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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:637.)
© 2004 American Heart Association, Inc.

Brief Reviews

The "CholesteROR" Protective Pathway in the Vascular System

Fatiha Boukhtouche; Jean Mariani; Alain Tedgui

From the Laboratoire Développement et Vieillissement du Système Nerveux (F.B., J.M.), Université P. & M. Curie-CNRS, UMR NPA 7102, case 14, 9 quai Saint Bernard, 75005 Paris, France; and U541 INSERM, Hôpital Lariboisière (A.T.), 41 boulevard de la Chapelle, 75475 Paris, France

Correspondence to Alain Tedgui, INSERM U541, 41 boulevard de la Chapelle, 75475 Paris cedex 10, France. E-mail Alain.Tedgui{at}larib.inserm.fr

	Abstract

Top Abstract Introduction Cellular Roles of Cholesterol Cholesterol Has Been Identified... ROR{alpha}, a Widely Expressed... Conclusion References

 
Retinoic acid receptor-related Orphan Receptor (ROR) is a member of the nuclear hormone receptor superfamily. ROR has long been considered as a constitutive activator of transcription in the absence of exogenous ligand; however, cholesterol has recently been identified as a natural ligand of ROR. The spontaneous staggerer (sg/sg) mutation is a deletion in the Rora gene that prevents the translation of the ligand-binding domain (LBD), leading to the loss of ROR activity. The homozygous Rora^sg/sg mutant mouse, of which the most obvious phenotype is ataxia associated with cerebellar degeneration, also displays a variety of other phenotypes, including several vascular ones; in particular, dysfunction of smooth muscle cells and enhanced susceptibility to atherosclerosis. Moreover, ROR appears to participate in the regulation of plasma cholesterol levels, and has been shown to positively regulate apolipoprotein (apo)A-I and apoC-III gene expression. Yet its activity is regulated by cholesterol itself, making ROR an intracellular cholesterol target.

Key Words: ROR • cholesterol • statins • atherosclerosis • lipid homeostasis

	Introduction

Top Abstract Introduction Cellular Roles of Cholesterol Cholesterol Has Been Identified... ROR{alpha}, a Widely Expressed... Conclusion References

 
Retinoic acid receptor-related Orphan Receptor (ROR), initially described as an orphan nuclear receptor, has recently been deorphanized by the identification of its natural ligand as cholesterol or a cholesterol derivative.^1,2 ROR has been shown to be implicated in the development and/or differentiation of many tissues, and provides protection against age-related degenerative processes, including atherosclerosis. In addition, ROR activates the apolipoprotein A-I (apoA-I) and apoC-III gene transcription,^3,4 modulates the vasomotor tone of small resistance arteries, and participates in the regulation of postischemic angiogenesis.^5,6 This review focuses on the link between ROR, vascular biology, and cholesterol homeostasis. Interestingly, recent findings suggest that ROR is an intracellular cholesterol target.¹

	Cellular Roles of Cholesterol

Top Abstract Introduction Cellular Roles of Cholesterol Cholesterol Has Been Identified... ROR{alpha}, a Widely Expressed... Conclusion References

 
Cholesterol is the predominant sterol present in the plasma membrane. It is a key component of membrane structure with functional roles, including modulation of membrane fluidity. The distribution of cholesterol throughout the membrane is heterogeneous, being the main component of the majority of membrane lipid domains (lipid rafts) involved in a variety of cell functions, including growth-factor signal transduction, cellular adhesion, axon guidance, vesicular trafficking, and membrane-associated proteolysis.^7–11 Cholesterol depletion may have deleterious cellular effects, especially in neurons, which display vast cellular membranes. During axonal growth and synaptogenesis, neurons require the constant supply of lipid molecules, including cholesterol, for the synthesis of new plasma membrane.^12,13

In vitro or in vivo cholesterol depletion can be achieved by treatment with statins, the competitive inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key enzyme in mevalonate metabolism. Statins prevent the conversion of HMG-CoA to mevalonic acid and the synthesis of bioactive sterol and nonsterol metabolic intermediates deriving from the cholesterol synthesis pathway.¹⁴ In vitro studies have shown that statins inhibit the growth and proliferation of various types of cells, indicating that cholesterol or nonsteroid isoprenoid products are required in the maintenance of cell functions. Using squalestatin, an inhibitor of squalene synthase that selectively inhibits cholesterol biosynthesis without interfering with other critical reactions involving the generation of nonsterol metabolites,¹⁵ it has been shown that the viability of neurons,¹⁶ as well as the synaptic plasticity,¹⁷ depend on the intracellular cholesterol content.

