Current Topics in Medicinal Chemistry, 2012 000 1 Current ... and Moln… · 2 Current Topics in...

Current Topics in Medicinal Chemistry, 2012, 12, 000-000 1

1568-0266/12 $58.00+.00 © 2012 Bentham Science Publishers

Current Status of Vitamin D Signaling and Its Therapeutic Applications

Carsten Carlberg1,* and Ferdinand Molnár2

Departments of Biosciences1 and Pharmacy

2, University of Eastern Finland, FIN-70211 Kuopio, Finland

Abstract: Vitamin D and in particular its biologically most active metabolite, 1 ,25-dihydroxyvitamin D3 (1 ,25(OH)2D3), are central endocrine molecules that influence many aspects of human physiology, which are not only the well-known calcium and phosphorus up-take and transport controlling bone formation, but also the control of immune functions and of cellular growth and differentiation. Basically all actions of 1 ,25(OH)2D3 are mediated by the transcrip-tion factor vitamin D receptor (VDR). The crystal structure of the VDR and detailed knowledge on its molecular interac-tions with the ligand provide significant insight into the mechanisms of vitamin D signaling. This applies also on the ac-tion of the huge number of synthetic 1 ,25(OH)2D3 analogues, which have been developed with the goal of a therapeutic application in hyper-proliferative diseases, such as psoriasis, benign prostate hyperplasia and different types of cancer, in immune functions, such as autoimmune diseases and microbial infections, or in bone disorders, such as osteoporosis. Moreover, detailed investigations on many VDR target genes and in particular the recently available genome-wide view on vitamin D signaling allows a more complete view on the potential of the nuclear hormone. In this review we discuss the latest insight into vitamin D signaling in context with the most prominent 1 ,25(OH)2D3 analogues.

Keywords: Crystal structure, gene regulation, genome-wide view, nuclear receptor, vitamin D, vitamin D analogues, vitamin D receptor.

1. INTRODUCTION

Since 40 years the biologically active form of vitamin D, the secosteroid 1 ,25(OH)2D3 Fig. (1), is known to act at sub-nanomolar concentrations as an endocrine hormone [1, 2]. At about the same time there were first indications on a receptor for vitamin D in the nucleus [3], but it took nearly further 20 years to clone the gene for the VDR and to charac-terize it as a nuclear receptor for 1 ,25(OH)2D3 [4]. In this pre-genomic period the receptors for all other nuclear hor-mones, such as estradiol, testosterone, cortisol and retinoic acid, had been cloned and were found to belong to the same transcription factor family [5]. In the following years it be-came clear that the nuclear receptor superfamily has 48 hu-man members, of which only 12, including the VDR, are classical endocrine receptors, whereas the others were or-phans at the moment of their discovery [6]. For a number of the latter, such as peroxisome proliferator-activated receptor (PPAR) , and or liver X receptor and , diet-derived metabolites were identified as their ligands and they were termed adopted orphan receptors [7].

In general, the members of the nuclear receptor superfa-mily have a similar mode of action, in particular those that have a natural ligand [8]. Therefore, a number of mecha-nisms that were identified, for example with estrogen recep-tors (ERs) or retinoic acid receptors (RARs), apply also for the VDR. However, since from the point of view of endocri-nology the actions of most nuclear hormones are clearly dis-tinct, there are also for vitamin D and the VDR mechanisms that explain this specificity. Firstly, these are the specific

*Address correspondence to this author at the Department of Biosciences, University of Eastern Finland, P.O. box 1627, FIN-70211 Kuopio, Finland; Tel:+358-40-3553062; Fax: +358-17-2811510; E-mail: [email protected]

interactions of 1 ,25(OH)2D3 and its analogues with the ligand-binding pocket (LBP) within the ligand-binding do-main (LBD) of the VDR that are not achieved by any other natural compound [9] Fig. (2). Secondly, genome-wide the VDR has a few thousand chromosomal target sides that regulate in a cell-specific fashion several hundred 1 ,25(OH)2D3 target genes [10, 11].

Vitamin D and its hormonal form 1 ,25(OH)2D3 are es-sential for maintenance of health. 1 ,25(OH)2D3 is a plei-otropic hormone that interferes with many physiological processes, such as the control of metabolism, cellular growth and immune functions. Thus, 1 ,25(OH)2D3 may be suited rather as a preventive agent than as a therapeutic compound. The most striking effect of severe vitamin D deficiency is rickets, since 1 ,25(OH)2D3 is essential for adequate Ca2+ and Pi absorption from the intestine and hence for bone for-mation [12]. Therefore, most clinical trials with 1 ,25(OH)2D3 or its metabolic precursors have focused on bone or on musculoskeletal functions, but they have also shown preventive and therapeutic potential for extra-skeletal chronic diseases, such as cardiovascular and autoimmune diseases, cancer, diabetes, and infections. Since the natural compounds vitamin D and 1 ,25(OH)2D3 are not patentable, pharmaceutical companies focused on the development of more than 3,000 1 ,25(OH)2D3 analogues. However, most of this work has not progressed beyond the preclinical stud-ies and only a few analogues are presently on the market.

This review is an update and extension of our previous summary in this series [9]. Based on the general understand-ing of transcriptional regulation and the structure and func-tion of nuclear receptors, we will discuss the signaling of 1 ,25(OH)2D3 and its most prominent analogues on the level of ligand-VDR interactions and of genomic VDR targets with a view on their therapeutic application.

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2 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 6 Carlberg and Molnár

Fig. (1). Structure of 1 ,25(OH)2D3 and its most prominent analogues. Central the structure of 1 ,25(OH)2D3 is shown and underlayed in salmon color are the different sites of modification. Prominent example analogues are shown for each type of modification. For analogues that have been co-crystallized with the VDR-LBD the respective Protein Data Bank (PDB) accession number is indicated in brackets. More details are displayed in Fig. 6.

2. VDR AS A MEMBER OF THE NUCLEAR RECEP-

TOR SUPERFAMILY

2.1. The impact of Chromatin and Co-Regulators on Nu-

clear Receptor Action

Eukaryotes have in their nucleus a complex of genomic DNA and nucleosomes, referred to as chromatin, that per se prevents access of DNA-binding proteins, such as transcrip-tion factors, to their genomic targets [13]. This intrinsic re-pressive potential of chromatin is essential for long-lasting regulatory decisions, such as terminal differentiation of cells [14]. However, the epigenetic landscape can also be highly dynamic and lead to short-lived states, such as a response of chromatin to stress and other endocrine signals [15]. One

major component of epigenetic changes are reversible post-translational modification of histone proteins, such as acetylation and methylation, that are directed by a class of co-regulatory proteins with either histone acetyltransferase (HAT), histones deacetylase (HDAC) and histone methyltransferase (HMT) or demethylase (HdM) activity [16]. Some of these histone modifications are associated with genes that are actively transcribed, whereas others are a sign of repressed genes [17], i.e. the post-translational modifications of histone correlate with either positive or negative regions of the chromatin. A second class of co-regulators has ATP-dependent remodeling activity and induces plasticity of chromatin by rearranging the organization of nucleosomes [18].

VDR Ligands and Their Therapeutic Potential Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 6 3

Most of the dynamic nature of transcriptional regulation in the context of chromatin has been described first for nu-clear receptors and their target genes [19-21]. Nuclear recep-tors recruit positively and negatively regulating proteins, referred to as co-activators (CoAs) [22] and co-repressors (CoRs) [23], respectively. In a simple view Fig. (3), in the absence of ligand most nuclear receptors interact with CoR proteins, which in turn associate with HDACs leading to a locally more compact chromatin packaging [24]. As dis-cussed in more detail below, ligand binding induces the dis-sociation of CoRs and the association of CoAs [25]. Some CoAs have HAT activity or are complexed with proteins harboring such activity, which in net effect results in local chromatin relaxation [26]. In a subsequent step, nuclear re-ceptors interact with another class of CoAs, which are mem-bers of the mediator complex that builds a bridge to the basal transcriptional machinery [27]. In this way, gene activation by nuclear receptor can be separated into three phases, in each of which the nuclear receptor interacts with a different class of regulatory proteins. Only in one of these phases the basal transcription machinery with RNA polymerase II as its core is activated and mRNA of target genes is produced (more details see in chapter 3.6).

