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Metabolism of HDL and its Regulation

D. Kardassis*, I. Mosialou, M. Kanaki, I. Tiniakou and E. Thymiakou

Laboratory of Biochemistry, Department of Basic Sciences, University of Crete Medical School and Institute of Molecu-

lar Biology and Biotechnology of Crete, Heraklion 71003, Greece

Abstract: Epidemiological studies have shown that low plasma levels of High Density Lipoprotein Cholesterol (HDL-C)

are associated with an increased risk for myocardial infarction. These studies suggested that by increasing HDL-C levels

one could reduce cardiovascular risk. However, emerging evidence from studies in animals and humans indicate that high

levels of HDL-C are not sufficient to confer atheroprotection but that the functionality of the HDL particles is equally im-

portant. The picture is complicated further by the finding that HDL functionality is compromised in patients with chronic

inflammatory diseases such as Coronary Artery Disease (CAD), diabetes and rheumatoid arthritis. Despite these obstacles,

HDL raising is still a promising strategy for the reduction of CAD risk. Low HDL-C can be caused by inactivating muta-

tions in apoA-I, ATP Binding Cassette Transporter A1 (ABCA1) or Lecithin-Cholesterol Acyl Transferase (LCAT) which

affect HDL biogenesis and maturation whereas high HDL-C can be caused by mutations in Cholesteryl Ester Transfer

Protein (CETP) or Scavenger receptor Class B Type I (SR-BI). Recent studies suggest that heterogeneity in HDL levels in

the population is polygenic in origin. One approach to raise plasma HDL-C is to increase the rate of HDL biosynthesis by

capitalizing on the mechanisms that control the transcription of genes that play key roles in HDL biogenesis. We review

some of the genetic and non-genetic factors that affect plasma HDL levels and functions and discuss the mechanisms that

regulate HDL metabolism at the level of gene transcription in the liver focusing on apoA-I, ABCA1 and apoM.

Keywords: ABCA1, apoA-I, apoM, atherosclerosis, gene transcription, HDL, HDL-based therapies, hepatocyte nuclear fac-tors, hormone nuclear receptors.

HDL METABOLISM: AN OVERVIEW

HDL metabolism begins in the liver and in the intestine. Lipid-free apolipoprotein A-I (apoA-I), which is produced and secreted by the above tissues, interacts physically and extracellularly with the membrane lipid transporter ATP Binding Cassette Transporter A1 (ABCA1) and this interac-tion promotes the transfer of phospholipids and cholesterol to apoA-I thus forming lipid-poor apoA-I [1]. These lipid-poor apoA-I particles acquire additional phospholipids and cholesterol from peripheral cells such as the macrophages of the arterial wall via interaction with ABCA1 and are con-verted first to discoidal pre- HDL and subsequently to spherical HDL, via the action of the enzyme LCAT (Leci-thin:Cholesterol Acyl Transferase). The recently identified apolipoprotein M (apoM) which associates with HDL as well as with LDL, also contributes to HDL remodeling as re-vealed by experiments in mice but the mechanisms still re-main unknown [2]. HDL particles can also be generated by similar mechanisms via the action of other apolipoproteins including apoE and apoA-IV as demonstrated in apoA-I

-/-

mice using adenovirus-mediated gene transfer [3, 4]. The functionality of these HDL-E or HDL-A-IV particles (which are mainly formed in the absence of apoA-I) and their con-tribution to HDL-mediated atheroprotection under physio-logical or pathological conditions is still a subject of

*Address correspondence to this author at the Department of Basic Sci-

ences, University of Crete Medical School, Heraklion 71003 Greece;

Tel: +30-2810-394549; Fax: +30-2810-394530; E-mail: [email protected]

investigation. Finally, -HDL particles are remodeled by the Scavenger Receptor class B type I (SR-BI) [5] which facili-

tates selective cholesterol ester uptake by the liver (for secre-

tion into the bile via the ABCG5/G8 transporters) or by the adrenal cells (for the synthesis of steroid hormones). While

the majority of bile acids are reabsorbed in the intestine, a

proportion is eliminated in the feces, thereby ridding the body of excess cholesterol [6]. Remodeling of HDL particles

is also facilitated by the ABCG1 transporter (and the SR-BI)

in macrophages which promotes the efflux of cellular choles-terol to mature spherical -HDL particles and by the hy-

drolysis of HDL lipids by lipases such as the lipoprotein li-

pase (LPL), the hepatic lipase (LIPC) and the endothelial lipase (LIPG) [7]. Exchange of lipids between HDL particles

and apoB-containing lipoproteins (VLDL, LDL) is facilitated

by the phospholipid transfer protein (PLTP) and the choles-terol ester transfer protein (CETP) regenerating the pre-

HDL particles that initiate another cycle of HDL maturation

and remodeling via the mechanisms described above [8]. Cholesterol from HDL can also be transported back to the

intestine via a process called TICE (Trans Intestinal Choles-

terol Efflux) [9]. Recently, the ecto-F1-ATPase was identi-fied as a novel receptor of apoA-I in the liver [10]. Binding

of apoA-I on ecto-F1-ATPase increases its hydrolase activity

and leads to enhanced HDL endocytosis whereas the purinergic P2Y13 receptor was identified as the target of

ADP produced by ecto-F1-ATPase [10]. (Fig. 1) is a sche-

matic overview of the HDL pathway, the proteins (apolipo-proteins, plasma enzymes, membrane transporters and recep-

wasim

Final

2 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Kardassis et al.

tors) involved, the physiologically relevant tissues and the

various functions that contribute to atheroprotection.

The population of HDL particles in plasma is very het-erogeneous in terms of size, shape and protein/lipid content. ApoA-I is the main protein component of all HDL particles, whereas apo - , apoE, apo -IV, apoM and apoC are only found in certain HDL sub-populations [11]. Proteomic studies identified more than 50 different proteins that are associated with HDL, some of which are proteins that are implicated in the anti-inflammatory functions of HDL [12, 13]. HDL lipids are also heterogeneous and include bioactive lipids such as phospho-sphingolipids, with sphingosine 1 phosphate (S1P) being the most physiologically important lipid molecule [14]. HDL also carries a great variety of quan-titatively minor but bioactive lipids such as oxysterols and lysolipids. Plasma HDL can be separated by two dimensional gel electrophoresis into discrete subpopulations: pre- 1, pre-

2, 4, 3, 2 and 1. The larger 1, 2 particles may be associated with protection from cardiovascular disease [11].

The pathway of HDL metabolism has been termed Re-verse Cholesterol Transport (RCT) and this refers to the abil-ity of HDL particles to transfer excess cholesterol from macrophages of the arterial wall to the liver for excretion to the feces thus contributing to atheroprotection. Although this is an attractive model that could account for the strongly negative association between plasma HDL cholesterol levels and risk for CAD, its value and especially the relationship between plasma HDL cholesterol and fecal cholesterol ex-cretion, has been challenged by findings in both mice and humans (reviewed in [15]). RCT can be measured experi-mentally both in vivo and ex vivo. For example, using a

whole body RCT assay it was shown that ligands of liver X receptors and overexpression of apoA-I are potent stimula-tors of RCT in mice without affecting plasma HDL choles-terol levels [16, 17]. The method of whole body RCT devel-oped by de Goma et al. (30) is using macrophages which are first loaded with radiolabeled cholesterol and subsequently are injected into the peritoneal cavity of mice. The accumu-lation of radiolabeled cholesterol in plasma, liver, and feces is then quantitated. An in vitro method to study RCT is the macrophage cholesterol efflux assay in which macrophages in culture are first loaded with radiolabeled cholesterol and then treated with purified apoA-I or reconstituted HDL (rHDL). The amount of radioactivity in the cells and the me-dium is then counted. The different approaches used to measure RCT in vitro and in vivo have been reviewed else-where [18]. The macrophage RCT assay has been proven to be valuable for the identi fication of proteins and lipids that play a role in the cholesterol efflux process, to evaluate the effect of drugs on HDL levels and in a clinical setting for the assessment of the functionality of HDL particles isolated from patients with diseases such as CAD, diabetes and rheumatoid arthritis.