On the other hand, cholesterol is the precursor of many steroids, and inborn errors of cholesterol synthesis are associated with malformation syndromes,^18,19 such as Smith-Lemli-Optiz Syndrome (SLOS) or holoprosencephaly. Cholesterol is required during the posttranslational modification of Sonic hedgehog (Shh): cholesterol must be covalently attached to the N-terminal component of the protein²⁰ to allow its auto-cleavage and its morphogenic function.²¹ Thus, absence of cholesterol impairs Shh signaling during embryogenesis. The hedgehog signaling pathway is fundamental in early embryonic patterning, and Shh mutations are associated with holoprosencephaly in humans.²²

The cellular concentration of cholesterol is regulated by the control of its biosynthesis (de novo synthesis from acetyl CoA) and uptake of circulating low density lipoproteins (LDL) cholesterol. Reverse cholesterol transport (from peripheral cells to the liver) also occurs through high density lipoproteins (HDL). An abnormal cholesterol metabolism may have detrimental consequences, especially during ageing when the membrane fluidity decreases.²³

In vivo, altered cholesterol metabolism or transport is associated with cardiovascular and neurodegenerative diseases, including atherosclerosis and Alzheimer’s disease (AD).

AD has been directly linked with cholesterol transport. Apolipoprotein (apo)E is a major apolipoprotein in the nervous system with a key role in cholesterol transport, implicated in the removal of remnant lipoproteins by the liver and in cholesterol efflux from peripheral cells. ApoE is a major risk factor for AD since 4 allele dose (apoE4) has been genetically associated with the occurrence of the disease in patients with sporadic²⁴ and late-onset familial AD.^25–27 Atherosclerosis is the most common vascular disease in which intimal accumulation of cholesterol and lipids constitutes a key event in atherosclerotic plaque formation.²⁸

Cholesterol is essential for cellular viability, and homeostasis of intracellular cholesterol is crucial to various cell functions. In addition to its role in cell membrane structure and function, cholesterol may regulate gene expression through its binding to a ligand-dependant nuclear receptor; recent studies have shown that cholesterol is a ligand of ROR, which permits its transcriptional activity.^1,2 This is the first assertion that native cholesterol can activate a transcription factor. Cholesterol derivatives (including steroid hormones and vitamin D) and oxidized cholesterol derivatives (oxysterols) are known to be signaling molecules,^29–31 but this is the first time that native cholesterol has been proposed to activate a transcription factor.

	Cholesterol Has Been Identified as the Natural Ligand of ROR

Top Abstract Introduction Cellular Roles of Cholesterol Cholesterol Has Been Identified... ROR{alpha}, a Widely Expressed... Conclusion References

 
ROR, also called NR1F1,³² is a member of the superfamily of the nuclear receptors^33,34 that includes receptors for thyroid and steroid hormones, retinoids, and vitamin D, as well as several "orphan" receptors of unknown ligands. Ligands for two of these receptors (peroxisome proliferator-activated receptors [PPARs] and liver X receptor [LXR])^31,35 show that products of lipid metabolism such as fatty acids,^36,37 prostaglandins,^38,39 or oxidized cholesterol derivatives³⁰ can regulate gene expression by binding to nuclear receptors.

