Control of gene expression by the retinoic acid-related orphan receptor alpha in HepG2 human hepatoma cells.

Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin.

Characterization of the transcriptional signature of C/EBPbeta isoforms (LAP/LIP) in Hep3B cells: implication of LIP in pro-survival functions.

BACKGROUND & AIMS: C/EBPbeta is an important mediator of several cellular processes, such as differentiation, proliferation, and survival of hepatic cells. However, a complete catalog of the targets of C/EBPbeta or the mechanism by which this transcription factor regulates certain liver-dependent pathways has not been clearly determined. Two major natural isoforms of this transcription factor exist: the liver-enriched activating protein (LAP) and the liver-enriched inhibitory protein (LIP), a functional LAP antagonist. In this study, we used the opposing transcriptional effects driven by LAP and LIP to determine the genuine C/EBPbeta molecular signature in the Hep3B human hepatoma cell line. We subsequently investigated the role of each of the LAP and LIP isoforms in drug-induced Hep3B cell death. METHODS: We engineered Hep3B cells with regulated LAP or LIP expression using the Tet-off expression system. The genes that showed inverse regulation by LAP and LIP were identified by cDNA array analysis. The cohort of direct-C/EBPbeta-targets was distinguished from indirect-targets by ChIP-on-chip analysis. RESULTS: We characterized 676 genes by this approach. Among these genes, 39 are novel direct targets of C/EBPbeta. Eleven of these new direct targets are involved in cell survival, suggesting critical roles for LAP/LIP isoforms in this cellular process. Therefore, we examined the effects of LAP and LIP over-expression on cell survival. We show that LIP promotes survival in staurosporine- or taxol-induced Hep3B cell death. CONCLUSIONS: Our study provides new molecular and cellular insights into the role of C/EBPbeta in cells of hepatic origin.

Induction of early Purkinje cell dendritic differentiation by thyroid hormone requires RORα.

BACKGROUND: The active form (T3) of thyroid hormone (TH) controls critical aspects of cerebellar development, such as migration of postmitotic neurons and terminal dendritic differentiation of Purkinje cells. The effects of T3 on early dendritic differentiation are poorly understood. RESULTS: In this study, we have analyzed the influence of T3 on the progression of the early steps of Purkinje cell dendritic differentiation in postnatal day 0 organotypic cerebellar cultures. These steps include, successively, regression of immature neuritic processes, a stellate cell stage, and the extension of several long and mature perisomatic protrusions before the growth of the ultimate dendritic tree. We also studied the involvement of RORalpha, a nuclear receptor controlling early Purkinje cell dendritic differentiation. We show that T3 treatment leads to an accelerated progression of the early steps of dendritic differentiation in culture, together with an increased expression of RORalpha (mRNA and protein) in both Purkinje cells and interneurons. Finally, we show that T3 failed to promote early dendritic differentiation in staggerer RORalpha-deficient Purkinje cells. CONCLUSIONS: Our results demonstrate that T3 action on the early Purkinje cell dendritic differentiation process is mediated by RORalpha.

Re-evaluation of the role of STOX1 transcription factor in placental development and preeclampsia.

Preeclampsia is a common disease of pregnancy, characterized by high blood pressure and proteinuria appearing from the second trimester of gestation. Preeclampsia has been shown to have a strong genetic component. In 2005 a positional cloning project led to the discovery of the STOX1 transcription factor, and mutations of this gene were proposed as causal for preeclampsia in Dutch families. Despite the publication of three contradictory studies, we have shown by analyzing the functional effects of STOX1 that its overexpression in choriocarcinoma cells recapitulates several transcriptomic aspects of preeclampsia. In this review, the current literature is analyzed to evaluate the possible involvement of STOX1 in the pathogenesis of this disease. While preeclampsia obviously cannot be considered as a disease caused by mutation in a single gene, we argue that STOX1 may be at the center of common pathways leading to preeclampsia.

Inhibition of adipocyte differentiation by RORalpha.

