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Objective: Liver X receptors (LXRs) are oxysterol-activated nuclear receptors regulating reverse cholesterol transport, in part by modulating cholesterol efflux from macrophages to apoAI and HDL via the ABCA1 and ABCG1/ABCG4 pathways. Moreover, LXR activation increases intracellular cholesterol trafficking via the induction of NPC1 and NPC2 expression. However, implication of LXRs in the selective uptake of cholesteryl esters from lipoproteins in human macrophages has never been reported., Methods and Results: Our results show that (1) selective CE uptake from HDL(3) is highly efficient in human monocyte-derived macrophages; (2) surprisingly, HDL(3)-CE uptake is strongly increased by LXR activation despite antiatherogenic effects of LXRs; (3) HDL(3)-CE uptake increase is not linked to SR-BI expression modulation but it is dependent of proteoglycan interactions; (4) HDL(3)-CE uptake increase is associated with increased expression and secretion of apoE and LPL, two proteins interacting with proteoglycans; (5) HDL(3)-CE uptake increase depends on the integrity of raft domains and is associated with an increased caveolin-1 expression., Conclusions: Our study identifies a new role for LXRs in the control of cholesterol homeostasis in human macrophages. LXR activation results in enhanced dynamic intracellular cholesterol fluxes through an increased CE uptake from HDL and leads to an increased cholesterol availability to efflux to apoAI and HDL.

The farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily and is activated by bile acids such as chenodeoxycholic acid, or synthetic ligands such as GW4064. FXR is implicated in the regulation of bile acid, lipid, and carbohydrate metabolism. Posttranslational modifications regulating its activity have not been investigated yet. Here, we demonstrate that calcium-dependent protein kinase C (PKC) inhibition impairs ligand-mediated regulation of FXR target genes. Moreover, in a transactivation assay, we show that FXR transcriptional activity is modulated by PKC. Furthermore, phorbol 12-myristate 13-acetate , a PKC activator, induces the phosphorylation of endogenous FXR in HepG2 cells and PKCalpha phosphorylates in vitro FXR in its DNA-binding domain on S135 and S154. Mutation of S135 and S154 to alanine residues reduces in cell FXR phosphorylation. In contrast to wild-type FXR, mutant FXRS135AS154A displays an impaired PKCalpha-induced transactivation and a decreased ligand-dependent FXR transactivation. Finally, phosphorylation of FXR by PKC promotes the recruitment of peroxisomal proliferator-activated receptor gamma coactivator 1alpha. In conclusion, these findings show that the phosphorylation of FXR induced by PKCalpha directly modulates the ability of agonists to activate FXR.

A role of the nuclear receptor Rev-erbalpha in the regulation of transcription pathways involving other nuclear receptors is emerging. Indeed, Rev-erbalpha is a negative regulator of transcription by binding to overlapping response elements shared with various nuclear receptors, including the peroxisome proliferator-activated receptors and the retinoid-related orphan receptor alpha (RORalpha). Here, we show that Rev-erbalpha is expressed in primary human macrophages and that its expression is induced by synthetic ligands for the liver X receptors (LXRs), which control cholesterol homeostasis, inflammation, and the immune response in macrophages. LXRalpha binds to a specific response element in the human Rev-erbalpha promoter, thus inducing Rev-erbalpha transcriptional expression. Interestingly, Rev-erbalpha does not influence basal or LXR-regulated cholesterol homeostasis. However, Rev-erbalpha overexpression represses the induction of toll-like receptor (TLR)-4 by LXR agonists, whereas Rev-erbalpha silencing by short interfering RNA results in enhanced TLR-4 expression upon LXR activation. Electrophoretic mobility shift, chromatin immunoprecipitation, and transient transfection experiments demonstrate that Rev-erbalpha represses human TLR-4 promoter activity by binding as a monomer to a RevRE site overlapping with the LXR response element site in the TLR-4 promoter. These data identify Rev-erbalpha as a new LXR target gene, inhibiting LXR-induction of TLR-4 in a negative transcriptional feedback loop, but not cholesterol homeostasis gene expression.

The role of the nuclear receptor FXR in adaptive thermogenesis was investigated using FXR-deficient mice. Despite elevated serum bile acid concentrations and increased mRNA expression profiles of thermogenic genes in brown adipose tissue, FXR-deficiency did not alter energy expenditure under basal conditions. However, FXR-deficiency accelerated the fasting-induced entry into torpor in a leptin-dependent manner. FXR-deficient mice were also extremely cold-intolerant. These altered responses may be linked to a more rapid decrease in plasma concentrations of metabolic fuels (glucose, triglycerides) thus impairing uncoupling protein 1-driven thermogenesis. These results identify FXR as a modulator of energy homeostasis.