So far, three ROR isotypes (-, -ß, -) have been described.^40–43 No ligand has been identified for ROR yet, whereas all-trans retinoic acid has recently been described as a ligand of RORß.⁴⁴

ROR was initially described as an orphan receptor and has long been considered a constitutive activator of transcription in the absence of exogenous ligand. Kallen et al¹ have recently succeeded in crystallizing the ROR ligand-binding domain (LBD) and revealed the presence of cholesterol in the ligand-binding pocket. Further experiments on purified ROR LBD have shown the presence of a fortuitous ligand: cholesterol and 7-dehydrocholesterol (provitamin D₃) were shown to be the major ligands present in the LBD.²

ROR LBD, expressed in Spodoptera frugiperda 9 (Sf-9) insect cells, was in a liganded form with bound cholesterol, which stabilizes the receptor in an agonistic conformation.¹ Depletion of cholesterol in U20S osteosarcoma cells using the statin lovastatin in LDL-free serum dramatically decreases the transcriptional activity of ROR, suggesting that changes in intracellular cholesterol level are capable of modulating the transcriptional activity of ROR, which implies that cholesterol is a "real" ligand rather than just a structural cofactor.¹ Both ROR and RORß, two isotypes of the ROR receptor subfamily, thus appear to be ligand-regulated transcription factors, activated by different natural ligands.

	ROR, a Widely Expressed Nuclear Receptor Involved in Many Cellular Processes

Top Abstract Introduction Cellular Roles of Cholesterol Cholesterol Has Been Identified... ROR{alpha}, a Widely Expressed... Conclusion References

 
ROR is expressed in several tissues in which it activates the transcription of specific genes. ROR, like all nuclear receptors, is composed of a variable N-terminal region (A/B region), a conserved DNA-binding domain (DBD) (C region), a linker, or hinge domain (D region), and a conserved E region that contains the LBD (Figure 1). The LBD contains an activation function motif (AF-2) responsible for ligand-dependant transcriptional activation (reviewed by Aranda et al⁴⁵).

View larger version (23K): [in this window] [in a new window]   Figure 1. The ROR protein. At the top is a schematic representation of a typical nuclear receptor. At the bottom is a depiction of the ROR protein conformation with its interaction with its RORE promoter site. NTD indicates N-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain.

ROR interacts as a monomer with the ROR response element (RORE) sequence within the promoter regions of target genes composed of a 6-bp A/T-rich region immediately preceding a consensus AGGTCA motif.^46–48 ROR is also able to bind as a homodimer at direct repeats of the RORE site separated by two base pairs, or direct repeat (DR2) sites.^49,50 An N-terminal modulator region varies between the four ROR splice isoformes (termed ROR1, -2, -3, and ROR4, also called RZR), allowing distinct interactions with AT-rich sequences just upstream of the AGGTCA motif, and distinct promoter recognition and transactivation properties through an identical DBD.⁴⁶

ROR is a widely expressed nuclear receptor. In situ hybridization has revealed ROR expression in the adult brain in cerebellar Purkinje cells, inferior olive, olfactory bulb, hippocampus, thalamus, cortex, suprachiasmatic nuclei of the hypothalamus, and retinal ganglion cells.^51,52 Outside the central nervous system, ROR mRNA have been detected in thymus, skeletal muscle, skin, heart, vessels, liver, lung, gut, kidney tubules, whisker follicles, and pancreas.^40,51,53,54

ROR Loss-of-Function Mutant Mice
ROR functions have been studied with the help of the staggerer (sg/sg) mutant mouse. sg/sg is a spontaneous mutation consisting in a 122-bp deletion in the Rora gene that shifts the reading frame and prevents the translation of the LBD of the protein.⁵³ A ROR deficient mouse (Rora^-/-) has been created by using a targeting vector in which a ß-Gal gene replaces the second zinc finger of the DBD of ROR.⁵¹ The Rora^-/- mouse displays a similar apparent cerebellar ataxic phenotype and massive cerebellar atrophy as the Rora^sg/sg mutant mouse, suggesting that the sg/sg mutation represents a loss of function of the ROR protein.

Cellular Roles of ROR
ROR plays a major role in cellular differentiation during development and growth of numerous tissues. In addition to the cerebellar phenotype, further studies of the sg/sg mutant mouse have shown a variety of age-related phenotypes beyond the cerebellum, including muscular atrophy, immunodeficiencies, osteoporosis, and atherosclerosis (reviewed by Jarvis et al⁵⁵) (Figure 2). Moreover, in the vascular system, ROR has been shown to be implicated in postischemic angiogenesis and in contractile functions of smooth muscle cells (SMC). We will briefly summarize these different abnormalities, and the vascular phenotype of the sg/sg mutant mouse will be described.

