Vitamin D Receptor Conformational Dynamics and Nongenomic Actions

C H A P T E R

16 Vitamin D Sterol/Vitamin D Receptor Conformational Dynamics and Nongenomic Actions Mathew T. Mizwicki, Anthony W. Norman University of California, Riverside, CA, United States

O U T L I N E Introduction269 1α,25(OH)2 Vitamin D3 Regulation of Genomic Versus Nongenomic Signaling Differentiating the Signals

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Vitamin D3 Sterol Chemistry 271 The Conformational Dynamics of the Vitamin D3 Sterol Seco-B-ring and A-rings 271 The Vitamin D3 Sterol Side-Chain Conformational Dynamics: Dot Maps 271 The Vitamin D3 Sterol Conformational Ensemble 273 Nongenomic Agonist Structural Features and Molecular Dynamics273 Nongenomic Antagonist Structural Features and Molecular Dynamics 275 1,25(OH)2D3-Mediated Rapid, Nongenomic Responses 275 Potentiation of Voltage-Gated Ion Channels 275 Control of Phosphatase, Kinase, and Phospholipase Activity278 Cross-Talk279

INTRODUCTION The classical understanding that the binding of 1α,25(OH)2 vitamin D3 (Fig. 16.1) to its nuclear receptor (NR), the vitamin D receptor (VDR), brings about the formation a VDR– retinoid X receptor (RXR) heterodimer in the nucleus of the cell, which then regulates gene expression proved to be an

Vitamin D, Volume 1: Biochemistry, Physiology and Diagnostics, Fourth Edition http://dx.doi.org/10.1016/B978-0-12-809965-0.00016-1

The Plasma Membrane Vitamin D Receptor 280 Structural/Scaffolding Proteins (e.g., Caveolins) 280 Other Signaling Factor/Scaffolding Proteins (e.g., PP1c and Src) 280 Covalent Modification (e.g., Palmitoylation) 281 Membrane Binding (e.g., PIP2/3)281 The Vitamin D Receptor Conformational Ensemble Model281 The Overlapping Two-Pocket Model 283 Experimental Support for the Vitamin D Receptor Alternative Ligand-Binding Pocket 284 Understanding the Nongenomic Agonist/Antagonist Affinity/Function Conundrum 285 Vitamin D Receptor Ligand Specificity: Does an Unliganded Vitamin D Receptor Ever Exist In Vivo?

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oversimplification for the VDR as well as other NRs. In fact it is now well established that 1,25(OH)2D3 alters cell physiology and function both through the regulation of the genome and the activity of extranuclear signaling factors localized in the plasma membrane, the cytosol, and/or intracellular organelles (e.g., the endoplasmic reticulum) [1–3]. Thus the shortand long-term effects of 1,25(OH)2D3 in in vitro, ex vivo, or

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© 2018 Elsevier Inc. All rights reserved.

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16.  VITAMIN D STEROL/VITAMIN D RECEPTOR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS



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FIGURE 16.1  Biogenesis of the vitamin D3 secosteroid. 7-dehydrocholesterol (7-DHC), also referred to as provitamin D3, in response to ultraviolet B irradiation, undergoes a photolytic ring opening to produce previtamin D3 (pre-D3). This process involves the scission of the carbon-10 (C10) and C-19 single bond. Next the vitamin D3 C10/C19 methylene and secosteroid triene system are produced by a [1,7] sigmatropic shift. Once hydroxylated at the 25-OH position, vitamin D3 becomes more bioavailable because 25-hydroxyvitamin D3 (25D3) has a high affinity for the serum vitamin D-binding protein (DBP) [179,180]. The DBP and the nuclear vitamin D receptor (VDR) are the two most well-defined binding proteins in the vitamin D endocrine system and have 1 nM affinity for 25(OH)D3 and 1,25(OH)2D3, respectively. Importantly, the conversion of 25(OH)D3 to 1,25(OH)2D3 reduces the affinity for DBP, while enhancing the affinity for the VDR [27].

in vivo environments involve dynamic, complex signaling mechanisms that include extranuclear (i.e., nongenomic) and nuclear (i.e., genomic) signaling; the relationship between the two signaling mechanisms is referred to as cross-talk in the vitamin D field [4,5]. 1,25(OH)2D3 extranuclear, nongenomic, rapid responses include the regulation of phospholipases, phosphatases, kinases, and ion channels. The signaling factors generated can themselves regulate both gene expression and transcribed/ translated products. A recent review article [6] thoroughly covers several of the model systems used to elucidate nongenomic actions of the nuclear VDR (as well as those of a membranederived receptor) to regulate intracellular signaling systems in various cellular and cell-free systems. The interaction between these nongenomic effects and the regulation of gene expression are also covered in this review. Our focus has been the study of vitamin D/VDR structure– function studies that differentiate nuclear and extranuclear signaling. Our model proposes the presence of two overlapping ligand-binding sites within the VDR, providing a physicochemical explanation for the analog-specific functions known for 1α,25(OH)2-lumisterol D3 (JN) and 1β,25(OH)2-vitamin D3 (HL) and the ability of 1,25(OH)2D3 to send signals dynamically by functioning as both a nongenomic and genomic agonist ligand.