Here we show that gene expression of the nuclear receptor RORalpha is induced during adipogenesis, with RORalpha4 being the most abundantly expressed isoform in human and murine adipose tissue. Over-expression of RORalpha4 in 3T3-L1 cells impairs adipogenesis as shown by the decreased expression of adipogenic markers and lipid accumulation, accompanied by decreased free fatty acid and glucose uptake. By contrast, mouse embryonic fibroblasts from staggerer mice, which carry a mutation in the RORalpha gene, differentiate more efficiently into mature adipocytes compared to wild-type cells, a phenotype which is reversed by ectopic RORalpha4 restoration.

Hypoxia and estrogen co-operate to regulate gene expression in T-47D human breast cancer cells.

Experimental and clinical studies have shown that both estrogen (E2) and hypoxia (H) are involved in tumor development and progression. A study was undertaken to determine whether these factors could interact to modulate gene expression using a microarray approach. We screened the transcript levels of over 8000 genes in the estrogen receptor (ERalpha) positive T-47D human breast cancer cell lines maintained at 21% O2 or 1% O2 with or without E2 co-treatment. Treatment by E2 or hypoxia alone altered the expression of 26 and 9 genes, respectively, whilst the expression of 31 genes was modulated by the H-E2 combination. The majority (21/31 genes) underwent a down-regulation. Microarray data was validated for 19 by quantitative real-time PCR and a good correlation noted (r2=0.8). Five out of these 19 genes were assayed for protein expression by Western blot. A correlation was also found between mRNA and protein levels. Statistical analysis showed that the gene expression modulation by the combined H and E2 treatment was additive in most cases, but for RasGRP2 and transferrin (TF) an antagonistic interaction was noted. The results demonstrate that hypoxic conditions and estrogen exposure interact to modulate the expression of a limited number of genes involved in cell growth and differentiation, angiogenesis, protein transport, metabolism and apoptosis.

Hypoxia-activated genes from early placenta are elevated in preeclampsia, but not in Intra-Uterine Growth Retardation.

BACKGROUND: As a first step to explore the possible relationships existing between the effects of low oxygen pressure in the first trimester placenta and placental pathologies developing from mid-gestation, two subtracted libraries totaling 2304 cDNA clones were constructed. For achieving this, two reciprocal suppressive/subtractive hybridization procedures (SSH) were applied to early (11 weeks) human placental villi after incubation either in normoxic or in hypoxic conditions. The clones from both libraries (1440 hypoxia-specific and 864 normoxia-specific) were spotted on nylon macroarrays. Complex cDNAs probes prepared from placental villi (either from early pregnancy, after hypoxic or normoxic culture conditions, or near term for controls or pathological placentas) were hybridized to the membranes. RESULTS: Three hundred and fifty nine clones presenting a hybridization signal above the background were sequenced and shown to correspond to 276 different genes. Nine of these genes are mitochondrial, while 267 are nuclear. Specific expression profiles characteristic of preeclampsia (PE) could be identified, as well as profiles specific of Intra-Uterine Growth Retardation (IUGR). Focusing on the chromosomal distribution of the fraction of genes that responded in at least one hybridization experiment, we could observe a highly significant chromosomal clustering of 54 genes into 8 chromosomal regions, four of which containing imprinted genes. Comparative mapping data indicate that these imprinted clusters are maintained in synteny in mice, and apparently in cattle and pigs, suggesting that the maintenance of such syntenies is requested for achieving a normal placental physiology in eutherian mammals. CONCLUSION: We could demonstrate that genes induced in PE were also genes highly expressed under hypoxic conditions (P = 5 x 10(-5)), which was not the case for isolated IUGR. Highly expressed placental genes may be in syntenies conserved interspecifically, suggesting that the maintenance of such clusters is requested for achieving a normal placental physiology in eutherian mammals.

The gene encoding fibrinogen-beta is a target for retinoic acid receptor-related orphan receptor alpha.

