c. elegans daf-12, nuclear hormone receptors and human

Review

C. elegans DAF-12, Nuclear Hormone Receptors

and human longevity and disease at old age

S.P. Mooijaart a,*, B.W. Brandt b, E.A. Baldal c, J. Pijpe c,M. Kuningas a, M. Beekman b, B.J. Zwaan c,

P.E. Slagboom b, R.G.J. Westendorp a, D. van Heemst a

a Department of Gerontology and Geriatrics, C-2-R, Leiden University Medical Center,

P.O. Box 9600, 2300 RC, Leiden, The Netherlandsb Section of Molecular Epidemiology, Department of Medical Statistics and Bioinformatics,

Leiden University Medical Center, The Netherlandsc Evolutionary Biology, Institute of Biology, Leiden University, The Netherlands

Received 7 January 2005; received in revised form 9 March 2005; accepted 11 March 2005

Abstract

In Caenorhabditis elegans, DAF-12 appears to be a decisive checkpoint for many life history traits

including longevity. The daf-12 gene encodes a Nuclear Hormone Receptor (NHR) and is member of

a superfamily that is abundantly represented throughout the animal kingdom, including humans. It is,

www.elsevier.com/locate/arr

Ageing Research Reviews

4 (2005) 351–371

Abbreviations: AR, Androgen Receptor; BLAST, Basic Local Alignment Search Tool; CAR, Constitutive

Androstane Receptor; CETP, Cholesterol Ester Transfer Protein; COUP-TF1/COUP-TF2, Chicken Ovalbumin

Upstream Promoter Transcription Factor 1/2; CYP7A1, cholesterol-7-alpha-hydroxylase; DAF, Dauer Formation

Influencing Genes; DAX1, DSS-AHC critical region on the X chromosome; DBD, DNA-binding domain;

EAR1A/EAR1B/EAR2, ERBA related 1 A/B/2; ERA/ERB, Estrogen Receptor Alpha/Beta; ERRA/ERRB/

ERRG, Estrogen Related Receptor Alpha/Beta/Gamma; FXR, Farnesoid X Receptor; GCNF1, Germ Cell

Nuclear Factor; GR, glucocorticoid receptor; HNF4A/HNF4G, Hepatocyte Nuclear Factor 4 A/G; IGF-1R,

Insulin-Like Growth Factor 1 Receptor; IR, Insulin Receptor; LBD, ligand binding domain; LRH1, Liver

Receptor Homologue; LXRA/LXRB, Liver X Receptor Alpha/Beta; MR, Mineralocorticoid Receptor; NGFIB,

Nerve Growth Factor-Induced Clone B; NHR, Nuclear Hormone Receptor; NOR, Neuron Derived Orphan

Receptor; NOT, Nuclear Receptor of T-cells; PNR, Putative Neurotransmitter Receptor; PPARA/PPARG/PPARD,

Peroxisome Proliferator Activated Receptor Alpha/Gamma/Delta; PR, Progesterone Receptor; PXR, Pregnane X

Receptor; RARA/RARB/RARG, Retinoid Acid Receptor Alpha/Beta/Gamma; RORA, RAR Related Orphan

Receptor Alpha; ROS, reactive oxygen species; RXRA/RARB/RARG, Retinoid X Receptor Alpha/Beta/Gamma;

SF1, Steroigenic Factor 1; SHP, Small Heterodimer Partner; SNP, single nucleotide polymorphism; THRA/

THRB, Thyroid Hormone Receptor Alpha/Beta; TLX, Tailless; TR2/TR4, Testicular Orphan Receptor 2/4; VDR,

Vitamin D Receptor

* Corresponding author. Tel.: +31 71 526 6640; fax: +31 71 524 8159.

E-mail address: [email protected] (S.P. Mooijaart).

1568-1637/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.arr.2005.03.006

however, unclear which of the human receptor representatives are most similar to DAF-12, and what

their role is in determining human longevity and disease at old age.

Using a sequence similarity search, we identified human NHRs similar to C. elegans DAF-12 and

found that, based on sequence similarity, Liver X Receptor A and B are most similar to C. elegans

DAF-12, followed by the Pregnane X Receptor, Vitamin D Receptor, Constitutive Andosteron

Receptor and the Farnesoid X Receptor. Their biological functions include, amongst others,

detoxification and immunomodulation. Both are processes that are involved in protecting the body

from harmful environmental influences. Furthermore, the DAF-12 signalling systems seem to be

functionally conserved and all six human NHRs have cholesterol derived compounds as their ligands.

We conclude that the DAF-12 signalling system seems to be evolutionary conserved and that

NHRs in man are critical for body homeostasis and survival. Genomic variations in these NHRs or

their target genes are prime candidates for the regulation of human lifespan and disease at old age.