1α,25(OH)2 VITAMIN D3 REGULATION OF GENOMIC VERSUS NONGENOMIC SIGNALING The principal goal of this chapter is to describe the structural/molecular details associated with the ability of the VDR to send signals dynamically. In addition, we will speculate on how some of the 1,25(OH)2D3 extranuclear, nongenomic, rapid responses are initiated through binding the VDR.

Differentiating the Signals A number of different methods and postulates are used to differentiate the relative role of each type of 1,25(OH)2D3dependent response in a specific function being addressed. The first and most common criterion used to differentiate 1,25(OH)2D3 genomic and nongenomic signaling is the time required to observe a measurable response following the administration of 1,25(OH)2D3 [7]. It is generally accepted in the hormone/NR fields that hormone-dependent effects observed within seconds to minutes involve only extranuclear signaling cascades [1]. These rapid, cellular effects do not require transcription of new mRNA, i.e., 1,25(OH)2D3–VDR regulation of genes [8].

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VITAMIN D3 STEROL CHEMISTRY

The advancement of biophysical instrumentation, molecular biology reagents, and knockout/in animal models has ushered in additional methods used to differentiate genomic and nongenomic responses that are not strictly dependent on time. For example, immunohistochemistry and confocal microscopy have led to the demonstration that the VDR and other NRs can be found localized or trafficked to the plasma membrane and/or various cytoplasmic compartments in response to hormone (e.g., 1,25(OH)2D3) treatment [9–13]. Importantly, knockdown or knockout technologies, in conjunction with phosphor-specific western analysis (e.g., activation of MAPK), whole-cell patch clamp, and/ or polymerase chain reaction can now be used to verify the role VDR-dependent nongenomic signaling plays in a given cellular function. However, the most telling, powerful, and widely used technique to differentiate 1,25(OH)2D3 nongenomic and genomic signaling is synthetic chemistry [14–19].

VITAMIN D3 STEROL CHEMISTRY The chemistry of vitamin D3 sterols (VDS) (e.g., 1,25(OH)2D3, Fig. 16.1) is unique among NR ligands that have been shown to regulate both genomic and nongenomic signaling through the same receptor molecule. Specifically, the VDS chemistry allows for the molecule to rapidly sample many different conformations and therefore 3D shapes (Figs. 16.1 and 16.2) [15,20,21]. Thus unlike traditional steroid hormones changes in the chemistry of the vitamin D3, carbon scaffold significantly alters both the conformational dynamics and the physicochemical properties of the ligand. The major message conveyed in this section is that ultimately the chemical change to the VDS underpins the change in function of the ligand observed, whether that be in the setting of a nongenomic, genomic, and/or cross-talk assay platform.

The Conformational Dynamics of the Vitamin D3 Sterol Seco-B-ring and A-rings Vitamin D3 (VD3) is a secosteroid hormone derived from 7-dehydrocholesterol (7-DHC) (Fig. 16.1) [22,23]. The term seco refers to the fact that VD3 has a fractured B-ring. The seco-Bring (i.e., previtamin D3 (pre-D3, Fig. 16.1)) is generated in the skin in response to exposure of 7-DHC to ultraviolet B (UVB) irradiation (Fig. 16.1) [24]. Following the production of preD3, VD3 is formed by a thermal [1,7] sigmatropic shift [25,26]. This shift allows for 360 degrees rotation about the carbon-6,7 single bond (Fig. 16.2A), allowing the molecule to sample cisoid and transoid conformations (Fig. 16.1, compare pre-D3 and VD3). Said differently, the A-ring (Fig. 16.2B) is free to rotate above and below the CD-ring plain of the VD3 molecule [27] (Fig. 16.2A). Thus, the sun provides the UVB and the heat required for endogenous production of the vitamin D seco-Bring (i.e., triene system), thereby conferring on the VD3 molecule increased entropy (i.e., disorder) when compared with its metabolic precursors 7-DHC and pre-D3 (Figs. 16.1 and 16.3).

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Opening of the B-ring also frees the A-ring of VD3 to equilibrate between the α-chair and β-chair conformations and the intermediates known to exist for cyclohexane in this process (e.g., twist boats and boats; Fig. 16.2B). The chair equilibrium has been shown by solution [28] and solid state [29] NMR and confirmed by computation [30] to be nearly identical for VD3, 25(OH)D3, and 1,25(OH)2D3 (∼50:50). The two chair conformations differ in the spatial orientation of the exocyclic methylene (C19) and the C1- and/or C3-OH groups (Fig. 16.2B). It is noted that the spatial orientation of the C19 atom in the 6-s-trans conformations is used to define the α-chair (below the CD-ring plain, referred to as the α-face, Fig. 16.2A) and the β-chair (above the CD-ring plain, referred to as the β-face, Fig. 16.2A) rotomers of VD3. A common synthetic modification used in vitamin D drug design is to remove C19 (i.e., the exocyclic methylene of VD3) [31]. The 19-nor modification makes it energetically easier for the molecule to transition between the 6-s-cis and 6-s-trans conformation (Fig. 16.2A) and the α-chair and β-chair A-ring chair flips (Fig. 16.2B) [32] (see next paragraph). The 19-nor synthetic modification also removes the most common site on the VD3 molecule known to react with reactive oxygen species (ROS) [33]. Perhaps these changes or those remaining unidentified account for the reduced calcemic effects of 19-nor, 1,25(OH)2D3 analogs [34]. Thus the A-ring and seco-B-ring molecular dynamics provide potential useful and under-investigated sites for future vitamin D therapeutic design when compared with the exhaustively modified and studied 1,25(OH)2D3 side chain [14].