The ligand-triggered dynamic exchange of CoR and CoA proteins binding to nuclear receptors, such as VDR, is also the basis for a molecular understanding of the action of ago-nists and antagonists/inverse agonists (see chapters 4.2 to 4.8). However, the switch between gene repression and acti-vation is more complex than a simple alternative recruitment

of different regulatory complexes [28]. Most co-regulator proteins are co-expressed in the same cell type at relatively similar levels, which raises the possibility of their concomi-tant recruitment to regulatory regions of specific genes. In some cases co-regulators already mark active regulatory elements independent of DNA-binding transcription factors, i.e. not always they require active recruitment by transcrip-tion factors, such as nuclear receptors, to target genomic regions. Both aspects emphasize the spatio-temporal context, i.e. the nuclear organization and the timing of the association of transcription factors and their co-regulators, of transcrip-tional regulation [10]. As discussed in chapter 3.6, the dy-namics of transcriptional regulation plays an important role in controlling VDR target genes.

2.2. Structure and Dynamics of Nuclear Receptors

The VDR is a member of the largest family of metazoan transcription factors, the nuclear receptors superfamily [29]. The members of this protein family are characterized by the presence of a highly conserved DNA-binding domain (DBD) and a structurally conserved LBD [6] Fig. (4). The 66 to 70 amino acids of the DBD are structurally coordinated by two zinc atoms that create a structure, in which one short -helix is interacting directly with the major groove of the DNA [30, 31]. The LBD is formed by a three-layer anti-parallel -helical sandwich composed of 11-13 -helices [32] Fig. (2A). Nuclear receptor ligands have the approximate size of cholesterol, share in common a mainly non-polar character,

Fig. (2). Structure of the human VDR-LBD. (A) The human VDR-LBD represents an example for the structurally conserved nuclear recep-tor LBD and is formed by 12 -helices (H1-H12). (B) The 1 ,25(OH)2D3 molecule is specifically bound via interaction of its three hy-droxyl-groups to a pair of amino acids, each.


Fig. (3). Phases of gene activation. The activation of mRNA transcription by a nuclear receptor dimeric complex can be subdivided into three phases. The first repression phase (also called de-activation phase), the nuclear receptor is in contact with CoRs and HDACs leading to locally repressed chromatin and inactive target genes. In the second initiation phase the nuclear receptor is part of a complex of CoAs, HATs and chromatin remodeling factors, which leads to local opening of chromatin but leave the target genes inactive. In the third activation step the nuclear receptor contacts via mediator proteins and general transcription factors (GTFs) the basal transcriptional machinery leading to mRNA synthesis of the target genes.

Fig. (4). VDR as example for the general structure of a nuclear receptor. A nuclear receptor is composed of a DBD and a LBD, which are linked by a hinge region. The DBD is based on the VDR homodimer on the mouse ONP DR3-type RE (structure 1KB2) and the LBD on structure 1DB1.


but vary greatly in their chemical structure. Accordingly, the internal structure of the LBDs of the respective specific nu-clear receptors shows a similar diversity. The lower part of the LBD of all ligand-activated nuclear receptors contains a LBP of 400 to 1400 Å3 in volume, in which the various ligands are specifically bound Fig. (2A) [33]. The interior surface of these pockets is formed by the side chains of mostly non-polar amino acids and thereby complements the lipophilic character of the ligands. Specificity is achieved through a limited number of stereo-specific polar contacts, the so-called anchoring points Fig. (2B), and the actual shape of the cavity.

Endocrine members of the nuclear receptor superfamily, such as ERs [34], bind with high affinity to a specific ligand and have a small LBP. In contrast, the adopted orphan mem-bers of the family, such as PPARs [35], are more promiscu-ous in the binding of numerous ligands with lower affinity and have much larger LBPs. Biochemical and biophysical methods showed that there is a significant stabilization of the LBD in the presence of ligand and co-regulators, resulting in a more compact and rigid structure. Interestingly, the lower portion of the LBD (helices 4, 5, 6, 7, 11, 12 and parts of helix 3) is less rigid than the upper portion (helices 1, 2, 8, 9, 10 and the other half of helix 3) Fig. (2A).

2.3. Nuclear Receptor LBDs as Molecular Switches

The LBDs of true endocrine nuclear receptors, such as VDR, undergo more dynamic changes than that of adopted orphans, such as PPARs [36]. The most carboxy-terminal helix of the LBD, helix 12 [37], is rather dynamic in apo-structures, but shows significantly slower dynamics after ligand and co-regulator binding. In the ligand-bound agonis-tic conformation, one side of the LBP is sealed by helix 12, which serves as a molecular switch by allowing the LBD in its agonistic conformation to interact with CoA proteins. In the absence of an agonistic ligand, most nuclear receptors interact with CoR proteins. CoA and CoR proteins both con-tain multiple, short receptor interaction domains, composed of the sequence LXXLL (L = leucine, X = any amino acid) in case of CoAs [38] and LXX(I/H)IXXX(I/L) in case of CoRs [39]. The interaction domains make contacts with heli-ces 3, 4 and 12 of the nuclear receptor LBD. This interaction is stabilized by an evolutionary conserved charge clamp that is formed between a glutamate in helix 12 and either a lysine in helix 3 (in the case of CoA binding) or a lysine in helix 4 (for contacting CoRs). CoAs and CoRs interact with the same hydrophobic groove on the surface of the LBD [32]. This provides the advantage that binding of the two co-regulators is mutually exclusive and creates a bipolar �“switch�” of two extremes.

The "mouse-trap" model [40] proposes that helix 12 acts as a lid to the LBP of the LBD, which has to be closed in order to allow nuclear receptor interaction with CoAs and open, when the receptor contacts CoRs. This implies that while helix 12 can take only one defined position in the ago-nist-bound receptor, multiple positions of the helix are pos-sible in antagonist-bound or apo-receptors. The dynamic behavior of the LBD can also explain how selective modula-tors can fine-tune the activity of the receptor, i.e. there is more than a simple on-off light switch, but many dimmer-

type intermediate states. The latter may in particular apply in living cells, where many endogenous co-regulators are pre-sent and nuclear receptors are part of large multi-subunit complexes and bind to DNA in a multiplex/composite chro-matin environment. In addition, the relative �“strength�” of a ligand�’s ability to activate its nuclear receptor may reflect its tightness of binding and probability of LBD occupancy.

2.4. The VDR-LBD is the Molecular Switch of 1 ,25(OH)2D3 Signaling

1 ,25(OH)2D3 can interact with the vitamin D binding protein (DBP) and some cytochrome P450 (CYP) enzymes, but its only high affinity binding protein in the nucleus is the VDR [41]. The VDR gene shows highest expression in metabolic tissues, such as kidneys, bone and intestine, but low to moderate expression is found in nearly all other of the approximately 250 different human tissues [42]. All physio-logical actions of 1 ,25(OH)2D3, such as its role in calcium homeostasis and bone mineralization [43], but also its cell- and immune-regulatory effects [42, 44], have to be mediated via the gene regulatory actions of the VDR. As discussed above in general for nuclear receptors, the VDR-LBD hereby acts as the molecular switch of 1 ,25(OH)2D3 signaling.

In contrast to other members of the nuclear receptor su-perfamily, there is only one gene coding for the VDR. How-ever, the human VDR gene shows a polymorphism (detect-able through the presence or absence of a FokI restriction site), where 37% of the human population are homozygous (and 48% heterozygous) for a three amino acid shorter VDR amino-terminus [45]. This shorter VDR isoform seems to lead to a more active protein, at least concerning the classical actions of the VDR in regulating bone mineral density [46]. Although VDR is a transcription factor acting in the nucleus, VDR protein has also been detected in the cytosol and is also associated with Caveolin-3 and Serca-2, a sarcoplasmic re-ticulum Ca2+-ATPase, in plasma membranes [47, 48]. This may explain some of the rapid, non-genomic actions of 1 ,25(OH)2D3, such as the up-take of calcium.

The crystal structure of the human VDR-LBD [49] dem-onstrated that it is closely related to the LBD of other endo-crine nuclear receptors, such as RAR [50] Figs. (2A and 4). The most noticeable difference between both LBDs concerns the connection between helices 1 and 3, which is shifted outward in VDR, and the loop between helices 6 and 7, which is also shifted to the surface. Both differences explain, why the LBP of the VDR is significantly larger (697 Å3) than that of ER (369 Å3) or RAR (421 Å3). Subsequently, the 1 ,25(OH)2D3 occupies only 56% of the volume of the LBP compared to 63 and 66% for estradiol and all-trans retinoic acid, respectively.

