Advance Publication Journal of Atherosclerosis and JournalThrombosis of Vol Atherosclerosis.21, No. ● and Thrombosis 1 Review Accepted for publication: February 10, 2014 Published online: April 1, 2014 Liver X : A Cardinal Target for Atherosclerosis and Beyond

Mihir Parikh1, Kirti Patel2, Sachin Soni1 and Tejal Gandhi1

1Department of Pharmacology, Anand Pharmacy College, Anand, Gujarat, India 2Pharmacy Department, Faculty of Technology and Engineering, M.S University, Baroda, Gujarat, India

The [LXR] is activated by endogenous oxidized derivatives of cholesterol. It constitutes a critical receptor in the regulation of various physiological functions related to the development of metabolic and cardiovascular diseases, such as atherosclerosis and dia- betes, as well as various other disorders. Both isoforms of LXR, LXRα [NR1H3] and LXRβ [NR1H2], form heterodimers with the isoforms of the [RXR], which then regu- late the expression by binding to DNA sequences associated with target . LXR acts as a cholesterol sensor in response to an increased concentration of cholesterol in cells and induces the transcription of genes that protect cells from cholesterol overload. LXRs play numerous roles in con- trolling cholesterol homeostasis via their actions on synthesis and metabolism/excretion, reverse cholesterol transport and cholesterol absorption/excretion in the intestines. Therefore, these receptors show great potential as pharmacological targets for anti-atherosclerotic activities. Recent discoveries have also emphasized the important involvement of LXRs in the pathogenesis of diabetes, Alzheimer’s disease, inflammation, adrenal steroid synthesis, skin aging and male fertility. However, LXR activation has also been shown to stimulate via sterol regulatory element binding protein-1c, leading to liver steatosis and hypertriglyceridemia. This review summarizes recent scien- tific discoveries and the biological actions of LXR with a special focus on the involvement of this type of receptor in important diseases and conditions.

J Atheroscler Thromb, 2014; 21:000-000.

Key words: Liver X receptor, LXR, Lipid, Atherosclerosis, Steroid

LXR, being a member of the nuclear receptor LXR superfamily, shares the ability to promote or inhibit The liver X receptor [LXR] was first discovered the transcription of target genes by binding to specific in 1995 as a new member of the nuclear receptor sites in the promoter regions of these genes, called superfamily1) and was subsequently found to be acti- response elements. LXR acts as a ligand-activated tran- vated by oxygenated cholesterol derivatives2). scription factor, and the initiation or blockade of tar- Initially, this receptor was identified in tissue get gene transcription is determined by the presence obtained from a rat liver3), with no known endoge- of coactivator or corepressor complexes4). LXR forms nous ligands, and was named LXR. Later, LXR was heterodimers with another nuclear receptor, retinoid termed an ‘adopted’ nuclear receptor with the discov- X receptor [RXR]. The formed heterodimer then ery of oxysterols2) as endogenous ligands for this binds with specific DNA sequences known as LXR- receptor. responsive elements [LXREs], which regulate the tran- scription of target genes1). LXR interacts with several Address for correspondence: Mihir Parikh, Department of Pharmacology, Anand Pharmacy College, Anand-388001, coregulators, including PGC-1b, RIP140, GPS2 and Gujarat, India. ACS-2, which have been linked to specific metabolic 5-8) E-mail: [email protected] processes . Received: May 26, 2013 The LXR/RXR heterodimer inhibits the tran- Accepted for publication: February 10, 2014 scription machinery of a number of target genes Advance Publication 2 Parikh etJournal al. of Atherosclerosis and Thrombosis Accepted for publication: February 10, 2014 Published online: April 1, 2014 through interactions with corepressor complexes, such involved in various metabolic processes and have lipo- as the nuclear receptor corepressor [NcoR] and the lytic/lipogenic effects on lipid metabolism. Clinical related factor SMRT [silencing mediator of retinoic case reports have documented high cholesterol levels acid and thyroid hormone receptors], in the absence in hypothyroid patients and low cholesterol levels in 9-10) 21, 22) of receptor activation . The binding of a ligand hyperthyroid patients . The T4 thyroid hormone is induces a conformational change in the protein, which eventually converted to T3, the active moiety of which leads to the displacement of