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

Evolution of a New Function by Degenerative Mutation in Cephalochordate Steroid Receptors

Gene duplication is the predominant mechanism for the evolution of new genes. Major existing models of this process assume that duplicate genes are redundant; degenerative mutations in one copy can therefore accumulate close to neutrally, usually leading to loss from the genome. When gene products dimerize or interact with other molecules for their functions, however, degenerative mutations in one copy may produce repressor alleles that inhibit the function of the other and are therefore exposed to selection. Here, we describe the evolution of a duplicate repressor by simple degenerative mutations in the steroid hormone receptors (SRs), a biologically crucial vertebrate gene family. We isolated and characterized the SRs of the cephalochordate Branchiostoma floridae, which diverged from other chordates just after duplication of the ancestral SR. The B. floridae genome contains two SRs: BfER, an ortholog of the vertebrate estrogen receptors, and BfSR, an ortholog of the vertebrate receptors for androgens, progestins, and corticosteroids. BfSR is specifically activated by estrogens and recognizes estrogen response elements (EREs) in DNA; BfER does not activate transcription in response to steroid hormones but binds EREs, where it competitively represses BfSR. The two genes are partially coexpressed, particularly in ovary and testis, suggesting an ancient role in germ cell development. These results corroborate previous findings that the ancestral steroid receptor was estrogen-sensitive and indicate that, after duplication, BfSR retained the ancestral function, while BfER evolved the capacity to negatively regulate BfSR. Either of two historical mutations that occurred during BfER evolution is sufficient to generate a competitive repressor. Our findings suggest that after duplication of genes whose functions depend on specific molecular interactions, high-probability degenerative mutations can yield novel functions, which are then exposed to positive or negative selection; in either case, the probability of neofunctionalization relative to gene loss is increased compared to existing models

Modulation of the far upstream enhancer of the rat a-fetoprotein gene by members of the RORalpha, Rev-erbalpha and Rev-erbbeta groups of monomeric orphan nuclear receptors.

International audienceExpression of the oncodevelopmental α-fetoprotein (AFP) gene is tightly regulated and occurs in the yolk sac, fetal liver and intestine, and cancerous liver cells. Transcription of the AFP gene is under the control of three enhancers that are very tissue specific. We have shown that the most upstream of these enhancers, located at - 6 kb, works through the combined action of liver-enriched factors and nuclear receptors that bind to three regions of this DNA regulatory element. This study showed that orphan nuclear receptors of the RORα, Reverbα, and Rev-erbβ groups can bind as monomers with high affinity and specificity to an evolutionarily conserved AGGTCA motif in the functionally important region 1 of this AFP enhancer. Transient transfection experiments performed with human HepG2 hepatoma cells showed that overproduction of RORα4 stimulated the activity of the AFP enhancer in a dose-dependent manner, while that of Rev-erbα and Rev-erbβ had the opposite effect. These effects were highly specific and required the integrity of the AGGTCA motif. The action of these nuclear receptors also occurred in the context of the entire 7-kb regulatory region of the rat AFP gene. These results suggest that altering the amounts or activities of these orphan receptors in cells of hepatic or endodermal origin could modulate AFP gene expression in response to a variety of developmental or carcinogenic stimuli

Dietary protein metabolism and body-weight regulation: Dose-response effects

Body-weight management requires a multifactorial approach. Recent findings suggest that an elevated protein intake seems to play a key role herein, through (i) increased satiety related to increased diet-induced thermogenesis; (ii) its effect on thermogenesis; (iii) body composition; and (iv) decreased energy-efficiency, all of which are related to protein metabolism. Supported by these mechanisms, relatively larger weight loss and subsequent stronger body-weight maintenance have been observed. Increased insulin sensitivity may appear, but it is unclear whether this is due to weight loss or type of diet. The phenomenon of increased satiety is utilized in reduced energy-intake diets, mainly in the ad libitum condition, whereby sustained satiety is achieved with sustained absolute protein intake in grams, despite lower energy intake. Elevated thermogenesis and glucagon-like peptide-1 (GLP-1) appear to play a role in high-protein induced satiety. Under conditions of weight maintenance, a high-protein diet shows a reduced energy efficiency related to the body composition of the body weight regained, that is, in favor of fat-free mass. Indeed, during body-weight loss, as well as during weight regain, a high-protein diet preserves or increases fat-free mass and reduces fat mass and improves the metabolic profile. In the short-term this may be supported by a positive protein and a negative fat balance, through increased fat oxidation. As protein intake is studied under various states of energy balance, absolute and relative protein intake needs to be discriminated. In absolute grams, a normal protein diet becomes a relatively high-protein diet in negative energy balance and at weight maintenance. Therefore, 'high protein negative energy balance diets' aim to keep the grams of proteins ingested at the same level as consumed at energy balance, despite lower energy intakes.M S Westerterp-Plantenga, N Luscombe-Marsh, M P G M Lejeune, K Diepvens, A Nieuwenhuizen, M P K J Engelen, N E P Deutz, D Azzout-Marniche, D Tome and K R Westerter