# 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: DAF-12; Nuclear Hormone Receptors; Longevity; Lipid metabolism; Detoxification

1. Introduction

The nematode worm Caenorhabditis elegans is a valuable experimental model for

research into ageing, because its lifespan is influenced by signalling systems that are well

characterised and highly conserved throughout evolution. In C. elegans, environmental

conditions, such as food availability, temperature and population density, are monitored by

sensory systems. To prevent damage and to maintain homeostasis the individual worm

constantly has to adjust itself to its changing environment through alterations in essential

traits, such as metabolic rate, developmental time and reproduction. Under favourable

conditions, C. elegans develops through four larval stages and two adult stages. Under

unfavourable conditions, C. elegans turns into an alternative highly resistant diapausal

dauer larva. During this optional life stage, all major life history events (development,

growth, metabolic rate and reproduction) are slowed down and although the worm is

capable of movement, it is mainly inactive and non-feeding. When conditions turn

favourable again, the larva resumes its normal developmental program to an adult, without

loss of post-dauer lifespan, adding up to 60 days to the normal maximum lifespan of 15

days from egg to adult (Klass and Hirsh, 1976). This suggests that during the dauer state the

worm does not age. Many genes in the worm’s signalling systems influence dauer

formation and are therefore called daf genes. Mutations in these genes can prolong the

lifespan of the worm by favouring dauer formation. Strikingly, some of these mutations

also extend adult lifespan up to three-fold (Gems et al., 1998), indicating that these

mutations also influence the rate of ageing in the adult worm.

Since the worm’s signalling systems are also present in other species, and are therefore

believed to be functionally conserved, their role in longevity in other species is subject of

intense research. The genetic mutations extending lifespan in the worm also extend

lifespan in D. melanogaster and mice. For example, it was first discovered that the C.

elegans daf-2 mutant was longlived (Kenyon et al., 1993). Later, it was discovered that the

daf-2 gene shows homology to the mammalian genes encoding the Insulin Receptor (IR)

and Insulin-Like Growth Factor 1 Receptor (IGF-1R) (Kimura et al., 1997), which are

S.P. Mooijaart et al. / Ageing Research Reviews 4 (2005) 351–371352

conserved throughout evolution. Extended lifespan was then also demonstrated in IR

mutants in D. melanogaster (Tatar et al., 2001) and in IR and IGF-1R mutants in mice

(Bluher et al., 2003; Holzenberger et al., 2003). This exemplifies that C. elegans is a

suitable model organism for research into ageing and that similarity searches in other

organisms can yield interesting results.

In C. elegans, DAF-12 is the decisive factor for the choice between the highly

resistant dauer diapause or the developmental program (Antebi et al., 1998) and DAF-12

orchestrates the cascade of events in the dauer larva formation. DAF-12 is a member of

the superfamily of Nuclear Hormone Receptors (NHRs) (Antebi et al., 2000). It is active

near the end of the dauer signalling pathway (Antebi et al., 1998) and integrates

information from various signalling systems (Tatar et al., 2003). NHRs are localized in

the nucleus, where they act as transcription factors. When activated, they stimulate or

repress the transcription of target genes. The longevity phenotype of some daf-2 mutants

is doubled by mutations in daf-12 (Larsen et al., 1995). daf-12 thus is a key player in the

regulation of adaptations that are associated with ageing and longevity. The superfamily

of the Nuclear Hormone Receptors is ubiquitously represented in phylogenetically

distant organisms (Mangelsdorf et al., 1995). Although in C. elegans the NHR

superfamily has more than 200 predicted genes (Sluder et al., 1999) and the superfamily

has undergone extensive proliferation and diversification, daf-12 belongs to the

phylogenetically conserved classes of C. elegans NHRs. In humans, the NHR

superfamily has more than 50 members (Robinson-Rechavi et al., 2001). However, it is

unclear which human NHR is most similar to DAF-12 and the role of NHRs in human

longevity has not been investigated.

In this review, we aim to identify human NHRs similar to C. elegans DAF-12, to analyse

in which biological pathways they are involved and to discuss their potential role in human

longevity and disease at old age. In Section 2, we briefly discuss the common and

discriminating characteristics of NHRs. In Section 3, we report on the systematic search for

human NHRs similar to C. elegans’ DAF-12 and the selection of the six most similar

human NHRs. In Section 4, we review the biological pathways in which the human

homologues are engaged. Special attention is paid to the presence of common gene variants

in components of these human pathways. In Section 5, we envisage the potential role of the

identified pathways on human longevity and disease at old age and draw conclusions.

2. Characteristics of human Nuclear Hormone Receptors

The superfamily of NHRs comprises hundreds of members throughout metazoan life.

Here, some of their common and discriminative features are briefly discussed. For more

detail, we refer to the reviews of Laudet (1997) and Mangelsdorf et al. (1995), and

references therein.

2.1. Structural features

Nuclear Hormone Receptors are composed of four functional domains (Fig. 1): the

modulator domain, the DNA-binding domain (DBD), the hinge region and the ligand

S.P. Mooijaart et al. / Ageing Research Reviews 4 (2005) 351–371 353

binding domain (LBD) (Giguere, 1999). The modulator domain displays the largest

variability of the four domains. It specifies different splice variants of the NHR resulting in

different isoforms that all contain the same LBD and DBD. Each isoform has distinct

biological functions and a specific expression pattern. The Activation Function (AF1)

within the modulator domain determines the biological activity of the receptor isoform, for

instance with regard to tissue specificity. The DBD is the most conserved domain. It

contains two zinc fingers to bind the DNA of the NHRs’ target genes. These target genes

contain a Hormone Response Element (HRE) of which the organisation differs between

different subfamilies of receptors (see below). The hinge region, located between the DBD

and LBD, is very variable. It has been suggested that it can function as a docking site for

(co-)repressor proteins. The LBD is a multifunctional region that mediates ligand binding,

dimerisation, transactivation functions and interaction with chaperones, such as Heat

Shock Proteins. It contains a highly conserved Activation Function 2 (AF2), which has a

smaller effect on transcriptional regulation than the AF1.