corepressors, thus result- then binds as a ligand to thyroid hormone receptors ing in the recruitment of coactivator proteins, such as (TRs). While two isoforms of TRs, TR-α and TR-β steroid receptor coactivator [SRC]-1, Grip1 [a p160 (Nr1a1 and Nr1a2), with different subtypes (α-1, α family coactivator] and peroxisome-proliferator-acti- -2, β-1, β-2 and β-3)23) are found in the liver, TR-β vated receptor [PPAR]γ coactivator 1a [PGC-1α]11). has been found to play a major role in gene regulation Two LXR isotypes have been identified: LXRα and is involved in crosstalk with LXR as shown in [NR1H3] and LXRβ [NR1H2]1, 12). Fig.224, 25). The human LXRα and LXRβ genes are located TRs and LXRs are both nuclear receptors, with on 11p11.2 and 19q13.3, respectively. different receptor subgroups showing similarity in The LXRα expression is found to be particularly high molecular mechanisms, target genes and physiological in organs and components such as the liver, small roles26). Similar to LXR, TR forms a heterodimer with intestine, kidneys, macrophages and adipose tissue, RXR upon activation. The TR-RXR heterodimer then while the LXRβ expression is more ubiquitous13). binds with DNA-responsive elements, TREs, as LXR- Human LXRα and LXRβ share almost 80% amino RXR binds to LXREs. TREs and LXREs exhibit struc- acid identity in their DNA-binding and ligand-bind- tural similarity, with direct repeat of the basic nucleo- ing domains. In addition, human LXRα and rat tide sequence AGGTCA spaced by four nucleotides LXRα show close to 100% homology in amino acid (DR-4)1, 26). sequences in their DNA-binding and ligand-binding Cholesterol catabolism by CYP7A1 in the liver domains14). produces bile acids. TRs and LXRs both regulate cho- Oxysterols, oxidized derivatives of cholesterol, lesterol catabolism into bile acids by CYP7A1, a rate including 22[R]-hydroxycholesterol, 24[S]-hydroxy- limiting enzyme, in rodents27). It was demonstrated in cholesterol, 24[S], 25-epoxycholesterol (most potent), one study that hypothyroid mice have undetectable 20[S]-hydroxycholesterol and 27-hydroxycholesterol, levels of CYP7A1 mRNA, while treatment with T 3 but not cholesterol15), are ligands for LXR. thyroid hormone increased the level of CYP7A128). 22[R]-hydroxycholesterol and 20[S]-hydroxy- LXR activation also regulates CYP7A1 transcription cholesterol are intermediates in the process of steroid via LXREs. Impaired CYP7A1 levels have been hormone synthesis. 24[S]-hydroxycholesterol is pro- reported in LXRα−/− mice, resulting in hepatic choles- duced in the brain and represents the most abundant terol accumulation29). Furthermore, activation of the circulating oxysterol, while 24[S], 25-epoxycholesterol CYP7A1 is induced by crosstalk is primarily found in the liver16). All but one of these between TR-β and LXR-α, thereby controlling cho- oxysterols species17) have been reported to activate lesterol metabolism30). The LXR-dependent regulation both LXRα and LXRβ2). Interestingly, it has been of CYP7A1 is limited to rodents and has no effect in demonstrated that glucose and glucose- 6-phosphate humans31). are able to bind and activate LXR in vitro18). The identification of cholesterol metabolites as endogenous LXR and PPAR: Crosstalk ligands already hints toward the physiological pro- cesses regulated by LXRs. Upon their activation, LXRs There is a potential interaction or crosstalk induce the transcription of multiple genes involved in between LXR and PPAR. Both of these nuclear recep- cholesterol efflux, conversion and transport19). How- tors form heterodimers with RXR (retinoid X recep- ever, LXRs also modulate the expression of genes tor), which enhances binding to their respective DNA involved in glucose and fatty acid metabolism while response elements 1, 32). The LXR ligand activates transrepressing those involved in inflammatory pro- hepatic LXRα, stimulating crosstalk with PPARα/ cesses20). RXR and thus leading to suppression of the expression of PPARα target genes involved in lipid degradation, while PPARα activation inhibits LXR ligand-induced LXR and Thyroid Hormone Crosstalk signaling in hepatocytes involved in lipid synthesis. , such as T4 and T3, are The expression of LXRα is subject to a number Advance Publication LXR in Dyslipidemia andJournal other Diseases of Atherosclerosis and Thrombosis 3 Accepted for publication: February 10, 2014 Published online: April 1, 2014 of regulatory mechanisms. These mechanisms were mice. For example, it was initially reported that an first examined in a study that determined the