Fibrinogen is a plasma protein synthesized by the liver. It is composed of three chains (alpha, beta, gamma). In addition to its main function as a coagulation factor, this acute phase protein is also a risk marker for atherosclerosis. Retinoic acid receptor-related orphan receptor (ROR)alpha is a nuclear receptor modulating physiopathological processes such as cerebellar ataxia, inflammation, atherosclerosis, and angiogenesis. In this study, we identified RORalpha as a regulator of fibrinogen-beta gene expression in human hepatoma cells and in mouse liver. A putative RORalpha response element (RORE) was identified in the human fibrinogen-beta promoter. EMSA showed that RORalpha binds specifically to this RORE, and cotransfection experiments in HepG2 hepatoma cells indicated that this RORE confers RORalpha-dependent transcriptional activation to both the human fibrinogen-beta and the thymidine kinase promoters. Stable transfection experiments in HepG2 and Hep3B hepatoma cells demonstrated that overexpression of RORalpha specifically increases endogenous fibrinogen-beta mRNA levels. Chromatin immunoprecipitation experiments revealed that the fibrinogen-beta RORE is occupied by RORalpha in HepG2 cells. Thus, the human fibrinogen-beta gene is a direct target for RORalpha. Furthermore, fibrinogen-beta mRNA levels in liver and plasma fibrinogen concentrations are specifically decreased in staggerer mice, which are homozygous for a deletion invalidating the Rora gene. Taken together, these data add further evidence for an important role of RORalpha in the control of liver gene expression with potential pathophysiological consequences on coagulation and cardiovascular risk.

Profiling of oxygen-modulated gene expression in early human placenta by systematic sequencing of suppressive subtractive hybridization products.

Villi from first-trimester human placenta were exposed to oxygen concentrations of either 2 or 20% during 3 h to construct two reciprocally subtracted libraries using the suppressive subtractive hybridization (SSH) methodology. After cloning, sequencing, and gene identification, the genes (1,071 clones corresponding to 822 different sequences) were classified according to 1) the subtracted library from which they originated and 2) within 58 groups of gene functions. We then developed a logarithm of the odds (LOD) test to identify a possible excess of genes in each group. We show that genes involved in angiogenesis are significantly overrepresented in the "hypoxic" condition (2% O2), whereas apoptotic genes are overrepresented in the "normoxic" condition (20% O2). Furthermore, we observed an excess of kinases relative to phosphatases and an excess of genes involved in proliferation over genes involved in cell growth in the hypoxic condition. To validate our results, we used quantitative RT-PCR to analyze the set of eight genes involved in angiogenesis on six independent placentas. Finally, we studied the distribution of gene clusters on human chromosomes to check whether their chromosomal distribution was random or not. We observed on human chromosome 11 a clear clustering of genes regulated similarly by O2 tension, and we also discovered indications that such clustering exists on chromosomes 6 and 12.

The gene encoding human retinoic acid-receptor-related orphan receptor alpha is a target for hypoxia-inducible factor 1.

Retinoic acid-receptor-related orphan receptor (ROR) alpha is a nuclear receptor involved in many pathophysiological processes such as cerebellar ataxia, inflammation, atherosclerosis and angiogenesis. In the present study we first demonstrate that hypoxia increases the amount of Rora transcripts in a wide panel of cell lines derived from diverse tissues. In addition, we identified a functional promoter sequence upstream of the first exon of the human Rora gene, spanning -487 and -45 from the translation initiation site of RORalpha1. When cloned in a luciferase reporter vector, this sequence allowed the efficient transcription of the luciferase gene in several cell lines. Interestingly, the activity of the Rora promoter was enhanced by hypoxia in HepG2 human hepatoma cells, and this effect was dependent on an HRE (hypoxia response element) spanning from -229 to -225. Using electrophoretic-mobility-shift assays, we showed that HIF-1 (hypoxia-inducible factor 1), which plays a key role in the transcriptional response to hypoxia, bound to this HRE. Overexpression of HIF-1alpha increased the activity of the Rora promoter through the HRE. Overexpression of a dominant-negative form of HIF-1alpha producing transcriptionally inactive HIF-1alpha/HIF-1beta dimers abolished hypoxic activation of the Rora promoter. This indicated that HIF-1 is involved in the response of RORalpha to hypoxia. Taken together, our data reveal Rora as a new HIF-1 target gene. This illustrates, at the molecular level, the existence of cross-talk between signalling pathways mediated by HIF-1 and those mediated by nuclear receptors.