In the agonistic conformation of the human human VDR-LBD helix 12 makes two direct van der Waals contacts via V418 and F422 with the methyl-group of 1 ,25(OH)2D3 [49]. The position of helix 12 is further stabilized by several hydrophobic contacts involving its residues T415, L417, V418, L419, V421 and F422 in connection with the helix 3 residues N232, V234, S235, I238 and Q239, the helix 5 resi-dues A267 and I268 and the helix 11 residues H397 and Y401. In addition, helix 12 is stabilized by two polar interac-tions involving the conserved salt bridge K246 (helix 3) be-


tween E420 (helix 12) and a hydrogen bond between S235 (helix 3) and T415 (helix 12). Since some of these residues (V234, I268, H397 and T401) also interact with 1 ,25(OH)2D3, the ligand has a direct control of helix 12�’s position and by this on the interaction with co-regulatory proteins.

In the VDR-LBD the A-ring of 1 ,25(OH)2D3 adopts a -chair conformation with the 1-OH- and 3-OH-groups in

equatorial and axial orientations, respectively [51]. The three anchoring points of the ligand within the LBP are hydrogen bonds between the 1-OH-group and S237 (helix 3)/R274 (helix 5), the 3-OH-group and S278 (helix 5)/Y143 (helix 1) and the 25-OH-group and H305 (loop between helices 6 and 7)/H397 (helix 11) Fig. (2B). The conjugated triene connect-ing the A- and C-ring is tightly fitted in a hydrophobic chan-nel sandwiched between S275 (loop between helix 5 and a -sheet) and W286 ( -sheet) on one side and L233 (helix 3) on the other side, while exclusively hydrophobic residues sur-round the CD-ring.

3. TRANSCRIPTIONAL REGULATION BY VITA- MIN D

3.1. DNA Binding of the VDR

Each eukaryotic gene is under the control of a large set of transcription factors that can bind up- and downstream of its

transcription start site (TSS). An essential prerequisite for a direct modulation of transcription via 1 ,25(OH)2D3-triggered protein-protein interactions is the location of acti-vated VDR close to the basal transcriptional machinery. This is achieved through the specific binding of the VDR to a 1 ,25(OH)2D3 response element (VDRE) in the regulatory region of a primary 1 ,25(OH)2D3 target gene and looping of genomic DNA carrying this VDR-VDRE complex towards the basal transcriptional machinery [29] Fig. (5).

The VDR-DBD contacts the major grove of the core binding motif with the consensus sequence RGKTSA (R = A or G, K = G or T, S = C or G). The affinity of monomeric VDR to a single core binding motif is not sufficient for the formation of a stable protein-DNA complex and thus VDR requires the formation of homo- and/or heterodimeric com-plexes with a partner nuclear receptor in order to allow effi-cient DNA binding [52]. The predominant heterodimeric partner of VDR is the retinoid X receptor (RXR). The pro-tein-DNA complex of a VDR-RXR heterodimer binding to a VDRE therefore can be considered as a molecular switch for primary 1 ,25(OH)2D3 responding genes.

Most nuclear receptor are able to dimerize in solution via their LBDs, but their DBDs dimerize only in the presence of DNA [53]. The DBD and the LBD of all nuclear receptors are linked by a hinge region of 35 to 50 amino acid residues that form a long -helical structure [54] Fig. (4). Steric con-

Fig. (5). Cellular actions of 1 ,25(OH)2D3. Bound to DBP 1 ,25(OH)2D3 is transported to the cytoplasma membrane. After passing the membrane the compound is either bound by the VDR (in the cytoplasma or the nucleus) or metabolized to calcitroic acid. Ligand-activated VDR activates or inhibits mRNA synthesis of its primary target genes, of which some of the most prominent are listed. These genes can be associated with three cellular functions, the control of metabolism, cellular growth and differentiation and immunity, which mediate the physiological functions of 1 ,25(OH)2D3.


straints allow dimerization of DBDs only on response ele-ments (REs) with properly spaced core binding motifs. Mod-eling of the DBDs of VDR and RXR on DNA [54] suggested that an asymmetric arrangement, i.e. head-to-tail, as a direct repeat with three intervening nucleotides (DR3) provides the most efficient interface of the core DBDs. This fits with the 3-4-5 rule of Umesono et al. [55], in which VDR-RXR het-erodimers show optimal binding to DR3-type REs, whereas other nuclear receptors prefer DR4-type REs (for example thyroid hormone receptors) and DR5-type REs (for example RARs). On DR3-, DR4- and DR5-type REs, different het-erodimers bind with the same polarity, in which RXR always binds to the upstream hexamer and the partner receptor, for example VDR, to the downstream hexamer [56, 57]. This specific and directed dimerization of the DBDs appears to be the major discriminative parameter for selective RE recogni-tion [58, 59]. In vitro VDR was shown to bind also to other RE types, such as DR4s [60] or to everted repeats of spac-ings between 6 and 10 nucleotides [57, 61], but the genome-wide analysis of VDR binding sites did not show any evi-dence for the use of such REs in the regulation of VDR tar-get genes [62] (more details in chapter 3.5).

3.2. VDRE Identification

The specificity of VDR for its DNA binding sites allows a description of VDRE properties that can be used to predict potential binding sites in genomic sequences. For this the VDR binding preference, often expressed as position weight matrix, has to be described on the basis of experimental data, such as a series of gel shift assays with a large number of natural binding sites [63, 64]. However, VDR-RXR het-erodimers do not only recognize a pair of the core binding motif RGKTSA, but also a number of variations to it. De-pending upon the individual position weight matrix descrip-tion, this leads to a prediction of VDREs every 1 to 10 kb of genomic sequence. This is certain to contain many false positive predictions, due to scoring methodology and limita-tions that are imposed by chromatin and supported by all available experimental data.

Internet-based software tools, such as TRANSFAC [65], screen DNA sequences with databases of matrix models. The accuracy of such methods can be improved by taking the evolutionary conservation of the binding site and that of the flanking genomic region into account. However, for a de-tailed analysis of the regulatory regions of primary nuclear receptor target genes and for the confirmation of the binding of a nuclear receptor to a given RE in living cells, chromatin immunoprecipitation (ChIP) analysis has become the gold standard. For example, for the VDR target genes CYP24A1 [66], CYP27B1 [67], cyclin C (CCNC) [68] and cyclin-dependent kinase inhibitor 1A (CDKN1A, also called p21) [69, 70] some 7-10 kb of their promoter regions were indi-vidually investigated by using a set of 20-25 overlapping genomic regions. This approach identified four functional REs for both the CYP24A1 and the CCNC gene, three in the CDKN1A promoter and two in the CYP27B1 gene. Each of the multiple 1 ,25(OH)2D3-responsive promoter regions is able to contact independently the basal transcriptional ma-chinery. This suggests that the simultaneous communication of the individual promoter regions with the RNA polymerase II complex occurs through a discrete 3-dimensional organi-

zation of the promoter. Such a model could resemble the traditional �„DNA looping model�“ discussed previously to explain the activity of upstream enhancer elements [71].

An alternative approach to the identification of primary nuclear receptor target genes has been conducted with the six members of the insulin-like growth factor binding protein (IGFBP) gene family. Here, an in silico screen for VDR-RXR binding was performed first, and was then followed by the analysis of candidate 1 ,25(OH)2D3-responsive se-quences in ChIP assays [72]. By using this approach, the genes IGFBP1, IGFBP3 and IGFBP5 were demonstrated to be primary VDR target genes. In a comparable study the human arachidonate 5-lipoxygenase (ALOX5) gene was also analyzed and shown to be a primary 1 ,25(OH)2D3 target gene [73]. From 22 in silico derived REs identified in the whole ALOX5 gene sequence (-10 kb to +74 kb), two have been validated to be functional in vitro and in the living cells. One of these REs is located far downstream of the TSS (+42 kb) [73], i.e. this study revealed candidate REs located more than 30 kb from their target gene�’s TSS.

Interestingly, the number of REs within a promoter does not correlate with the inducibility of a nuclear receptor target gene, since the average short-term transcriptional response of most primary nuclear receptor target genes is only 2-fold or less [74]. However, most of them are simultaneously under the control of other transcription factors, such as p53 in case of the CDKN1A gene [69], and therefore possess significant basal levels of transcription.