poten- increase in the level of the ATP-binding cassette trans- tial for crosstalk between peroxisome proliferator-acti- porter [ABC] superfamily, subfamily A, member 1 vated receptors [PPARs] and LXRs33). In rat hepato- [ABCA1] gene expression in the intestine inhibits cytes, both in vivo and in vitro, unsaturated fatty acids cholesterol absorption9). However, it was later demon- have been found to strongly increase the mRNA and strated that ABCA1 is present on the basolateral mem- protein expression levels of LXRα, while causing no brane of enterocytes and, ergo, does not mediate changes in the expression of LXRβ. This upregulated dietary cholesterol absorption, although it is involved expression of LXRα is primarily due to an increase in in the generation of HDL via cholesterol efflux39). the transcriptional rate and binding of the nuclear ABCG5 and ABCG8 play key roles in prevent- receptor PPARα to a PPAR [PPRE] ing the intestinal absorption of excess dietary choles- in the 5’ flanking region of the murine LXRα gene33). terol from the gut and enhancing cholesterol efflux Subsequently, a PPRE was identified in the human from hepatocytes into bile as ABC transporters40). The LXRα 5’-flanking region34). In conjunction with the ABCG5 and ABCG8 mRNA levels, present in both observation that unsaturated fatty acids are competi- hepatocytes and enterocytes, are increased by the acti- tive inhibitors of LXR ligands35), these studies indicate vation of LXR. No changes are observed in the the complex role of unsaturated fatty acids in LXR ABCG5 or ABCG8 gene expression in mice lacking regulation, as these compounds can act via PPARα to LXRα and LXRβ, indicating that ABCG5 and induce the LXRα expression and may also act directly ABCG8 are direct targets of LXRα and LXRβ41). to decrease the LXR transcriptional activity. Niemann-Pick C 1 like 1 [NPC1L1] is a protein Peroxisome proliferator activated receptor present in the brush border membrane of enterocytes [PPAR]α regulates energy depletion via fatty acid deg- in the small intestine and has been identified to be an radation36) ,while sterol regulatory element-binding enteric transporter of dietary cholesterol42). NPC1L1 protein-1c, which is activated by liver X receptor is a major contributor to the regulation of intestinal [LXR], is involved in fatty acid synthesis37). cholesterol absorptio43). Many studies have ostensibly Gel shift assays have demonstrated that LXR proven that synthetic LXR activators enhance free reduces the binding of PPAR α/retinoid X receptor cholesterol transport to the plasma membrane and [RXR] α to peroxisome proliferator response ele- inhibit intestinal cholesterol absorption by reducing ments. The addition of increasing amounts of RXR α the expression of NPC1L1. has been found to restore these inhibitory effects in One study showed that intestinal cholesterol both luciferase and gel shift assays, thus suggesting the absorption is required for LXR agonists to increase the presence of RXR α competition. Meanwhile, in vitro plasma HDL-C level by treating mice lacking protein binding assays have demonstrated that activa- NPC1L1 [L1-KO mice] with the LXR agonist tion of LXR by an LXR agonist promotes the forma- TO-901317 for seven days. In that study, treatment tion of LXR/RXR α and, more importantly, LXR/ with TO-901317 failed to raise the plasma HDL-C PPAR α heterodimers, leading to the reduction of level, while inducing greater fecal cholesterol excretion PPAR α/RXR α formation. In support of these find- in L1-KO mice compared with that observed in con- ings, the in vivo administration of the LXR ligand in trol mice44). mouse and rat primary hepatocytes substantially A recent study also demonstrated that LXR ago- decreases the hepatic mRNA levels of PPAR α-tar- nists decrease the NPC1L1 gene expression in human geted genes under both basal and PPAR α agonist- Caco-2/TC-7 enterocytes 45). These results indicate induced conditions. In one study, the amount of that NPC1L1 is essential for LXR agonists to increase nuclear PPAR α /RXR heterodimers in the mouse liv- the plasma HDL-C level and that LXRs decrease ers was increased by treatment with a PPAR α ligand intestinal cholesterol absorption via downregulation of and suppressed by treatment with a superimposed the intestinal NPC1L1 expression. LXR ligand38). LXR and RCT LXR and Dietary Cholesterol Absorption RCT, reverse cholesterol transport, includes a LXR activation results in a reduction in intestinal repertoire of reactions in which cholesterol moves cholesterol absorption