2.2. Function

Most nuclear hormone receptors are constitutively localized in the nucleus. When no

ligand is bound, the receptor can act as a potent repressor or activator of transcription.

Ligand binding changes the transcriptional activity of the NHR by either activating or

repressing it. In addition, the NHRs are highly dependent on the interaction with co-

regulatory proteins, which can also either enhance or repress their activity.

2.3. Classification and nomenclature

The first classification of NHRs in subfamilies was suggested by Mangelsdorf et al.

(1995) and based on DNA-binding and dimerisation properties of the NHRs. The

classification distinguishes four classes: a class of steroid receptors, a class of receptors that

heterodimerise with the Retinoid X Receptor, a class of homodimeric receptors and a class

of monomeric receptors.

Laudet (1997) investigated the evolution of NHRs. Based on his phylogenetic analyses

(with NHRs from nematodes, arthropods and vertebrates), he describes six subfamilies of

NHRs. Interestingly, four of the phylogenetic classes (I–IV) that Laudet proposes based on

his phylogeny coincide with the classification that Mangelsdorf et al. (1995) proposed based

on dimerisation and DNA-binding properties. Since the two additional classes that Laudet

describes do not contain any human NHRs, which are subject of the present study, the details

of the differences between the two classifications are beyond the scope of this review.

The current nomenclature of the NHRs is based on the subfamilies of Laudet (Nuclear

Receptors Committee, 1999). Each gene name starts with ‘‘NR’’, followed by the Arabic

number corresponding to the subfamily, then a single letter for the group and then an


Fig. 1. Schematic structure of NHR.

Arabic number again for each individual NHR. For instance, NR1A1 belongs to subfamily

1, group A and is member number 1: Thyroid Hormone Receptor A. A representative

phylogenetic tree of the classification is depicted in Fig. 2.

3. Human homologues of DAF-12

Sequence similarity searches are an effective tool to aid in the prediction of orthology of

proteins from different species. It is unclear which human NHRs are most similar to C.

elegans DAF-12. The Pregnane X Receptor and the Vitamin D Receptor have been

indicated as homologues of DAF-12 (Antebi et al., 2000; Mangelsdorf et al., 1995), but

other sources indicate the Liver X Receptor Alpha as the most similar human protein

(http://www.wormbase.org) or the Liver X Receptor Beta as the most likely human


Fig. 2. Schematic representation of the classification of human NHRs. For each protein, NHR classification name

and trivial name is given. Subfamilies are presented in different colors. Amino acid sequences of human NHRs in

the NUREBASE database (Duarte et al., 2002) were retreived from RefSeq. A consensus neighbour-joining tree

(1000 bootstrap trials, excluding gap positions) was produced from a multiple sequence alignment using ClustalX

1.83. The presented tree shows that members of the subfamlies and groups are clustered according to their

classification. Bootstrap values (not shown) were high for nodes within groups but lower for more distant nodes.

This implies that different phylogenetic algorithms will result in different positions of subfamilies relative to each

other but little changes in the relative positions of the group members within the subfamilies.

http://www.wormbase.org/

homologue (http://www.ensembl.org). Although Mangelsdorf et al. included C. elegans

DAF-12 in their analyses, they restricted the sequence comparison to the DBD of the

NHRs. Their conclusions were drawn based on a dendrogram, which is not suitable for

drawing phylogenetic conclusions. More recent phylogeny searches, such as that of

Laudet, also include the LBD, but did not include DAF-12. Furthermore, since the

classifications of Mangelsdorf and Laudet, in 1995 and 1997, respectively, the sequencing

of the human genome has been completed and new NHRs have emerged. In this section, we

searched for human NHRs most similar to C. elegans DAF-12 using Basic Local

Alignment Search Tool (BLAST) sequence similarity searches. Since the function of a

NHR depends on both domains, we chose to use the entire amino acid sequence containing

both domains for the sequence similarity.

3.1. Sequence alignment

In C. elegans, three isoforms of the DAF-12 protein are present. The longest two contain

a DBD and LBD, the short transcript contains an LBD only. We used the complete amino

acid sequence of the longest of the three isoforms (F11A1.3a, 753 amino acids) and

performed a BLAST against the Ensembl annotated human protein database. Amino acid

sequence alignment was performed with WuBLAST2.0 on Ensembl (www.ensembl.org).

The following parameters, which Ensembl uses in their orthology predictions, were used:

no optimisation, no filter, span 1, postsw, the BLOSUM62 matrix, gap opening penalty 9

and gap extension penalty 2. The 20 most similar human NHRs, as identified by sequence

similarity search are listed in Table 1.

In bio-informatics, the criterion of bidirectional best hit is often used in orthology

predictions. A bidirectional best hit means that the members of the pair are each other’s best

hit in the other species. Therefore, a reverse BLAST against the Ensembl annotated C.

elegans protein database was performed using each of the 20 most similar human proteins as

queries. For each of the 20 proteins we noted the rank of C. elegans DAF-12 and the E-value.

3.2. Identification of most similar NHRs

The human proteins retrieved using C. elegans DAF-12, were sorted based on ascending

E-value. The E-values express the probability that the score of the observed alignment(s)

within the protein occurred as a result of chance. A lower E-value indicates a lower

probable contribution of chance and thus a higher probability for the contribution of

homology. We listed both the NCBI Reference Sequence accession number and the NHR

classification number. Multiple alignments within one protein are listed as separate rows

for each alignment. For each alignment length, sequence identity and score are listed. The

percentage identity indicates the fraction of amino acids within the alignment that are

similar for the aligned proteins. The E-value for the total protein, as reported by

WuBLAST, is also listed in Table 1.