3.3. Physiological Impact of VDR Target Genes

VDR has been shown to induce expression of the gene encoding for the major Ca2+ channel in intestinal epithelial cell, transient receptor potential cation channel, subfamily V, member 6 (TRPV6) [75] or the sodium-phosphate transport protein 2B (SLC34A2) gene [76]. 1 ,25(OH)2D3 also down-regulates the expression of the parathyroid hormone (PTH) gene that opposes 1 ,25(OH)2D3 in regulation of serum Ca2+ and Pi levels, but up-regulates the fibroblast growth factor 23 (FGF23) gene, whose gene product, like PTH, lowers serum Pi levels [77]. The induction of the tumor necrosis factor ligand superfamily, member 11 (TNFSF11, also called RANKL) gene leads to stimulation of osteoclast precursors to fuse and form new osteoclasts, resulting in enhanced re-sorption of bone [78].

The main anti-proliferative effect of 1 ,25(OH)2D3 on cells is a cell cycle block at the G1 phase. This can be ex-plained by changed expression of multiple cell cycle regula-tor genes. Among the first targets described, expression of cyclin-dependent kinase inhibitor (CDKI) genes CDKN1A and CDKN1B (also called p27) were found to be up-regulated by ligand treatment [79, 80]. As described above, the CDKN1A gene was shown to be a primary 1 ,25(OH)2D3 target gene [28, 69, 70]. Additional CDKIs, such as CDKN2B (p15), CDKN2A (p16), CDKN2C (p18) and CDKN2D (p19), also show transcriptional response to 1 ,25(OH)2D3, although the CDKN2A response appears to be secondary as it can be blocked by protein synthesis inhibi-tors [81, 82]. Furthermore, the genes cyclin E1 (CCNE1), cyclin D1 (CCND1) and cyclin-dependent kinase 2 (CDK2) were also found to be down-regulated by 1 ,25(OH)2D3


[83]. Another interesting 1 ,25(OH)2D3 target gene is CCNC. The cyclin C-CDK8 complex was found to be asso-ciated with the RNA polymerase II basal transcriptional ma-chinery and is considered to be a functional part of mediator protein complexes that are involved in gene repression [84]. The fact that the CCNC gene, being located in chromosome 6q21, is deleted in a subset of acute lymphoblastic leuke-mias, suggests that it may be involved in tumorigenesis [85]. In addition, growth arrest and DNA-damage-inducible, alpha (GADD45A) and members of the IGFBP gene family also respond to 1 ,25(OH)2D3 [72, 86]. GADD45A plays an es-sential role in DNA repair and GADD45 proteins displace cyclin B1 from Cdc2 and thus inhibit the formation of M phase-promoting factor that is essential for G2/M transition [87]. GADD45A has been shown to be a direct transcrip-tional target of 1 ,25(OH)2D3 with a functional VDRE within the fourth exon of the gene [88]. IGFBPs modulate the activity of the circulating insulin-like growth factors (IGF) I and II. The IGFBP3 gene was first discovered to be up-regulated by 1 ,25(OH)2D3 and contains a functional VDRE [89]. As described above, IGFBP1 and IGFBP5 are also primary 1 ,25(OH)2D3 target genes [72]. Another inter-esting primary 1 ,25(OH)2D3 target is the PPARD gene, which contains a potent DR3-type VDRE in close proximity to its TSS [90]. PPAR and VDR proteins are widely ex-pressed and in an apparent overlap in the physiological ac-tion of the two nuclear receptors, both are involved in the regulation of cellular growth, particularly neoplasms. High PPARD expression in tumor seems to be positive for the prognosis of associated cancers [91]. Overall, 1 ,25(OH)2D3 restricts cell cycle progression in several phases via multiple and partially redundant targets on parallel pathways that when combined, provide a robust anti-proliferative effect.

3.4. Transcriptome-Wide Analysis of 1 ,25(OH)2D3

Signaling

The effect of 1 ,25(OH)2D3 on mRNA expression, i.e. 1 ,25(OH)2D3-induced changes of the transcriptome, has been assayed by multiple microarray experiments in cellular models (either an established cell line or primary cells) or in in vivo models (mostly rodents). While the focus is on identi-fication of primary VDR target genes, the stimulation times were short (2 to 6 h), but when the overall physiological ef-fects are the center of the study, longer treatment times were used (24 to 72 h). For a limited number of putative VDR target genes, quantitative PCR can be applied, but for a tran-scriptome-wide perspective on VDR signaling, microarrays or RNA-Seq have to be used.

A few years ago, cDNA arrays with an incomplete num-ber of genes were used and rather short lists of VDR target genes from colon [92], prostate [74, 93-95], breast [96] and osteoblasts were obtained [97, 98]. However, despite these limitations in squamous cell carcinoma already more than 900 genes responded within 12 h to a stimulation with 1 ,25(OH)2D3 [99]. Also more recent microarray analyses in various tissues and cells from different species have sug-gested a long list of VDR target genes. For example, in hu-man monocytes 638 genes responded to a 4 h treatment with 1 ,25(OH)2D3 [62], while a 36 h stimulation of human lym-phoblasts let only 229 genes move [100]. However, the over-lap between these two 1 ,25(OH)2D3 target gene lists is only

5.6%. Although the setups of all these microarray analyses were different in treatment times and probe sets, there is the overall impression that most VDR target genes respond to 1 ,25(OH)2D3 in a very tissue-specific fashion and may manifest only a rather transient response. A number of these genes may not be primary 1 ,25(OH)2D3 targets, but never-theless they contribute to the physiological effects of 1 ,25(OH)2D3. The induction of these genes may be delayed by a few hours or even days and were probably mediated by primary 1 ,25(OH)2D3-responding gene products, such as transcription factors or co-regulator proteins [92].

3.5. Genome-Wide View on VDR Binding

In some cases VDREs can be in a distance of 100 kb and more, either up- or downstream of the TSS. As discussed above, most primary VDR target genes seem to use multiple VDREs for full functionality [101]. These VDREs are typi-cally arranged near binding sites for other transcription fac-tors into collections of neighboring sites, so-called modules or enhancers. Modules of transcription factors that act on focused genomic regions have been shown to be far more effective than individual factors at isolated locations. The combination of ChIP assays with hybridization of the result-ing chromatin fragments on microarrays, so-called ChIP-chip assays, provided an additional step for a larger scale analysis of VDR target genes. The ChIP-chip technology has been applied to the analysis of the VDR gene [102], the CYP24A1 gene [103], the intestinal calcium ion channel gene TRPV6 [75], the Wnt signaling co-regulator low den-sity lipoprotein receptor-related protein 5 (LRP5) [104] and the TNF receptor ligand gene TNFSF11 [78]. For all those genes, a number of VDR-associated chromatin regions were identified, some of which were far upstream or downstream of the gene�’s TSS. These studies also confirmed that many, if not all, VDR target genes have multiple VDR-associated regions.

Recently published massive parallel sequencing of ChIP templates (ChIP-Seq) studies reported genome-wide 5,000 to 10,000 binding sites per nuclear receptor [105, 106], while microarrays showed some 10-fold less genes being primary target genes within the same cell type [106, 107]. All ChIP-Seq studies agree on that the majority of identified transcrip-tion factor binding sites are distal to promoters [108, 109]. This also confirms the above-mentioned concept that the regulatory unit of a gene involves multiple transcription fac-tor binding sites at various positions and that the spatial or-ganization of the unit is important to bring via DNA looping at least one activated nuclear receptor protein close to the TSS of the respective primary nuclear receptor target gene.

The first ChIP-Seq results for VDR in human lym-phoblasts confirmed reported 2776 genomic VDR binding sites and 229 target genes [100]. A second, very recently published ChIP-Seq analysis of the human monocytic cell line THP-1 reported 2340 genomic VDR binding sites and 638 target genes [62]. However, the first study analyzed VDR binding and gene regulation 36 h after ligand stimula-tion, while the second study looked on VDR binding 40 min after ligand treatment and mRNA regulation of 4 h. Interest-ingly, 520 of the VDR binding sites in monocytes are exclu-sive to unstimulated THP-1 cells. Still, the number of the


remaining 1820 1 ,25(OH)2D3-treated genomic VDR bind-ing sites in THP-1 cells is comparable with the 2776 VDR binding sites in 1 ,25(OH)2D3-treated lymphoblasts, but the overlap is only 18.2%.

