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INVITED REVIEW
Vitamin D receptor signaling and its therapeutic implications:
Genome-wide and structural view1
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Carsten Carlberg and Ferdinand Molnár
Abstract: Vitamin D3 is one of the few natural compounds that has, via its metabolite 1␣,25-dihydroxyvitamin D3 (1,25(OH)2D3)
and the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. For efficiently applying the therapeutic
and disease-preventing potential of 1,25(OH)2D3 and its synthetic analogs, the key steps in vitamin D signaling need to be
understood. These are the different types of molecular interactions with the VDR, such as (i) the complex formation of VDR with
genomic DNA, (ii) the interaction of VDR with its partner transcription factors, (iii) the binding of 1,25(OH)2D3 or its synthetic
analogs within the ligand-binding pocket of the VDR, and (iv) the resulting conformational change on the surface of the VDR
leading to a change of the protein–protein interaction profile of the receptor with other proteins. This review will present the
latest genome-wide insight into vitamin D signaling, and will discuss its therapeutic implications.
Key words: vitamin D, vitamin D analogs, chromatin immunoprecipitation, crystal structure, VDR partner proteins, vitamin D
signaling in vivo.
Résumé : La vitamine D3 appartient à la courte liste de composés naturels qui exerce, par l’intermédiaire de son métabolite, la
1␣,25-dihydroxyvitamine D3 (1,25(OH)2D3), et de son récepteur (VDR), un facteur de transcription, un effet direct sur la régulation
génique. Afin d’exploiter de manière efficace le potentiel thérapeutique et préventif de la 1,25(OH)2D3 et ses analogues synthétiques, les étapes clés de la signalisation de la vitamine D doivent être comprises. Le VDR est engagé dans différents types
d’interactions moléculaires comme (i) la formation d’un complexe impliquant le VDR et l’ADN génomique, (ii) l’interaction du
VDR avec des facteurs de transcription partenaires, (iii) la liaison de la 1,25(OH)2D3 ou de ses analogues synthétiques dans la poche
de liaison du ligand du VDR et (iv) le changement de conformation à la surface du VDR qui en résulte et qui mène à un
changement du profil d’interaction protéine–protéine du récepteur avec d’autres protéines. Cet article de synthèse présentera
un aperçu des données les plus récentes à l’échelle du génome de la signalisation de la vitamine D et il discutera de ses
implications thérapeutiques. [Traduit par la Rédaction]
Mots-clés : vitamine D, analogues de la vitamine D, immunoprécipitation de chromatine, structure cristalline, protéines partenaires du
VDR, signalisation de la vitamine D in vivo.
Introduction
The secosteroid vitamin D3 is a pleiotropic signaling molecule
that was already being used by early unicellular organisms to
protect their DNA against UV-B irradiation (Holick 2011). Even in
humans, vitamin D3 is still linked to UV-B, since its energy is
essential to convert 7-dehydrocholesterol in the skin into previtamin D3. The step-wise depigmentation of human skin that
occurred when Homo sapiens moved from equatorial regions in
Africa to higher latitudes in Asia and Europe, demonstrates that
the need for endogenous vitamin D3 production acted as an evolutionary driver (Hochberg and Templeton 2010). Vitamin D3
started to get used as a signaling molecule when the first fish
developed bones, for which calcium uptake needed to be regulated (Bouillon and Suda 2014). Today, the endocrinology of vita-
min D3 is still tightly connected with calcium homeostasis and
bone formation. It is undisputed that a sufficient vitamin D status
is essential for bone health, such as in the prevention of osteoporosis (Institute-of-Medicine 2011), but in parallel tissues, the greatest fear for a side-effect that might be caused by overdosing with
natural and synthetic vitamin D analogs is calcification (Cheskis
et al. 2006). However, the physiological impact of vitamin D is
broader than just controlling proper bone mineralization (Carlberg
2014b), as it is involved in the regulation of cellular growth and
differentiation (Feldman et al. 2014) as well as innate and adaptive
immunity (Chun et al. 2014). This provides vitamin D not only
with a therapeutic and disease-preventive potential in osteoporosis, but also in different types of cancer, in several infectious
diseases such as tuberculosis, and in autoimmune diseases, such
as multiple sclerosis (Holick 2007).