Macrophages play a central role in host defense against pathogen microbes by recognizing bacterial components, resulting in the activation of an arsenal of anti-microbial effectors. Toll-like receptor (TLR)-4 mediates the recognition of lipopolysaccharide, a pathogen-associated molecular pattern from gram-negative bacteria. Activation of the TLR-4 signaling pathway by lipopolysaccharide increases antibacterial effects by inducing secretion of cytokines that activate an immune inflammatory response and by generating bactericidal reactive oxygen species via the NADPH oxidase system. Liver X Receptors (LXRs) are nuclear receptors controlling cholesterol homeostasis and inflammation in macrophages. In addition, LXRs are critical for macrophage survival and play a role in the innate immune response in the mouse. In this study, we investigated whether LXR activation also regulates host defense mechanisms in human macrophages. In primary human macrophages, oxidized LDL and synthetic LXR ligands increased TLR-4 gene expression. Transient transfection assays, gel shift and chromatin immunoprecipitation analysis indicated that LXRs induce human TLR-4 promoter activity by binding to a DR4-type LXR response element. LXR induction of TLR-4 mRNA was followed by an induction of TLR-4 protein expression. Moreover, although short-term pretreatment with LXR agonists significantly reduced the inflammatory response induced by lipopolysaccharide, pretreatment of macrophages for 48 hours with LXR agonists resulted in an enhanced lipopolysaccharide response. Finally, LXR activation increased reactive oxygen species generation by enhancing the expression of NADPH oxidase subunits. These data provide evidence for an immunomodulatory function of LXRs in human macrophages via mechanisms distinct from those previously identified in mouse macrophages.

Liver X receptors (LXRs) are members of the nuclear receptor superfamily, which have been implicated in lipid homeostasis and more recently in glucose metabolism. Here, we show that glucose does not change LXRalpha protein level, but affects its localization in pancreatic beta-cells. LXRalpha is found in the nucleus at 8 mM glucose and in the cytoplasm at 4.2 mM. Addition of glucose translocates LXRalpha from the cytoplasm into the nucleus. Moreover, after the activation of LXR by its synthetic non-steroidal agonist (T0901317), insulin secretion and glucose uptake are increased at 8 mM and decreased at 4.2 mM glucose in a dose-dependent manner. Furthermore, at low glucose condition, okadaic acid reversed LXRalpha effect on insulin secretion, suggesting the involvement of glucose signaling through a phosphorylation-dependent mechanism.

Retinoic acid receptor-related orphan receptor alpha (RORalpha) is a member of the nuclear receptor family. Recently, cholesterol (derivatives) has been identified as an RORalpha ligand and deorphanized this receptor. RORalpha is expressed in many tissues and is therefore a regulator of multiple biological processes. Studies of staggerer mice and in vitro assays indicate a beneficial modulatory role of RORalpha in the pathogenesis of dyslipidemia, inflammation and atherosclerosis. This paper provides an overview on the role of RORalpha in lipid metabolism and discusses its potential therapeutic option for treating lipid and inflammatory disorders leading to atherosclerosis., (Copyright 2006 Prous Science)

Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and liver X receptors (LXRalpha and LXRbeta) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPARalpha and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPARalpha ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis.

Objective: The newly identified apolipoprotein A5 (APOA5), selectively expressed in the liver, is a crucial determinant of plasma triglyceride levels. Because elevated plasma triglyceride concentrations constitute an independent risk factor for cardiovascular diseases, it is important to understand how the expression of this gene is regulated. In the present study, we identified the retinoic acid receptor-related orphan receptor-alpha (RORalpha) as a regulator of human APOA5 gene expression., Methods and Results: Using electromobility shift assays, we first demonstrated that RORalpha1 and RORalpha4 proteins can bind specifically to a direct repeat 1 site present at the position -272/-260 in the APOA5 gene promoter. In addition, using transient cotransfection experiments in HepG2 and HuH7 cells, we demonstrated that both RORalpha1 and RORalpha4 strongly increase APOA5 promoter transcriptional activity in a dose-dependent manner. Finally, adenoviral overexpression of hRORalpha in HepG2 cells led to enhanced hAPOA5 mRNA accumulation. We show that the homologous region in mouse apoa5 promoter is not functional. Moreover, we show that in staggerer mice, apoa5 gene is not affected by RORalpha., Conclusions: These findings identify RORalpha1 and RORalpha4 as transcriptional activators of human APOA5 gene expression. These data suggest an additional important physiological role for RORalpha in the regulation of genes involved in lipid homeostasis and probably in the development of atherosclerosis.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor. PPARalpha regulates lipid and glucose metabolism and controls the inflammatory response. Recently, we have shown that PPARalpha is a short-lived protein degraded by the ubiquitin-proteasome system. In this study, we have analysed the effects of interaction with RXRalpha, CBP, and N-CoR and also the implication of phosphorylation on ubiquitination and stability of PPARalpha. Our results show that interaction of PPARalpha with RXRalpha or CBP leads to an increase in the turnover of the protein. In contrast, interaction with the corepressor N-CoR, which inhibits its transcriptional activity, leads to a stabilization of the protein. Interestingly, treatment of cells with an inhibitor of Ser/Thr phosphatases known to lead to hyperphosphorylation of PPARalpha induces its transcriptional activity which is accompanied by a stabilization of the protein. These data indicate that heterodimerization, recruitment of cofactors, and post-translational modifications can modulate the stability of PPARalpha.