In the absence of ligand, VDR has been shown to ac-tively repress its target genes, likely via a mechanism involv-ing an interaction with CoR proteins [110, 111]. Interest-ingly, only 14% of the 520 genomic VDR binding locations that occur in THP-1 cells uniquely in the absence of ligand contain a DR3-type VDRE [62]. In contrast, up to 90% of the best 1 ,25(OH)2D3-dependent VDR binding regions in these cells contain at least one DR3-type VDRE. This means that upon ligand treatment the VDR occupancy is strongly shifted from non-DR3 locations to DR3-type RE locations. It is possible that the non-DR3 locations may serve as a nuclear store of VDR to be utilized rapidly upon the introduction of the ligand, partly substituting for the need to transport VDR into the nucleus from outside. Interestingly, for 300 (73.5%) of the 408 up-regulated 1 ,25(OH)2D3 target genes in THP-1 cells showed VDR binding within 400 kb of their TSS, while this applied only for 104 (45.2%) of the 230 down-regulated genes [62]. This suggests that the mechanisms of down-regulation of VDR target genes are rather complex and/or varied, and may require gene-specific investigations as dem-onstrated for the CYP27B1 gene [67].

In both VDR ChIP-Seq studies [62, 100] DR3-type REs were detected by de novo motif search as the most dominat-ing sequence in the genomic VDR peak regions. However, in THP-1 cells only 31.7% of all VDR binding regions contain a DR3-type RE, i.e. the majority of genomic VDR binding sites do not use this type of VDRE. Since the most dominant ligand-induced VDR binding sites are highly enriched for DR3-type REs that preferentially locate to the neighbor-hoods of up-regulated 1 ,25(OH)2D3 target genes, it is likely that DR3-type REs are primarily used in gene activation. However, this leaves the question about the nature of VDR binding at those 68.3% of locations that lack a DR3-type VDRE. Although the de novo motif search could not identify any non-DR3-type VDRE that would explain any significant proportion of the VDR binding, it does not exclude the pos-sibility that a low number of such sites exist. Other possible explanations include sites where VDR binds indirectly to genomic DNA via other transcription factors. A possible explanation for both the VDR-peak-less target genes as well as the target gene-less VDR peaks comes from the recent demonstration that gene regulation by VDR is a very dy-namic process with rapid changes of VDR binding site occu-pancy as discussed below. Therefore, the time points chosen in each study represent only snap-shots of VDR�’s actions. Due to the fast dynamics of VDR binding site occupancy and resulting fluctuations in target gene mRNA amounts, it is likely that without time-course data a considerable propor-tion of all transient VDR binding sites remains unknown.

Genome-wide analysis of VDR in THP-1 cells showed that there are only about 20 genes that show a single VDR location close to the target gene TSS [62]. More common are situations where either one target gene has multiple VDR binding sites in its regulatory region or a pair of closely lo-cated VDR target genes share one or more VDR binding sites. The first constellation were already demonstrated for

the above mentioned VDR target genes, while the second scenario applies for the members of the IGFBP gene family [72]. The most complex regulatory situations was found in the clusters around the VDR target genes thrombomodulin (THBD) and myosin IXB (MYO9B), which each contains five or six VDR binding sites of different characteristics. In summary, ChIP-Seq analysis provides regulatory explana-tions for a large majority of 1 ,25(OH)2D3 target genes.

3.6. Transcriptional Dynamics

Using time-resolved ChIP, Shang et al. [112] demon-strated that several CoA proteins were recruited in a cyclical fashion to an estrogen responsive chromatin region of the human trefoil factor 1 (TFF1, also called pS2) gene. Metiv-ier et al. [113] showed on the same chromatin region the sequential and ordered recruitment of ER , RNA polym-erase II and many chromatin factors, such as CoAs, CoRs, HATs, HDACs and HMTs, which defined the direction of cycling on this chromatin region. Similar observations were made with the androgen receptor on the human kallikrein 3 (KLK3, also called PSA) gene [114], with thyroid hormone receptor on the human thyroxine deiodinase type I (DIO1) gene [115] and with VDR on the human genes CYP24A1 [66, 116], CDKN1A [70], IGFBP3 [117] and MYC [118]. All these examples show cyclical association of co-regulators and, in part, also of the respective nuclear receptor with a periodicity of 30 to 60 min. Interestingly, the more recently published reports on CDKN1A and IGFBP3 also demonstrate the cycling of mature mRNA [70, 117].

Generally, transcriptional cycling is a phenomenon that depends on stimulus availability, association and dissociation of the transcription factor and its co-regulators and finally their possible removal through proteasomal degradation. Transcriptional cycling of mature mRNA can be observed only with those genes, whose half-life of the induced mRNA transcript is shorter than the periodicity of cyclical association of transcription factors and their co-factors, i.e. in average 60 min or less. Only under this condi-tion there is within one transcription cycle enough mRNA degradation, in order to observe cycling of transcript levels [10]. This reduces the list of genes that show transcriptional cycling to those that encode short-lived regulatory proteins, such as transcription factors and kinases. Moreover, in order to see transcriptional cycling at the cell population level, cells must be synchronized. Stimulation with a nuclear re-ceptor ligand has been shown to be sufficient for a popula-tion level synchronization of cells [70, 117], although in some studies [112, 113] pre-treatment with the RNA polym-erase II inhibitor -amanitin was employed.

Transcriptional cycling allows for better control of gene transcription. A gene can be silenced far quicker, when it has to confirm every 60 min, if its transcription is still required, than without this control mechanism. As discussed above, transcription is a dynamic process with high mobility of transcription factors. The action of reusable factors, such as transcription factors and their co-regulators, and of the chromatin activation status is intrinsically cyclic, since they act as catalysts or scaffolds. Ensembles of such systems can subsequently display synchronized cycles depending on the stochastic distribution functions of their cycling time. For


example, low frequency stimulations of cells with tumor necrosis factor induces cycling of the abundance of the tran-scription factor NF- B in the nucleus [119]. Moreover, pul-sative exposure of cells with ultradian release of cortisol stimulates transcriptional cycling of the glucocorticoid re-ceptor [120]. Interestingly, these transcriptional cycles are not observed, when the synthetic glucocorticoid receptor ligand dexamethasone is used, which stabilizes the receptor for longer periods than the natural ligand cortisol [153]. A similar observations was made with the VDR agonist Gem-ini, which failed to induce transcriptional cycling of the hu-man IGFBP3 gene, while the natural ligand does [117]. These observations may have implications for the therapeu-tic application of synthetic nuclear receptor ligands and may explain some of their side effects.

4. MOLECULAR ACTIONS OF VITAMIN D AND ITS ANALOGUES

4.1. Vitamin D Metabolism

Vitamin D can be either taken up from diet, such as fish oil, or it is synthesized in the skin from 7-dehydrocholesterol under the exposure of the UV component of sunlight [121]. In the liver vitamin D is then hydroxylated at its 25 position producing the main circulating form, 25-hydroxyvitamin D3 (25(OH)D3), followed by 1 -hydroxylation in the kidney leading to the hormonal form 1 ,25(OH)2D3 [122]. Both steps are enzymatically controlled by CYP enzymes. The major 25-hydroxylase is CYP2R1 located in microsomes [123], while the 1 -hydroxylation is carried out by mito-chondrial CYP27B1 enzyme [124]. The degradation of 1 ,25(OH)2D3 starts with the action of the mitochondrial 24-hydroxylase CYP24A1 yielding in multiple steps to either calcitroic acid or to 1 ,25(OH)2D3-26,23S-lactone [125]. Also 1 ,25(OH)2D3 analogues are metabolized by these CYP enzymes, but often with different efficiency leading to a different metabolic profile. Some tissues, such as keratino-cytes and monocytes, express all enzymes for the metabolic conversion of vitamin D, i.e. in these tissues 1 ,25(OH)2D3 acts a paracrine hormone [42].

In the serum all vitamin D compounds are transported by the DBP [126]. The affinity of DBP for the various vitamin D compounds differs, being for example higher for 25(OH)D3 than for 1 ,25(OH)2D3 [127]. The biological effi-cacy of 1 ,25(OH)2D3, its natural metabolites and synthetic analogues therefore does not only depend on the efficiency of their binding to the VDR-LBD but also on their interac-tion with CYP enzymes and DBP. In particular for a clinical application of 1 ,25(OH)2D3 analogues their metabolic sta-bility is of high impact.

4.2. 1 ,25(OH)2D3 Analogues

More than 3,000 synthetic analogues of 1 ,25(OH)2D3 are presently known and the majority of them carry a modi-fication in their aliphatic side chain [128] Fig. (1). 1 ,25(OH)2D3 analogues have been developed with the goal to improve the biological profile of the natural hormone for a therapeutic application either in hyper-proliferative diseases, such as psoriasis and different types of cancer, or in bone disorders, such as osteoporosis [51]. Most of the analogues

described to date are agonists, with a few having been identi-fied as antagonists. Only the two side chain analogue Gemini and some of its derivatives act under restricted conditions as inverse agonists [129, 130]. All these compounds bind to the VDR-LBD, which agonistic and antagonistic/inverse agonis-tic conformations explain well the functional profile of the different types of ligands.