Most of the 20 human proteins show alignment with C. elegans DAF-12 on two

domains: the DBD and the LBD. Two NHRs (ERA and ERB) align on the DBD only, since

they do not contain an LBD. One NHR (EAR1A) aligns on three locations, with a small

alignment in the hinge region besides in the DBD and LBD.


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Table 1

Result of sequence similarity search of C. elegans DAF-12 with human Nuclear Hormone Receptors

Human protein characteristics Alignment characteristics Reverse BLAST

Protein name NHR subfamily

and group

RefSeq accession

number

Location Length ID

(%)

E-value Rank of

DAF-12

E-value of

DAF-12

LXRA NR1H3 NP_005684 DBD 119 41 1.2 e�27 1 5.7 e�28

LBD 223 26

LXRB NR1H2 NP_009052 DBD 95 35 3.8 e�26 1 2.5 e�26

LBD 75 29

PXR NR1I2 NP_003880 DBD 129 45 6.5 e�26 1 6.9 e�26

LBD 175 25

VDR NR1I1 NP_000367 DBD 109 44 7.2 e�25 2 3.4 e�25

LBD 94 23

CAR NR1I3 NP_005113 DBD 88 47 3.8 e�23 3 1.8 e�23

LBD 179 26

EAR1A NR1D1 NP_068370 DBD 402 27 1.1 e�21 11 7.0 e�22

LBD 37 46

EAR1B NR1D2 NP_005117 DBD 131 37 6.6 e�20 11 2.9 e�20

Hinge 15 53

LBD 179 27

THRA NR1A1 NP_003241 DBD 153 33 1.2 e�19 10 5.9 e�20

LBD 105 28

THRB NR1A2 NP_000452 DBD 89 45 4.9 e�19 16 2.4 e�19

LBD 62 34

RARB NR1B2 NP_057236 DBD 157 36 1.0 e�18 20 6.6 e�19

LBD 40 33

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Table 1 (Continued )

Human protein characteristics Alignment characteristics Reverse BLAST

Protein name NHR subfamily

and group

RefSeq accession

number

Location Length ID

(%)

E-value Rank of

DAF-12

E-value of

DAF-12

ESRRG NR3B3 NP_996317 DBD 135 36 3.6 e�18 31 1.8 e�18

LBD 246 23

ESRRA NR3B1 NP_004442 DBD 155 39 7.0 e�18 25 4.6 e�18

LBD 90 27

FXR NR1H4 NP_005114 DBD 99 44 7.5 e�18 20 3.7 e�18

LBD 33 33

RARG NR1B3 NP_000957 DBD 95 42 2.2 e�17 43 1.1 e�17

LBD 40 35

ERA NR3A1 DBD 76 46 4.1 e�17 25 2.4 e�17

RORA NR1F1 NP_599023 DBD 114 34 4.3 e�17 32 2.1 e�17

LBD 44 32

ESRRB NR3B2 NP_004443 DBD 141 35 1.2 e�16 44 6.2 e�17

LBD 110 27

ERB NR3A2 NP_001428.1 DBD 224 29 1.3 e�16 25 2.1 e�16

RARA NR1B1 NP_000955.1 DBD 86 43 1.5 e�16 55 7.4 e�17

LBD 110 22

HNF4A NR2A1 NP_849181 DBD 103 37 4.5 e�16 73 2.3 e�16

LBD 51 31

For abbreviations, see ‘‘list of abbreviations’’.

Most of the 20 human proteins in Table 1 are members of the subfamily of the NHRs that

heterodimerise with RXR (subfamily 1), suggesting that DAF-12 also belongs to this

subfamily. The Liver X Receptor Alpha and Beta are the most similar to DAF-12, followed

by the PXR, the VDR and the CAR. Both LXRs belong to group H of subfamily 1, the

VDR, PXR and CAR belong to group I. Although the FXR is number 13 in the table, it

belongs to the same group as both Liver X Receptors. Repeating the sequence similarity

search with different BLAST parameters retrieved the same proteins, although in a

different order (data not shown).

To limit our review, we selected the human members of groups H (LXRA, LXRB and

FXR) and I (VDR, PXR and CAR). As can be seen in Fig. 2, these two groups together form

one branch of the phylogenic tree. In the following two sections, we will evaluate their

biological function and putative role in longevity and disease in humans.

4. Characteristics and function of selected human NHRs

Biological characteristics of the six selected human NHRs are listed in Table 2. For each

of the selected NHRs, we evaluated in which biological processes they are active. We then

assessed to what extent genetic variation may influence their function. In humans common

gene variants are useful to elucidate the influence of variation in the function of a gene in

biological processes in vivo and phenotypes on a whole-organism level in the population at

large. We used Pub Med to look for literature reporting whether in humans functional


Table 2

Biological features of the selected human NHRs

Full names and synonyms Ligands Tissues Biological processes

Liver X Receptor Alpha (LXRA) Oxysterols Liver, macrophages Cholesterol sensor

Liver X Receptor Alpha (LXRB) Oxysterols Ubiquitous Cholesterol sensor

Pregnane X Receptor (PXR) Drugs (rifampicine,

phenobarbital, more)

Liver and intestine Detoxification and excretion

of steroids and xenobiotics

Steroid hormones

Dietary compounds

Vitamin D Receptor (VDR) Vitamin D hormone

1,25(OH)2D3

Ubiquitous Bone metabolism and

calcium homeostasis

Cell differentiation

and proliferation

Apoptosis

Genomic stability

Immunomodulation

Detoxification

Constitutive

Androstane Receptor (CAR)

Phenobarbital-like

substances

Liver and intestine Detoxification and excretion

of steroids and xenobiotics

Farnesoid X Receptor (FXR) Bile acids Terminal ileum Bile acid synthesis

Vascular smooth

muscle

Bile acid re-uptake

variants exist in the selected NHRs, and in what way these variants influence the processes

the NHRs play a role in and affect phenotypes. If mice knockout models were present, we

briefly reviewed their phenotype(s).