Plasma homocysteine levels increase in humans treated with fibrates but the molecular mechanisms are unknown. The goal of the present study was to determine the mechanism of this increase using animal models. Firstly, an increase in homocysteine was observed in mice treated with fenofibrate irrespective of the genetic background C57BL/6 or SV129. Secondly, as the effect of fenofibrate on gene expression is mediated through activation of the peroxisome proliferator-activated receptor alpha (PPARalpha), a transcription factor belonging to the nuclear receptor family, it was determined whether the effect of fenofibrate on homocysteine levels were modulated through PPARalpha activation. Using PPARalpha-deficient mice, it was shown that the homocysteine increase after fenofibrate treatment was completely abolished in these animals. It can be concluded that fibrates increase homocystinemia through a PPARalpha-mediated mechanism and that mice constitute an animal model for analyzing the molecular mechanisms behind the homocysteine increase after fibrate therapy in dyslipidemic patients.

Purpose of Review: This review focuses on recent advances on the roles of peroxisome proliferator-activated receptors in the control of lipid metabolism, and the inflammatory response of macrophages and the potential participation of these actions in the modulation of atherogenesis., Recent Findings: Altered macrophage functions contribute to the pathogenesis of many infectious, immunological and inflammatory disease processes. Pharmacological modulation of macrophage gene expression therefore represents an important strategy for the prevention and treatment of inflammation-related diseases, such as atherosclerosis. Peroxisome proliferator-activated receptors are lipid-activated transcription factors that control lipid and lipoprotein metabolism, glucose and energy homeostasis, as well as cellular differentiation and proliferation. Recent data suggest that peroxisome proliferator-activated receptor alpha and peroxisome proliferator-activated receptor gamma activators may decrease the incidence of cardiovascular disease, not only by correcting metabolic disorders, but also by directly acting at the level of the vascular wall. In this context, ligand-activated peroxisome proliferator-activated receptors control cellular functions by regulating gene expression in different cell types, including monocytes, macrophages and foam cells., Summary: These findings identify the crucial roles of peroxisome proliferator-activated receptors in macrophages, improving the comprehension of the patho-physiological mechanisms of atherogenesis. Moreover, a scientific rationale for the evaluation of peroxisome proliferator-activated receptor activators in the treatment of inflammatory disorders such as atherosclerosis is thus provided.

Rev-Erbalpha (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor alpha gene. Rev-Erbalpha mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor gamma (PPARgamma) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbalpha expression is induced by PPARgamma activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro. Furthermore, activated PPARgamma induces Rev-Erbalpha promoter activity by binding to the direct repeat (DR)-2 response element Rev-DR2. Mutations of the 5' or 3' half-sites of the response element totally abrogated PPARgamma binding and transcriptional activation, identifying this site as a novel type of functional PPARgamma response element. Finally, ectopic expression of Rev-Erbalpha in 3T3-L1 preadipocytes potentiated adipocyte differentiation induced by the PPARgamma ligand rosiglitazone. These results identify Rev-Erbalpha as a target gene of PPARgamma in adipose tissue and demonstrate a role for this nuclear receptor as a promoter of adipocyte differentiation.

Glucuronidation, a major metabolic pathway for a large variety of endobiotics and xenobiotics, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT2B4 conjugates a large variety of endogenous and exogenous molecules and is considered to be the major bile acid conjugating UGT enzyme in human liver. In the present study, we identify UGT2B4 as a novel target gene of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha), which mediates the hypolipidemic action of fibrates. Incubation of human hepatocytes or hepatoblastoma HepG2 and Huh7 cells with synthetic PPAR alpha agonists, fenofibric acid, or Wy 14643 resulted in an increase of UGT2B4 mRNA levels. Furthermore, treatment of HepG2 cells with Wy 14643 induced the glucuronidation of hyodeoxycholic acid, a specific bile acid UGT2B4 substrate. Analysis of UGT2B mRNA and protein levels in PPAR alpha wild type and null mice revealed that PPAR alpha regulates both basal and fibrate-induced expression of these enzymes in rodents also. Finally, a PPAR response element was identified in the UGT2B4 promoter by site-directed mutagenesis and electromobility shift assays. These results demonstrate that PPAR alpha agonists may control the catabolism of cytotoxic bile acids and reinforce recent data indicating that PPAR alpha, which has been largely implicated in the control of lipid and cholesterol metabolism, is also an important modulator of the metabolism of endobiotics and xenobiotics in human hepatocytes.

Cardiovascular diseases (CVD) remain the leading cause of mortality in the western societies. Several risk factors predispose to CVD including diabetes, obesity, insulin resistance, dyslipidemia and hypertension. Various pharmacological therapies have been developed to control the risk factors associated to CVD. Fibrates are able to correct dyslipidemia, therefore decreasing CVD risk. Thiazolidinediones (TZD) or glitazones by increasing insulin sensitivity decrease plasma glucose levels in diabetic patients. Both fibrates and TZD activate the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors that play a central role in the control of lipid and glucose metabolism. In this review, we will discuss the mode of action of fibrates and TZD and we will present an overview on PPAR ligands under development.