as depicted in Fig.19). from macrophages and other peripheral tissue compo- Studies have demonstrated the intestinal choles- nents, back to the liver. Lipoprotein HDL plays a key terol absorption inhibition effects of LXRα agonists in role in this process by acting as an acceptor and trans- Advance Publication 4 Parikh etJournal al. of Atherosclerosis and Thrombosis Accepted for publication: February 10, 2014 Published online: April 1, 2014 porter of cholesterol. In addition, ABCA1 initiates the concomitant decrease in the hepatic ABCA1 gene process of RCT by inducing the efflux of deposited expression53). In addition, recent data demonstrate excess cholesterol from peripheral cells to lipid-poor that pharmacological activation of LXRs in vivo pro- apolipoproteins [apo], such as apo A-Ⅰ and apo E46). motes RCT from cholesterol injected-loaded macro- Effluxed cholesterol is then esterified by lecithin and phages, accompanied by the upregulation of ABCA1, cholesterol acyltransferase and is subsequently moved ABCG1, ABCG5 and ABCG8 in both the liver and to the core of the HDL lipoprotein particle, where it intestines54). transforms nascent discoidal HDL particles into spherical mature HDL particles that are ultimately LXR and Bile Acids taken up by the liver. LXRs control RCT via several potential mecha- Bile fluid contains water, cholesterol, phospho- nisms. lipids [primarily phosphatidylcholine], bile salts, bili- For example, LXRs stimulate intracellular choles- rubin and bicarbonate ions. Bile acid synthesis and terol mobilization to the plasma membrane, thus secretion constitute the major route of elimination of enhancing the availability of cholesterol for efflux to cholesterol from the body. Bile acids are produced in extracellular acceptors. Several apolipoproteins and the liver from cholesterol. Furthermore, the classical lipid-modulating enzymes involved in cholesterol pathway of bile acid synthesis is initiated by the 7α efflux and HDL remodeling are transcriptional targets -hydroxylation of cholesterol catalysed by cytochrome for LXRs. P450 cholesterol 7α-hydroxylase [CYP7A1], which Indeed, LXRs stimulate the expression of apoE encodes the rate-limiting enzyme of this pathway55). in murine macrophages and adipose tissue, but not Bile acids are then conjugated to glycine or tau- the liver47). Previous studies have also demonstrated rine prior to secretion into the bile. The biliary secre- that LXR activation in cultured HepG2 cells and/or tion of lipid and bile salts is controlled by hepatic cho- in vivo in mice results in an increased expression and lesterol, phospholipids and bile salt transporters plasma activity of the enzyme phospholipid transfer located on the hepatocyte canalicular membrane. The protein [PLTP], which catalyzes the exchange of phos- human multidrug-resistant 3p-glycoprotein, also pholipids between HDL particles and triglyceride-rich known as ABCB4 [corresponding to the murine Mdr2 lipoproteins, resulting in an increased level and size of p-glycoprotein], translocates phosphatidylcholine HDL-c48). Furthermore, LXRs activate the human molecules from the inner to outer leaflet of the cana- cholesterol ester transfer protein [CETP] gene expres- licular membrane. Finally, the functional complex sion49), which facilitates the transfer of cholesterol ABCG5/G8 pumps cholesterol into the bile. Choles- ester and triglycerides between HDL and apoB-con- terol is only slightly soluble in aqueous solutions; taining lipoprotein particles. As LXR is not expressed however, it is made soluble in bile by bile salts and in rodents, but rather in humans, a previous study phospholipids. showed that LXR agonist treatment does not increase During a meal, bile acids are released into the the HDL-cholesterol levels in human CETP trans- duodenum. The vast majority of bile acids are subse- genic mice50). The expression of lipoprotein lipase quently reabsorbed throughout the intestine and [LPL], an enzyme catalyzing the hydrolysis of lipopro- return via the portal blood to the liver, where they tein triglycerides that is also involved in the matura- exert a negative retro feedback loop on bile acid syn- tion of HDL, is regulated by LXR51). LXRs enhance thesis in which recirculated bile acids are again RCT via direct actions in macrophages. In contrast, secreted into the bile after a meal. Only a fraction of ABCA1 does not directly interact with mature HDL bile acids escape intestinal absorption and are secreted particles, although two other members of the ABC in feces. This constitutes the