Received 4 October 2014. Accepted 27 December 2014.
Abbreviations: 1,25(OH)2D3, 1␣,25-dihydroxyvitamin D3; 25(OH)D3, 25-hydroxyvitamin D3; AR, androgen receptor; ChIP, chromatin immunoprecipitation;
ChIP-seq, ChIP coupled with massive parallel sequencing; CYP24A1, cytochrome P450, family 24, subfamily A, polypeptide 1; DBD, DNA-binding domain;
DR3, direct repeat spaced by 3 nucleotides; ER, estrogen receptor; ESRRB, estrogen-related receptor ␤; FAIRE-seq, formaldehyde-assisted isolation of
regulatory elements sequencing; FXR, farnesoid X receptor; GABPA, GA binding protein transcription factor, alpha subunit 60 kDa; GR, glucocorticoid
receptor; HDAC, histone deacetylase; HOMER, Hypergeometric Optimization of Motif EnRichment; IGV, Integrative Genomics Viewer; LBD, ligand-binding
domain; LBP, ligand-binding pocket; PBMC, peripheral blood mononuclear cell; PRMT10, protein arginine methyltransferase 10; PXR, pregnane X receptor;
RAR, retinoid acid receptor; RXR, retinoid X receptor; SH2B1, SH2B adaptor protein 1; SPI1, spleen focus forming virus (SFFV) proviral integration oncogene;
TR, thyroid hormone receptor; TsA, trichostatin A; TSS, transcription start site; VDR, vitamin D receptor.
C. Carlberg. School of Medicine, Institute of Biomedicine, University of Eastern Finland, POB 1627, FI-70211 Kuopio, Finland.
F. Molnár. School of Pharmacy, Institute of Biopharmacy, University of Eastern Finland, FI-70211 Kuopio, Finland.
Corresponding author: Carsten Carlberg (e-mail: carsten.carlberg@uef.fi).
1This Invited Review is part of a Special Issue entitled “Pharmacology of vitamins and beyond: Vitamin D.”
Can. J. Physiol. Pharmacol. 93: 311–318 (2015) dx.doi.org/10.1139/cjpp-2014-0383
Published at www.nrcresearchpress.com/cjpp on 21 January 2015.
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Fig. 1. Molecular basis of vitamin D signaling. The complex of vitamin D receptor (VDR; in green) with its partner transcription factor
retinoid X receptor (RXR; in blue) bound to DNA is shown in the central panel. (A) The details of the contact of the DNA-binding domains of
VDR and RXR with DNA (based on Protein Database (PDB) file 1YNW). The sequence logo of a DR3-type VDR binding site is shown on the
bottom. (B) Demonstration of the heterodimerization of the ligand-binding domains (LBDs) of VDR and RXR (based on a model derived from
1RK3 cryo-electron microscopic studies with full length receptors (Orlov et al. 2012)). (C) VDR’s ligand-binding pocket filled with a 1,25(OH)2D3
molecule (based on PDB file 1DB1). The key amino acids contacting the 3 hydroxyl groups (in red) of 1,25(OH)2D3 are indicated. (D) Detail of the
surface of the VDR LBD (in green), where a rather minor movement of helix 12 (in red) after ligand binding allows the binding of the LXXLL
motif of the co-activator TRAP220 (also called MED1, in orange; based on PDB file 1RK3).