The Vitamin D3 Sterol Side-Chain Conformational Dynamics: Dot Maps The most conformationally dynamic region of the VDS carbon scaffold is the cholesterol-like side chain (Fig. 16.2C). Hydroxylation of the C25 atom of 25(OH)D3 by the cytochrome P-450 enzyme, commonly referred to as the 25-OHase [35], produces a more bioavailable form of vitamin D3, 25(OH)-vitamin D3 (25(OH)D3) (Figs. 16.1 and 16.3). The most common method used to assess the molecular dynamics of this region of the VDS is a conformational search calculation, commonly referred to as a dot map calculation [36]. Early dot map calculations performed in the Okamura and Yamada laboratories indicated that the 25-OH group of 1,25(OH)2D3 samples a large steric space, where most of the dots (i.e., 3D position of the 25-OH group) were located to the right of the D-ring [37,38] (Fig. 16.2C). When C20 of 1,25(OH)2D3 is epimerized (20-epi-1,25(OH)2D3; IE), it was observed in the dot map that the side chain preferred occupying steric space above the CD-ring [36,39] (Fig. 16.2C), a relative steric position later shown by X-ray crystallography to be the location of the 25-OH group with respect to the CD-ring, when bound to the VDR ligand-binding pocket [40,41]. Thus the dot map and the X-ray side-chain orientation were consistent with the results provided by a number of laboratories demonstrating that IE was a superagonist VDR ligand because its chemistry favors the “active” side-chain trajectory (i.e., C16dC17dC20dC22 dihedral; Fig. 16.2C).

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FIGURE 16.2  Illustration of the vitamin D sterol (VDS) conformational ensemble model. (A) The seco-B-ring: the diagram shows 360 degrees rotation about the C6dC7 single bond of vitamin D3 (i.e., vitamin D sterol (VDS)) triene. This is indicated by the curved arrow in the 1,25(OH)2D3 structure shown in the upper left of the figure. Specific C5dC6dC7dC8 dihedral angles are highlighted in the figure that has been observed in the vitamin D-binding protein (DBP) and vitamin D receptor (VDR) X-ray complexes and also in the in silico flexible docking complexes obtained when 1,25(OH)2D3

II.  MECHANISM OF ACTION

VITAMIN D3 STEROL CHEMISTRY

Superagonists are able to activate VDR transcription of reporter constructs and cell differentiation at a ≥10-fold lower concentration with respect to 1,25(OH)2D3 [42,43]. More recently we showed that the initial dot map parameters/conditions replicated the side-chain rotomers observed when bound to the serum vitamin D-binding protein (DBP) (Pop. C; Fig. 16.2D) and the two overlapping VDR ligandbinding pockets (Pop. B and Pop. A; Fig. 16.2D) [37]. In the population A rotomers, the 1,25(OH)2D3 side-chain (carbons C16,17,20,22) adopts a gauche+ conformation. This rotomer is favored by the VDR alternative ligand-binding pocket (VDR-AP) and is observed 73% of the time in the 1,25(OH)2D3 dot map calculation (262 total conformers in this particular calculation). In contrast, the side chain conformers observed in the X-ray crystallographic analysis are low in population in the 1,25(OH)2D3 dot map [37] (Pop. B; Fig. 16.2D). It is noted that oxidation (i.e., metabolism; Fig. 16.3) of the 25(OH)D3 and 1,25(OH)2D3 side-chain C23 or C24 atoms (Fig. 16.3) reduces the free conformational space (i.e., entropy) sampled by the side chain [32]. Thus in vivo 25(OH)D3 and 1,25(OH)2D3 are the most conformationally dynamic of the over 30 natural VDS isolated and characterized in vivo (Fig. 16.3) [14]. This fact is supportive of the concept that the molecular dynamics of 1,25(OH)2D3 underpins its ability to stimulate both genomic and nongenomic functions. It also strongly suggests that assuming the conformation of 1,25(OH)2D3 observed in the VDR X-ray structure is the only physiologically relevant shape of 1,25(OH)2D3 is incorrect.