The most detailed information about the molecular mechanisms of the analogues can be obtained from crystal structures. In addition to the above discussed complex of the VDR-LBD with the natural hormone [49], it has been crys-tallized with a number of synthetic analogues Figs. (1, 2, 4 and 6). However, docking of a ligand into the VDR-LBP is a much more dynamic process than the �“static�” crystal struc-tures can monitor. Therefore, several VDR ligands were also analyzed by molecular dynamics simulations.

4.3. 20-epi VDR Agonists

The 20-epi analogues MC1288 (20-epi-1 ,25(OH)2D3) and KH1060 (1 ,25(OH)2-(20S)-22-oxa-24,26,27-trihomovitamin D3) Figs. (1 and 5) were the first synthetic ligands that were co-crystallized with the VDR [131]. Inter-estingly, the VDR-MC1288 and VDR-KH1060 complexes show deviations on the positions of their C atoms of only 0.08 Å and 0.14 Å, respectively, when compared with the VDR-1 ,25(OH)2D3 complex [131]. These variations con-cern only some side chains located at the surface of the pro-tein. Contrary to the belief that the 20-epi analogues are in-ducing a different agonist conformation of the VDR, the overall conformation and especially the position of helix 12 are strictly maintained in all three complexes. Furthermore, the deviations of all atoms comprising the LBP are only 0.09 Å for the VDR-MC1288 complex and 0.12 Å for the VDR-KH1060 complex. Whilst the sizes of 1 ,25(OH)2D3, MC1288 and KH1060 are 381 Å3, 375 Å3 and 392 Å3, re-spectively, the volume of the LBP remains unaltered in the three complexes (697 Å3), so that the ligands occupy only 55-56 % of its volume.

The A-, seco-B and CD-rings of MC1288 and KH1060 form identical contacts as described above for the VDR-1 ,25(OH)2D3 complex as well as the OH-groups use the same three anchoring points [131]. Also when comparing other details of the receptor-ligand complexes, the interac-tion between the 20-epi agonists and the VDR does not result in significant variations from the LBD conformation induced by the natural ligand. However, the 20-epi analogues make new contacts with the VDR-LBD as a consequence of the different conformation adopted by their side chain. Addi-tional interactions of KH1060 due to the 20-epimerization and the extra methyl-groups could explain the higher stabil-ity and longer half-life of its complex with VDR. The VDR-MC1288 complex is energetically more favorable than the more tense conformation of the natural ligand. VDR-20-epi analogue complexes are more resistant to digestion over time [132, 133] suggesting that the half-life of the active confor-mation is the main factor responsible for the formation of a more potent complex with CoA and subsequent higher tran-scriptional activity. In addition, the hydrophobic core located in the loop 6-7 showing the major rearrangement of these residues and moving towards MC1288, which subsequently


decreases the volume of the LBP leading to a ligand fill it up of 66% [134]. Moreover the temperature-b-factors for C -atoms, which reflect the molecular oscillation or vibration, of the 20-epimer analogue-bound-VDR-LBD are lower sug-gesting the higher stabilizations of these complexes com-pared to the 1 ,25(OH)2D3-bound VDR-LBDs.

4.4. Other VDR Superagonists

The human VDR-LBD has also been crystallized in complex with the analogues MC903 (calcipotriol) [135], EB1089 (seocalcitol) [135] and TX522 (14-epi conforma-tion) [136] Figs. (1 and 6). In addition, the rat VDR-LBD was crystallized with 1 ,25(OH)2D3 and the 2-substituted analogues 2-methylene-19-nor-(20S)-1 ,25(OH)2D3 (2MD), 1 -hydroxy-2-methylene-19-nor-(20S)-bishomopregnacalciferol (2MbisP) and 2 -methyl-19-nor-1 ,25(OH)2D3 (2AM20R) [137] Figs. (1 and 6). In all crystal structures a single conformation with a deviation of their C -atom positions between 0.09 and 0.37 Å in comparison to the VDR-1 ,25(OH)2D3 complex was observed. The LBDs of human and rat VDR show high similarity with a deviation of their backbone C -atoms of only 0.53 Å [137]. Moreover, also with these analogues the position and conformation of helix 12 was strictly maintained.

The varying side chain length of the ligands resulted only in small differences and degrees of freedom in the distances between the OH-groups at the anchoring points S237/R274, Y143/S278 and H305/H397 Fig. (2B). Despite an increased rigidity and length of the side chain of EB1089 its 25-OH-group is shifted by only 0.4 Å. The triple bond of the side chain of TX522 causes a shift of carbon 21 by 0.4 Å and because of its rigidity the side chain takes another pathway in the pocket and makes an additional contact with I268 (he-lix 5) [136]. Due to the 14-epi-configuration, the CD ring of TX522 is shifted by 0.5 Å and carbon 12 makes closer con-tacts with V300 (helix 6) [136]. These closer and additional contacts between the analogue and the LBP may cause TX522 to dissociate slower from the VDR than the natural hormone although this is in contrast to an analysis by limited protease digestion [138]. A remarkable feature of MC903 is the absence of a direct contact with helix 12, while its cyclo-propyl-group interacts with the C -atom of A231 (helix 3) [135].

The binding of 2MD, 2MbisP and 2AM20R to the rat VDR-LBD provides another example that the positions of the amino acids in the LBP do not change in comparison to the complex with the natural hormone [137] Fig. (6). The cavity is quite large and the VDR accepts the structural modifications at carbon 2 without any conformational dis-ruption. The CD-ring of 2MD is tilted and the 17 -aliphatic side chain is permitted substantial flexibility within the pocket, while the 25-OH-group still occupies the same posi-tion as that of the natural ligand. The conformation of the entire core portion of 2MbisP closely resembles that of 1 ,25(OH)2D3, but due to the absence of the 25-OH-group in the truncated side chain a water molecule provides the link to the two anchoring histidine residues. However, the lack of a direct contact with the anchoring points increases the mobil-ity of 2MbisP and decreases the half-life of the complex pro-portionally. The A-ring of the analogue 2AM20R takes the

-chair conformation and the 1-OH-group is positioned equatorial. However, the expected differences in the biologi-cal activity of the carbon 2-modified analogues are not re-flected by the crystal structure data.

A new structure-based approach was taken, when an oxo-lane ring was incorporated at C20 with the aim to optimize the aliphatic side chain conformation with a subsequent en-tropy benefit yielding to AMCR277A with in vitro super-agonistic properties while showing the calcemic effect of 1 ,25(OH)2D3 in vivo [139]. The binding mode of AMCR277A is very similar to that of 1 ,25(OH)2D3 taking a half-boat conformation without further tension of the side chain with the observed bond angle of C23-C24-C25 (118º), highly similar to those of 1 ,25(OH)2D3 (121º) and KH1060 (118º) Fig. (6). Moreover, the additional van der Waals con-tact to V300 [139] increases the stability of the hydrophobic core in the helices and loop 6-7 [134] helping to further sta-bilize the ligand-protein complex.

An extra improvement in the superagonistic properties is provided by addition of a 2-methyl-group to the A-ring of AMCR277A. The basis for this modification is that 2-methyl-1 ,25(OH)2D3 has better binding and biological properties compared to 1 ,25(OH)2D3. The 2-methyl-AMCR277A-bound VDR-LBD supports the inheritance from both parent molecules the 2-methyl-1 ,25(OH)2D3 and AMCR277A, especially the unique van der Waals contacts of the 2 -methyl moiety with F150, L233, S237 and the oxo-lane ring with V300 [140]. The additional van der Waals contacts of 2-methyl-AMCR277A may explain in part its higher transactivation properties, and more potent ability to inhibit HL60 cell proliferation and promote differentiation into monocytic cells.