Background & Aims: Increased serum triglyceride levels constitute a risk factor for coronary heart disease. Apolipoprotein CIII (Apo CIII) is a determinant of serum triglyceride metabolism. In this study, we investigated whether activators of the nuclear farnesoid X receptor (FXR) modulate Apo CIII gene expression., Methods: The influence of bile acids and synthetic FXR activators on Apo CIII and triglyceride metabolism was studied in vivo by using FXR wild-type and FXR-deficient mice and in vitro by using human primary hepatocytes and HepG2 cells., Results: In mice, treatment with the FXR agonist taurocholic acid strongly decreased serum triglyceride levels, an effect associated with reduced Apo CIII serum and liver messenger RNA levels. By contrast, no change was observed in FXR-deficient mice. Incubation of human primary hepatocytes and HepG2 cells with bile acids or the nonsteroidal synthetic FXR agonist GW4064 resulted in a dose-dependent down-regulation of Apo CIII gene expression. Promoter transfection experiments and mutation analysis showed that bile acid-activated FXR decrease human Apo CIII promoter activity via a negative FXR response element located in the I(4) footprint between nucleotides -739 and -704. Chromatin immunoprecipitation experiments showed that bile acid treatment led to binding of FXR/retinoid X receptor heterodimers to and displacement of HNF4alpha from this site. Bile acid treatment still repressed liver Apo CIII gene expression in hepatic HNF4alpha-deficient mice, suggesting an active rather than a competitive mechanism of Apo CIII repression by the FXR., Conclusions: We identified bile acid and synthetic activators of the nuclear FXR as negative regulators of Apo CIII expression, an effect that may contribute to the triglyceride-decreasing action of FXR agonists.

The treatment of ischemic strokes is limited to the prevention of cerebrovascular risk factors and to the modulation of the coagulation cascade during the acute phase. A new therapeutic strategy could be to preventively protect the brain against noxious biological reactions induced by cerebral ischemia such as oxidative stress and inflammation to minimize their neurological consequences. Here, we show that a peroxisome proliferator-activated receptor (PPAR-alpha) activator, fenofibrate, protects against cerebral injury by anti-oxidant and anti-inflammatory mechanisms. A 14 d preventive treatment with fenofibrate reduces susceptibility to stroke in apolipoprotein E-deficient mice as well as decreases cerebral infarct volume in C57BL/6 wild-type mice. The neuroprotective effect of fenofibrate is completely absent in PPAR-alpha-deficient mice, suggesting that PPAR-alpha activation is involved as a mechanism of the protection against cerebral injury. Furthermore, this neuroprotective effect appears independently of any improvement in plasma lipids or glycemia and is associated with (1) an improvement in middle cerebral artery sensitivity to endothelium-dependent relaxation unrelated to an increase in nitric oxide synthase (NOS) type III expression, (2) a decrease in cerebral oxidative stress depending on the increase in numerous antioxidant enzyme activities, and (3) the prevention of ischemia-induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in cerebral vessels without any change in NOS II expression. These data demonstrate that PPAR-alpha could be a new pharmacological target to preventively reduce the deleterious neurological consequences of stroke in mice and suggest that PPAR-alpha activators could preventively decrease the severity of stroke in humans.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. Three PPARs isoforms have been characterized: PPARalpha, beta/delta and gamma. As other nuclear receptors, the PPARs are organized in distinct functional domains: A/B, C or DNA binding domain (DBD), D, E or ligand binding domain (LBD) and F. The A/B domain contains the activation function 1 (AF-1) which is transcriptionally active in absence of ligands. The DBD and the LBD of the PPARs determine the specificity of promoter DNA sequence recognition and ligand recognition, respectively. An activation function 2 (AF-2) is contained in the E domain, which mediates the ligand-dependent activation of the receptor. The transcriptional activity of the PPARs is regulated by post-translational modifications, such as phosphorylation and ubiquitination. Phosphorylation of PPARs is controlled by environmental factors activating different kinase pathways leading to the modulation of their activities. PPARs degradation by the ubiquitin-proteasome system modulates the intensity of the ligand response by controlling the level of PPAR proteins in the cells. PPARs also control the expression of genes implicated in the inflammatory response via negative interference with different inflammatory pathways, such as NFkappaB, AP-1, C/EBP beta, STAT-1 and NFAT. As such, PPARs influence inflammatory cytokine production and cell recruitment to the inflammatory sites. A better understanding of the mechanism of action of PPARs could improve the design of more specific and more efficient novel drugs. Molecules with dissociated effects could be useful for the treatment of lipid disorders or inflammation.

Background & Aims: Bile acids are essential for bile formation and intestinal absorption of lipids and fat-soluble vitamins. However, the intrinsic toxicity of hydrophobic bile acids demands a tight control of their intracellular concentrations. Bile acids are ligands for the farnesoid X receptor (FXR) that regulates the expression of genes controlling bile acid synthesis and transport. The human uridine 5'-diphosphate-glucuronosyltransferase 2B4 (UGT2B4) converts hydrophobic bile acids into more hydrophilic glucuronide derivatives. In this study, we identify UGT2B4 as an FXR target gene., Methods: Human hepatocytes or hepatoblastoma HepG2 cells were treated with chenodeoxycholic acid or the synthetic FXR agonist GW4064, and the levels of UGT2B4 messenger RNA, protein, and activity were determined by using real-time polymerase chain reaction, Western blot, and glucuronidation assays., Results: Treatment of hepatocytes and HepG2 cells with FXR agonists resulted in an increase of UGT2B4 messenger RNA, protein, and activity. A bile acid response element in the UGT2B4 promoter (B4-BARE) to which FXR, but not retinoid X receptor, binds, was identified by site-directed mutagenesis, electromobility shift, and chromatin immunoprecipitation assays. Retinoid X receptor activation abolished the induction of UGT2B4 expression and inhibited binding of FXR to the B4-BARE, suggesting that retinoid X receptor modulates FXR target gene activation. Overexpression of UGT2B4 in HepG2 cells resulted in the attenuation of bile acid induction of the FXR target gene small heterodimeric partner., Conclusions: These data suggest that UGT2B4 gene induction by bile acids contributes to a feed-forward reduction of bile acid toxicity and a decrease of the activity of these biological FXR activators.