HDL metabolism as described above can be regulated pharmacologically [19, 20]. Fibrates such as clofibrate and

gemfibrozil have been used for decades as anti-lipidemic

drugs but their beneficial effects on mortality is controversial [21, 22]. On average, fibrates increase HDL-C by 4.5% [22].

Their effects are mediated by the Peroxisome Proliferator

Activated Receptors (PPARs) which regulate the transcrip-tion of various genes including apoA-I as described below.

In a clinical study involving 234 patients with combined

hyperlipidemia, both fenofibrate (FF) and gemifibrozil (GF)

Fig. (1). HDL metabolism and functions. Schematic representation of the pathway of HDL metabolism. The scheme shows: a) the proteins

that play important roles in discrete steps of the pathway such as HDL biogenesis, remodeling and catabolism; b) the different sub-

populations of HDL particles that are formed (poorly lipidated apoA-I, pre , , 1- 4); c) the physiologically relevant organs or cell types

(liver, intestine, macrophages, adrenals, endothelium); and d) the functions of HDL in each organ or cell type that contribute to atheroprotec-

tion.

Metabolism of HDL and its Regulation Current Medicinal Chemistry, 2014, Vol. 21, No. 1 3

reduced triglycerides and increased HDL-C to a similar ex-

tent but only FF treatment increased apoA-I plasma levels

and this was in agreement with previous clinical trials [90, 93-95]. Niacin increases HDL-C levels by 15-30% but the

mechanisms that are responsible for this upregulation are not

very well understood [23]. Various models have been pro-posed based on ex vivo and in vivo studies such as niacin-

mediated inhibition of ATP synthase chain and HDL parti-

cles uptake by hepatocytes [24], increase in the expression of PPAR , Conserved Domain 36 (CD36) and ABCA1 [25, 26]

as well as reduction in hepatic expression and plasma levels

of CETP [27]. Statins typically increase HDL-C levels by approximately 5% to 10% [28]. Although the mechanism(s)

by which statins raise HDL-C remains unclear, by decreasing

the number of apoB-containing lipoproteins statins appear to reduce the rates of CETP-mediated cholesteryl ester transfer

to HDL [29]. Statins were also shown to increase apoA-I

gene transcription by inhibiting the activity of the small GTPase RhoA and by activating PPAR [30]. The concept

that CETP inhibition could be a promising HDL-C raising

therapy was based on the observation that gene mutations in CETP were shown to increase HDL-C and apoA-I in humans

with no evidence for premature atherosclerosis [31]. As dis-

cussed above, inhibition of CETP prevents the exchange of cholesteryl esters from HDL to VLDL/LDL particles and of

triglycerides from VLDL/LDL to HDL causing a disturbance

in HDL remodeling (Fig. 1). CETP inhibitors such as anace-trapib, dalcetrapib and torcetrapib were shown to cause a

drastic elevation in HDL-C levels in recent large clinical

trials such as DEFINE (138% increase), dal-OUTCOMES (31-40% increase) and ILLUMINATE (72% increase) re-

spectively but these studies failed to demonstrate a clinical

benefit by CETP inhibition. These studies have been recently reviewed [20]. Through an epigenetic mechanism (compet-

ing with proteins bound to acetylated lysines in chromatin)

the compound RVX-208 was shown to increase the produc-tion of apoA-I in hepatic cells [32]. However, in a recent trial

(ASSERT) this compound had a minor impact on HDL-C

levels (8% increase relative to placebo) and the primary out-come was not met [20].

At the transcriptional level, members of the hormone nu-

clear receptor superfamily and notably the Liver X Receptors (LXRs) and the Peroxisome Proliferator Activated Receptor

(PPARs) coordinate the expression of several genes in this

pathway (e.g. apoA-I, ABCA1, ABCG1, apoE, CETP, SR-BI) in response to cholesterol or fatty acid accumulation [19,

20, 33]. Additional hormone nuclear receptors and hepatic

transcription factors including Hepatocyte Nuclear factors 1, 4 and 3 also play coordinating roles in cholesterol metabo-

lism as revealed by knock out or overexpression studies in

mice. Their mechanisms of action are discussed extensively below. A novel mechanism of HDL regulation has recently

been revealed with the identification of microRNAs (miRs)

involved in lipid and lipoprotein metabolism at the posttran-scriptional level and this topic has been covered by recent

reviews [34-36]. Multiple genes in the HDL pathway have

been shown to be under control of miRs including those af-fecting HDL biogenesis, cellular cholesterol efflux, selective

cholesterol uptake and bile acid transport. HDL particles

have also been shown to carry miRs along with them in the

plasma suggesting that they may play novel roles in cell-cell

communication [37].

Most tissues are unable to biochemically degrade choles-terol and depend on efflux mechanisms via HDL to get rid of

excess cholesterol and maintain a normal cholesterol homeo-

stasis. Moreover, HDL exerts several protective effects on the endothelial cells of the arteries including inhibition of the

expression of adhesion molecules and monocyte chemoat-

tractant proteins such as MCP-1, induction of eNOS, inhibi-tion of inflammatory cytokine signaling and inhibition of

apoptosis [14, 38-40]. These effects were shown to be medi-

ated by the interaction of apoA-I with SR-BI and by the in-teraction of S1P with its receptors (S1PR1-3) on the endothe-

lial membranes (Fig. 1) [39, 41, 42]. HDL also protects LDL

from oxidation in the intima by enzymes secreted by the macrophages [43, 44]. Importantly, all or some of the above

functions of HDL are compromised in patients with CAD,

diabetes and chronic inflammation [45-47]. In addition to having dysfunctional HDL, patients with CAD or diabetes

are frequently characterized by dyslipidemia including high

LDL and triglycerides (TG) and low HDL [48]. It was re-cently shown that the direct effects of HDL on endothelial

cells isolated from patients with CAD and diabetes are mark-

edly altered when compared to HDL from healthy subjects. Patients with diabetes have low levels of HDL cholesterol.

HDL from these patients failed to stimulate endothelial cell

NO production and to promote endothelial repair in a carotid artery injury model in mice [49]. The molecular mechanisms

linking diabetes to low HDL levels are incompletely under-

stood. Insulin signals via a cascade of intracellular effectors including Insulin Receptor Substrate (IRS), Phosphatidyl

Inositol 3 Kinase (PI3K) and AKT [50]. Events downstream

of AKT involve the activation of Forkhead Box (Fox) tran-scription factors such as Foxo1 and Foxa2 in macrophages

and hepatocytes [51, 52]. Little is known about the effect of

insulin on macrophages. The few available studies show that HDL-mediated cholesterol efflux from macrophages is in-

hibited by insulin a finding that could be translated to an

enhanced foam cell formation in the atherosclerotic plaques of patients with hyperinsulinemia [53].