Vitamin D acts as a pre-hormone that is converted in 2 hydroxylation steps via 25-hydroxyvitamin D3 (25(OH)D3) into its
biologically active metabolite 1,25(OH)2D3 (Norman 2008). The latter molecule is the only natural high-affinity ligand to the VDR
(Haussler et al. 1997). VDR is not only one of approximately 1900
human transcription factors (Vaquerizas et al. 2009), but it also
belongs to the nuclear receptor superfamily (Evans and Mangelsdorf
2014). Like other endocrine nuclear receptors, such as the receptors for estrogen (ER), testosterone (AR), and cortisol (GR), which
specifically bind lipophilic molecules the size of cholesterol
(Carlberg and Molnár 2012), VDR is also directly activated by
1,25(OH)2D3. VDR is rather ubiquitously expressed, i.e., tissues and
cell types that are not related to calcium homeostasis and bone
formation, such as cancer cells and cells of the immune system,
are also responsive to 1,25(OH)2D3 (Wang et al. 2012). The molecular actions of 1,25(OH)2D3 are largely identical to those of its receptor, but VDR also has additional ligand-independent functions
(Polly et al. 2000).
The different types of molecular interactions of the VDR (Fig. 1)
are the mechanistic core of vitamin D signaling. These are (i) the
protein–DNA interaction of VDR’s DNA-binding domain (DBD)
with genomic sequences, (ii) the protein–protein interaction of
VDR with its partner proteins, (iii) the protein–ligand interaction
of 1,25(OH)2D3 or its synthetic analogs with the ligand-binding
pocket (LBP) within the ligand-binding domain (LBD), and (iv) the
resulting conformational changes on the surface of the LBD,
which change the protein–protein interaction profile of the receptor with nuclear adaptor proteins, such as co-repressors, co-
activators, and the Mediator complex. The latter proteins attract
(i) chromatin remodeling complexes that re-arrange the local positioning of nucleosomes, and (ii) chromatin modifying enzymes
that read, write, or erase post-translational marks to histone proteins, such as the transfer of acetyl and methyl groups of the local
nucleosomes. These epigenomic changes facilitate looping of the
VDR-marked genomic regions, i.e., 1,25(OH)2D3-inducible enhancers, towards the basal transcriptional machinery that has assembled on accessible transcription start sites (TSSs) (Carlberg and
Campbell 2013). This assembly of enhancer and TSS regions with
RNA polymerase II, other nuclear adaptor proteins, and ligandactivated VDR finally leads to an increase or decrease in the transcription of primary vitamin D target genes.
In this review, we will provide more details on vitamin D signaling by describing the latest (epi)genome-wide and structural
insight, and will discuss its therapeutic implications.
Genome-wide VDR binding
Nuclear receptors, such as VDR, are characterized by a highly
conserved DBD, which is formed by 2 zinc fingers specifically
contacting the sequence RGKTSA (R = A or G, K = G or T, S = C or G)
within the major groove of genomic DNA (Shaffer and Gewirth
2002) (Fig. 1A). Like most other transcription factors, VDR increases
its DNA binding affinity and specificity via heterodimerization with
a partner transcription factor (Fig. 1B). In most studies, this protein partner turned out to be the nuclear receptor retinoid X
receptor (RXR) (Sone et al. 1991; Carlberg et al. 1993), but VDR has
also been shown to form homodimers (Cheskis and Freedman
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Fig. 2. Different modes of vitamin D receptor (VDR) interacting with genomic DNA. The Integrative Genomics Viewer (IGV) browser
(Robinson et al. 2011) was used to visualize the binding of VDR in unstimulated LX2 cells and cells that were stimulated with the 1,25(OH)2D3
analog MC903 (Ding et al. 2013). The input lane serves as a negative control. (A) Example of a VDR binding site close to the transcription start
site (TSS) of the CYP24A1 gene, which is drastically up-regulated by the treatment with VDR ligand. At this locus a DR3-type sequence was
identified below the peak summit, providing strong evidence that VDR binds DNA as a heterodimer with retinoid X receptor (RXR).
(B) Example of a site close to the TSS of the PRMT10 gene. At this site, VDR binding does not change after ligand stimulation and no DR3-type
sequence is found below the peak summit. (C) Example of a DR3-free site close to the TSS of the SH2B1 gene where ligand treatment leads to a
significant reduction of VDR binding. The DNA binding mode of VDR to the sites in (B) and (C) are not known, and it can only be speculated
that VDR eithers bind directly in a complex with an alternative partner transcription factor, or indirectly, i.e., piggybacks onto an undefined
“carrier” transcription factor.