main physiological path- superfamily, ABCG1 and ABCG4, have been demon- way of cholesterol elimination from the body. The strated to be mediators of cholesterol efflux from cells enterohepatic circulation and feedback regulatory loop to HDL, which stimulates the atheroprotective activ- of bile acid synthesis and excretion constitute the ity of HDL52). ABCG1 is highly expressed in macro- essential mechanisms underlying the maintenance of phages [more so than ABCG4], and its activity likely cholesterol and bile acid homeostasis, thus preventing underlies the relationship between the HDL level and bile acid accumulation and avoiding toxicity as well as the risk of atherosclerosis. Moreover, in mice fed a cholestasis. The different steps involved in bile acid low-fat diet, LXRα- and LXRβ-deficiency results in metabolism are tightly controlled by the complemen- the significant accumulation of cholesterol in macro- tary and coordinated actions of LXR and the Farne- phages of the spleen, lungs and arterial walls, with a soid X receptor [FXR], which acts as a bile acid sen- Advance Publication LXR in Dyslipidemia andJournal other Diseases of Atherosclerosis and Thrombosis 5 Accepted for publication: February 10, 2014 Published online: April 1, 2014 sor56). vating lipolysis in adipocytes, exhibits an increased In rodents, LXRα stimulates the expression of expression, while apoA-V is downregulated, following CYP7A1 by binding to an LXRE present in the LXR activation66). CYP7A1 promoter. Therefore, rats and mice have the capacity to convert dietary cholesterol into bile Diabetes and LXR acids57). In contrast to that observed in rats and mice, LXRα agonist treatment suppresses the expression of The apparent role of LXR in glucose homeostasis CYP7A1 in primary human hepatocytes58), which has been documented in various studies demonstrat- may be due to the direct induction of a small het- ing potent glucose-lowering and insulin-sensitizing erodimer partner, a gene that with a repressive effect effects of synthetic LXR activators. In one study, the on CYP7A1 via liver receptor homolog 1 [LRH1; also administration of T0901317, an LXR activator, nor- called FIF in rats and CPF in humans]59). malized the plasma glucose levels in db/db mice and The excretion of free cholesterol into the bile is Zucker diabetic fatty [ZDF] rats67), while leaving gly- another major route of elimination of excess choles- cemia unaffected in non-diabetic animal models68). terol from the liver. In the liver, ABCG5 and ABCG8 GW3965 and T0901317 also improve insulin sensi- have been proposed to transport cholesterol from tivity in db/db mice and Zucker fatty rats, as well as hepatocytes to the bile canaliculi. ABCG5 and high-fat diet-fed rodents67-71). Several potential mecha- ABCG8 are half transporters that form obligate het- nisms for the antidiabetic actions of LXR agonists erodimers and are both regulated by LXR activa- have been proposed. For example, LXR activation tion60). ABCG5 and ABCG8 are also expressed in the reduces the mRNA level of peroxisome proliferator- apical membrane of enterocytes and at the canalicular activated receptor γ coactivator-1α (PGC-1α) in the membrane of hepatocytes. These transport proteins rodent liver68, 71), the main controller of hepatic glu- promote the secretion of hepatic cholesterol into the cose biogenesis72). In addition, a high expression of bile. Mice that lack ABCG5 and/or ABCG8 demon- SREBP-1c induced by LXR activation downregulates strate a significant decrease in the biliary cholesterol the expression of phosphoenolpyruvate carboxyki- level and cholesterol accumulation in the liver after nase73). These two explanatory mechanisms of LXR cholesterol feeding40). activation can account for the induced inhibition of hepatic glucogenesis. Furthermore, LXR agonists have been shown to increase the GLUT4 expression in adi- LXR and Fatty Acids pose tissue, but not skeletal muscle69, 71, 74), as well as LXR activation increases the expression of genes raise the insulin-mediated glucose uptake and oxida- of fatty acid biosynthesis [i.e. lipogenesis], whereby it tion levels in human differentiated myotubes75). induces fatty liver and raises the plasma triglyceride level [hypertriglyceridemia] by promoting the secre- LXR and Alzheimer’s Disease tion of large TAG-rich VLDL particles in mice61). It has been reported that the induction of the fatty acid Cholesterol accumulation has been demonstrated transporter CD36 in response to pharmacological to be one of the prime culprits of the pathology of