Moreover, HDL from patients with either stable CAD or

an acute coronary syndrome inhibited endothelial cell NO production and lost the ability to inhibit endothelial inflam-

matory activation as well as to promote endothelial repair in

vivo [54]. During acute or chronic inflammation, several changes occur in HDLs. As part of the acute phase response,

several plasma proteins carried in HDLs are decreased in-

cluding apoA-I, Paraoxonase (PON), LCAT, CETP, and PLTP. The levels of HDL-associated serum amyloid A

(SAA) and apoJ (clusterin) also increased several-fold in

these subjects. In agreement with these biochemical findings, the prevalence of atherosclerosis is increased in rheumatic

diseases, with the highest prevalence being in Systemic Lu-

pus Erythematosous (SLE), followed by Reumatoid Arthritis (RA) (reviewed in [55]).

Inflammatory bowel diseases (IBD) are a heterogeneous

group of chronic inflammatory intestinal disorders which may also lead to systemic/extra-intestinal manifestations

[56]. Several inflammatory mediators are involved in the


pathogenesis of both IBD and atherosclerosis [57]. Studies

have demonstrated that patients with IBD have a higher risk

of developing early atherosclerosis and increased carotid intima-media thickness when compared with the general

population [58]. Patients with IBD are characterized by

chronic inflammation which is associated with lipid abnor-malities including low levels of HDL cholesterol (HDL-C).

REGULATION OF HDL METABOLISM BY apoA-I:

STUDIES IN EXPERIMENTAL ANIMALS

Mutations in the genes that participate in the early steps of the HDL pathway such as the apoA-I, ABCA1 and LCAT

have a strong impact on HDL metabolism and affect greatly

the structure and the concentration of HDL in plasma. In humans, inactivating mutations in apoA-I cause familial

hypo-alpha lipoproteinemia by affecting one or more of the

functional properties of apoA-I which include: a) activation of the enzyme LCAT which is responsible for HDL choles-

terol esterification; b) interaction with ABCA1 and ABCG1

membrane transporters which is important for the transfer of cellular cholesterol to premature and mature HDL particles

respectively; and, c) interaction with SR-BI which facilitates

the selective HDL cholesterol uptake by hepatic and extra-hepatic cells [11].

Studies that were pioneered by V. Zannis group using adenovirus-mediated gene transfer of human apoA-I (wt and mutant) in apoA-I deficient mice were instrumental for the identification of residues in apoA-I that are critical for the above functions and for the functional characterization of natural or artificial mutation in apoA-I. These studies led to the identification of discrete steps where the pathway of bio-genesis of HDL was inhibited or impaired [1, 11, 59]. For example, two naturally occurring mutants, the apoA-I[L141A]Pisa and apoA-I[L159R]Fin were thoroughly char-acterized in mice using adenovirus-mediated gene transfer [60, 61]. Both mutations are localized inside the LCAT acti-vation domain and were anticipated to disrupt the interaction of apoA-I with LCAT and to inhibit HDL maturation to spherical particles. Human subjects hemizygotes for apoA-I I(L141R)Pisa had greatly decreased plasma apoA-I levels and near absence of HDL cholesterol, a profile very similar to Tangier patients [62]. First, using in vitro assays it was shown that both mutants had normal ability to promote ABCA1 mediated cholesterol efflux but greatly compro-mised capacity to activate LCAT. Adenovirus-mediated gene transfer showed that apoA-I

-/- mice expressing either of the

two mutants had greatly reduced HDL cholesterol and apoA-I levels. By two-dimensional gel electrophoresis of the plasma it was shown that only pre- and small size 4-HDL subpopulations were formed in these mice [60, 61]. Impor-tantly, co-infection of apoA-I

-/- mice with adenoviruses ex-

pressing either of the two apoA-I mutants and human LCAT increased plasma HDL cholesterol and apoA-I, generated spherical HDL and restored the normal pre- - and -HDL sub-populations [60, 61]. The same methodology was used to characterize a large panel of bioengineered mutations in apoA-I and to provide novel insights into the structure-function relationship in apoA-I and other apolipoproteins such as apoE [1, 59]. In order to assess the effect of the two naturally occurring mutations (Pisa and Fin) in atherosclero-

sis, we recently generated transgenic mice expressing either wt or mutant human apoA-I in a mouse apoA-I

-/- background.

Compared with the adenovirus system, transgenic mice offer unique opportunities to study not only short term but also the long term effects of apoA-I dysfunction in cholesterol me-tabolism and in atherosclerosis following a high fat diet. Our preliminary studies show that the lipid and lipoprotein phe-notype of these Tg mice mimics the phenotype in humans with the same apoA-I mutations (Tiniakou et al., unpub-lished).

Several lines of evidence coming from studies in humans and experimental animals support the notion that plasma levels of HDL cholesterol are not a reliable predictor of CAD risk and premature atherosclerosis. For example, Tang-ier patients with inactivating mutations in ABCA1 have total absence of HDL in plasma but the predisposition to athero-sclerosis is not as severe as anticipated and the same holds true for patients with mutations in the LCAT gene [15]. Re-cently, this notion was supported by the findings of a study in which three non-synonymous mutations in apoA-I (V11X, L144R, A164S) were identified by sequencing the coding region and exon-intron boundaries of the gene in 190 indi-viduals with the 1% highest and 1% lowest HDL levels [63]. In the Copenhagen City Heart Study, twenty four, four and one unrelated individuals (carrier frequencies: 0.23%, 0.04% and 0.01%) were heterozygous for A164S, L144R and V11X, respectively. Heterozygotes for A164S had normal levels of HDL and apoA-I whereas in the other two het-erozygotes plasma HDL and apoA-I levels were reduced by 40-50%. Using adenovirus-mediated gene transfer in mice we showed that mice expressing the A164S mutation had normal lipid and lipoprotein profile in plasma whereas mice expressing the L144R mutation had very low total choles-terol levels and decreased cholesteryl ester-to-total choles-terol ratio (CE/TC) indicating reduced LCAT activity [63]. Two-dimensional gel electrophoresis showed that the mutant apoA-I L144R promoted the formation of less mature, lipid-poor pre- and 4 HDL particles whereas WT apoA-I and the mutant apoA-I A164S both promoted the formation of nor-mal pre- and a HDL subpopulations ( 1, 2, 3 and 4). Simultaneous treatment of mice with the adenovirus express-ing apoA-IL144R and human LCAT normalized the total cholesterol level and the CE⁄TC, normalized the levels and the distribution of apoA-I in the HDL, restored normal pre- and HDL subpopulations and promoted the formation of spherical HDL particles. In summary, this was the first report of a mutation in apoA-I which predicts an increased risk of ischemic heart disease, myocardial infarction and total mor-tality in the general population independently of HDL cho-lesterol levels [63].