4.1. Liver X Receptor Alpha and Beta (LXRA and LXRB)

The Liver X Receptor Alpha and Beta are two different proteins transcribed from two

different genes. Since they have a high sequence identity (Willy et al., 1995) and since their

biological function is the same, they will be discussed together. The LXRA is expressed in

liver, kidney, intestine and macrophages, whereas the LXRB is expressed ubiquitously.

Both are involved in lipid metabolism and cholesterol sensing mechanisms. Their ligands

are oxysterols (Janowski et al., 1999), which are oxidized derivatives of cholesterol that

serve as intermediary substrates in the rate-limiting steps of steroid hormone and bile acid

synthesis.

The LXRs are associated with a number of steps in cholesterol metabolism (Repa et al.,

2002). Activation of LXRs leads to up-regulation of the reverse cholesterol transporter

cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis.

Up-regulation of CYP7A1 in the liver activates the conversion of cholesterol in bile acids

(Peet et al., 1998). LXRs also up-regulates ATP Binding Cassete 1 (ABC1) (Repa et al.,

2000). In peripheral cells, such as macrophages, this results in the efflux of cholesterol for

transport back to the liver by high density lipoproteins. ABC1 up-regulation in the small

intestine inhibits cholesterol absorption by stimulating cholesterol efflux from enterocytes

back into the intestinal lumen. As a result of these combined actions, the balance between

cholesterol absorption and excretion shifts in favour of its excretion.

Oxysterols accumulate in the liver when there is a surplus of cholesterol, causing tissue

damage. Oxysterols also accumulate in arterial foam cells and may be generated by oxidation

of lipoproteins by smooth endothelial muscle cells, endothelial cells and macrophages.

Exposure of vascular cells to oxysterols leads to changes in gene expression and cellular

morphology which are thought to enhance the development of atherosclerosis. Interestingly,

LXR was shown to be an important endogenous inhibitor of atherosclerosis in murine models

(Tangirala et al., 2002). In addition, in mice, LXR activation was found to negatively regulate

macrophage inflammatory gene expression in vivo and in vitro (Joseph et al., 2003). These

findings identify LXRs as lipid-dependent regulators of inflammatory gene expression that

may serve to link lipid metabolism and immune functions in macrophages.

LXR null mice have an impaired cholesterol catabolism, leading to accumulation of

hepatic cholesterol when fed a high cholesterol diet (Peet et al., 1998). However, the

concentration of cholesterol in these diets was far higher than normal.

4.2. Pregnane X Receptor (PXR)

The Pregnane X Receptor is involved in detoxification and removal of xenobiotic

compounds as well as endogenous hormones. It is a broad specificity, low affinity sensing

receptor that triggers metabolising enzymes. It is expressed in liver, small intestine and

colon (Bertilsson et al., 1998; Blumberg et al., 1998). Ligands are mainly steroids, such as

glucocorticoids, but also include drugs like rifampicine and phenobarbital (Lehmann et al.,


1998). Target genes include cytochrome P450 enzymes (CYPs), of which CYP3A1 and

CYP3A4 are the major forms, and multiple drug resistance gene 1 (MDR1) (reviewed in

Wang and LeCluyse, 2003). Because of its role in these biological pathways, research on

the PXR has focussed on adverse drug effects and drug–drug interactions.

The PXR is also associated with inflammatory disease. For instance, expression of the

PXR is negatively regulated by IL-6 (Pascussi et al., 2000b), a pro-inflammatory cytokine.

Inflammatory bowel disease is associated with dysregulation of PXR target genes

(Langmann et al., 2004).

Four studies report on natural variants in the PXR in humans (Hustert et al., 2001;

Koyano et al., 2002; Uno et al., 2003; Zhang et al., 2001). PXR genes of a total of several

hundreds of individuals from different races were sequenced, revealing a small number of

infrequent mutations but no common variants. A six base pair deletion in the promoter

region influences PXR transcription in vitro (Uno et al., 2003). However, no associations

were found with a specific phenotype.

PXR null mice are viable and fertile in the normal laboratory environment (Xie et al.,

2000). Although there is no loss of basal expression of CYP3A, expression is no longer

induced by specific ligands, such as pregnenolone, which is a key intermediate in steroid

hormone synthesis.

4.3. Vitamin D Receptor (VDR)

The Vitamin D Receptor is expressed in almost all tissues of the human body (Minghetti

and Norman, 1988). It is activated by the Vitamin D hormone 1,25(OH)2D3, a secosteroid

hormone that is synthesised in the skin from 7-dehydrocholesterol under the influence of

sunlight. Target genes of the VDR are active in different processes discussed below

(Minghetti and Norman, 1988).