View larger version (33K): [in this window] [in a new window]   Figure 2. Physiological roles of ROR. The schema describes the main target tissue of the ROR action with known associated protein implicated. "+" indicates a positive regulation of the protein indicated, whereas "-" indicates a decrease.

Cerebellar Degeneration
In the cerebellum, the loss of function of the ROR protein causes a cell-autonomous developmental defect of the Purkinje cell,⁵⁶ leading to the absence of about 80% of the Purkinje cells at 2 months of age.⁵⁷ Purkinje cells seem to be generated in a normal number at the embryonic stage, but undergo cell death most likely between postnatal ages of 0 and 5 days.⁵⁸

The sg/sg mutation was initially described as recessive, but a semi-dominant effect of the mutation has been highlighted. Despite a lack of overt clinical phenotype in heterozygous sg/sg mice (Rora^sg/+), Purkinje cell loss and a progressive atrophy of the dendrites of the surviving cells have been demonstrated in the cerebellum,^59–61 and clear abnormalities in motor behavior have been revealed by using the rotarod test.⁶² The molecular mechanisms underlying the cerebellar sg/sg phenotype are still not understood, but both homozygous and heterozygous cerebellar phenotypes suggest that ROR plays a neuroprotective role during development and ageing.

Osteoporosis
Susceptibility to osteoporosis has been revealed in sg/sg mutant, and ROR has been implicated in bone formation and maintenance. Bones of Rora^sg/sg mice are thin, long, and osteopenic compared with the wild-type C57BL/6 mouse. ROR expression in mesenchymal stem cells derived from bone marrow is increased during the osteogenic differentiation. A direct control by ROR on mouse bone sialoglycoprotein and osteocalcin gene promoter has been characterized and may account in part for the mechanism of action of ROR in bone.⁶³

Skeletal Muscle
ROR is expressed in skeletal muscle and in proliferating myoblasts during their differentiation to postmitotic multinucleated myotubes, at least in vitro. ROR has a positive influence on muscular development via the upregulation of MyoD and p300 expression.⁶⁴ MyoD activates muscle-specific gene transcription and promotes cell-cycle exit after the induction of differentiation.^65–67 This study provides evidence that ROR1 functions to positively regulate myogenesis (ie, muscle differentiation).

Roles of ROR in the Vascular System
ROR has been shown to be involved in the differentiation of SMC, in the control of the vascular tone of small arteries, in ischemia-induced angiogenesis, in lipid metabolism, and in inflammation.

In the vascular system, ROR mRNA have been detected in human SMC, endothelial cells (EC), as well as in mammary arteries.^54,68 ROR expression is significantly decreased in human atherosclerotic plaques, whereas increased expression is observed after treatment with interleukin (IL)-1ß, tumour necrosis factor (TNF), and lipopolysaccharide (LPS) in both EC and human aortic SMC.⁵⁴

ROR Function and SMC Differentiation
ROR appears to be involved in the differentiation of SMC of small arteries.⁵ In Rora^sg/sg mutant mice, the expression of the SMC differentiation markers, calponin and h-caldesmon, are significantly decreased in mesenteric arteries. Moreover, ROR is involved in the regulation of vascular tone of small resistance arteries. Mesenteric resistance arteries of Rora^sg/sg mice display lower mean blood pressure, as well as reduced flow-induced dilation and pressure-induced myogenic tone. The vascular reactivity of mesenteric resistance arteries in response to vasoconstrictors and to endothelium-dependent or -independent vasodilators is also impaired. Furthermore, SMC from Rora^sg/sg mice display a reduced expression of the contractile protein SM-myosin, which might account for the observed decrease in contractile function in Rora^sg/sg mice. Thus, ROR seems to be essential in the differentiation and contractile function of SMC.