LXR activation contributes to hepatic lipid accumula- Alzheimer’s disease. An increased brain cholesterol tion, presumably by increasing the hepatic fatty acid content and/or irregular distribution increases the β uptake62). and γ secretase activities76, 77), which cleaves amyloid Severe hepatic steatosis develops in mice upon precursor protein78), thus leading to Aβ production79), LXR agonist treatment; many studies have explained whereas a decreased intracellular cholesterol concen- this effect based on the direct upregulation of tration promotes the non-amyloidogenic activity of α SREBP1c, the major SREBP1 isoform, in the liver, secretase80). Both in vivo and in vitro studies have which stimulates the transcription of lipogenic genes, highlighted the role of LXR activation in reducing Aβ such as fatty acid synthase [FAS], acetyl-coA carboxyl- production. Moreover, Lxr α−/− and Lxr β−/− have ase [ACC] and stearoyl-coenzyme A desaturase 1 been found to enhance Aβ plaque production and [SCD1], a rate-limiting enzyme necessary for the bio- thus further deteriorate the pathology of Alzheimer’s synthesis of monounsaturated acids63, 64). In one study, disease in an animal model81). ABCA-1, a transporter it was found that angiopoietin-like protein 3 [Ang- in the brain, pumps cholesterol out of cells, resulting ptl3]65), a liver-secreted protein that increases the in lipid efflux and helping to achieve the proper redis- plasma triglyceride level by inhibiting the LPL activity tribution of lipids in the brain. Increased transcription in different tissues and the free fatty acid levels by acti- of the ABCA-1 gene is considered to be beneficial, as Advance Publication 6 Parikh etJournal al. of Atherosclerosis and Thrombosis Accepted for publication: February 10, 2014 Published online: April 1, 2014 it upregulates the ABCA-1 transporter expression, TNF-α and MMP-9 and reduced the expression of thereby regulating the cholesterol levels in the brain82). class Ⅱ MHC antigens, thereby suppressing immu- In one preclinical study, the LXR agonist, T0901317, nity91). increased the ABCA-1 expression and decreased the Meanwhile, in a rat liver inflammatory model, Aβ production83). Hence, the modulation of APP LXR agonist treatment achieved a reduction in processing and reduction of Aβ production induced increased bilirubin and alanine aminotransferase by LXR makes it a promising target for the treatment plasma levels, decreased mast cell infiltration and sup- of Alzheimer’s disease. pressed the inflammatory gene expression, thus ame- Cholesterol is responsible for maintaining vari- liorating an inflammatory liver condition92). ous neuronal functions in the brain. For example, cholesterol prevents β-amyloid accumulation. The LXR and the Skin final catalytic synthesis of cholesterol is carried out by 24-dehydrocholesterol reductase, an enzyme coded by Chronological aging and photoaging are the two the Selective Alzheimer’s disease indicator-1 (Sela- biological processes of skin aging. Chronological din-1) gene, the overexpression of which increases the aging, in which physiological changes occur in the de novo synthesis of cholesterol in neurons and has skin over time, results in dry, thin, wrinkled and easily been found to protect neuronal cells against oxidative injured skin. This process is characterized by cellular stress and β-amyloid accumulation, ultimately pre- senescence, leading to decreased keratinocyte prolifer- venting neuronal apoptosis. Recent studies have dem- ation, improper terminal differentiation, reduced neu- onstrated crosstalk between TR and LXR in the regu- tral lipid and collagen synthesis and an increased pro- lation of the Seladin-1 expression. Both TR and LXR duction of prostaglandin E2 [PGE2] and MMP as upregulate the Seladin-1 expression. However, in well as oxidative damage. Solar UV radiation damages mice, it has been demonstrated that LXR-α upregu- the skin, causing it to age prematurely, which results lates the Seladin-1 expression only under hypothyroid in the photoaging process involving epidermal kerati- conditions or in the presence of mutant TR-β, other- nocytes, dermal fibroblasts and infiltrating neutro- wise the Seladin-1 expression is predominately regu- phils. UV exposure induces the activation of activator lated by TR-β. In contrast, in humans, both TR-β protein 1 [AP-1] and nuclear factor-κB [NF-κB] in and LXR-α competitively bind to specific sites on the the skin, leading to the expression of matrix metallo- Seladin-1 promoter, thus upregulating the Seladin-1 proteinases [MMPs] and cytokines in keratinocytes, expression84, 85). which consequently damages the extracellular matrix. Repeated exposure results in wrinkle formation due to incompletely degraded collagen accumulation in the LXR and Inflammation dermis. In the skin, LXRs are expressed in keratino- A well-recognized driving force behind the devel- cytes [LXRα and -β in human keratinocytes and opment of atherosclerotic lesions is chronic inflamma- LXRβ in murine keratinocytes], and their natural tion. Inflammation plays a major role in both the ini- ligands have been shown to induce keratinocyte differ- tiation and progression of atherosclerotic lesion for- entiation and improve the epidermal barrier function. mation. As an anti-inflammatory , Topical treatment with an LXR ligand, T1317, signifi- LXR regulates the innate and adaptive immune cantly reduces UV-induced abnormal skin thickening responses, apoptosis and phagocytosis. In addition, in vivo in a dose-dependent manner. LXR is thus a LXR agonists demonstrate anti-inflammatory effects potential target for treating skin aging and photoaging in various animal models of inflammation, and LXRs problems93). antagonize the cytokine-mediated expression of proin- flammatory genes in macrophages via the transcrip- LXR and Adrenal Steroids tional silencing of proinflammatory transcription fac- tor nuclear factor (NF)-κB86). It has also been proven LXRα and LXRβ are both found in all three lay- that LXR activation inhibits the gene transcription of ers of the adrenal cortex (the zona glomerulosa, fascic- various inflammatory genes, including TNF-α, COX- ulata and reticularis), with a higher expression of 2, IL-1β, MMP-9 and iNOS87-89). Such effects are not LXRβ94). Human adrenal cell lines have been shown observed in LXR α/β knockout animals90). In one to possess LXR-responsive genes involved in adrenal experimental autoimmune encephalitis (EAE) mouse hormone biosynthesis. LXR activation has different in model, an LXR agonist suppressed the expression of vivo and in vitro effects on adrenal steroidogenesis. For IFN-γ, intercellular adhesion molecule-1(ICAM-1), example, the adrenal cortex contains multiple ste- Advance Publication LXR in Dyslipidemia andJournal other Diseases of Atherosclerosis and Thrombosis 7 Accepted for publication: February 10, 2014 Published online: April 1, 2014 roidogenesis enzymes, including: a) CYP11A1 (cho- mone production. Increased ACTH production in the lesterol side chain cleavage enzyme), which converts pituitary cancels the inhibitory effects of LXR activa- cholesterol to pregnenolone, the first step towards ste- tion on adrenal hormone biosynthesis. Therefore, roid hormone biosynthesis95), b) StAR (steroidogenic LXR agonists with BBB-penetrating properties have acute regulatory protein), which transports cholesterol different effects than those without, as LXR activates from the outer to inner mitochondrial membrane, ACTH production in the pituitary and increases adre- determining the availability of cholesterol for steroid nal steroidogenesis. hormone production, thus making it a rate-limiting enzyme96), and c) SF1 (), which LXR and Male Fertility is involved in the transcription of various steroido- genic enzymes, including CYP11A195). In addition to The cardinal functions of the testis are testoster- these steroidogenic enzymes, other genes in the adre- one production and spermatogenesis. Leydig and Ser- nal cortex are involved in lipid homeostasis, primarily toli cells are testicular cells. Leydig cells secrete testos- HMGCR, ABCA1 and LDL-R, which play a role in terone, while Sertoli cells provide structural and nutri- cholesterol biosynthesis, efflux and uptake, respec- tional support for developing germ cells101). tively. Furthermore, Leydig cells express LXRα, while In vitro LXR activation inhibits all steroidogenic Sertoli cells LXRβ, whereas germ cells express both genes in the adrenal cortex, thereby suppressing ste- LXRs. LXRα regulates basal testosterone synthesis and roid hormone production. Oxysterols, including is involved in the control of germ cell apoptosis. In 22R-hydroxycholesterol and 20S-hydroxycholesterol, contrast, LXRβ controls lipid metabolism in Sertoli are produced as intermediates during cholesterol con- cells by regulating cholesterol export, as well as germ version to pregnenolone by CYP11A1; these oxyster- cell proliferation. Moreover, both LXRs together regu- ols are well-recognized to be LXR activators. It has late ligand-induced steroidogenesis, fatty acid metabo- been suggested that