The Vitamin D3 Sterol Conformational Ensemble A limitation of the original dot map protocol was that only the transhydrindane CD-ring/side chain portion of the VDS could be used in the conformational search calculation. Fortunately, since the dot map protocols were first published, advancements in computational hardware/software have allowed for inclusion of the entire VDS molecule in the same type of conformational search calculation [44,45]. This type of calculation allows for all combinations of A-ring, seco-B-ring, and side-chain rotomers to be generated that range from 6-s-cis planar-like to 6-s-trans bowl and planar-like shapes (Fig. 16.4). In fact, the 1,25(OH)2D3 conformational ensemble generated using PC_Model v9.2 was recently combined with a flexible docking simulation (Discovery Studio v2.0) to blindly replicate the 1,25(OH)2D3 pose, energetics in molecular dynamics

273

observed in the 1,25(OH)2D3–VDR (aa118–427; Δ165–215) X-ray cocrystal [44,45]. Thus, the combination of PC_Model and DS2.0 may be useful in future VDR drug design efforts. Calculations that consider the dynamics of the vitamin D sterol A-ring, seco-B-ring, and side chain demonstrate that oxidation of vitamin D3 (i.e., the production of 1,25(OH)2D3 (Fig. 16.3)) in the liver (25-OHase) and the kidney (1-OHase) does not alter significantly the conformational dynamics of the vitamin D sterol molecule when compared with vitamin D3. As stated previously, this is not true for photolysis of the secoB-ring. Importantly, the reactions required for conversion of 7-DHC to 1,25(OH)2D3 (Fig. 16.1) have been observed in other tissues, namely the skin [46], where biogenesis begins and can be continued to form 1,25(OH)2D3. Metabolic epimerization of the C3-OH group of 1,25(OH)2D3, by an as yet to be physically identified epimerase, forms 3-epi-1,25(OH)2D3 (Fig. 16.3) and alters the A-ring chair–chair equilibrium (Fig. 16.2B) [47–49]. According to computational studies, a 3αdOH group causes the 1,25(OH)2D3 A-ring to favor the α-chair conformation, given the introduction of a syn-1,3-diol intramolecular hydrogen bond (see Fig. 16.2B) [50,51]. 3-epi-1,25(OH)2D3 can function as a VDR agonist ligand, is produced in vivo [49], and shows activity in multiple cell types [52,53]. Thus mimicking the natural C3-epimerization may prove to be a useful modification in future drug design efforts in the vitamin D field.

Nongenomic Agonist Structural Features and Molecular Dynamics The unique conformational heterogeneity of 1,25(OH)2D3 has provided researchers in the vitamin D field a unique tool used to differentiate genomic and nongenomic signaling because it has been discovered that different shapes of 1,25(OH)2D3 initiate genomic and nongenomic signaling [54]. This theory is based in large on the results from multiple independent researchers that the 6-s-cis locked analog of 1,25(OH)2D3, JN (Fig. 16.5), functions as a potent nongenomic agonist and rather weak genomic agonist in vitro and ex vivo, when compared with 1,25(OH)2D3 [4,32,37]. The A-ring of the JN molecule is locked in the α-chair conformation, where the 1α-OH group is axial and the 3β-OH is equatorial (Fig. 16.2B). This is the 1,25(OH)2D3 chair conformation that was shown to be a preferred conformation, capable of selectively binding to the VDR-AP [30,37] and to the serum DBP [55] (Figs. 16.2A,B and 16.4).

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was docked to the VDR genomic pocket (VDR-GP) and the VDR alternative ligand-binding pocket (VDR-AP); see text for more details. These conformations were first generated by a conformational search calculation using a modified PC_Model v9.2 GMMX protocol (see [1,44,45]). Thus the “active” seco-B-ring conformations of 25(OH)D3 and 1,25(OH)2D3 and other vitamin D sterols can be produced in silico [1,37,44]. (B) The A-ring: because of the [1,7] sigmatropic shift, the A-ring of 1,25(OH)2D3 is capable of undergoing a chair flip reorienting the axial and equatorial position of the C1 and/or C3 hydroxyl groups and orientation of the C19 methylene, either above (β-chair) or below (α-chair) the plain of the CD-ring (see panel A). The equilibrium between the two chairs is roughly 50:50, but can be altered by natural C-3 or synthetic C-1 epimerization, see text. (C) The side-chain: the cholesterol-like side-chain contains only sp3-hybridized sigma bonds and is therefore highly flexible. This flexibility was first simulated by Midland and Okamura [36] and referred to as a dot map. The black lines in the dot map diagrams highlight the side-chain region that must be populated [181] to form hydrogen bonds with H305 and H397 of the VDR (see Fig. 16.9B). (D) Population distribution of 1,25(OH)2D3 side-chain rotomers observed in the dot map calculation; ∼95% of the side-chain conformers possessed C16dC17dC20dC22 dihedral angles that fall into three 20 degrees dihedral angle windows [37]. Empirical (X-ray) and theoretical (computation) structure–function analyses show that Population A (Pop. A) is the most abundantly observed side-chain conformation in the VDR-AP (see text), Pop. B in the VDR-GP (see Fig. 16.9A) or X-ray ligand-binding pocket, and Pop. C in the serum DBP [1,37].