ROR and Ischemia-Induced Angiogenesis
The role of ROR in angiogenesis has been assessed by studying Rora^sg/sg mice with induced hindlimb ischemia.⁶ Femoral artery ligation induces a rapid and transient increase in ROR mRNA in ischemic tissue. In Rora^sg/sg mice, angiogenesis is markedly enhanced after ischemia induced by ligation of the femoral artery: capillary density is increased in the ischemic hindlimb, and leg perfusion is improved. Endothelial nitric oxide synthase (eNOS) protein expression is increased in Rora^sg/sg mice, whereas the levels of antiangiogenic cytokine IL-12 are significantly reduced. These results demonstrate that ROR is a potent negative regulator of ischemia-induced angiogenesis.

ROR, Lipid Metabolism, and Atherosclerosis
Interestingly, the sg/sg mutation has been linked with several aspects of atherosclerosis, including impairment of lipid metabolism. ROR has been shown to bind to a RORE in the promoter of the murine apoA-I and apoC-III genes, and to directly regulate gene transcription,^3,4 suggesting that ROR is a regulator of triglyceride and lipoprotein metabolism. ApoA-I is the major constituent of HDL, and apoC-III is a component of both triglyceride-rich lipoproteins and HDL. Rora^sg/sg mutants show significantly reduced plasma triglyceride levels associated with a strong decrease in apoC-III plasma concentrations,⁴ suggesting a physiological role of ROR in the regulation of plasma triglyceride metabolism in the mouse. This finding is likely to also apply to humans, since human ROR1 overexpression in HepG2 cells activates human apoC-III promoter activity in cotransfection assays.⁴ Although elevated triglycerides likely affect atherosclerosis in humans, the effect of hypertriglyceridemia in mice is modest.⁶⁹ This might explain why, despite their decreased hepatic apoC-III gene expression,⁷⁰ Rora^sg/sg mice fed an atherogenic diet develop more severe atherosclerosis than wild type mice. In fact, Rora^sg/sg mice show a decrease in HDL levels related to a specific reduction of apoA-I gene expression in the intestine and decreased plasma apoA-I levels, compared with wild type controls.⁷⁰ The increased susceptibility to atherosclerosis was highly correlated with the hypoalphalipoproteinemia. Interestingly, the susceptibility to atherosclerosis observed in Rora^sg/sg fed an atherogenic diet resembles the exacerbated atherosclerosis development in female apoA-I–deficient mice crossed with human apoB transgenic mice and fed a high fat diet to increase total and LDL cholesterol.⁷¹

Inflammation has been shown to play a major role in atherogenesis,⁷² particularly through nuclear factor B (NF-B) activation.⁷³ Interestingly, ROR exerts antiinflammatory activities through inhibition of the NF-B signaling pathway (this aspect is described under "Inflammation"). It is therefore likely that the enhanced atherosclerotic susceptibility of Rora^sg/sg mice is related both to low HDL plasma levels and to exaggerated inflammatory response to the high-fat, high-cholesterol diet.

Inflammation
Inflammation is a key element in both atherosclerosis⁷² and angiogenesis.⁷⁴ Interestingly, abnormalities in immune-inflammatory responses have been described in Rora^sg/sg mice. Homozygous sg/sg mutants have a delayed thymic development and a defect in terminating T-cell responses.⁷⁵ Moreover, peripheral macrophages of sg/sg mutants produce an abnormally high amount of proinflammatory cytokines IL-1, IL-6, and TNF on LPS stimulation,⁷⁶ demonstrating a general condition of macrophage hyperexcitability. Interestingly, abnormal IL-1ß cytokine production has been also described in the brain after peripheral LPS treatment.⁷⁷

ROR has been reported to inhibit inflammatory responses in vascular SMC by transcriptional regulation of I-B gene, the inhibitor of NF-B transcription factor activity. ROR negatively regulates the TNF-induced inflammatory response in SMC. Ectopic expression of ROR1 with an adenoviral vector in human primary SMC leads to a decreased expression in IL-6, IL-8, and cyclooxygenase-2 (COX-2) in response to TNF.⁶⁸ Antiinflammatory activities of ROR result from the inhibition of NF-B signaling pathway by inducing I-B gene expression. Furthermore, mRNA levels of I-B are significantly reduced in aortas from Rora^sg/sg mice compared with controls, indicating an in vivo control of I-B transcription by ROR. Antiinflammatory effects of ROR may contribute to the prevention of chronic inflammatory diseases in conjunction with regulation of specific target genes such as apoA-I in atherosclerosis (Figure 3).