GENETIC CAUSES OF HDL HETEROGENEITY

Mutations in the three genes described (apoA-I, ABCA1, LCAT) account for only 10% of the heterogeneity in plasma HDL levels [64]. Genome Wide Association Studies (GWAS) identified additional genetic loci that could influ-ence plasma HDL levels. One of the genes that was identi-fied in GWAS was the gene encoding for GALNT2 (ppGal-NAc-T2). SNPs in intron 1 of GALNT2 were found to be associated with plasma HDL-C and triglyceride levels [65]. The enzyme belongs to a family of ppGalNAc transferases


comprising 20 members in humans all catalyzing the transfer of GalNAc residues onto proteins, and thereby initiating mucin-type O-glycan synthesis on threonine and/or serine residues. It was shown that hepatic overexpression and si-lencing of GALNT2 in mice reduced and increased HDL-C levels, respectively [66]. Elegant work by the group of J. A. Kuivenhoven in Amsterdam showed that mutations in GALNT2 cause abnormal glycosylation of apoC-III which is an inhibitor of Lipoprotein Lipase (LPL) [67]. The abnor-mally lipidated apoC-III binds to LPL less efficiently and as a consequence LPL is more active to hydrolyze triglycerides in lipoproteins thus decreasing triglyceride rich lipoproteins in plasma and increasing HDL [67]. In a very recent interest-ing study by the same group it was shown that HDL hetero-geneity in the human population has a polygenic origin [68]. They sequenced 195 genes with either established or impli-cated roles in lipid and lipoprotein metabolism plus 78 lipid-unrelated genes in patients with HDL-C <1st (n=40) or >99th (n=40) percentile and the results were compared with 498 individuals representative of the Dutch general popula-tion and 95 subjects with normal HDL-C. The extreme HDL cohort carried more rare non-synonymous variants in the lipid gene set than both the general population and normal HDL-C cohorts. In the extreme HDL cohort, however, there was enrichment of rare non-synonymous variants in the lipid versus the control gene set and 70% of the lipid-related vari-ants altered conserved nucleotides. In the most extreme cases, individuals were found to carry 12 or 14 rare variants simultaneously some of which are predicted to be potentially deleterious. This study indicated that even very extreme HDL-C phenotypes can have a polygenic origin. This com-plexity may explain why efforts to identify new HDL genes through linkage analyses have not been successful. Obtain-ing significant logarithm of odds (LOD) scores can be diffi-cult in the face of genetic heterogeneity which characterizes the extreme HDL-C cohorts.

As discussed above, a promising strategy to increase HDL levels without compromising HDL functions is to up-regulate apoA-I and ABCA1 genes in the liver which is the major organ of HDL synthesis contributing approximately 70% to plasma HDL levels. In the following paragraphs we will review the current knowledge on the mechanisms that regulate these two genes at the transcriptional level. The regulation of the apoM gene in the liver will also be re-viewed in light of recent findings that apoM is the sole car-rier of the bioactive phospholipid sphingosine 1 phosphate in HDL which is an important component of HDL-mediated atheroprotection [42]. The role of certain transcription fac-tors in the regulation of HDL metabolism in general will also be discussed.

TRANSCRIPTIONAL REGULATION OF THE apoA-I GENE

The hypothesis that apoA-I overexpression could posi-tively influence plasma HDL-C concentration has been vali-dated experimentally in transgenic mice expressing human apoA-I under homologous or heterologous regulatory se-quences. These mice have significantly elevated levels of HDL-C containing human apoA-I [69, 70]. These “human-ized” apoA-I transgenic mice are valuable tools for the study of apoA-I gene regulation in vivo. Furthermore, it was dem-

onstrated that overexpression of apoA-I in apoE KO or LDLR KO mice via transgene- or adenovirus-mediated gene transfer reduced atherosclerosis development confirming the anti-atherogenic role of apoA-I up regulation [71-73].

In humans, the apoA-I gene is expressed abundantly in liver and intestine, and to a lesser extent in other tissues [74]. Previous studies had established that several members of the hormone nuclear receptor superfamily of transcription fac-tors are major determinants of hepatic and intestinal apoA-I gene expression. Hormone nuclear receptors belong to a su-perfamily of transcription factors that are activated by steroid hormones (estrogens, androgens, glucocorticoids etc), reti-noids, thyroids and products of intermediate metabolism such as bile acids, fatty acids, cholesterol derivatives etc [75]. Certain members of this family do not need ligand binding to regulate transcription and are classified as “or-phans”. Nuclear receptors are structurally highly conserved. The main structural features of hormone nuclear receptors are: an N-terminal transactivation domain called Activation Function 1 (AF-1), a conserved DNA binding domain that contains two zinc fingers, and a second transactivation do-main called AF-2 located inside to the ligand binding domain (LBD) [76]. Nuclear receptors can bind to Hormone Re-sponse Elements (HREs) on the promoters of target genes either as homodimers or as heterodimers with the Retinoid X Receptor (RXR). The HREs consist of direct repeats (DRs), inverted repeats (IRs) or palindromic repeats (PRs) of the consensus sequence 5’ AG(G/T)TCA 3’. The repeats are separated by 1, 2, 3, 4 or 5 nucleotides and are designated DR1, DR2 etc (for the direct repeats), IR1, IR2 etc (for the inverted repeats) and PR1, PR2 etc (for the palindromic re-peats) [77, 78]. (Fig. 2) shows the mechanism of action of hormone nuclear receptors, their basic structural features and the consensus sequence of a typical HRE.

The proximal human apoA-I promoter spanning a region of 250 bp upstream of the transcription start site of the gene is sufficient to drive liver-specific gene expression both in cell cultures and in mice [79-81]. This promoter region is rich in nuclear factor binding sites and responds to various intracellular as well as extracellular ligands [82, 83]. Promi-nent role in the regulation of the apoA-I promoter play two Hormone Response Elements (HREs) located at positions -210/-190 and -132/-120 that bind members of the hormone nuclear receptor superfamily in a competitive manner (Fig. 3A) [81]. One of the factors that plays a prominent role in apoA-I gene regulation and HDL metabolism in general is Hepatocyte Nuclear Factor 4 (HNF-4) [81].

THE ROLE OF HEPATOCYTE NUCLEAR FACTOR 4

IN HDL METABOLISM

HNF-4 was discovered as a factor present in rat liver nu-clear extracts that interacts with the promoters of various liver-specific genes including transthyretin and apo-CIII [84]. In the adult organism, HNF-4 is expressed in the liver, kidney, intestine and pancreas [84]. In humans, heterozygous mutations in the HNF-4 gene are associated with an early-onset form of type II diabetes called maturity onset diabetes of the young 1 (MODY 1) [85]. HNF-4 binds as a homodi-mer to the promoters of various genes involved in lipid and lipoprotein metabolism.


Experiments in genetically manipulated mice have illu-minated the important role of HNF-4 in the generation and the maintenance of hepatic differentiation as well as for lipid and lipoprotein metabolism. Total disruption of the HNF-4 gene in mice leads to an embryonic lethal phenotype due to impairment of endodermal differentiation and gastrulation [86]. This early developmental arrest was rescued by the complementation of the HNF-4

-/- embryos with a tetraploid

embryo-derived wild type visceral endoderm [87]. Analysis of the rescued mice showed that the expression of the apoA-I gene as well as of other apolipoprotein genes, shown previ-ous to be regulated by HNF-4 including apoA-II, apoB, apoC-III and apoC-II, was abolished confirming the cell cul-ture data [87]. Experiments in mice in which the HNF-4 gene was disrupted in the adult liver using Alb-Cre revealed that HNF-4 is essential not only for the establishment but also for the maintenance of hepatic differentiation [88]. HNF-4 loss caused an increase in liver weight mainly due to the accumu-lation of lipid droplets. This fatty liver phenotype could be accounted for by the loss of the expression of both apoB and microsomal triglyceride transfer protein (MTTP), two pro-teins that play key roles in VLDL secretion. Lipid and lipo-protein analysis of plasma of these mice revealed a dramatic reduction in total cholesterol, HDL cholesterol and triglyc-erides and a dramatic increase in the concentration of bile acids [88]. Furthermore, FPLC analysis showed that HDL cholesterol from the HNF-4 Liv KO mice eluted later than

that from controls indicative of the presence of smaller HDL populations. Interestingly, the expression of the two essential genes for HDL biogenesis namely apoA-I and ABCA1 was not affected in the livers of the HNF-4 Liv KO suggesting that the reduction in the plasma HDL levels was the result of altered HDL remodeling rather than reduced biosynthesis. In agreement with this, the expression levels of the HDL recep-tor SR-BI was dramatically increased [88]. The increased levels of bile acids in the plasma could be accounted for by altered levels in the expression of genes of bile acid traffick-ing including bile acid export pump (BSEP) and sodium taurocholate co-transporter (NTCP). It was concluded that the main role of HNF-4 is to promote the net efflux of cho-lesterol from the liver and to control bile acid trafficking. It was subsequently shown that HNF-4 regulates the expression of the bile acid transporters ABCG5 and G8 in a coordinated fashion [89].