The biological functions of the VDR are diverse (reviewed in Lin and White, 2004),

including roles in bone metabolism, cellular proliferation and differentiation, chemopreven-

tion, neuroprotection and immunomodulation. Knowledge on the role of Vitamin D in bone

metabolism and calcium homeostasis comes from the observation that rickets is the result of

poor dietary intake and low exposure to sunlight. Low levels of circulating Vitamin D cause a

decrease in calcium absorption from the intestine and re-absorption from the kidney, thereby

decreasing circulating calcium levels. This leads to a decreased calcium content of bone, that

becomes deformed and more vulnerable to fractures. The role of the VDR in bone mineral

density has been confirmed by the observation that VDR knockout mice develop

osteomalacy, a disease that can be reversed by calcium supplementation (Yoshizawa et al.,

1997). In humans, polymorphisms in the VDR have been associated with decreased bone

mineral density and increased fracture risk. For instance, in a meta-analysis the Cdx-2

polymorphism was associated with hip fracture risk (Fang et al., 2003).

The active hormone has been suggested to have anticancer properties (Hutchinson et al.,

2000) in several different ways. Active Vitamin D was shown to inhibit cell proliferation

while it stimulates cellular differentiation and apoptosis in a number of tissues expressing

VDR. When the level of apoptosis is limited cells that have accumulated damage can

evolve into cancerous cells. Furthermore, the VDR participates in maintaining genomic

stability. The target genes are involved in DNA repair mechanisms in response to reactive


oxygen species (ROS). This role for the VDR is especially important in the skin, where

ultraviolet radiation causes ROS. In clinical studies, both serum Vitamin D levels and VDR

polymorphisms have been associated with susceptibility to, and outcome of malignancies,

such as breast cancer, prostate cancer, colon cancer and malignant melanoma. On the other

hand, metastatic growth of lung cancer cells is extremely reduced in VDR knockout mice

(Nakagawa et al., 2004).

The VDR is also implicated in immunomodulation. For instance, knockout mice fail to

develop experimental asthma (Wittke et al., 2004), suggesting a role for the receptor in

inflammation at least in the lung. Immunological factors are of great importance in late life

survival (van den Biggelaar et al., 2004). There is also a role for VDR in detoxification

processes. For instance, dehydroepiandrosterone-sulfotransferase (SULT2A1), which is a

target gene of the VDR, mediates the inactivation and excretion of steroid hormones,

amongst others (Echchgadda et al., 2004). Other functions of the VDR have yet to be

explained. For instance, VDR knockout mice also showed gonadal deformities, implying a

role for the receptor in gonadal development (Yoshizawa et al., 1997).

Research on polymorphisms in the VDR is abundant and includes a large number of

polymorphisms and their effect on a large number of phenotypes, such as bone mineral

density and cancer risk. Uitterlinden et al. (2004) reviewed the genetics and biology of

common VDR polymorphisms. The associations found with the polymorphisms also

indicate roles for the VDR that have to be established. For instance, a polymorphism in the

VDR gene has been associated with the onset of diabetes (Motohashi et al., 2003).

4.4. Constitutive Androstane Receptor (CAR)

The Constitutive Androstane Receptor is closely related to PXR and has a comparable

but distinct function in xenobiotic detoxification (Maglich et al., 2002). There are

indications that the PXR and CAR arose from a common ancestor (Handschin et al., 2004).

Like the PXR, CAR is expressed mainly in the liver and intestine. Ligands include

androstanol, which inhibits constitutive activity. CAR is induced by the binding of

phenobarbital-like substances which activates the transcription of CYP genes.

Dexamethasone also enhances expression of CYPs through CAR (Pascussi et al.,

2000a), illustrating the overlapping functions of CAR and PXR.

Together with the PXR CAR is involved in the transcription of UDP-glucuronosyl-

transferase gene UGT1A1 (reviewed in Wang and LeCluyse, 2003). CAR is responsible for

the conjugation of bilirubin (a breakdown product of haemoglobin) which makes the

compound suitable for excretion and thus accelerates its clearance. High levels of

unconjugated bilirubin are toxic. Furthermore, CAR was identified as a key regulator of

acetaminophen metabolism and hepatotoxicity (Zhang et al., 2002).

However, apart from xenobiotic metabolism CAR exerts other functions (Goodwin and

Moore, 2004). CAR participates in the molecular mechanisms contributing to homeostatic

resistance to weight loss, as CAR influences thyroid metabolism during caloric restriction

(Maglich et al., 2004). Furthermore, CAR appears to be a primary glucocorticoid receptor-

response gene (Pascussi et al., 2003a).

The loss of CAR expression in knockout mice did not result in any overt phenotype in

the normal laboratory environment (Wei et al., 2000). However, loss of CAR function


altered sensitivity to toxins (such as cocaine), increasing or decreasing it depending on the

compound (Wei et al., 2000).

4.5. Farnesoid X Receptor (FXR)

The Farnesoid X Receptor functions in bile acid metabolism and transport as well as

cholesterol and lipid homeostasis (Kuipers et al., 2004). Bile acids are the product of

cholesterol degradation, involving a large number of enzymatic steps. The FXR regulates


Fig. 3. Schematic representation of the biochemical processes that involve the selected NHRs, emphasizing the

central role for cholesterol.

the transcription of several of these enzymes. For instance, it represses CYP7A1, the rate-

limiting step in the conversion of cholesterol into bile acids, thereby influencing both

cholesterol and bile acid homeostasis. This repression was shown to be dominant over the

CYP7A1 induction by the LXR (Lu et al., 2000).