The recently discovered APOA5 gene has been shown in humans and mice to be important in determining plasma triglyceride levels, a major cardiovascular disease risk factor. apoAV represents the first described apolipoprotein where overexpression lowers triglyceride levels. Since fibrates represent a commonly used therapy for lowering plasma triglycerides in humans, we investigated their ability to modulate APOA5 gene expression and consequently influence plasma triglyceride levels. Human primary hepatocytes treated with Wy 14,643 or fenofibrate displayed a strong induction of APOA5 mRNA. Deletion and mutagenesis analyses of the proximal APOA5 promoter firmly demonstrate the presence of a functional peroxisome proliferator-activated receptor response element. These findings demonstrate that APOA5 is a highly responsive peroxisome proliferator-activated receptor alpha target gene and support its role as a major mediator for how fibrates reduce plasma triglycerides in humans.

Peroxisome proliferator-activated receptor (PPAR) alpha and gamma are ligand-activated transcription factors belonging to the nuclear receptor family. PPAR alpha mediates the hypolipidemic action of the fibrates, whereas PPAR gamma is a receptor for the antidiabetic glitazones. In the present study, the UDP-glucuronosyltransferase (UGT) 1A9 enzyme is identified as a PPAR alpha and PPAR gamma target gene. UGTs catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT1A family enzymes, UGT1A9 metabolizes endogenous compounds, including catecholestrogens, and xenobiotics, such as fibrates and to a lesser extent troglitazone. Treatment of human hepatocytes and macrophages and murine adipocytes with activators of PPAR alpha or PPAR gamma resulted in an enhanced UGT1A9 expression and activity. In addition, disruption of the PPAR alpha gene in mice completely abolished the PPAR alpha agonist-induced UGT1A9 mRNA and activity levels. A PPAR response element was identified in the promoter of UGT1A9 at positions -719 to -706 bp by transient transfection and electromobility shift assays. Considering the role of UGT1A9 in catecholestrogen metabolism, PPAR alpha and PPAR gamma activation may contribute to the protection against genotoxic catecholestrogens by stimulating their inactivation in glucuronide derivatives. Furthermore, since UGT1A9 is involved in the catabolism of fibrates, these results suggest that PPAR alpha and PPAR gamma may control the intracellular level of active fibrates.

Purpose of Review: The liver X receptors (alpha and beta) are oxysterol-activated nuclear receptors. A large number of liver X receptor target genes with functions in lipid homeostasis have been identified. Increasing evidence indicates that these receptors play a beneficial role in avoiding cholesterol overload by intervening at several steps of cholesterol metabolism. In this review, we describe the most recent developments concerning their functions in cholesterol and lipid metabolism and their impact in atherogenesis., Recent Findings: Although potentially undesirable effects of liver X receptor activation on triglyceride metabolism have been observed, recent in-vivo studies confirm that the overall trend is a reduction of atherogenesis., Summary: Consequently, liver X receptors are becoming therapeutic targets of great interest for the treatment of atherosclerosis, especially if their action on triglyceride and cholesterol metabolism can be dissociated and isoform-specific ligands identified.

The liver X receptors (LXRalpha and LXRbeta) are nuclear receptor transcription factors that are activated by certain oxysterol derivatives of cholesterol. As such, LXR activity may be up-regulated by cellular lipid loading or dietary cholesterol intake. Intensive research interest in the LXRs has led to the identification of an expanding list of LXR target genes. The identity of these genes, and their response to LXR activation, indicates that the LXRs play an important role in the response to excess cholesterol, and that their activation may protect against tissue cholesterol overload. In this review, we highlight the multiple roles of the LXRs in controlling cholesterol homeostasis via their coordinated effects on cholesterol synthesis, dietary cholesterol absorption, reverse cholesterol transport, and bile acid synthesis and excretion. We discuss the therapeutic interest of developing LXR agonists, in view of their apparent protective effects against atherosclerosis. However, we also draw attention to the possible undesirable side-effects of LXR activation, and thus the potential interest of developing target gene-specific LXR agonists, or agonists that are specific for only one LXR isoform.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. PPARalpha is also reported to influence bile acid formation and bile composition. Farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that mediates the effects of bile acids on gene expression and plays a major role in bile acid and possibly also in lipid metabolism. Thus, both PPARalpha and FXR appear to act on common metabolic pathways. To determine the existence of a molecular cross-talk between these two nuclear receptors, the regulation of PPARalpha expression by bile acids was investigated. Incubation of human hepatoma HepG2 cells with the natural FXR ligand chenodeoxycholic acid (CDCA) as well as with the nonsteroidal FXR agonist GW4064 resulted in a significant induction of PPARalpha mRNA levels. In addition, hPPARalpha gene expression was up-regulated by taurocholic acid in human primary hepatocytes. Cotransfection of FXR/retinoid X receptor in the presence of CDCA led to up to a 3-fold induction of human PPARalpha promoter activity in HepG2 cells. Mutation analysis identified a FXR response element in the human PPARalpha promoter (alpha-FXR response element (alphaFXRE)] that mediates bile acid regulation of this promoter. FXR bound the alphaFXRE site as demonstrated by gel shift analysis, and CDCA specifically increased the activity of a heterologous promoter driven by four copies of the alphaFXRE. In contrast, neither the murine PPARalpha promoter, in which the alphaFXRE is not conserved, nor a mouse alphaFXRE-driven heterologous reporter, were responsive to CDCA treatment. Moreover, PPARalpha expression was not regulated in taurocholic acid-fed mice. Finally, induction of hPPARalpha mRNA levels by CDCA resulted in an enhanced induction of the expression of the PPARalpha target gene carnitine palmitoyltransferase I by PPARalpha ligands. In concert, these results demonstrate that bile acids stimulate PPARalpha expression in a species-specific manner via a FXRE located within the human PPARalpha promoter. These results provide molecular evidence for a cross-talk between the FXR and PPARalpha pathways in humans.