Specific protein-1 is a universal regulator of UDP-glucose dehydrogenase expression: its positive involvement in transforming growth factor-beta signaling and inhibition in hypoxia.

UDP-glucose dehydrogenase (UGDH) is a key enzyme of the unique pathway for the synthesis of UDP-glucuronate, the substrate for the numerous glucuronosyl transferases, which act on the synthesis of glycosaminoglycans and glucuronidation reaction of xeno- and endobiotics. Using the bacterial artificial chromosome approach, we have cloned and characterized the human UGDH promoter. The core promoter of -644 nucleotides conferred reporter gene activity in transient transfection assay of a variety of cell types, including MRC5 fibroblasts and the HepG2 hepatoma cell line. The minimal promoter of -100 nucleotides contains a functional inverted TATA box. No consensus CAAT sequence was found up to -2133 nucleotides. The expression of UGDH was up- and down-regulated by transforming growth factor (TGF)-beta and hypoxia, respectively. TGF-beta enhanced the activity of all the deletion constructs, except the minimal promoter. Hypoxia slightly increased the activity of the short promoter-containing constructs but decreased that of the -374 nucleotides and core promoter constructs. The core promoter contained numerous GC-rich sequences for the binding of Sp1 transcription factor. Bisanthracycline, an anti-Sp1 compound, decreased UGDH mRNA expression and inhibited the core promoter constructs activity. Gel mobility shift and supershift assays after TGF-beta stimulation demonstrated an increased DNA binding of the nuclear extract proteins to the two Sp1 sequences located in the -374-bp promoter. By contrast, nuclear extract proteins from hypoxia-treated cells demonstrated a decreased binding of the consensus Sp1 sequence. These results indicate that numerous Sp1 cis-acting sequences of the UGDH core promoter are responsible for up- and down-regulation of the gene after TGF-beta stimulation and in hypoxic conditions, respectively.

Hepatocyte nuclear factor-6 stimulates transcription of the alpha-fetoprotein gene and synergizes with the retinoic-acid-receptor-related orphan receptor alpha-4.

The rat alpha-fetoprotein ( afp ) gene is controlled by three enhancers whose function depends on their interaction with liver-enriched transcription factors. The afp enhancer III, located at -6 kb, is composed of three regions that act in synergy. Two of these regions, called s1 and s2, contain a putative binding site for hepatocyte nuclear factor-6 (HNF-6). This factor is the prototype of the ONECUT family of cut-homoeodomain proteins and is a known regulator of liver gene expression in adults and during development. We show here that the two splicing isoforms of HNF-6 bind to a site in the s1 region and in the s2 region. The core sequence of the s1 site corresponds to none of the known HNF-6 binding sites. Nevertheless, the binding properties of the s1 site are identical with those of the s2 site and of previously characterized HNF-6 binding sequences. The HNF-6 consensus should therefore be rewritten as DRRTCVATND. Binding of HNF-6 to the s1 and s2 sites requires both the cut and the homoeo domains, is co-operative and induces DNA bending. HNF-6 strongly stimulates the activity of the afp enhancer III in transient transfection experiments. This effect requires the stereo-specific alignment of the two HNF-6 sites. Moreover, HNF-6 stimulates the enhancer in synergy with the retinoic-acid-receptor-related orphan receptor alpha (RORalpha), which binds to a neighbouring site in the s1 region. Thus expression of the afp gene requires functional interactions between HNF-6 molecules and between HNF-6 and RORalpha.

The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.