1994) and complexes with other nuclear receptors, such as retinoid acid receptor (RAR) (Schräder et al. 1993) and thyroid hormone receptor (TR) (Schräder et al. 1994). In-vitro experiments
have determined the optimal binding site of VDR–RXR heterodimers as a direct repeat of 2 hexameric nuclear receptor binding motifs spaced by 3 nucleotides (DR3, see the sequence logo on
the bottom of Fig. 1A) (Umesono et al. 1991; Shaffer and Gewirth
2004).
Nowadays, a more appropriate approach to determine the association of VDR with its genomic targets is to apply the chromatin
immunoprecipitation (ChIP) method (Orlando 2000). When combined with massive parallel sequencing (ChIP-seq) (Park 2009),
this technique can monitor, genome-wide, all binding sites of a
transcription factor in living cells. For human VDR, public ChIPseq data are available from the following cell lines: (i) GM10855
and GM10861 (B cells) (Ramagopalan et al. 2010), (ii) THP-1 (monocytes) (Heikkinen et al. 2011), (iii) lipopolysaccharide-differentiated
THP-1 (macrophages) (Tuoresmäki et al. 2014), (iv) LS180 (colon
cancer) (Meyer et al. 2012), and (v) LX2 (hepatic stellate cells) (Ding
et al. 2013). A harmonized re-analysis of all these ChIP-seq datasets
resulted in 23 409 non-overlapping VDR binding loci (Tuoresmäki
et al. 2014), of which more than 70% are unique for one of the
analyzed cellular models. Importantly, de novo search of the
genomic sequence below VDR peak summits (±100 bp) confirmed
DR3-type elements as the most abundant motifs. However, motif
screening algorithms, such as HOMER (Heinz et al. 2010) at a moderate score of 7, could only identify DR3-type elements at 2686 VDR
peaks (11.5% of total) (Tuoresmäki et al. 2014). This DR3 percentage
differs significantly between the analyzed cellular models, ranging between 38.2% in macrophages and 9.0% in B cells. Interestingly, in every cell type investigated, the top 200 VDR sites (ranked
by binding strength) show a DR3 rate of more than 60%, i.e.,
DR3-type motifs are found preferentially at highly ligand-responsive
VDR loci, such as that close to the TSS of the gene cytochrome
P450, family 24, subfamily A, polypeptide 1 (CYP24A1, Fig. 2A).
Therefore, the genes that are controlled by this type of VDR binding site seem to be the prime targets of vitamin D, and may be the
first to be addressed by a therapeutic intervention with a synthetic VDR ligand. This finding also implies that the mechanistic
basis of VDR’s action is independent of the cell type and the total
number of identified binding sites (Carlberg 2014a).
Taken together, human cells have between 1000 and 10 000 genomic
VDR binding loci. This is clearly more than the 100–500 primary
1,25(OH)2D3 target genes per cell type. Not only in vitro, but also in
living cells, DR3-type sites are the preferred VDR binding motifs,
but such an element is far from being found at all VDR loci.
Nevertheless, VDR sites with a DR3-type motif show more pronounced responsiveness to ligands, and may be the preferred targets for a therapy with 1,25(OH)2D3 analogs.