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are oxidized at the C1 and C25 positions [182] (see Fig. 16.1 and text). This figure shows the structures of over 30 different vitamin D metabolites that have been identified in mammalian tissues, primary cells, and/or transformed cell lines. The figure highlights the three major forms of vitamin D3, vitamin D3 (VD3), 25(OH)D3 (25D3), and 1,25(OH)2D3 (1,25D3) (see Fig. 16.1) and their C1, C3, C8, C10, C23, C24, C25, and/or C26 metabolites. The label for each metabolite uses VD3, 25D3, and 1,25D3 as the root of the name and calls out the specific carbon number(s), stereochemistry, and functionality of each of their side-chain (see Fig. 16.2C and D) metabolites. Those labels that are bold highlight the metabolites that have been most extensively studied in our laboratory and by the work of others. In general, the 25D3 or 1,25(OH)2D3 can be further stereospecifically metabolized by the CYP24 side-chain hydroxylase at the C23, C24, and/or C26 positions [35]. The C23 pathway ends with production of the 25R–OH–26,23-lactone of 25D3 or 1,25(OH)2D3. The end of the C24 pathway produces calcitroic acid. It is largely assumed that the side-chain metabolism of 1,25(OH)2D3 is catabolic [35]; however, there exists increasing evidence that metabolism of the side-chain directly alters VDR conformation and therefore signaling [32] and can temper 1,25(OH)2D3-induced hypercalcemia in vivo [183–185]. The metabolism of vitamin D sterols has been recently made more complex by the finding that C3 of 1,25(OH)2D3 can be epimerized to form 3-epi-1,25(OH)2D3 in many different cell types. 3-epi-1,25(OH)2D3 is discussed in more detail in the text.

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FIGURE 16.4  Shapes of vitamin D sterols (VDS) produced by conformational search calculations and observed in bound vitamin D-binding protein (DBP) and vitamin D receptor (VDR) complexes. The three structures in the figure are rendered in ball and stick format, with hydrogen atoms removed. The structures represent different shapes of vitamin D sterols observed in 1,25(OH)2D3–VDR X-ray complex [40], in PC_Model conformational search calculations (see text), and in an in silico simulation of 1,25(OH)2D3 binding to the VDR alternative and genomic overlapping ligand-binding pockets (i.e., the VD-AP and VDR-GP, see Fig. 16.9A) complexes [1]. Moving from left to right, they represent the 25-hydroxyvitamin D3 (25(OH)D3) J-shape, a conformation accepted by the DBP binding cleft [27], the 1α,25(OH)2 vitamin D3 (1,25(OH)2D3) planar-like shape, a conformation preferred by the VDR-AP [37] (see Fig. 16.9A and C), and the bowl shape of 1,25(OH)2D3, preferred by the VDR-GP, which is a conformation proven by many to be strongly associated with 1,25(OH)2D3–VDR cis regulation of the genome. Carbon atoms referred to throughout the text are labeled in each panel for reference.

It is noted that the VDR genomic pocket (VDR-GP) is highly selective regarding the A-ring and seco-B-ring geometry required for strong interaction with the VDR-GP, X-ray pocket, where a β-chair and 6-s-trans configuration are required (Figs. 16.2A,B and 16.4). The PC_Model calculations show that the side-chain atoms of 1,25(OH)2D3 and JN sample a similar molecular space based on their similar Pop. A–C ensemble distributions. This indicates that the similarities and changes in JN and 1,25(OH)2D3 function are underpinned by their similar A-ring but different seco-B-ring chemistries.

Nongenomic Antagonist Structural Features and Molecular Dynamics In addition to ligand shape differentiating 1,25(OH)2D3 genomic and nongenomic function, researchers have discovered that HL (Fig. 16.5) is a nongenomic specific antagonist that is equipotent to JN in its ability to stimulate an osteocalcin vitamin D DNA response element reporter plasmid in CV-1 and COS-1 cells cotransfected with human VDRwt [30,32,37,38]. Like 3-epi-1,25(OH)2D3, epimerization of C-1 changes the A-ring chair equilibrium. For HL, the β-chair is favored and stabilized by the C-1 and C-3 diaxial intramolecular hydrogen bond [30] (see Fig. 16.2B). Importantly, in the preferred HL (β-chair) and JN (α-chair) chair conformations, the 1-OH is axial; however, the steric space it occupies with respect to the rest of the VDS differs dramatically (see Fig. 16.2B). As we will describe in more detail below, comparison of 25(OH)D3, 1,25(OH)2D3, JN and HL chemistries, conformational dynamics, and structure–function profiles shows that at least for ion channels the 1-OH group is not a required feature for a ligand to be capable of functioning as a nongenomic, rapid response agonist. However, it is required for HL to block all known nongenomic responses shown to be activated by all of the following: 1,25(OH)2D3, JN, and 25(OH)D3. Perhaps most importantly, this is the exact opposite of the A-ring hydroxyl requirements that are

well defined for genomic agonists, where the 1α-OH group is essential, whereas the 3β-OH group is not [56,57]. The molecular details that underlie the importance of A-ring hydroxyl groups and their role in dictating VDR structure–function are the focus of a later section in this chapter.