The Rev-erb and retinoic acid-related orphan receptors (ROR) are two related families of orphan nuclear receptors that recognize similar response elements but have opposite effects on transcription. Recently, the Rev-erbalpha gene promoter has been characterized and shown to harbor a functional Rev-erbalpha-binding site known as Rev-DR2, responsible for negative feedback down-regulation of promoter activity by Rev-erbalpha itself. The present study aimed to investigate whether Rev-erbalpha gene expression is regulated by RORalpha. Gel shift analysis demonstrated that in vitro translated hRORalpha1 protein binds to the Rev-DR2 site, both as monomer and dimer. Chromatin immunoprecipitation assays demonstrated that binding of RORalpha to this site also occurred in vivo in human hepatoma HepG2 cells. The Rev-DR2 site was further shown to be functional as it conferred hRORalpha1 responsiveness to a heterologous promoter and to the natural human Rev-erbalpha gene promoter in these cells. Mutation of this site in the context of the natural Rev-erbalpha gene promoter abolished its activation by RORalpha, indicating that this site plays a key role in hRORalpha1 action. Finally, adenoviral overexpression of hRORalpha1 in HepG2 cells led to enhanced hRev-erbalpha mRNA accumulation, further confirming the physiological importance of RORalpha1 in the regulation of Rev-erbalpha expression.

Several clinical and angiographic intervention trials have shown that fibrate treatment leads to a reduction of the coronary events associated to atherosclerosis. Fibrates are ligands for peroxisome proliferator-activated receptor alpha (PPARalpha) that modulate risk factors related to atherosclerosis by acting at both systemic and vascular levels. Here, we investigated the effect of treatment with the PPARalpha agonist fenofibrate (FF) on the development of atherosclerotic lesions in apolipoprotein (apo) E-deficient mice and human apoA-I transgenic apoE-deficient (hapoA-I Tg x apoE-deficient) mice fed a Western diet. In apoE-deficient mice, plasma lipid levels were increased by FF treatment with no alteration in the cholesterol distribution profile. FF treatment did not reduce atherosclerotic lesion surface area in the aortic sinus of 5-month-old apoE-deficient mice. By contrast, FF treatment decreased total cholesterol and esterified cholesterol contents in descending aortas of these mice, an effect that was more pronounced in older mice exhibiting more advanced lesions. Furthermore, FF treatment reduced MCP-1 mRNA levels in the descending aortas of apoE-deficient mice, whereas ABCA-1 expression levels were maintained despite a significant reduction of aortic cholesterol content. In apoE-deficient mice expressing a human apoA-I transgene, FF increased human apoA-I plasma and hepatic mRNA levels without affecting plasma lipid levels. This increase in human apoA-I expression was accompanied by a significant reduction in the lesion surface area in the aortic sinus. These data indicate that the PPARalpha agonist fenofibrate reduces atherosclerosis in these animal models of atherosclerosis.

Elevated serum levels of triglyceride-rich remnant lipoproteins (TRL) are a major risk factor predisposing a subject to atherosclerosis. Apolipoprotein C-III (apoC-III) is a major constituent of TRL that impedes triglyceride hydrolysis and remnant clearance and, as such, may exert pro-atherogenic activities. In the present study, transient cotransfection experiments in rat hepatocytes in primary culture and rabbit kidney RK13 cells demonstrated that overexpression of Rev-erbalpha specifically decreases basal and HNF-4 stimulated human apoC-III promoter activity. A Rev-erbalpha response element was mapped by promoter deletion, mutation analysis, and gel-shift experiments to a AGGTCA half-site located at position -23/-18 (downstream of the TATA box) in the apoC-III promoter. Finally, Rev-erbalpha-deficient mice displayed elevated serum and liver mRNA levels of apoC-III together with increased serum VLDL triglycerides. Taken together, our data identify Rev-erbalpha as a regulator of apoC-III gene expression, providing a novel, physiological role for this nuclear receptor in the regulation of lipid metabolism.