thus activated LXR switches off lism and, surprisingly, the retinoic acid signaling path- the steroidogenesis pathway in the adrenal glands by way in the testis102). inhibiting various genes involved in steroidogenesis97). Spermatogenesis is maintained by a delicate bal- In contrast to that observed in the in vitro set- ance between cell proliferation, differentiation and ting, LXR activation in vivo increases the corticoste- death103, 104). It has been hypothesized that alterations rone level in the plasma in mice, which suggests that in these processes result in spermatogenic impairment LXR has a meddling effect on the HPA (hypothala- and thus infertility. Interestingly, proliferation and mus-pituitary-adrenal) axis. Several LXR-responsive apoptosis are altered in LXRβ- and LXRα-deficient genes are located in the pituitary gland, which mice, respectively, although these mice do not exhibit increases the ACTH (adrenocorticotropic hormone) any fertility problems. Indeed, in lxrβ−/− mice, a lower production upon LXR activation. 11β-HSD1 (11β rate of proliferation is associated with a compensatory -hydroxysteroid dehydrogenase type 1) enzyme in the decline in apoptosis. In contrast, among lxrα−/− mice, pituitary converts inactive glucocorticoid hormone to a higher level of apoptosis is associated with a com- active cortisol, while increased active cortisol signals pensatory increase in proliferation. These compensa- the feedback inhibition of ACTH in pituitary cells. tory effects may thus explain the normal fertility LXR activation therefore inhibits the 11β-HSD1 observed in single LXR knockout mice. Meanwhile, enzyme and thus conversion to active cortisol, remov- the lack of both LXRs leads to a dramatic decline in ing the stop signal on ACTH production 97, 98). proliferation and increase in apoptosis. This may be Recently, the effects of LXR in increasing ACTH pro- one explanation for the complete loss of germ cells, duction were confirmed in a study that proved that resulting in infertility102). LXR-α positively regulates the gene expression of Retinoic acid, a metabolite of vitamin A, medi- proopiomelanocortin (POMC), from which ACTH is ates the growth and development of vitamin A and cleaved, in the pituitary gland99). Further demonstrat- appears to play a vital role in various testicular func- ing the involvement of LXR-α, the mRNA levels of tions. It has been demonstrated that excess vitamin A LXR-α were found to be higher than those of LXR-β leads to the formation of testicular lesions and sper- in ACTH-secreting pituitary adenomas compared to matogenic disorders, whereas vitamin A deficiency that noted in other pituitary adenomas and normal induces the early cessation of spermatogenesis and pituitary tissues100). adversely affects testosterone production105). Further- ACTH binds to the ACTH-R (ACTH-receptor) more, the lack of LXR results in an increased RARα, in the adrenal cortex, where it induces steroid hor- RARβ and RALDH-2 expression, which consequently Advance Publication 8 Parikh etJournal al. of Atherosclerosis and Thrombosis Accepted for publication: February 10, 2014 Published online: April 1, 2014 induces retinoic acid signaling, as demonstrated by the aging, Alzheimer’s disease and male fertility. expression patterns of known RAR target genes, e.g. dmc1 and scp3, potentially causing spermatogenic dis- Conflicts of Interest orders. Moreover, lipid accumulation is observed in the Sertoli cells of rats with hypervitaminosis A106), None. suggesting a link between retinoids and lipids. 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Fig.1. Effects of LXR on lipids LXR activation increases the conversion of choles- terol (CH) in the liver to bile acid (BA) by induc- ing a higher expression of CYP7A1 enzymes, thus increasing BA excretion. In addition, LXR upreg- ulates the ABCG5/G8 expression on the basolat- eral membrane of intestinal cells, thereby causing the efflux of excess absorbed cholesterol. It also downregulates the NPC1L1 expression on the luminal membrane of intestinal cells and hence decreases cholesterol absorption. In macrophages, LXR activates the ABCA1 expression, thus stimu- lating the egress of excess deposited cholesterol from the cell.

Fig.2. LXR and TR crosstalk LXR and thyroid hormone receptor (TR) form heterodimers with RXR upon activation, which then bind to DNA-responsive elements, including TREs and LXREs. TREs and LXREs exhibit structural similarity, with a direct repeat of the basic nucleotide sequence AGGTCA spaced by four nucleotides (DR-4).