THE ROLE OF PEROXISOME PROLIFERATOR

ACTIVATED RECEPTOR A IN HDL METABOLISM

Studies in transgenic mice expressing the human apoA-I gene under its own regulatory sequences and clinical studies in humans have shown that fibrates exert a positive effect on apoA-I gene transcription as well as on plasma HDL levels [90]. The increase in human apoA-I gene transcription by fibrates is mediated by peroxisome proliferator-activated receptor (PPAR ) which binds to the HREs on the proxi-

Fig. (2). Mode of action of hormone nuclear receptors. Panel A: schematic representation of the mechanism of activation and function of a

sub-family of hormone nuclear receptors, the heterodimers of Retinoid X receptors (RXRs) which includes several members such as the re-

ceptors for retinoic acid (RAR), thyroid hormone (T3), oxysterols (LXRs), fatty acids (PPARs) and bile acids (FXRs).All these receptors

bind to DNA as obligatory heterodimers with RXR. Panel B: Domain structure of a typical hormone nuclear receptor. The structure is modu-

lar and consists of a n-terminal ligand-independent Activation Function 1 (AF-1) domain, a DNA binding domain of the zinc finger type and

a composite domain consisting of the ligand binding, the dimerization and the Activation Function 2 (AF-2) domain. Nuclear receptors bind

to DNA elements called Hormone Response Elements (HREs) in the promoters of target genes consisting of direct, inverted or palindromic

repeats. The consensus sequence of a DR (Direct Repeat) HRE is shown at the bottom. Each receptor in the heterodimer occupies one half-

repeat and the type of the repeat determines binding specificity among nuclear receptors (for instance DR4 is preferred by LXR/RXR het-

erodimers).


mal apoA-I promoter as a heterodimer with RXR (Fig. 3A) [81, 91]. In vivo this was confirmed by experiments in mice that express human apoA-I under the control of its own pro-moter but lack the expression of PPAR . When these mice were given fenofibrate (FF) or gemfibrozil (GF) for 17 days an increase in plasma HDL-C levels was observed by FF and to a lesser extent by GF only in the mice that express en-dogenous PPAR [92]. The fibrate-treated mice had larger HDL particles possibly due to the up-regulation of phosphol-ipid transfer protein and down-regulation of SR-BI [92]. Interestingly, the apoA-I gene cannot be up-regulated by fibrates in rodents due to a 3 base pair difference in the PPRE rendering the rodent apoA-I PPRE non-functional [93]. In line with the above findings, liver-specific inactiva-tion of the PPAR heterodimer partner Retinoid X Receptor

(RXR ) gene in mice was associated with increased ex-pression of the apoA-I gene [94].

LIVER RECEPTOR HOMOLOGUE 1 AND SMALL

HETERODIMER PARTNER

In addition to HNF-4 and PPAR , the HREs of the proximal human apoA-I promoter bind apoA-I Regulatory Protein 1 (ARP-1) and Liver Receptor Homologue 1 (LRH-1) which repress and activate the apoA-I promoter, respec-tively (Fig. 3 ) [95, 96]. LRH-1 is a member of the Ftz-F1 subfamily of the hormone nuclear receptor gene superfamily [97]. It belongs to the orphan nuclear receptors, although its ligand-binding pocket can bind various phospholipids. It interacts with its cognate target sequence 5’-(Py)CAAGG(Py)C(Pu)-3’as a monomer [98]. It is highly expressed in liver, intestine, pancreas and ovary [98-100]. In the liver, LRH-1 plays a key role in cholesterol homeostasis, through the control of the expression of genes that are impli-cated in bile acid biosynthesis and enterohepatic circulation (CYP7A1, CYP8B1, ABCG5/8) [99, 101], reverse choles-terol transport (SR-BI, apoA-I) and HDL remodeling (CETP) [96, 100, 102, 103] as well as in early development through the control of the expression of genes that are in-volved in embryonic development (Oct4) and of the hepatic nuclear factors HNF-3 , HNF-4 and HNF-1 [104, 105].

OTHER NUCLEAR RECEPTORS IN apoA-I REGU-

LATION

The two HREs of the apoA-I promoter also mediate the response of apoA-I to thyroids, retinoids and bile acids via heterodimers of Retinoid X Receptor (RXR ) with thyroid hormone receptor (TR ), Retinoic acid Receptor (RAR ) and farnesoid X receptor (FXR ), respectively (Fig. 3A) [106, 107]. Although retinoids activate apoA-I gene expres-sion, thyroids have dual effects on apoA-I promoter activity whereas bile acids inhibit apoA-I gene expression [81, 108-110]. It was shown that in response to bile acids FXR down-regulates apoA-I gene transcription both by binding to the apoA-I HRE as well as by inducing small heterodimer part-ner (SHP) which in turn represses the activity of LRH-1 that binds to the same region [96].

Ligands of the vitamin D receptor (VDR) were also shown to affect negatively apoA-I gene expression in hepatic cells [111]. In VDR KO mice serum HDL-C levels were 22% higher and the mRNA levels of ApoAI were 49.2%

higher compared with WT mice. The mechanism by which VDR ligands affect HDL levels remains unclear [112].

THE ROLE OF THE DISTAL ENHANCER IN apoA-I

GENE EXPRESSION IN THE LIVER AND THE IN-TESTINE

The strength of the proximal apoA-I promoter in hepatic

and intestinal cells was increased several-fold when it was

fused to a 200 bp fragment upstream of the adjacent apoC-III gene [113]. This transcriptional enhancer domain contains

HREs that bind HNF-4 and different combinations of ligand-

dependent nuclear receptors as well as Specificity protein 1 (Sp1) [113, 114]. The contribution of the HREs and the Sp1

sites in the tissue-specific expression of the apoA-I gene in

vivo was addressed using transgenic mice expressing either wild type or mutant forms of the proximal promoters and the

enhancer [69, 115, 116]. A mutation in one of the two HREs

of the enhancer (element I4) abolished the intestinal expres-sion and reduced the hepatic expression of apoA-I to 20% of

the WT control. Mutations in the two HREs of the proximal

apoA-I promoter reduced the hepatic and intestinal expres-sion of the apoA-I gene to approximately 15% of the WT

control. Finally, combined mutations in the two HREs of the

proximal apoA-I promoter and the HRE of the enhancer abolished the intestinal and hepatic expression of the apoA-I

gene [117]. Studies in cell cultures established that HNF-4

and Sp1 factors are both required for the apoA-I pro-moter/enhancer communication by physically interacting

with each other and forming transcriptional complexes in

order to facilitate the recruitment of the basal transcriptional machinery [118].