Peroxisome proliferator activated-receptor alpha (PPARalpha) is a ligand-activated transcription factor belonging to the nuclear receptor family. PPARalpha is implicated in the regulation of lipid and glucose metabolism and in the control of inflammatory response. Recently, it has been demonstrated that a number of nuclear receptors are degraded by the ubiquitin-proteasome pathway. Since PPARalpha exhibits a circadian expression rhythm and since PPARalpha is rapidly regulated under certain pathophysiological conditions such as the acute phase inflammatory response, we hypothesized that PPARalpha protein levels must be under tight control. Here, we studied the mechanisms controlling PPARalpha protein levels and their consequences on the transcriptional control of PPARalpha target genes. Using pulse-chase experiments, it is shown that PPARalpha is a short-lived protein and that addition of its ligands stabilizes this nuclear receptor. By transient cotransfection experiments using expression vectors for PPARalpha and hemagglutinin-tagged ubiquitin, it is demonstrated that PPARalpha protein is ubiquitinated and that its ligands decrease the ubiquitination of this nuclear receptor, thus providing a mechanism for the ligand-dependent stabilization observed in pulse-chase experiments. In addition, treatment with MG132, a selective proteasome inhibitor, increases the level of ubiquitinated PPARalpha and inhibits its degradation in transfected cells. Furthermore, MG132 treatment enhances the level of endogenous PPARalpha in HepG2 cells. Finally, transient transfection and quantitative reverse transcription-PCR show that inhibition of PPARalpha degradation increases its transcriptional activation and expression of target genes such as apoA-II and fatty acid transport protein (FATP). Taken together, these data demonstrate that PPARalpha is degraded by the ubiquitin-proteasome system in a ligand-dependent manner. Regulation of its degradation provides a novel regulatory mechanism of transcriptional activity of this nuclear receptor.

Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors that regulate lipid and lipoprotein metabolism, glucose homeostasis and inflammation. The PPAR family consists of three proteins, alpha, beta/delta and gamma. Recent data suggest that PPAR alpha and gamma activation decreases atherosclerosis progression not only by correcting metabolic disorders, but also through direct effects on the vascular wall. PPARs modulate the recruitment of leukocytes to endothelial cells, control the inflammatory response and lipid homeostasis of monocytes/macrophages and regulate inflammatory cytokine production by smooth muscle cells. Experiments using animal models of atherosclerosis and clinical studies in humans strongly support an anti-atherosclerotic role for PPAR alpha and gamma in vivo. Thus, PPARs remain attractive therapeutic targets for the development of drugs used in the treatment of chronic inflammatory diseases such as atherosclerosis. Future research will aim for the development of more potent drugs with co-agonist activity on PPAR alpha, PPAR beta/delta and/or PPAR gamma as well as tissue and target gene-selective PPAR receptor modulators (SPPARMs).

The use of cyclosporin A (CsA) in solid organ transplantation has been shown to be associated with the development of hypertension and nephrotoxicity. Several mechanisms, including endothelin (ET)-1-mediated systemic vasoconstriction, are considered to be responsible for CsA-induced hypertension. This study shows that: (i) incubation of CsA (1 microg) with bovine aortic endothelial cells leads to increased ET secretion by+40%; (ii) the use of compactin, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and fibric acid, the peroxisome-proliferator-activated receptor (PPAR)-alpha activator, inhibit the CsA-induced ET secretion to the level below the basal ET secretion, by -32% and -26% respectively; (iii) both inhibitions were reversed by the addition of mevalonate, suggesting communication between the HMG-CoA reductase product and PPAR-alpha pathway. The present findings may be of significant clinical relevance, since statins and fibrates beyond their hypolipidaemic action may represent a potential therapeutic tool in the treatment or prophylaxis of CsA-associated side effects. Furthermore, we suggest that the mevalonate metabolism would interfere with PPAR-alpha activity.

Nuclear receptors are transcription factors that are activated by ligands and subsequently bind to regulatory regions in target genes, thereby modulating their expression. Nuclear receptors thus allow the organism to integrate signals coming from the environment and to adapt by modifying the expression levels of relevant genes. The peroxisome proliferator-activated receptors (PPARs) alpha, beta/delta, and gamma constitute a subfamily of nuclear receptors. PPARalpha has been shown to bind and to be activated by leukotriene B4 and the hypolipidemic drugs of the fibrate class; PPARbeta/delta ligands are polyunsaturated fatty acids and prostaglandins; while prostaglandin J2 derivatives and the antidiabetic glitazones are, respectively, natural and synthetic ligands for PPARgamma. Upon binding and activation by their ligands, they regulate the transcription of numerous genes involved in intracellular lipid metabolism, lipoprotein metabolism, and reverse cholesterol transport in a subtype- and tissue-specific manner. PPARs therefore constitute interesting targets for the development of therapeutic compounds useful in the treatment of disorders of lipid and lipoprotein metabolism.