FXR is expressed in the terminal ileum where it regulates the re-uptake of bile acids

from the intestine, as a part of the so-called entero-hepatic circulation of bile acids. Bile

acids lower triglyceride levels via a pathway involving FXR (Watanabe et al., 2004).

Recently, FXR has been shown to induce very low density lipoprotein expression (Sirvent

et al., 2004a) and to repress hepatic lipase gene expression (Sirvent et al., 2004b),

emphasizing the role for FXR in cholesterol metabolism. The FXR is also expressed in

vascular smooth muscle. In an in vitro study (Bishop-Bailey et al., 2004), stimulation of the

FXR by its ligands resulted in apoptosis of the smooth muscle cells. This hints at an

important role for the FXR in atherogenic processes.

FXR null mice develop normally and are outwardly identical to wild-type littermates.

However, they have elevated serum bile acid, cholesterol and triglycerides, increased

hepatic cholesterol and triglycerides and a proatherogenic serum lipoprotein profile (Sinal

et al., 2000).

4.6. Summary

The NHRs are engaged in various biological pathways. A simplified scheme of the

biochemical mechanisms that involve the six selected NHRs is shown in Fig. 3 and

highlights the central role for cholesterol. For both LXRs, FXR and CAR, we were unable

to retrieve any article on common gene variants. For some of the genes mouse models are

available, but usually without a clear phenotype in relation to ageing and longevity.

However, potential effects may become visible when tested in environments more

mimicking the natural environment of the mouse (Zwaan, 2003).

5. Discussion and conclusions

The human NHRs that were selected based on their structural similarity to C. elegans

DAF-12 orchestrate a large number of processes that are essential for maintaining body

integrity. They all play a role in detoxification and immunomodulation. Furthermore,

cholesterol is a key component in these biochemical pathways.

5.1. Detoxification and immunomodulation

All six selected NHRs are involved in detoxification and immunomodulation.

Detoxification decreases the harmful effects of a wide range of endogenous and exogenous

compounds, such as bilirubin, cholesterol and its metabolites, steroid hormones, drugs and

xenobiotics. Regulation of detoxifying genes has recently been implicated in longevity in

C. elegans (McElwee et al., 2004). The transcriptional profile of longlived daf-2 mutants in

C. elegans showed up-regulation of detoxification enzymes, such as cytochrome P450,

short-chain dehydrogenase/reductase, UDP-glucuronodyltransferase and glucagon S-


transferase. Some of these genes were recently identified as target genes of DAF-12

(Shostak et al., 2004). Strikingly, human NHRs have similar target genes. In humans,

decreased detoxification by the PXR in the intestine has been associated with inflammatory

bowel disease (Langmann et al., 2004). Other studies report on the association of NHRs

with immune function and inflammation (Joseph et al., 2003; Lin and White, 2004). The

immune system is one of the major lines of defence against harmful environmental

influences. At old age, a reduced inflammatory response is associated with increased

morality risk (van den Biggelaar et al., 2004). Taken together, NHRs are involved in

biological processes that involve protection against harmful environmental influences.

These processes have been associated with longevity in either model organisms, humans or

both. However, no systematic research has been performed into the specific role of NHRs

or their target genes in these processes and their association with human longevity and

disease at old age.

5.2. Cholesterol

Cholesterol is a common component in the biochemical pathways in which the selected

NHRs are involved (Fig. 3). Cholesterol is a structural component of cell membranes and a

precursor in hormones synthesis. Cholesterol metabolism includes production, uptake,

transport to target tissues and the excretion of toxic (by)products of cholesterol and its

derivatives. Because of its hydrofobic nature, cholesterol is transported in lipoprotein

particles consisting of cholesterol, triglycerides and apolipoproteins.

In C. elegans, cholesterol is a substrate in DAF-12 signalling. DAF-9, a member of the

cytochrome P450 superfamily, is involved in the conversion of cholesterol into the

potential ligand for DAF-12, which is believed to be a steroid (Jia et al., 2002). Conversely,

DAF-12 negatively regulates DAF-9 expression in a feedback loop (Gerisch and Antebi,

2004). Recently, DIN-1 was discovered as an important cofactor for DAF-12 (Ludewig

et al., 2004). It interacts with DAF-12 to form a transcriptionally active complex. In

parallel, the selected human NHRs all have cholesterol derivatives as ligands and

Cytochrome P450s are involved in the production of these ligands. The LXRs and FXR are

involved in the metabolism, transport and excretion of cholesterol. The PXR, VDR and

CAR have cholesterol derived hormones as ligands. Furthermore, the expression of some

major cytochromes is regulated by a network of nuclear receptors, including the PXR,

CAR and VDR (Pascussi et al., 2003b). The C. elegans cofactor DIN-1 also has distinct

homologues in humans. These homologues act as regulators of transcription by interaction

with NHRs (Ariyoshi and Schwabe, 2003; Shi et al., 2001). Taken together, these data

suggest that both the sequence and the function of the components of DAF-12 signalling

are conserved throughout evolution, hinting at a critical role of such components in the

processes that they regulate. Besides putative homology, there are more arguments that hint

at essential functions of the NHR signalling systems. The fact that there are two LXRs

duplicates with highly similar function and that PXR and CAR have strongly overlapping

function, indicates functional redundancy. Secondly, although research in this area is far

from complete, gene variants or polymorphism do not appear to be common.