THE ROLE OF HEPATOCYTE NUCLEAR FACTOR

3 /FOXA2 IN HDL GENE REGULATION

The proximal apoA-I promoter contains one element that binds Hepatocyte Nuclear Factor 3 /Foxa2 [119, 120]. Foxa2 is a liver-enriched forkhead box transcription factor with a winged-helix DNA binding domain that binds to its consensus DNA sequence 5’- T(G/A)TTT(A/G)(C/T)T-3’ as a monomer [121, 122]. In response to insulin signaling, Foxa2 transcriptional activity is inhibited through a mecha-nism that involves phosphorylation at threonine residue 156 by AKT/PKB kinase and nuclear exclusion [123, 124].

Foxa2 is expressed mainly in the liver and also in pan-creas, stomach, intestine and lung [125] and plays important roles in fetal development. Mice homozygous for a Foxa2 null mutation die in early embryogenesis shortly after gastru-lation, show defects in node development and lack a noto-chord which leads to secondary defects in neural and foregut morphogenesis [126, 127].

In addition to its critical role in embryonic development, Foxa2 also regulates lipid and glucose homeostasis in liver and pancreatic cells of adult mice [124, 128]. In the liver, Foxa2 has been shown to enhance fatty acid oxidation, keto-genesis and increased secretion of very low density lipopro-tein and high density lipoprotein (VLDL and HDL) during fasting by activating the expression of key enzymes of these pathways [2, 124, 129, 130]. Using liver-specific gene abla-tion, it has been shown that Foxa2 plays an important role in


bile acid homeostasis because it prevents cholestatic liver injury and ER stress after cholic acid diet by regulating mul-tiple genes involved in bile acid metabolism [131].

Several groups have used genome wide analysis such as ChIP-on-chip (chromatin immunoprecipitation followed by microarrays) and ChIP-Seq (chromatin immunoprecipitation followed by sequencing) to investigate the in vivo binding sites of Foxa2 in the adult mouse liver or liver-derived cells (HepG2).

Wolfrum et al., generated gene chip expression profiles from livers of wild type mice that had been infected with adenoviruses expressing either GFP or a constitutively active form of Foxa2 that cannot be phosphorylated by AKT kinase (Foxa2-T156A) and they observed robust increases in the expression of genes involved in HDL metabolism (SR-BI, HNF-4 ), triglyceride degradation (LPL, LIPC), -oxidation, ketogenesis and glycolysis [124]. In another ChIP-Seq analysis, approximately 11,000 Foxa2 binding sites were identified in the adult mouse liver. The majority of these binding sites were localized within 10 kb upstream of the transcription start sites of genes and within the first introns. It was found that 43.5% of genes expressed in the liver were also associated with at least one Foxa2 binding site, includ-ing genes of HDL metabolism (apoA-I, apoB, HNF-4 , LPL), with the greatest enrichment of 30-fold for apoA-II [132].

Studies by the group of K. Kaestner have used ChIP-Seq and Chip-on-chip analysis in samples of livers of wild type and hepatocyte specific Foxa2 knockout mice (Foxa2loxP/loxPAlfp-Cre) [131, 133, 134]. They identified 574 enhancer and promoter regions, corresponding to 484 unique genes, occupied by Foxa2 and clustered in categories of genes involved in lipid and steroid metabolism such as ABCA1, ABCC2, apoA-I, apoE, LIPC, LDLR [131]. Fur-thermore, in Foxa2-deficient mice fed with CA diet, the ex-pression of genes involved in lipid metabolism-transport (ABCG8, LCAT, LIPC, LPL, PLTP) was significantly re-duced where as the expression of apoB gene was increased [133].

ChIP-Seq studies in HepG2 cells identified binding sites for Foxa2 in genes associated with HDL metabolism such as apoA-I , apoA-II, apoA-IV, hepatic lipase (LIPC), LDL re-ceptor (LDLR), phospholipid transfer protein (PLTP) and nuclear receptors HNF1 , HNF1 , HNF4 and RXR thus confirming the role of Foxa2 in HDL metabolism [122, 135].

TRANSCRIPTIONAL REGULATION OF THE ABCA1

GENE BY LXRs

The gene encoding the ABCA1 is expressed in the liver, small intestine, macrophages, kidney and various other tis-sues [136-139]. ABCA1 is an important regulator of HDL biogenesis in the liver and facilitates the removal of excess cholesterol from macrophages. Thus, studies on the control of its expression have been mostly focused on these two physiologically relevant cell types.

The expression of the ABCA1 gene is controlled primar-ily by the levels of intracellular cholesterol. When the levels of cellular cholesterol increase, certain products of choles-terol hydroxylation such as 22(R)-hydroxycholesterol, 24(S)-

hydroxycholesterol, 24(S),25-epoxycholesterol, 20(S)-hydroxycholesterol, and 27-hydroxycholesterol act as natural agonists of Liver X Receptors (LXRs). LXRs along with SREBPs are the key cholesterol sensing transcription factors in the cell and as a consequence, most of their target genes have major roles in intracellular and plasma cholesterol ho-meostasis [33, 140-142]. There are two LXR isoforms, LXR and LXR , sharing almost 80% amino acid identity in their DNA-binding and ligand-binding domains. LXRs bind to LXR response elements (LXREs) of the DR4 type (direct repeats separated by 4 nucleotides) as obligatory heterodi-mers with the retinoid receptor RXR [141]. Synthetic LXR ligands such as T0901317 and GW3965 are currently used widely as tools for the assessment of LXR signaling and for the identification of novel LXR target genes [143, 144]. Experiments in mice have shown that synthetic LXR ligands up-regulate ABCA1 gene transcription, increase plasma HDL levels and promote cholesterol efflux from macrophage cultures [145]. However, LXRs are strong inducers of he-patic lipogenesis due to the transcriptional up-regulation of SREBP-1c gene [146, 147]. Thus, treatment of mice with synthetic LXR agonists elevates triglyceride levels in the liver and in the plasma [143]. On top of their role as choles-terol sensors, in macrophages LXRs have been shown to suppress inflammatory signalling pathways via a mechanism of transrepression [148]

In HDL metabolism LXRs play an important role by co-ordinately regulating the expression of several genes includ-ing ABCA1, ABCG1, SR-BI, PLTP and CETP [33]. Given the strong impact of LXRs on Reverse Cholesterol Transport [16], it should be anticipated that natural or synthetic LXR agonists should increase HDL-C levels in plasma. However, LXRs also activate the transcription of CETP leading to a cholesterol transfer from HDL to LDL and VLDL particles and a reduction in HDL concentration [149]. Therefore, stud-ies in mice on the effects of LXR agonists on the lipid and lipoprotein profiles have to be interpreted with caution be-cause mice lack CETP. Thus, T0901317 was able to elevate HDL-C levels in wild-type mice while it decreased HDL-C levels in CETP transgenic mice and cynomolgus monkeys [149-151].