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VDR partner proteins
Can. J. Physiol. Pharmacol. Vol. 93, 2015
Although the most prominent genomic VDR binding sites preferentially carry DR3-type sequences on which VDR heterodimerizes
with RXR, there are a significant number of important VDR loci
(some 40% of the top 200 and 80%–90% of the lower ranking sites)
where no DR3-type element is available. Interestingly, at the latter
sites, VDR ligands have either no significant effect on VDR binding,
such as shown for the site close to the TSS of the gene protein arginine methyltransferase 10 (PRMT10, Fig. 2B), or even reduce VDR association, such as demonstrated for the gene SH2B adaptor protein 1
(SH2B1, Fig. 2C). It is not yet known how VDR associates at such sites
with DNA, but it is most likely that either VDR binds (i) directly to
DNA by using a different protein partner than RXR, or (ii) indirectly
piggybacks onto a DNA-binding “carrier” protein (Fig. 2). In both
scenarios the specific DNA binding site would be different to a DR3type sequence, and either composed of a single nuclear receptor
binding motif and the motif of the partner protein, or the specific
motif of the carrier protein. Interestingly, HOMER motif screening of
the VDR ChIP-seq datasets from hematopoietic cells found significant enrichment of binding sites for the transcription factors spleen
focus forming virus (SFFV) proviral integration oncogene (SPI1, also
called PU.1), estrogen-related receptor ␤ (ESRRB, also called NR3B2),
and GA binding protein transcription factor, alpha subunit 60 kDa
(GABPA) (Tuoresmäki et al. 2014). SPI1 is a well-known pioneer factor
(Zaret and Carroll 2011) that is known to work together with VDR in
monocytic differentiation (Novershtern et al. 2011).
The lack of a dominant non-DR3 binding sequence below VDR
peak summits suggests that there is no single alternative protein
partner of the receptor, but rather a larger set of proteins
(Tuoresmäki et al. 2014). These VDR partnering proteins will have
a cell-specific expression pattern and may explain some of the
cell-specific actions of VDR and its natural ligand 1,25(OH)2D3.
Importantly, VDR already binds to a number of its genomic targets, such as those close to the genes PRMT10 and SH2B1 (Figs. 2B
and 2C) in the absence of ligand. Since these ligand-independent
genomic VDR loci have a clearly lower DR3 rate than liganddependent sites (Heikkinen et al. 2011), they associate preferentially
with proteins related to gene repression, such as is demonstrated for
the example of the MYC gene (Toropainen et al. 2010). Although so
far no direct protein–protein interaction between VDR and SPI1,
ESRRB, or GABPA has been reported, the concept of alternative
VDR binding modes has the potential to modulate VDR signaling
via the activation of signal transduction pathways that affect
VDR’s alternative protein partners. However, in the case of the
well-characterized VDR–RXR heterodimers, there is no significant
effect of the RXR ligand 9-cis retinoid acid when VDR is not ligandactivated at the same time, i.e., VDR–RXR heterodimers are nonpermissive (Schräder et al. 1995; Schulman et al. 1997).
In summary, VDR ChIP-seq data indicated that there are alternative VDR binding motifs to VDR–RXR heterodimer complexes
on DR3-type sites. However, we do not know the identity of these
alternative VDR partner proteins. Therefore, further investigations are necessary before a potential therapeutic impact of these
proteins on vitamin D signaling can be evaluated.
nuclear receptors pregnane X receptor (PXR) and farnesoid X receptor (FXR), which share this evolutionary precursor with VDR
(Makishima 2005). Bile acids are cholesterol derivatives and therefore structurally related to vitamin D3 and its metabolites, but
they bind with inverse orientation to the LBP (Fig. 3A) (Masuno
et al. 2013). They are positioned within the LBP by the same 3 pairs
of polar amino acids, but owing to their more rigid structure they
need to use 2 or more water molecules to contact these fixation
points. However, non-polar amino acids also stabilize VDR ligands
within the LBP via hydrophobic interactions. Interestingly, when
comparing the complexes of 1,25(OH)2D3 and 3-keto lithocholic
acid within the LBP (Fig. 3B), it becomes obvious that additional
hydrophobic interactions contribute to 1,25(OH)2D3 aliphatic sidechain stabilization (namely L223 and Y397). All interactions are
relatively conserved, but there is a difference in the position of the
interacting amino acids with respect to ligand orientation with
the 2 types of natural ligands. Therefore, the finding on the reverse binding mode of the bile acids may stimulate the design of
new types of 1,25(OH)2D3 analogs.