In humans, the role of cholesterol itself in disease at old age is well established. High

levels of cholesterol cause cardiovascular disease at middle age. At old age, high levels of


total cholesterol are associated with decreased mortality from cardiovascular disease and

infectious disease (Weverling-Rijnsburger et al., 1997). The genetic determinants of

cholesterol homeostasis are currently being uncovered, as is the role of cholesterol

metabolism in longevity. Strikingly, a number of the discovered genes are under regulation

of NHRs: CETP and APOE are target genes of the LXR, whereas MTP is a target gene of

the FXR. A linkage analysis of longlived subjects identified a linkage region on

chromosome 4 (Puca et al., 2001). Further association analysis of the region identified

variations in the gene encoding Microsomal Transfer Protein (MTP) to associate with

longevity (Geesaman et al., 2003). MTP is a rate-limiting protein in the production of

lipoproteins (Jamil et al., 1998). In a group of longlived subjects a single nucleotide

polymorphism (SNP) in the gene encoding the Cholesterol Ester Transfer Protein (CETP)

was enriched in a group of longlived subjects compared to young controls (Barzilai et al.,

2003). CETP plays a role in reverse cholesterol transport. The SNP was associated with

alterations in HDL and LDL particle size, and it was therefore concluded that CETP affects

longevity by influencing cholesterol metabolism. Another example is the gene encoding

apolipoprotein E (APOE). A genetic variant causing variation in the structure of the ApoE

protein affects its biological activity, resulting in alteration in circulating levels of

cholesterol. The variant ‘‘e2’’ allele has been associated with decreased cardiovascular

mortality and the variant ‘‘e4’’ allele with early cognitive decline, although the results have

been inconsistent. Although the serum levels of ApoE are highly heritable (Beekman et al.,

2002), the APOE e2/e3/e4 genotype only explains 15% of this genetic component

suggesting that variation in other genes regulating ApoE transcription, such as the LXRs,

largely contribute to these serum levels. Taken together, these data show that cholesterol

metabolism is a key process in human longevity and disease at old age, and that NHRs play

a crucial role in cholesterol metabolism. Similarly, in a sequence similarity analysis across

species, the vitellogenin gene family has been identified as a conserved pathway regulating

energy storage and lipid metabolism in species from nematodes to humans (Brandt et al.,

2005). All this fits the suggestion that the mechanisms that regulate human longevity are

conserved throughout evolution.

5.3. Evolutionary background

The disposable soma theory on ageing states that each individual has to allocate limited

energy resources to either maintenance of the soma or reproduction (Kirkwood, 1977).

This trade-off between reproduction and longevity was shown to exist also in humans

(Westendorp and Kirkwood, 1998). Therefore, it is likely that human longevity necessitates

more than average investments in somatic maintenance. Toxic compounds or radiation can

generate excess ROS in the body. ROS cause damage to organelles and the important

macromolecules, such as lipids and DNA, ultimately leading to cell death. High levels of

oxidative stress have been associated with accelerated ageing (Finkel and Holbrook, 2000).

Genetic factors influencing the maintenance processes may influence the rate of damage

accumulation in the cell and thus the rate of ageing. Loss-of-function mutations lead to

inefficient adaptation and accumulation of damage, causing accelerated ageing and

disease. Gain-of-function mutations lead to more efficient adaptation and decreased

accumulation of damage, and may thus slow the rate of ageing and therefore promote


longevity. For example, mice lacking the LXRA have impaired cholesterol and bile acid

metabolism when fed a high cholesterol diet, leading to cholesterol accumulation in the liver

and eventually hepatic failure (Peet et al., 1998). Conversely, a synthetic LXR ligand inhibits

the development of atherosclerosis in mice (Joseph et al., 2002). Mice knockout models of the

selected NHRs show no overt phenotype under standard laboratory conditions. However,

when challenged with changing environmental conditions the null mice are less able to cope,

resulting in disease and early mortality. This implies an essential role for NHRs in

maintaining of homeostasis under changing environmental conditions, in line with the

function of C. elegans DAF-12. In C. elegans, DAF-12 is downstream of signalling systems

that translate environmental cues, as well as germline signals (Tatar et al., 2003).

Interestingly, the longlived phenotype exhibited by germline ablated worms is dependent on

DAF-12 and DAF-9 (Hsin and Kenyon, 1999). This positions DAF-12 at the crossing point of

maintenance of the soma and germline signalling. In humans, the PXR, CAR and VDR are

involved in homeostasis of steroid hormones and detoxification of xenobiotic compounds,

implicating a role for these NHRs in both germline signalling and maintenance. This

observation identifies these NHRs as candidates for longevity regulation.

5.4. Conclusions

Our results indicate that cholesterol is a key player in the regulation of NHR activity. We

have limited our review to the two groups of human NHRs that are most similar to DAF-12,

but we do not exclude the possibility that other groups are equally interesting. It remains

possible that during evolution specific NHRs have changed into forms different from DAF-

12 without loosing their function in longevity associated processes. Also, cross-talk

between different NHRs may play a role in orchestrating the effector pathways. We

conclude that the approach we used is powerful to detect signalling pathways involved in

essential processes, yielding a new list of candidate genes. The approach can be extended to

more, if not all NHRs. We think it likely that human longevity and disease at old age

depend on NHR function.

Acknowledgement

This work was supported by an IOP grant (Innovative Oriented Research) from the

Dutch Ministery of Economic Affairs (grant number IGE01014).

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c. elegans daf-12, nuclear hormone receptors and human

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