The present study was aimed at determining whether hepcidin, a recently identified peptide involved in iron metabolism, plays a role in conditions associated with both iron overload and iron deficiency. Hepcidin mRNA levels were assessed in two models of anemia, acute hemolysis provoked by phenylhydrazine and bleeding provoked by repeated phlebotomies. Hepcidin response to hypoxia was also studied, both ex vivo, in human hepatoma cells, and in vivo. Anemia and hypoxia were associated with a dramatic decrease in liver hepcidin gene expression, which may account for the increase in iron release from reticuloendothelial cells and increase in iron absorption frequently observed in these situations. A single injection of turpentine for 16 hours induced a sixfold increase in liver hepcidin mRNA levels and a twofold decrease in serum iron. The hyposideremic effect of turpentine was completely blunted in hepcidin-deficient mice, revealing hepcidin participation in anemia of inflammatory states. These modifications of hepcidin gene expression further suggest a key role for hepcidin in iron homeostasis under various pathophysiological conditions, which may support the pharmaceutical use of hepcidin agonists and antagonists in various iron homeostasis disorders.

Retinoic acid receptor-related orphan receptor (ROR) alpha4 is the predominant isoform of the nuclear receptor RORalpha in the liver and is up-regulated by hypoxia in HepG2 human hepatoma cells.

The retinoic acid receptor-related orphan receptor alpha (RORalpha) is critically involved in many physiological functions in several organs. We find that the main RORalpha isoform in the mouse liver is the RORalpha4 isoform, in terms of both mRNA and protein levels, while the RORalpha1 isoform is less abundant. Because hypoxia is a major feature of liver physiology and pathology, we examined the effect of this stress on Rora gene expression and RORalpha transcriptional activity. HepG2 human hepatoma cells were cultured for 24 h under normoxia (20% O2) or hypoxia (10, 2, and 0.1% O2) and the abundance of the Rora transcripts measured by Northern blot and semi-quantitative RT-PCR. Hypoxic HepG2 cells contained more Rora mRNA than controls. This was also observed in rat hepatocytes in primary culture. Cobalt chloride and desferrioxamine also increased the amount of Rora mRNA in HepG2 cells. It is likely that these treatments increase the amount of the RORalpha4 protein in HepG2 cells as evidenced by Western blotting in the case of desferrioxamine. Transient transfection experiments indicated that hypoxia, cobalt chloride, and desferrioxamine all stimulate RORalpha transcriptional activity in HepG2 cells. Hence, we believe that RORalpha participates in the control of gene transcription in hepatic cells and modulates gene expression in response to hypoxic stress.

Repression of alpha-fetoprotein gene expression under hypoxic conditions in human hepatoma cells: characterization of a negative hypoxia response element that mediates opposite effects of hypoxia inducible factor-1 and c-Myc.

Hypoxia is an important component of many pathological processes including cancerogenesis and cirrhosis. We have attempted to identify additional hepatic genes sensitive to hypoxia by postulating that genes with possible binding sites for hypoxia inducible factor-1 (HIF-1) are regulated by hypoxia. A computer analysis identified the oncodevelopmental alpha-fetoprotein gene (afp) as one of them. The amounts of both alpha-fetoprotein mRNA and protein were decreased under hypoxic conditions in HepG2 hepatoma cells. Stability of afp mRNA was not altered, and de novo synthesis of proteins was required. Transfection experiments in HepG2 cells showed that both hypoxia and overproduction of HIF-1alpha specifically repressed the transcriptional activity of the rat afp regulatory region through the sequence 5'-CACGTGGG-3' located at -3625 to -3619. Mutation in this sequence strongly impaired these repressions. Interestingly, this sequence was a functional stimulatory target for c-Myc, suggesting that c-Myc regulates afp gene expression. Lastly, the amounts of c-myc mRNA and protein were reduced when these cells were grown under hypoxic conditions. Taken together, these results suggest the existence of a possible competition between HIF-1 and c-Myc that could modulate the transcriptional activity of the afp gene in response to hypoxia.