Further evidence for the flexibility of the LBP are provided by a
comparison of the crystal structures of VDR’s LBD complexed
with 1,25(OH)2D3 and 3 structurally, rather divergent synthetic
analogs (Fig. 4). The goal of the development of synthetic 1,25(OH)2D3
analogues is to improve their biological profile for a therapeutic
application in one of the pleiotropic functions of the natural
hormone in bone disorders, such as osteoporosis, and in hyperproliferative diseases, such as psoriasis and different types of cancer (Bouillon et al. 1995). Preferential sides for a modification of
1,25(OH)2D3 are the aliphatic side chain, the A-ring, the CD-rings,
and the triene system (Carlberg et al. 2012). One strategy for the
design of 1,25(OH)2D3 analogs is the addition of extra carbons in
form of aliphatic, cyclic, or even aromatic structures. A prominent
example is the 2 side-chain analog Gemini (1,25-dihydroxy-21-(3methyl-3-hydroxy-butyl)-cholecalciferol) (Herdick et al. 2000), which
has a significantly increased volume (25%) but still fits into the LBP
(Fig. 4B). One of the 2 side chains of Gemini takes the same location as in the natural hormone, whereas for the second side chain,
an extra subcavity is opened within the LBP (Vaisanen et al. 2003).
A second analog design strategy is to aim for an increased LBP
binding affinity via replacing hydrogens by more electronegative
atoms, such as fluorines. Moreover, side-chain fluorinations often
make the compounds resistant to degradation by CYP24A1. An
example of this is the semi-steroidal D-ring analogue CD578
(Fig. 4C) (Eelen et al. 2008), which is missing the C-ring within the
secosteroid structure. The bis-aromatic molecule CD3938 exemplifies a third strategy, the design of pure nonsteroidal ligands
(Fig. 4D) (Ciesielski et al. 2012). The 4 structurally divergent VDR
ligands all fit to the LBP and demonstrate the flexibility of the
latter.
Taken together, VDR’s LBD binds the natural ligand 1,25(OH)2D3
with high affinity, but also with lower affinity, and in inverse
orientation bile acids. The LBP has a dynamic structure that can
adapt in its volume to the structure of a rather divergent set of
synthetic analogs.
Ligand binding to the VDR
Interaction of VDR with chromatin components
Resolving the crystal structure of the VDR’s LBD complexed
with 1,25(OH)2D3 (Rochel et al. 2000) created a new level in the
understanding of the molecular mechanisms of the receptor. The
inner surface of the VDR–LBD forms a cavity with a volume of
⬃700 Å3 (Molnár et al. 2006), which is created by a network of
around 40 mostly non-polar amino acids (Fig. 1C). Within this LBP
there are 3 pairs of polar amino acids, each of which fixes one of
the 3 hydroxyl groups of the natural VDR ligand 1,25(OH)2D3 via
hydrogen bonds (Fig. 3A). From an evolutionary point of view, bile
acids such as lithocholic acid were the first ligands of a VDR precursor molecule. Bile acids are still major natural ligands to the
The binding of VDR ligands to the LBP induces minor but significant conformational changes to the surface of the LBD that
result in an exchange of protein–protein interaction partners,
such as co-activator and co-repressor proteins (Carlberg 2003).
Agonistic VDR ligands have to effect both an efficient dissociation
of co-repressor proteins from the LBD surface as well as a specific
association with co-activator proteins, in order to lead to transcriptional activation. More than 50 nuclear proteins are known
to interact with VDR’s LBD (reviewed in (Molnar 2014)), which
include different components of the Mediator complex, which
builds a bridge to the basal transcription machinery assembled on
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Fig. 3. Binding mode of 1,25(OH)2D3 and bile acid to the vitamin D receptor (VDR) ligand binding pocket (LBP). (A) 1,25(OH)2D3 (in green; based
on Protein Database (PBD) file 1RK3) and the bile acid 3-keto lithocholic acid (in blue; based on PBD file 3W5P) in 2 different orientations.
