNF4alpha binding sites. The presence of HNFgamma in human and mouse implies that a complex transcriptional network exists in higher vertebrates involving a number of HNF4 members with overlapping yet distinct function and tissue distribution.

A Kir6.2 Mutation Causing Neonatal Diabetes Impairs Electrical Activity and Insulin Secretion From INS-1 β-Cells

ATP-sensitive K+ channels (KATP channels) couple β-cell metabolism to electrical activity and thereby play an essential role in the control of insulin secretion. Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of this channel, cause neonatal diabetes. We investigated the effect of the most common neonatal diabetes mutation (R201H) on β-cell electrical activity and insulin secretion by stable transfection in the INS-1 cell line. Expression was regulated by placing the gene under the control of a tetracycline promoter. Transfection with wild-type Kir6.2 had no effect on the ATP sensitivity of the KATP channel, whole-cell KATP current magnitude, or insulin secretion. However, induction of Kir6.2-R201H expression strongly reduced KATP channel ATP sensitivity (the half-maximal inhibitory concentration increased from ∼20 μmol/l to ∼2 mmol/l), and the metabolic substrate methyl succinate failed to close KATP channels or stimulate electrical activity and insulin secretion. Thus, these results directly demonstrate that Kir6.2 mutations prevent electrical activity and insulin release from INS-1 cells by increasing the KATP current and hyperpolarizing the β-cell membrane. This is consistent with the ability of the R201H mutation to cause neonatal diabetes in patients. The relationship between KATP current and the membrane potential reveals that very small changes in current amplitude are sufficient to prevent hormone secretion.

Distinct promoter elements mediate endodermal and mesodermal expression of the HNF1α promoter in transgenic Xenopus

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.

Tissue-specific transcription factor HNF4alpha inhibits cell proliferation and induces apoptosis in the pancreatic INS-1 beta-cell line.

Abstract Hepatocyte nuclear factor 4α (HNF4α) is a tissue-specific transcription factor expressed in many cell types, including pancreatic β-cells. Mutations in the HNF4α gene in humans give rise to maturity-onset diabetes of the young (MODY1) characterized by defective insulin secretion by β-cells. To elucidate the mechanism underlying this disease, we introduced the splice form HNF4α2 or HNF4α8 into the rat β-cell line INS-1. Upon tetracycline-induced expression, both HNF4α isoforms caused distinct changes in cell morphology and a massive loss of cell numbers that was correlated with reduced proliferation and induced apoptosis. This differential activity was reflected in oligonucleotide microarray analysis that identified more genes affected by HNF4α2 compared to HNF4α8, and suggests that both isoforms regulate largely the same set of genes, with HNF4α2 being a stronger transactivator. We verified the induction of selected transcripts by real-time RT-PCR, including KAI1 and AIF, both known to have apoptotic potential. By establishing cell lines with inducible expression of these target genes, we deduce that both factors are insufficient to induce apoptosis. We propose that the anti-proliferative and apoptotic properties of HNF4α may be an essential feature impaired in MODY1 and possibly also in type 2 diabetes.

Transcription factor HNF1β and novel partners affect nephrogenesis

Heterozygous mutations of the tissue-specific transcription factor hepatocyte nuclear factor (HNF)1beta, cause maturity onset diabetes of the young (MODY5) and kidney anomalies including agenesis, hypoplasia, dysplasia and cysts. Because of these renal anomalies, HNF1beta is classified as a CAKUT (congenital anomalies of the kidney and urinary tract) gene. We searched for human fetal kidney proteins interacting with the N-terminal region of HNF1beta using a bacterial two-hybrid system and identified five novel proteins along with the known partner DCoH. The interactions were confirmed for four of these proteins by GST pull-down assays. Overexpression of two proteins, E4F1 and ZFP36L1, in Xenopus embryos interfered with pronephros formation. Further, in situ hybridization showed overlapping expression of HNF1beta, E4F1 and ZFP36L1 in the developing pronephros. HNF1beta is present largely in the nucleus where it colocalized with E4F1. However, ZFP36L1 was located predominantly in the cytoplasm. A nuclear function for ZFP36L1 was shown as it was able to reduce HNF1beta transactivation in a luciferase reporter system. Our studies show novel proteins may cooperate with HNF1beta in human metanephric development and propose that E4F1 and ZFP36L1 are CAKUT genes. We searched for mutations in the open reading frame of the ZFP36L1 gene in 58 patients with renal anomalies but found none.

The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps

BackgroundThe three distinct types of kidneys, pronephros, mesonephros and metanephros, develop consecutively in vertebrates. The earliest form of embryonic kidney, the pronephros, is derived from intermediate mesoderm and the first expressed genes localized in the pronephros anlage are the transcription factors osr1, osr2, hnf1b, lhx1 and pax8, here referred to as the early nephrogenic transcription factors. However, the pathway inducing nephrogenesis and the network of theses factors are poorly understood. Treatment of the undifferentiated animal pole explant (animal cap) of Xenopus with activin A and retinoic acid induces pronephros formation providing a powerful tool to analyze key molecular events in nephrogenesis.ResultsWe have investigated the expression kinetics of the early nephrogenic transcription factors in activin A and retinoic acid treated animal caps and their potential to induce pronephric differentiation. In treated animal caps, expression of osr1, osr2, hnf1b and lhx1 are induced early, whereas pax8 expression occurs later implying an indirect activation. Activin A alone is able to induce osr2 and lhx1 after three hours treatment in animal caps while retinoic acid fails to induce any of these nephrogenic transcription factors. The early expression of the five transcription factors and their interference with pronephros development when overexpressed in embryos suggest that these factors potentially induce nephrogenesis upon expression in animal caps. But no pronephros development is achieved by either overexpression of OSR1, by HNF1B injection with activin A treatment, or the combined application of LHX1 and PAX8, although they influenced the expression of several early nephrogenic transcription factors in some cases. In an additional approach we could show that HNF1B induces several genes important in nephrogenesis and regulates lhx1 expression by an HNF1 binding site in the lhx1 promoter.ConclusionsThe early nephrogenic transcription factors play an important role in nephrogenesis, but have no pronephros induction potential upon overexpression in animal caps. They activate transcriptional cascades that partially reflect the gene activation initiated by activin A and retinoic acid. Significantly, HNF1B activates the lhx1 promoter directly, thus extending the known activin A regulation of the lhx1 gene via an activin A responsive element.