The ABCA1 promoter upstream of exon 1 contains a LXRE of the DR4 type at position -62/-47 (Fig. 3B) [152]. In addition to serving as a LXRE, the same DR4 element of the ABCA1 promoter has been shown to bind RXR/retinoic acid receptor (RAR) and RXR/thyroid hormone receptor (T3R) heterodimers and to mediate the activation or the re-pression of ABCA1 transcription by retinoic acid and thyroid hormone, respectively [153, 154]. Recently, it was shown that niacin treatment of HepG2 cells caused an increase in ABCA1 promoter activity and that the LXRE was required for niacin-mediated ABCA1 up-regulation [26]. The mecha-nism by which niacin activates ABCA1 gene transcription was not studied further but based on previous findings [25] it was hypothesized that niacin treatment increases oxysterols which subsequently activate LXRs. If this hypothesis is cor-rect, it would be anticipated that niacin should have a global impact on the regulation of LXR target genes.

We have shown that the oxysterol/retinoid-induced tran-scription of the ABCA1 gene in hepatic cells is modulated


by the ubiquitous transcription factor Sp1 that binds to the proximal ABCA1 promoter, adjacently to the LXRE [155]. Sp1 inhibition by mithramycin blocked the induction of the ABCA1 gene by oxysterols/retinoids as well as the ligand-inducible recruitment of Sp1 and RXR /LXR heterodimers to the ABCA1 promoter. LXR and Sp1 were shown to in-teract physically both in vitro and ex vivo. This study re-vealed a novel mechanism of regulation of the human ABCA1 transporter which involves synergistic interactions between oxysterol/retinoid-inducible hormone nuclear recep-tors and the transcription factor Sp1 [155]. An additional but not thoroughly characterized LXRE is present at the intron 1 promoter of the ABCA1 gene [156].

Whereas cholesterol accumulation leads to ABCA1 gene induction via the oxysterol/LXR pathway, cholesterol deple-tion which leads to activation of the sterol regulatory element binding protein 2 (SREBP-2), represses the activity of the ABCA1 promoter via binding of SREBP-2 to an E-box ele-ment [103]. The same E-box serves as a binding site for Up-stream Stimulatory Factors (USFs) 1 and 2, which also re-press the ABCA1 promoter [104, 105]. The ABCA1 pro-moter also contains a binding site for the zinc finger protein ZNF202 (GT box), which acts a repressor of ABCA1 gene expression (Fig. 3B) [106, 107].

MECHANISMS OF apoM GENE REGULATION IN

THE LIVER

Apolipoprotein M (apoM) is a glycoprotein that belongs to the lipocalin protein family [157, 158]. It is synthesized and secreted primarily by the liver and kidney and associates with HDL particles through its N-terminal signal peptide which is retained following secretion [159, 160]. In mice, silencing of the endogenous apoM gene via small interfering RNA technology was associated with the loss of pre- -HDL particles and the formation of larger HDL particles suggest-ing that apoM may play a role in HDL maturation [161]. Overexpression of apoM in LDL receptor knockout mice via adenovirus reduced atherosclerosis development [2, 162]. Furthermore, apoM-containing HDL particles isolated from human plasma or apoM transgenic mice have been shown to be more resistant to oxidation and more efficient in protect-ing against LDL oxidation as well as stimulating cholesterol efflux from macrophages [162, 163].

The expression of apoM in the liver is primarily con-trolled by Hepatocyte Nuclear Factor-1 (HNF-1 ) [164]. HNF-1 belongs to the homeodomain transcription factor family and regulates gene transcription via binding to target sites that are identical or homologous with the consensus sequence 5’-ATTAACCATTAA-3’ as a homodimer or het-erodimer with its isoform vHNF-1/HNF-1 [165]. HNF-1a is primarily expressed in the liver, intestine, kidney and the exocrine pancreas where it plays important roles in devel-opment, cell differentiation and metabolism [165]. In hu-mans, mutations in HNF-1a cause maturity-onset diabetes mellitus of the young type 3 (MODY 3) that is characterized by impaired insulin secretion due to defects in pancreatic cells [166].

HNF-1-/-

mice have complete absence of apoM in plasma whereas the concentration of other apolipoproteins is either similar (apoA-II, apoB, apoC) or increased (apoA-I, apoE)

compared to wild type mice but this was not due to the ab-sence of apoM as restoration of apoM gene expression in the liver via adenovirus-mediated gene transfer could not rescue the abnormal apolipoprotein profile [161].

HNF1-/-

mice exhibit hepatic, pancreatic and renal dys-functions such as liver enlargement and extensive hypercholesterolemia and hyperphenylalaninemia, defective bile acid transport and increased bile acid and hepatic choles-terol synthesis, impaired HDL metabolism and glucose homeostasis [165]. Shih et al. analyzed the plasma lipoprotein profile of HNF-1

-/- mice and found that plasma

cholesterol was distributed in the HDL fraction and to an abnormal apoE-enriched HDL fraction that was identified as HDLc or HDL1 (large HDL fraction) suggesting that this abnormal lipid profile in HNF-1

-/-mice may be due to the

lack of apoM [167].

HNF-1 regulates apoM gene expression through direct binding to a conserved DNA element located in the proximal apoM promoter region between nucleotides -55 to -41 in humans and -103 to -88 in mice [164]. Interestingly, this site overlaps with a DNA sequence element that is homologous to the consensus binding site for members of the pro-inflammatory transcription factors c-Jun and JunB (Fig. 3C).

ApoM gene expression is negatively regulated during in-

flammation and the acute phase response. Treatment of hepa-toma cells with stimuli that produce systemic inflammation

such as lipopolysacharide (LPS) or with pro-inflammatory

cytokines (Tumor Necrosis Factor or Interleukin 1 ) was shown to decrease apoM mRNA levels in the liver [168].

These data indicate that apoM, similarly to other apolipopro-

teins such as apoA-I or apoC-III is a negative acute response protein and its levels decrease during infection and inflam-

mation.

We showed recently that the HNF-1 binding element in

the proximal human apoM promoter serves as a dual speci-

ficity regulatory element that mediates activation or repres-sion of apoM promoter activity by HNF-1 and Jun proteins

respectively in hepatic cells (Fig. 3C) [169]. The experimen-

tal evidence that supports the dual functionality of this ele-ment of the apoM promoter was the following: a) Jun pro-

teins repressed apoM promoter activity only in cells express-

ing HNF-1 . Jun-mediated repression was abolished in HepG2 cells in which endogenous HNF-1 expression had

been silenced by specific shRNAs; b) the mouse apoM pro-

moter does not contain a functional AP-1 site due to single nucleotide difference but it retains a functional HNF-1 re-

sponsive element; c) competition experiments showed that

binding of Jun proteins and HNF-1 to the apoM promoter is mutually exclusive d) chromatin immunoprecipitation assays

established that AP-1 activation leads to the recruitment of c-

Jun and Jun B proteins to the proximal apoM promoter with the simultaneous displacement of HNF-1.

Interestingly an identical mechanism of Jun-mediated re-pression of promoter activity via dual specificity AP-1/HNF-1 responsive element operates on the promoter of the human apolipoprotein A-II gene, the expression of which is also under negative regulation during the acute phase response [169]. Furthermore, Jun proteins have been shown to repress the activity of the human apolipoprotein C-III [170], apoE


[171] and ABCA1 (Mosialou and Kardassis unpublished) promoters in hepatic cells suggesting a broader role of AP1 factors in lipoprotein metabolism in the liver during inflam-mation. The pathways leading to apolipoprotein gene down-regulation by pro-inflammatory Jun factors remain poorly characterized, however the existing evidence supports impor-tant roles of Protein Kinase C, C-Jun N Terminal Kinase (JNK) and Tpl2 (MEKK3) [169, 171].