Hydrogen bonds (broken lines) between polar amino acids of the LBP and hydroxyl groups of the ligands or stabilizing water molecules are
indicated. The data are based on X-ray analysis rat VDR crystal structures. (B) The same structures in a more schematic plane display
indicating that (i) in part, different amino acids of the LBP are involved in contacting the ligands, and (ii) lithocholic acid fills the LBP in
an inverse orientation to all other tested VDR ligands. The illustrations were created with the software PyMOL (The PyMOL Molecular Graphics
System, version 1.3, Schrödinger).
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Fig. 4. Shapes of vitamin D receptor (VDR) ligand binding pocket (LBP) in a complex with most divergent ligands. The central panel shows the
position of the LBP (in grey) inside a zebrafish VDR (in green). (A) Details of the shape of the LBP complexed with the natural VDR ligand
1,25(OH)2D3 in 2 different orientations. (B–D) The LBP in the same orientation but complexed with (B) the 2 side-chain analog Gemini, (C) the
fluorinated D-ring analog CD578, and (D) the bis-aromatic nonsteroidal analog CD3938. Broken boxes indicate regions of major (in blue) and
minor (in red) deviations between the LBP shapes. The Protein Database identifiers of the respective crystal structures are indicated in
brackets behind the VDR ligand name.
TSS regions. VDR-interacting co-activators and co-repressors act as
adaptor proteins that connect the receptor with chromatin modifying enzymes, such as histone acetyltransferases and histone
deacetylases (HDACs), respectively. This allows the VDR to initiate
specific changes to its local chromatin environment. In contrast,
HDAC inhibitors, such as trichostatin A (TsA), achieve a less specific but more global activation of chromatin (Marks et al. 2001;
Monneret 2005). Interestingly, 1,25(OH)2D3 and TsA act synergistically in their antiproliferative effect on malignant and non-malignant
human breast cancer cell lines (Liu et al. 1996; Warrener et al.
2003; Malinen et al. 2008) and in their global gene-regulatory
effect in THP-1 cells (Seuter et al. 2013a).
Chromatin has an intrinsic repressive potential with the purpose to conserve the epigenetic landscape of a differentiated cell,
i.e., by default it largely restricts the access of transcription factors
to promoter and enhancer regions, leaving only in the order of
50–100 000 accessible chromatin regions per cell type (ENCODEProject-Consortium et al. 2012). VDR-mediated histone modifications can change this pattern of accessible chromatin regions.
Experimentally, this can be monitored genome-wide by using the
Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-seq) method (Giresi et al. 2007). A detailed FAIRE-seq
time course in 1,25(OH)2D3-stimulated THP-1 cells demonstrated
that some 87% of VDR binding sites co-localize with open chromatin (Seuter et al. 2013b). At approximately 20% of these loci, a
stimulation with 1,25(OH)2D3 leads to a significant increase in
chromatin accessibility (Seuter et al. 2013b). These sites are strong
indications for the location of primary 1,25(OH)2D3 target genes
within the same genomic region. Thus, approaches to categorize
VDR binding sites are useful. For example, VDR loci that (i) carry a
DR3-type sequence, (ii) show ligand-stimulated VDR association,
(iii) co-locate with ligand-induced chromatin opening, and (iv) are
conserved between several cellular systems, may play a more im-
portant role in mediating the functions of 1,25(OH)2D3 than the
vast majority of other VDR sites that lack most of these properties.
In summary, the therapeutic effect of 1,25(OH)2D3 on, for example, the control of cellular proliferation can be combined with
that of chromatin modifiers, such as HDAC inhibitors. The molecular basis of this synergy is related to the fact that 1,25(OH)2D3
dynamically controls the epigenetic state of chromatin at approximately 1 in 6 of all genome-wide VDR binding sites.
Vitamin D signaling in vivo
The endocrinology of vitamin D3 is designed not to imply any
fast change in its physiological effects (DeLuca 2004; Norman 2008).
When the production of vitamin D3 in UV-B exposed skin or its
intake of from diet or supplements is sufficient, the serum 25(OH)D3
level, i.e., the indicator of the vitamin D3 status of the human body
(Hollis 2005), should be optimal. Seasonal variations in sun exposure cause changes in serum 25(OH)D3 concentrations by a factor
of 2–3, but only in the order of weeks and months (Virtanen et al.