1,25(OH)2D3-MEDIATED RAPID, NONGENOMIC RESPONSES 1,25(OH)2D3 has been shown to modulate a number of nongenomic, rapid responses that occur outside the nucleus of the cell. Three nongenomic case studies were presented in detail in a previous edition of this book. These are wholecell patch clamp analysis of ROS17/2.8 cells, 1,25(OH)2D3 stimulation of insulin secretion, and activation of phosphatidylinositol 3-kinase by 1,25(OH)2D3 [54]. Regulation of extranuclear signaling factors, in a rapid fashion, results in cellular responses that include but are not limited to increasing intracellular calcium, exocytosis/ATP secretion, vitamin D3 sidechain metabolism, and UV protection (Table 16.1). Recent evidence suggests that like the sex steroid hormones (e.g., 17β-estradiol), 1,25(OH)2D3-mediated nongenomic effects are modulated at least in part by the classic NR functioning outside the nucleus of the cell [2]. In this section we describe some rapid responses to 1,25(OH)2D3, highlight those that have been shown to require a functional extranuclear VDR protein, and present two case studies.

Potentiation of Voltage-Gated Ion Channels The Farack-Carson, Norman, and Zanello laboratories have demonstrated that in several transformed cell lines and primary cell cultures, 1,25(OH)2D3 treatment activates a rapid opening of voltage-gated chloride and calcium ion channels [58–62]. Both the CLC-type outwardly rectifying chloride channel (ORCC) and the L-type calcium channel, are activated

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16.  VITAMIN D STEROL/VITAMIN D RECEPTOR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS

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FIGURE 16.5  Chemical structures of the vitamin D receptor (VDR) ligands and/or ion channel blockers used in vitamin D sterol structure–function studies discussed in Table 16.1 and the text. All of the vitamin D sterols that are synthetic analogs are labeled with a two-letter code. All vitamin D sterols, with the exception of HL and HM (i.e., 1β-OH analogs), have been described to function as nongenomic agonists in at least one tissue/cell type (Table 16.1). All of the vitamin D sterols, with the exception of MK and ZK (i.e., genomic antagonists), function as genomic agonist ligands; however, they dramatically differ in their effective concentrations (see Tables 16.2 and 16.3). The L-type calcium channel blockers (isradipine, ISR; nifedipine, NIF; and verapamil, VER) are depicted in the middle, right panel of the figure. DIDS is a chloride channel blocker discussed further in the text and has been shown to bind specifically to the VDR. The chemical structures of nonsteroidal VDR botanical ligands are shown in the bottom panel of the figure. They are curcumin (CM), bisdemethoxycurcumin (BDC), and withaferin A (WFA).

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1,25(OH)2D3-MEDIATED RAPID, NONGENOMIC RESPONSES

TABLE 16.1  Summary of the Scope of Nongenomic Signaling Regulated in Various Tissues by 1,25(OH)2D3 (1,25D3) and/or Its Analogs Cell Type

Species

Signaling Factor(s)

Cellular Response

Agonist Ligand(s)

Inhibitor(s)

References

Adipocytes

R

[Ca2+]I/FAS

expression/ GADH activity

Lipid metabolism

1,25D3, JN

HL

[151]

Aortic smooth muscle

R

PI3K

VSMC migration

1,25D3, JN

HL

[152]

Bladder

R

L-type Ca2+ channel

Bladder contraction

EL, BK

Isa, Ver

[153]

Cardiac myocytes

R

Cav-3

Contractility

1,25D3

none

[111]

Cardiomyocytes

R

VDR/ion channels

Contractility

1,25D3

none

[154]

Colonic epithelial (Caco-2)

H

PP1c/PP2A/p70(S6K)

Antiproliferation

1,25D3

none

[72]

Colon cancer

H

[Ca2+]I/RhoA-ROCK/ p38MAPK-MSK1

Antiproliferation (↓Wnt)

1,25D3

none

[155]

Colonic epithelial (Caco-2)

H

ERK1/2-MED1 and ERK5-Ets-1

Antiproliferation

1,25D3

none

[156]

Colonic epithelial (Caco-2)

H

PKCα/IP3

[Ca2+]I

1,25D3

TPA, H7

[157]

Colonocytes

R

cSrc/PLCγ

PI-hydrolysis

1,25D3

none

[158]

Enterocytes

C

Tyr-kinase/MAPK

Structure–function study

1,25D3, JN

none

[116]

Fibroblasts

h*

L-type Ca2+ channel/ MEK1/2/Cyp24

[Ca2+]I and vitamin D metabolism

1,25D3

Ver

[58]

Hela and OB-6 cells

H

PI3K, Src, JNK, ERK, MEK

Antiapoptosis

1.25D3, JN

HL

[79]

HeLa/COS-7/ROS

h/m/r

CaMKV

VDR transactivation 1,25D3

none

[159]

Immune

R

[Ca2+]

EAE incidence

1,25D3

none

[160]

Intestine

C

Calcium channels

Transcaltachia

1,25D3, JN, JM, JP

HL

[161,162]

Keratinocytes

H

Raf/MAPK/Shc/Grb2/Ras

DNA synthesis

1,25D3

none

[163]

Psoratic keratinocytes

H

MEK–ERK/NFκB

Antimicrobial peptides (cAMP)

1,25D3, BT, ZK-series

none

[92]

Keratinocyte/fibroblasts H

CPD/p53

UV photoprotection 1,25D3, JN, JM, QW

HL

[164,165]