4.5. Analogues with Two Side Chains

The first two side chains analogue synthesized termed Gemini is a 1 ,25(OH)2D3 analogue with two identical side chains that despite its significantly increased volume (25%) binds to the VDR [141] Figs. (1 and 6). Molecular dynamics simulations of the Gemini-VDR complex demonstrated that one of the two side chains of Gemini takes the same location as in the natural hormone, whereas for the second side chain, two approximately equal positions were identified [142]. As agonist, Gemini prefers possibility 1, whereas as inverse agonist it takes possibility 2. Supramolar CoR concentrations shift Gemini from an agonist to an inverse agonist, which actively recruits CoR to the VDR and thus mediates repres-sion of 1 ,25(OH)2D3 target genes [130]. Molecular dynam-ics simulations indicated that the second side chain of Gem-ini creates tension within the VDR-LBD, which in excess of CoR proteins can be released by shifting helix 12 into an inverse agonistic/antagonistic position. However, a classical antagonist, such as ZK168281 (see chapter 4.7), can never convert to a superagonist, because its bulky side chains causes steric hindrance to helix 12 [141]. This suggests that Gemini may function as a sensor for cell-specific CoA/CoR ratios.

The second group of the analogues with two side chains are represented by eight derivatives with 22-butyl branching of the side chain [143]. Unusual and divers properties have been established for the two of the most active analogues


(Fig. 6) contd�….

Fig. (6). List of VDR-ligand complexes deposited in the PDB within the years 2000-2011. 1 ,25(OH)2D3 and selected analogues have been crystallized with the VDR-LBDs of human, rat and zebrafish. Extra references: [176-184].

namely 22S-butyl-20-epi-1 ,25(OH)2D3 and 22S-butyl-1 ,25(OH)2D3. The 22S-butyl-20-epi-1 ,25(OH)2D3 acts in all tested conditions, such as the activation of the rat osteo-pontin (ONP) gene promoter, the expression of CYP24A1 mRNA, the ligand-dependent recruitment of the RXR and CoA peptide, and in HL-60 cell differentiation and anti-proliferation model as a potent agonist similar to 1 ,25(OH)2D3. In contrast, its 20-epimer 22S-butyl-1 ,25(OH)2D3 behaves in all assays as a potent antagonist in a concentration-dependent manner, when also 1 ,25(OH)2D3 is present. Surprisingly, the two analogues have been crystal-ized in complex with the rat VDR-LBD and CoA peptide. For both of them the overall protein fold is similar to the canonical active conformation but with differences such as the 22-butyl chain of the agonist is oriented toward helix 12 and the 24-hydroxyl-group contacts the carbonyl oxygen of the main chain of V296 in the helix 6. This more tight ar-rangement of the side chains slightly expands the bottom region of the LBP.

The changes in the cavity of the LBP to accommodate the branched moiety of the antagonist�’s side chain are simi-lar to what has been seen for Gemini. The clear twisting of the residues L305 and S394 creates the expansion of the LBP in the region surrounded by helices 6, 7 and 11 [143].

The additional modification of the 22S-butyl-1 ,25(OH)2D3 antagonist to 22S-butyl-1 ,24R(OH)2D3, which contains the three carbons absent from the side chain of 22S-butyl-1 ,25(OH)2D3, shows a weak but restored ago-nistic activity [144]. The structural determinants of this re-versed property is attributed to the restoration of the con-served interactions of the ligand with residues (of the rat VDR-LBD), such as H301 and H393, the original position of L305 and a general increase in the stability of the complex.

4.6. Non-Steroidal VDR Agonists

Most 1 ,25(OH)2D3 analogues carry only rather minor modifications compared to the natural hormone and stabilize the same agonistic VDR conformation [131] Figs. (1 and 6). However, there are also semi-steroidal analogues, which either have significant changes in their A-, C- or D-rings,

such as CD ring deletion [145], or carry substantial addi-tions, such as the second side chain in Gemini [141, 146]. Non-steroidal ligands are known for several members of the nuclear receptor superfamily, such as flutamine for the an-drogen receptor [147] and raloxifene for the ER [148]. For the VDR initially only diphenylmethane derivatives, such as LG190178 and its derivatives [149-152] and the bis-aromatic molecule CD4528 and its derivatives [153], have been de-scribed as pure non-steroidal agonistic ligands. Molecular dynamics simulations demonstrated that both types of non-steroidal VDR ligands take a shape within the LBP that is very similar to that of the natural ligand. The fact that the non-steroidal ligands have a significant effect on the activity of the VDR, indicates that the seco-steroid backbone of 1 ,25(OH)2D3 is of less importance than the positioning of the three anchoring OH-groups see Fig. (2B). The more ex-actly the non-steroidal compounds place their OH-groups, the lower seem to be their respective EC50-values in the dif-ferent in vitro assays. CD4528 is mimicking the natural hormone best and was found to be in vitro at least 5-times more potent than LG190178 [153]. At an appropriate non-calcemic dose (150 g/kg in mice) CD4528 acts very much like 1 ,25(OH)2D3 both in vitro and in vivo [153]. The only to date published crystal structure of a non-steroidal VDR agonist is YR301 ((2S)-3-[4-(3-{4-[(2R)-2-hydroxy-3,3-dimethylbutoxy]-3-methylphenyl}pentan-3-yl)-2-methylphenoxy] propane-1,2- diol [154]), a stereoisomer of LG190178, which has been shown to be a potent agonist. In the rat VDR-LBP YR301 mimics the conformation of 1 ,25(OH)2D3, where its diethylmethyl-group takes the same spatial position than the C and D rings of 1 ,25(OH)2D3 Fig. (6). The two functional hydroxyl-groups of YR301, 20-OH and 2-OH, form hydrogen bonds with H301 and H393 and S233 and R270, respectively. In addition, the terminal hy-droxyl-group (3-OH) has a contact with R270 and a unique water mediated interaction with D144 and Y232 [154]. YR301 shows transcriptional activity comparable to 1 ,25(OH)2D3.

In the recent years new medicinal chemistry approaches have taken to improve the stability and efficacy of non-steroidal VDR analogue that leaded to phenyl-furan ana-logues [155]. These analogues display wide range of bio-


logical properties from binding affinities comparable to 1 ,25(OH)2D3 and at the same time greatly lowered calcemic properties in vivo.

4.7. Classical VDR Antagonists

Agonism and antagonism of nuclear receptor ligands are closely related processes. Both type of ligands bind to the same site within the LBP, but agonists stabilize the position of helix 12 in the agonistic conformation, while antagonists prevent this stabilization. Two classical types of VDR an-tagonists are well described [156]: 25-carboxylic esters, such as ZK159222 [157] and ZK168281 [158], and the 26,23-lactone TEI-9647 [159-161]. Compared to the natural hor-mone, both types of compounds have relatively bulky ring structures in their side chains. In addition, ZK159222 and ZK168281 carry much longer side chains than TEI-9647. ZK159222 and TEI-9647 were characterized as partial an-tagonists, whereas ZK168281 was found to be a pure an-tagonist [156, 162]. ZK159222 and ZK168281 have an affin-ity to the VDR that is comparable to that of 1 ,25(OH)2D3, while that of TEI-9647 is at least 10-fold lower [156].

Selective antagonism as exhibited by TEI-9647, ZK159222 or the synthetic ER ligands tamoxifen and raloxifene appears to be a complex phenomenon that arises through the interplay of a number of factors, such as differ-ential ligand effects on the transactivation of the nuclear re-ceptor, the type of co-regulator recruited as well as the cell and promoter context. The most critical parameter for judg-ing the quality of an antagonist appears to be the amount of its residual agonism, which showed to have for TEI-9647 a rather high level [156, 163]. Interestingly, in rat cells the residual agonistic activity of TEI-9647 is significantly higher than in human cells, because the rodent-specific VDR amino acids S403 and N410 display more and stronger interactions than C403 and C410 in human VDR [164].

The lactone ring of TEI-9647 is more bulky than the end of the side chain of the natural hormone and cannot interact effectively with the anchoring point H397 [164]. Moreover, the carbonyl-group of the lactone ring cannot interact di-rectly with F422 (helix 12), so that finally some steric hin-drance is created. This destabilizes the position of helix 12, induces flexibility to the helix and makes a stable interaction of the VDR-LBD with CoA proteins unlikely. However, TEI-9647 lacks an extended side chain and disturbs helix 12 less than ZK168281, so that only the 26,23-lactone but not the 25-carboxylic ester is affected by the increased interac-tion potential of S403 and N410 in rat VDR [164]. This re-sults in a loss of the antagonist function of TEI-9647 in ro-dents. The increasing agonistic action of TEI-9647 parallels to the gain of interaction of the VDR with CoA protein and the lack of stabilization of the antagonistic conformation of the receptor. Chemical modifications of TEI-9647, in par-ticular at carbon 2, significantly increase the VDR binding affinity [165, 166]. These compounds cannot sit as deep at the bottom of the LBP as an unsubstituted ligand. This de-creases the distance between the carbonyl-group of the lac-tone ring and F422 and provides the respective compounds with a higher antagonistic potential than the parental com-pound.