2011). This is in clear contrast to cell-culture experiments, where
often pharmacological doses of 1,25(OH)2D3 are applied in the time
frame of a few hours to days. This comparison brings to question
how far results from in-vitro experiments, such as most of those
described above, represent the physiological reality in vivo.
T cells isolated from 9 human individuals with variant serum
25(OH)D3 concentrations were the first primary human cells that
were analyzed by VDR ChIP-seq (Handel et al. 2013). Interestingly,
the number of identified VDR loci varied between 200 and 7000,
and correlated with the 25(OH)D3 levels of the individuals. In all,
9 subjects, together with more than 14 000 unique VDR loci were
identified, of which, however, only 3.1% co-localized with a DR3type sequence. An alternative approach was the study of peripheral blood mononuclear cells (PBMCs) and adipose tissue biopsies
from 71 elderly, pre-diabetic individuals, who were supplemented
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Carlberg and Molnár
over 5 months with vitamin D3 (Carlberg et al. 2013). The changes
in the mRNA expression of 12 primary 1,25(OH)2D3 target genes at
the start and the end of the vitamin D3 intervention correlated
with the alteration in 25(OH)D3 levels, but only for some 60% of
the study participants (Ryynänen et al. 2014; Wilfinger et al. 2014).
These individuals are considered as “responders”. The remaining
individuals may not have benefited from the vitamin D3 supplementation because they were already at their individual optimal
vitamin D status at the start of the intervention (Carlberg et al.
2013).
Taken together, an insufficient vitamin D status can contribute
to a large variety of diseases, such as osteoporosis, cancer, or
autoimmune diseases, reflecting the pleiotropic profile of the
micronutrient/pre-hormone. Genome- and transcriptome-wide studies using preferentially easily accessible samples, such as blood
cells, from human individuals, allow (i) a molecular profiling of
their vitamin D status, and (ii) their classification concerning responsiveness to vitamin D3.
Conclusions
Historically, vitamin D and its metabolite 1,25(OH)2D3 were understood to control calcium homeostasis and bone formation; but
at present, the genome-wide actions of the vitamin/nuclear hormone have been studied in most detail in the cells of the hematopoietic system. This emphasizes the impact of 1,25(OH)2D3 on the
function of innate and adaptive immunity. There are preliminary
indications that the core actions of 1,25(OH)2D3 and its receptor
VDR can be extrapolated from hematopoietic cells to other cell
types (Carlberg et al. 2013), suggesting that the vitamin D status
and the responsiveness of a human individual can be derived from
the response of, for example, PBMCs. ChIP-seq and FAIRE-seq studies on VDR locations and accessible chromatin in living cells provided initial insight on the genome-wide actions of 1,25(OH)2D3
and its nuclear receptor. The large number of vitamin D-modulated
genomic loci supported the view that vitamin D has pleiotropic
effects, and suggests many possibilities for therapeutic applications, such as addressing different VDR partner proteins. In general, this could be (i) the use of appropriate doses of vitamin D3 for
supplementing everyone to their individual optimal vitamin D
status, (ii) the combination of vitamin D with chromatin modifying compounds, such as HDAC inhibitors, or (iii) the application of
synthetic 1,25(OH)2D3 analogs. For designing and understanding
the latter, crystal structure information on the analog-VDR LBD
complex is indispensable. These data demonstrated the great
flexibility of VDR’s LBP, harboring a large variety of structurally
divergent secosteroidal and nonsteroidal 1,25(OH)2D3 analogs. Importantly, all known VDR–1,25(OH)2D3 analog crystal structures
display the active LBD conformation, with helix 12 tightly packed
towards the surface of the domain, i.e., the respective compounds
will initiate vitamin D signaling in a very comparable way.
Acknowledgements
C.C. thanks the Academy of Finland and the Juselius Foundation for support.
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