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.

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.

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.

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.

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.

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.

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.

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.