Recently, an interesting mechanistic insight on TEI-9647-mediated antagonism was provided from the published crystal structure [167]. The crystal structures of TEI-9647 bound to the rat VDR-LBD or to the human VDR-LBD mu-tants H305F and H305F/H397F indicated that the interaction of TEI-9647 with H305 is crucial for its antagonist activity. In addition, the presence of the two cysteine residues (C403 and C410) is critical for the inactivation of human VDR in the presence of TEI-9647. Based on the crystal structure, it is suggested that first TEI-9647 contacts H305, then the VDR-LBD undergoes a conformational change that results in the reorientation of C403 or C410 to close proximity of the ana-logue and finally a covalent adduct (1,4-Michael addition) between the thiol-group of C403 or C410 and the exo-methylene-group of TEI-9647 is formed [167].

Other newly synthesized analogues displaying antagonis-tic/partial agonistic profile, such as adamantyl and 22-butyl side chain modifications, are discussed in chapters 3.5 and 3.8.

4.8. Analogues with Partial Agonistic Profiles

A wide repertoire of modifications to the backbone of the secosteroid molecule is possible yielding to multiple distinct analogues. These modifications are introductions of aromatic rings, hetero-atoms, extra carbons/alkyls and various func-tional groups Fig. (1). One of the interesting analogues that have been created by introduction an adamantly-group at the side chain terminus [168]. The crystal structures of these 19-nor-vitamin D-adamantyl substituent analogues complexed with the rat VDR-LBD show a series of partial agonistic (10-75% efficacy)/antagonistic activities. Nevertheless, in all these complexes the LBD is in canonical active conforma-tion. Although the large adamantly-moiety does not directly displaces helix 12, it protrudes into the space formed by helices 3 and 11 and loops 6-7 and 11-12 creating an extra space within the LBP. Consequently, these changes lead to the shift of the equilibrium towards inactive conformations. The presence of a CoA peptide in the crystal structure shifts the equilibrium back to the active conformation enhancing the partial agonism of these molecules. Therefore, these ana-logues are suggested to sense the CoA/CoR ratio in the cell and change their agonistic/antagonistic properties accord-ingly.

5. THERAPEUTIC POTENTIAL OF VITAMIN D AND ITS ANALOGUES

The essential role of 1 ,25(OH)2D3 for bone health is known since long time and led to the supplementation of milk products and margarine with vitamin D [169]. Within the last years the appropriate levels of the major circulating form on vitamin D in the serum, 25(OH)D3, were discussed and recently in the US the Institute of Medicine (IoM) sug-gested a serum concentration of 20 ng/ml (50 nM) as suffi-cient [170]. Although this level is still claimed to be too low [171], already a significant proportion of the world popula-tion has to be considered as vitamin D deficient. These dis-cussions emphasize that the role of 1 ,25(OH)2D3 is rather of preventing diseases than of curing them. However, in case of diseases, which directly result from vitamin D deficiency,


such as rickets, a supplementation of the diet with vitamin D had been shown to have great beneficial effects.

Compared to other nuclear hormones, such as estrogens and retinoids, vitamin D and 1 ,25(OH)2D3 are rather save compounds, since the only known severe side effect of vita-min D treatment is hypercalcemia [172]. However, as known for many other drugs, low side effects are often paired with a rather low therapeutic potential. Whether this rule applies also for 1 ,25(OH)2D3 and its analogues, has not been fully explored. 1 ,25(OH)2D3 analogues, such as MC903, are used since many years in mild and moderate forms for hy-per-proliferative skin disorders, such as psoriasis [173]. However, in more severe disease forms VDR ligands need to be combined with other drugs, such as the glucocorticoid receptor ligand cortisol, in order to be effective. There had been a number of clinical trials for the treatment of different forms of cancer with 1 ,25(OH)2D3 and its analogues [174], but none of them suggested that a VDR ligand may be the first choice of therapy for such a severe disease. However, in benign forms of hyper-proliferative diseases, such as benign prostate hyperplasia, which effects nearly 50% of elderly men, 1 ,25(OH)2D3 and its analogues can be very effective both as therapeutic as a well as preventive compounds [175].

The question, whether 1 ,25(OH)2D3 analogues are any better than the natural compound cannot be easily answered. The main attempt for the development of 1 ,25(OH)2D3 analogues was the dissociation of beneficial cell- or immune-regulatory functions from undesired side effects on calcium metabolism. This goal was reached for a number of com-pounds and some, such as MC903, are on the market and others are in development. However, in most cases not those analogues were chosen that were the most potent in in vitro and in vivo assays, but those who showed in vivo the lowest calcemic potential. This selection is safe, but does not pro-vide the most effective drug to the patient. It can be assumed that in future personalized medicine approaches will allow a better matching of patients and drugs. This may then also bring very potent VDR agonists, such as Gemini and its de-rivatives, for a specialized group of patients to the market.

6. CONCLUSION

Crystallization of the VDR-LBD with many structurally variant VDR ligands provide significant insight into the mo-lecular actions of these compounds and explained their ago-nistic and antagonistic potential. However, these studies also indicated that most VDR agonists showed no significant dif-ferences in their mode of action, i.e. most of them act as the natural ligand 1 ,25(OH)2D3. Therefore, a differential in vivo profile of an average 1 ,25(OH)2D3 analogue is more likely based on its individual pharmacokinetic properties and its metabolic stability than on any special interaction with the VDR-LBD. Exceptions are the VDR antagonists and two-side chain analogues, such as Gemini, which display special properties.

The molecular actions of 1 ,25(OH)2D3 in many differ-ent tissues are recently far better understood based on large datasets obtained from genome- and transcriptome-wide in-vestigations. Moreover, the parameter time emerges as very critical due to the dynamic response of cells and tissues to ligand treatment. All these studies underline the broad

physiological profile of the VDR and its natural and syn-thetic ligands. This supports the view that 1 ,25(OH)2D3 can be of impact for nearly all human diseases, in particular in contribution to their prevention. However, this broad insight implements also a warning for potential side effects, when VDR ligands are overdosed or applied to human individuals with an inappropriate genomic profile. Like with all other compounds with a therapeutic potential, personalized medi-cine will become increasingly important. In this respect the detailed molecular understanding of the actions of the VDR and its ligands, both on the level of receptor-ligand interac-tions as well as on genome-wide actions, provides them with a special advantage as therapeutic drugs of the 21th century.

ACKNOWLEDGEMENTS

We thank the Academy of Finland, the Juselius Founda-tion and the Foundation for Advanced Technology of East-ern Finland for support.

ABBREVIATIONS

1 ,25(OH)2D3 = 1 ,25-dihydroxyvitamin D3

25(OH)D3 = 25-hydroxyvitamin D3

ALOX5 = Arachidonate 5-lipoxygenase

CCNC = Cyclin C

CDKI = Cyclin-dependent kinase inhibitor

CDKN1A = Cyclin-dependent kinase inhibitor 1A

CoA = Co-activator

CoR = Co-repressor

ChIP = Chromatin immunoprecipitation

CYP = Cytochrome P450

DBD = DNA-binding domain

DBP = Vitamin D binding protein

ER = Estrogen receptor

FGF23 = Fibroblast growth factor 23

GADD45A = Growth arrest and DNA-damage-inducible, alpha

HAT = Histone acetyltransferase

HDAC = Histone deacetylase

HMT = Histone methyltransferase

HdM = Histone demethylase

IGFBP = Insulin-like growth factor binding pro-tein

LBD = Ligand-binding domain

LBP = Ligand-binding pocket

LRP5 = Low density lipoprotein receptor-related protein 5

MYO9B = Myosin IXB

ONP = Osteopontin


PPAR = Peroxisome proliferator-activated re-ceptor

PTH = Parathyroid hormone

RAR = Retinoic acid receptor

RE = Response elements

RXR = Retinoid X receptor

SLC34A2 = Sodium-phosphate transport protein 2B

THBD = Thrombomodulin

TNFSF11 = Tumor necrosis factor ligand superfa-mily, member 11

TRPV6 = Transient receptor potential cation channel, subfamily V, member 6

TSS = Transcription start site

VDR = 1 ,25(OH)2D3 receptor

VDRE = 1 ,25(OH)2D3 response element

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