PPARs are ligand-activated transcription factors that regulate energy homeostasis. In addition, PPARs furthermore control the inflammatory response by antagonizing the nuclear factor-kappaB (NF-kappaB) signaling pathway. We recently demonstrated that PPARalpha activators increase IkappaBalpha mRNA and protein levels in human aortic smooth muscle cells. Here, we studied the molecular mechanisms by which PPARalpha controls IkappaBalpha expression. Using transient transfection assays, it is demonstrated that PPARalpha potentiates p65-stimulated IkappaBalpha transcription in a ligand-dependent manner. Site-directed mutagenesis experiments revealed that PPARalpha activation of IkappaBalpha transcription requires the NF-kappaB and Sp1 sites within IkappaBalpha promoter. Chromatin immunoprecipitation assays demonstrate that PPARalpha activation enhances the occupancy of the NF-kappaB response element in IkappaBalpha promoter in vivo. Overexpression of the oncoprotein E1A failed to inhibit PPARalpha-mediated IkappaBalpha promoter induction, suggesting that cAMP response element binding protein-binding protein/p300 is not involved in this mechanism. By contrast, a dominant-negative form of VDR-interacting protein 205 (DRIP205) comprising its two LXXLL motifs completely abolished PPARalpha ligand-mediated activation. Furthermore, cotransfection of increasing amounts of DRIP205 relieved this inhibition, suggesting that PPARalpha requires DRIP205 to regulate IkappaBalpha promoter activity. By contrast, DRIP205 is not involved in PPARalpha-mediated NF-kappaB transcriptional repression. Taken together, these data provide a molecular basis for PPARalpha-mediated induction of IkappaBalpha and demonstrate, for the first time, that PPARalpha may positively regulate gene transcription in the absence of functional PPAR response elements.

PPARalpha is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. The factors regulating human PPARalpha (hPPARalpha) gene expression remain largely unexplored. To study the mechanisms controlling hPPARalpha expression, the hPPARalpha gene promoter was identified and characterized. First, an alternatively spliced exon within the 5'-untranslated region of the hPPARalpha gene was identified by RT-PCR. Next, the transcription start site was mapped and the hPPARalpha gene promoter was cloned and functionally analyzed. Because PPARalpha levels are elevated in tissues expressing the hepatocyte nuclear factor-4 (HNF4), such as liver, the regulation of hPPARalpha by HNF4 was examined. Transient transfections in HepG2 and Cos cells showed that HNF4 enhances hPPARalpha promoter activity. 5'-Deletion and mutation analysis of the hPPARalpha promoter identified a regulatory element (RE) consisting of a degenerate hexamer repeat with a single nucleotide spacer (direct repeat 1), termed alphaHNF4-RE. Gel shift assays demonstrated that HNF4 binds to this alphaHNF4-RE. Furthermore, HNF4 increased the activity of a heterologous promoter driven by two copies of the alphaHNF4-RE. The nuclear receptor COUP-TFII also bound this site and down-regulated basal as well as HNF4-induced hPPARalpha promoter activity. Finally, PPARalpha was shown to bind the alphaHNF4-RE, leading to an induction of PPARalpha expression in hepatocytes. In summary, the organization of the 5'-flanking and untranslated region of the hPPARalpha gene was characterized and the hPPARalpha promoter region has been identified. Furthermore, these data demonstrate that the hPPARalpha gene is regulated by nuclear receptors, such as HNF-4, COUP-TFII, and PPARalpha.

Serum levels of HDL are inversely correlated with the risk of coronary heart disease. The anti-atherogenic effect of HDL is partially mediated by its major protein constituent apoA-I. In this study, we identify bile acids that are activators of the nuclear receptor farnesoid X receptor (FXR) as negative regulators of human apoA-I expression. Intrahepatocellular accumulation of bile acids, as seen in patients with progressive familial intrahepatic cholestasis and biliary atresia, was associated with diminished apoA-I serum levels. In human apoA-I transgenic mice, treatment with the FXR agonist taurocholic acid strongly decreased serum concentrations and liver mRNA levels of human apoA-I, which was associated with reduced serum HDL levels. Incubation of human primary hepatocytes and hepatoblastoma HepG2 cells with bile acids resulted in a dose-dependent downregulation of apoA-I expression. Promoter mutation analysis and gel-shift experiments in HepG2 cells demonstrated that bile acid-activated FXR decreases human apoA-I promoter activity by a negative FXR response element mapped to the C site. FXR bound this site and repressed transcription in a manner independent of retinoid X receptor. The nonsteroidal synthetic FXR agonist GW4064 likewise decreased apoA-I mRNA levels and promoter activity in HepG2 cells.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor family. The hypolipidemic fibrates and the antidiabetic glitazones are synthetic ligands for PPAR alpha and PPAR gamma, respectively. Furthermore, fatty acids and eicosanoids are natural PPAR ligands. PPARs function as regulators of lipid and lipoprotein metabolism and glucose homeostasis and influence cellular proliferation, differentiation and apoptosis. PPAR alpha is highly expressed in tissues such as liver, muscle, kidney and heart, where it stimulates the beta-oxidative degradation of fatty acids. PPAR alpha furthermore mediates the action of the hypolipidemic drugs of the fibrate class on plasma lipoprotein metabolism. PPAR gamma is predominantly expressed in intestine and adipose tissue. PPAR gamma triggers adipocyte differentiation and promotes lipid storage. In addition, PPARs play a role in inflammation control. PPAR activators inhibit the activation of inflammatory response genes by negatively interfering with the NF-kappa B and AP-1 signalling pathways. PPAR activators exert these anti-inflammatory activities in different immunological and vascular wall cell types such as monocyte-macrophages, endothelial, epithelial and smooth muscle cells in which PPARs are expressed. These findings indicate a modulatory role for PPARs in the control of the inflammatory response with potential therapeutic applications in inflammation-related diseases, such as atherosclerosis.