In addition to HNF-1 , apoM gene transcription in the liver has been shown to be controlled positively by Liver Receptor Homolog-1 (LRH-1) and Forkhead box A2 (FOXA2) transcription factors which bind to distinct sites on the proximal apoM promoter [2, 172]. LRH-1 stimulates apoM gene transcription by binding to an LRH-1 response element located in the proximal human apoM promoter [172]. Small heterodimer partner (SHP), an atypical orphan nuclear receptor that lacks a DNA-binding domain, inhibits the transactivation function of LRH-1 by direct interaction with its AF-2 activation domain thus competing with the binding of co-activators. Overexpression of SHP or induc-tion of its expression by bile acids (ligands for the nuclear receptor Farnesoid X Receptor, FXR), strongly increased SHP association to the apoM promoter resulting in reduced apoM gene expression levels [172]. Similarly, apoM expres-sion was reduced in the liver of mice fed with a diet supple-mented with cholic acid or in the liver of SHP transgenic mice [172]. Thus, bile acids suppress apoM expression in

vivo by inhibiting LRH-1 transcriptional activity via recruit-ment of SHP to the apoM promoter (Fig. 3C).

Haploinsufficient Foxa2 or hyperinsulinemic mice exhib-

ited decreased apoM expression and plasma pre- HDL lev-els due to inactivation of Foxa2. Leptin treatment of ob/ob

(leptin deficient) and db/db (leptin receptor deficient) mice

restored apoM expression through relocation of Foxa2 to the nucleus [2]. Foxa2 is recruited to a conserved binding site on

the human apoM promoter located between nucleotides -398

and -388 (Fig. 3C) [2]. Preliminary data from our group in-dicate that Foxa2 plays a wider role in HDL metabolism by

regulating in a positive or a negative manner, the expression

of several HDL genes in hepatic cells. Foxa2 negatively regulates the human ABCA1 promoter by binding to three

sites in the proximal region, one of which is the TATA box

(Thymiakou and Kardassis, submitted).

In agreement with the role of Foxa2 in apoM gene regu-lation, insulin, insulin-like growth factor I (IGF-I) and IGF-I potential peptide (IGF-IPP) significantly inhibited apoM gene expression in a dose- and time-dependent manner in HepG2 cells and in primary human and murine hepatocytes [173]. The inhibitory effect of insulin was mediated through the activation of phosphatidylinositol-3-kinase (PI3K) that in turn activates PKB resulting in inactivation of Foxa2 [173]. Furthermore, glucose strongly inhibited apoM gene expres-sion in HepG2 cells and the inhibitory effects of glucose and

Fig. (3). Summary of transcriptional regulation of the apoA-I (panel A), ABCA1 (panel B) and apoM (panel C) genes. For each promoter,

the corresponding scheme shows the regulatory elements, the transcription factors that bind to them and their mode of regulation by ligands

or conditions.


insulin were additive while in rats rendered hyperglycemic by short-term glucose infusion, resulting in enhanced insulin secretion, serum apoM concentrations and hepatic apoM mRNA levels were significantly reduced [174].

We showed that apoM gene is under the control of hor-mone nuclear receptors that play pivotal roles in lipid and glucose metabolism [175]. Among these nuclear receptors, HNF-4 seems to play a major role in the regulation of apoM gene expression in the liver. This is in agreement with a pre-vious cDNA microarray analysis of HNF-4-induced genes in human hepatoma cells that showed a 4.5-fold increase in apoM expression levels by overexpressed HNF-4 [176]. Overexpression via adenovirus mediated gene transfer and siRNA-mediated gene silencing established that hepatocyte nuclear factor 4 (HNF-4) is an important regulator of apoM gene transcription in hepatic cells. ApoM promoter deletion analysis combined with DNA affinity precipitation and chromatin immunoprecipitation assays revealed that HNF-4 binds to a hormone-response element in the proximal apoM promoter (nucleotides -33 to -21) [175]. Mutagenesis of this HRE decreased basal hepatic apoM promoter activity to 10% of control and abolished the HNF4-mediated transactivation of the apoM promoter. In addition to HNF-4, homodimers of retinoid X receptor and heterodimers of retinoid X receptor with receptors for retinoic acid, thyroid hormone, fibrates (peroxisome proliferator-activated receptor), and oxysterols (liver X receptor) were shown to bind with different affini-ties to the proximal HRE in vitro and in vivo. Ligands of these receptors strongly induced human apoM gene tran-scription and apoM promoter activity in HepG2 cells, whereas mutations in the proximal HRE abolished this in-duction. These findings provide novel insights into the role of apoM in the regulation of HDL by steroid hormones.

CONCLUSIONS

HDL is an attractive target for the development of thera-pies for CAD due to its multiple atheroprotective functions in several cell types such as the macrophages and the endo-thelium. The current status of HDL-based clinical trials has been recently reviewed [20]. Despite the recent disappoint-ments from clinical trials aiming to prevent CAD events by HDL-raising strategies (CETP inhibitors, niacin, RVX-208) and the failure of genetics to correlate HDL levels with CAD events [177] the HDL hypothesis has not been abandoned yet. The challenges now are to develop novel therapies that would raise HDL without compromising HDL functionality, to achieve as much specificity towards HDL as possible and to capitalize on the functions of HDL.

As discussed above, drugs that could specifically in-crease the expression of apoA-I or ABCA1 are anticipated to be of great clinical benefit because they will increase the rate of HDL biogenesis and of RCT and such drugs do not exist yet. All studies that have been performed to date in experi-mental animals (i.e. using Tg mice) indicate that HDL struc-ture or functionality is not affected by apoA-I or ABCA1 up-regulation. In order to develop such drugs, we need to under-stand in depth the molecular mechanisms (regulatory ele-ments and transcription factors) that control the expression of these two genes in hepatic cells. This knowledge on gene regulation will also allow us to understand better the signifi-cance of polymorphisms or mutations in regulatory regions

of HDL genes and their association with disease as we have done recently in the cases of ABCG1 and LXR [178, 179]. The identification of Foxa2 as a transcription factor that co-ordinately regulates the expression of genes involved in both lipid and glucose metabolism in response to insulin offers new opportunities to dissect the molecular mechanisms that link dyslipidemia with diabetes. Regarding LXR-based therapies, the current ligands greatly enhance RCT in vitro and in vivo but are of no clinical value due to their severe lipogenic effects in the liver. Thus the development of novel isoform and hopefully target-specific drugs is anticipated. Novel agonists of PPARs are also anticipated. Understanding how HDL genes are regulated by inflammatory factors will enable us to develop novel tools to reduce the atherosclerotic burden in patients with chronic inflammatory diseases. For example, the identification of AP1 (c-Jun, JunB) proteins as negative regulators of several genes involved in HDL me-tabolism (ABCA1, apoM, apoA-II, apoE) provides new op-portunities towards this goal.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

Research by the group of Dr Dimitris Kardassis is sup-ported by grants from the General Secretariat of Research and Technology of Greece (PENED 01, SYNERGASIA 09SYN-12-897) and the Ministry of Education of Greece (THALIS MIS 377286) as well as by COST Action BM0904. Efstathia Thymiakou is supported by a post-doctoral fellowship from fp7-REGPOT program “TranSPOT (Translational Potential)” awarded to the University of Crete Medical School.

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31(12), 2990-2996.

Received: January 26, 2014 Revised: February 04, 2014 Accepted: February 25, 2014

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