View larger version (21K): [in this window] [in a new window]   Figure 3. Mechanism of protection against atherosclerosis by ROR. The schema shows the action of ROR on lipid metabolism by the activation of the apoA-I gene transcription leading to the increase of HDL, in conjunction with its action on inflammation by the activation of the I-B gene transcription, leading to the decrease of the NF-B activity and the decrease of production of inflammatory cytokines.

	Conclusion

Top Abstract Introduction Cellular Roles of Cholesterol Cholesterol Has Been Identified... ROR{alpha}, a Widely Expressed... Conclusion References

 
ROR activates apoA-I transcription gene. ApoA-I, with HDL, carries cholesterol from peripheral cells to the liver, preventing its accumulation in arterial walls. ROR seems to be a key regulator of plasma cholesterol level. It also seems to be involved in triglyceride metabolism, because apoC-III gene expression is positively regulated by ROR.⁴ ROR, whose transcriptional activity is activated by cholesterol itself, is thus able to increase apoA-I expression, which leads to the decrease of intracellular cholesterol content, suggesting a negative feedback regulation of the intracellular cholesterol content by ROR. This mechanism describes a new role for ROR, which could act as an intracellular cholesterol level target.

ROR exerts an antiatherogenic role both by inducing apoA-I expression and by regulating inflammation. In mammalian cells, the concentration of cholesterol is relatively high and might allow ROR to be constitutively active. In vitro, ROR activity can be decreased by lowering intracellular cholesterol drugs in absence of LDL cholesterol, and can be reactivated by adding cholesterol in depleted cells.¹ Thus, this study suggests that conditions that affect the cellular content of cholesterol, such as pharmacologic treatment or diseases that affect the synthesis or metabolism of cholesterol, could modulate the ROR activity in vitro. Statins are widely used in treatment of coronary artery disease (CAD). Statins, which are inhibitors of the rate-limiting step of cellular cholesterol synthesis, are the most commonly used lipid-lowering drugs in the treatment of atherosclerosis. Statins are very effective in lowering cholesterol levels, mainly by reducing serum LDL cholesterol levels; by inhibiting intracellular cholesterol synthesis, statins enhance expression of LDL receptors at the cell surface and increase the uptake of cholesterol by cells, resulting in LDL clearance from the circulation⁷⁸ and intracellular cholesterol supply. Statins have also been shown to exert antiinflammatory actions.⁷⁹ Many of the pleiotropic effects of statins are mediated by their ability to block the synthesis of important isoprenoid intermediates, which serve as lipid attachments for a variety of intracellular signaling molecules.^80–82 The potential inhibition of the antiatherogenic ROR transcriptional activity by statins appears contradictory to the known beneficial effects of statins in the vascular system, through their antiinflammatory and antiatherogenic actions.⁷⁹

However, this apparent contradiction can be accounted for by the in vitro model used. The modulation of the ROR transcriptional activity by statins has been assessed in in vitro studies with LDL serum-free medium.¹ However, total depletion in intracellular cholesterol has detrimental effects, leading to cell death. In vivo inhibition of cellular synthesis of cholesterol by statins results in increased uptake of circulating LDL cholesterol, leading to a compensation of decreased intracellular cholesterol synthesis. Therefore, it is most likely that in vivo statin treatment does not result in total depletion of cellular cholesterol, allowing ROR to be active. Thereby, in vivo statin treatment is unlikely to impair the protective effects of ROR in the vascular system.

ROR is strongly implicated in the differentiation process of many cell types, and seems to have a protective role during ageing. Interestingly, ROR-deficient mice Rora^sg/sg and Rora^-/- display a cerebellar neurodegeneration and age-related pathologies caused by a defect of survival and/or differentiation of cells. If some effects of cholesterol on differentiation can be accounted for by the cellular need of this component in the vast plasma membrane, some effects of cholesterol could also be mediated by the activation of the nuclear receptor ROR, allowing the expression of target genes implicated in the differentiation and cell survival.

Received November 24, 2003; accepted January 15, 2004.

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