Keratinocytes (NHEK)

H

MEK–ERK

Cathelicidin expression

LCA

None

[166]

Leukemia (THP-1/ HL-60)

H

PP1c/PP2A/p70(S6K)

Cell differentiation

1,25D3

none

[73]

Leukemia (NB4)

H

Ser-Thr/Tyr kinases

Monocytic cell differentiation

1,25D3 ± TPA, HF, JN, HL, HM JM, JP

[58,167–170]

Leukemia (HL60)

H

MAPK

Differentiation

1,25D3, JN

HL

[171]

Macrophages (P388D1)

H

TNFα/NFκB/IκBα

Antiinflammation

1,25D3, 1,24RD3

none

[148]

Microsomes/microcytes C

cAMP

Ca2+-uptake/Protein 1,25D3 phosphorylation

Nif

[172]

Myoblasts

PKC/PLA2

[3H]-AA secretion

1,25D3

Nif, H7

[173]

L-type Cl− channels

[Ca2+]I/exocytosis/

25D3, 1,25D3, JN, Bay K

HL

[60–62,174]

ATP secretion

Osteoblasts

C R

I

Ca2+/CLC-3

Osteoblasts/kidney (CV1)

m/r

Akt/PI3K

Antiapoptosis

1,25D3

none

[80]

Pancreatic islets

R

L-type Ca2+ channel

Insulinotropic effect

1,25D3, JN

HL, Nif

[93] Continued

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16.  VITAMIN D STEROL/VITAMIN D RECEPTOR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS

TABLE 16.1  Summary of the Scope of Nongenomic Signaling Regulated in Various Tissues by 1,25(OH)2D3 (1,25D3) and/or Its Analogs—cont’d Cell Type

Species

Signaling Factor(s)

Cellular Response

Agonist Ligand(s)

Inhibitor(s)

References

Pancreatic β-cell

R

L-type Ca2+ channel/ Ryandodine receptor

[Ca2+]I/insulin secretion

1,25D3

Nif

[94]

Parathyroid

P

PLC

Biphasic ↑ in DAG, MG, and IP3

1,25D3

None

[175]

Sertoli

R

cAMP/PKC/PKA/CLC-3 Cl− channel

Exocytosis

25D3, 1,25D3, JN, MK, IE

HL, DIDS

[45,63]

Skeletal muscle

R

c-Src/ERK1/2/ p38-MAPK/Cav-1

Structure–function study

1,25D3, JN

HL

[176]

Skin

R

p53/NOS

UV/ROS/CPD formation

1,25D3

none

[99]

PI3K/ERK1/2

Apoptosis

1,25D3

None

[177]

Squamous cell carcinoma H

The column headings highlight the cell type, species (c, chicken; h, human; h*, human natural mutations; m, monkey; r, rodent), brief overview of the signaling factor(s) and the cellular response studied, and the small molecule agonist(s) or antagonist(s) (i.e., inhibitors) applied in each individual nongenomic case study. The data in the table are organized based on the left hand column (i.e., the cell type utilized in each published work). Bold entries are those studies that have been defined as vitamin D receptor (VDR)-dependent by the authors and/or experimental design. The chemical structures for all of the agonist and antagonist ligands are provided in Fig. 16.5, with the exception of TPA (12-O-tetradecanoylphorbol-13-acetate), H7 [1-(5-isoquinolinesulfonyl)-3-methylpiperazine], and Bay K-8644 (Bay-K). If none is entered in the inhibitor column, the authors commonly used siRNA, gene-KO (–,–), and/or other commercial agents to attenuate the production and/or the function of proteins listed in the signaling factor(s) column. Unconventional abbreviations present in the Cellular Response column are as follows: CPD, cyclobutane pyrimidine dimers; EAE, experimental autoimmune encephalomyelitis; MG, monoacylglycerol; and VSMC, ventricular smooth muscle cells.

within seconds to minutes following addition of ≤1.0 nM 1,25(OH)2D3 or JN (Table 16.1). In addition, in these studies all VDR agonist ligands were blocked by coincubation with equimolar HL (Table 16.1). In our nongenomic ORCC structure–function analysis we have demonstrated that 25(OH)D3 is as effective as 1,25(OH)2D3 in potentiating chloride currents in osteoblasts and TM4 Sertoli cells [63]. Given the link between changes in serum 25(OH)D3 levels and multiple physiological disorders [64,65], it would not be surprising if 25(OH)D3 functions as a nongenomic or perhaps even genomic VDR agonist ligand [66,67]. All current evidence suggests that in both of these cell types, the presence of an extranuclear VDR, localized to a caveolae lipid, raft microdomain, is involved in the regulation of channel activity by 1,25(OH)2D3, JN, and 25(OH)D3. Ion channels are classified based on the type of current regulated, ionophore selectivity, and the response to small-molecule channel activators and/or blockers [68–70]. Fig. 16.5 and Table 16.1 highlight the ion channel blockers used to attenuate 1,25(OH)2D3, JN, and 25(OH)D3 potentiation of ORCC and L-type calcium channels. Of these four blockers, we recently showed that DIDS is a VDR ligand with ∼10 μM affinity [45]. Whether DIDS blocks activation of 1,25(OH)2D3-sensitive ORCC currents through competitive inhibition remains to be determined. Nonetheless, it blocks 1,25(OH)2D3 activation of the ORCC currents and exocytosis in TM4 cells [63] and suggests that the CLC channel being regulated by 1,25(OH)2D3 in a VDR-dependent manner is CLC-3 and/or CLC-5 [71]. Given the similarities in agonist/antagonist profiles in other systems (e.g., osteoblasts), it is likely that VDR nongenomic regulation of CLC-3 is a common cellular pathway, linking

1,25(OH)2D3–VDR nongenomic actions to cell processes such as secretion [61,63] or phagocytosis, depending on the cell type.

Control of Phosphatase, Kinase, and Phospholipase Activity Two phosphatase signaling factors whose activity is impacted by 1,25(OH)2D3 in both colon cancer and leukemia cells are PP1c and PP2A. Both of these phosphatases are activated in a 1,25(OH)2D3–VDR dependent nongenomic manner and linked to cell differentiation/antiproliferation (Table 16.1). In both colon cancer and leukemia cells, the VDR is complexed to the catalytic subunits of PP1 and PP2A and is dissociated by addition of 1,25(OH)2D3, activating the phosphatase [72,73] (Fig. 16.6). Interestingly, the 1,25(OH)2D3-mediated activation of these two phosphatases leads to different effects on the p70(S6K) Ser/Thr protein kinase [74,75] in the two cell types. In colon cancer cells activation of PP1c leads to inhibition of p70(SK6) and G1-arrest (i.e., antiproliferation) [72], whereas in leukemia cells, phosphatase activation leads to activation of p70(S6K) and cell differentiation [73]. In theory, 1,25(OH)2D3 activation of PP1c could also provide a link to the rapid (∼5 s to min) increase in intracellular calcium in response to 1,25(OH)2D3 in fibroblasts, osteoblasts, pancreatic β-cells, immune cells (Table 16.1 and references therein), and in yet to be determined cell/tissue types (reviewed in Ref. [14]). This potential link emerges from the observation that PP1c dephosphorylates and activates IRBIT (inositol 1,4,5-triphosphate (IP3) receptor-binding protein) [76]. IRBIT is an endogenous repressor of the IP3R that blocks IP3 access

II.  MECHANISM OF ACTION

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1,25(OH)2D3-MEDIATED RAPID, NONGENOMIC RESPONSES

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FIGURE 16.6  A simplified model of vitamin D receptor (VDR) regulation of selected nongenomic signaling factors: a phosphatase (i.e., PP1c), a phosphoesterhydrolase (i.e., PLC), a kinase (PKCα), the cSrc/ERK1/2 signaling axis and their regulation of intracellular calcium, and cross-talk. The diagram represents the VDR as two concentric ellipses. The larger one is the VDR and the smaller the vitamin D ligand-binding surface. In the absence of nM levels of 1,25(OH)2D3, the VDR forms a complex with catalytic subunit of pp1 (see text). This releases PP1c allowing it to dephosphorylate multiple substrates that include IRBIT, a protein that blocks binding of inositol-1,4,5-triphosphate (IP3) to the IP3R, and mobilization of calcium from the endoplasmic reticulum (ER) lumen and into the cytoplasm ([Ca]c) [186]. The figure depicts how binding of 1,25(OH)2D3 to the VDR alternative ligand-binding pocket could stimulate PLCγ [14,187] and other hydrolysis reactions by exposing bound PIP2 or PIP3. An increase in intracellular calcium is well documented to stimulate the catalytic activity of PKCα [188]. The holo-1,25(OH)2D3–VDR complex has also been reported to bind to Src and the scaffolding proteins, caveolae, tuberin, and modulator of nongenomic activity of the estrogen receptor (MNAR) [189,190]. By stimulating Src, 1,25(OH)2D3–VDR can activate downstream ERK1/2. Both PKCα and ERK1/2 can function to activate and/or repress the transcription of genes through regulating the activity of second messengers, other transcription factors, and/or the nuclear receptor itself, a process referred to in the literature and the text as “cross-talk.”

to its binding site in the IP3R and thus efflux of calcium from endoplasmic reticulum (ER) stores [77] (Fig. 16.6). Of the kinases that have been shown to be regulated by 1,25(OH)2D3, the receptor tyrosine kinase Src is of considerable interest because it is often mutated in cancer cells and it links 1,25(OH)2D3 to yet another classical second messenger system that includes ras, raf, ERK1/2, MEK1/2, and/or p38-MAPK (Table 16.1 and Fig. 16.6). Kinases that have been shown to be activated by both 1,25(OH)2D3 and JN include Ser-Thr/Tyr kinases, PI3K, and PKA (Table 16.1). Of the kinases mentioned, the ability of HL to block the effect of 1,25(OH)2D3 has been demonstrated for Ser-Thr/Tyr-kinases and MAPK (Table 16.1). For example, PKC and PKA have been shown to be required for 1,25(OH)2D3 potentiation of CLC-3 in TM4 Sertoli cells [63] (Table 16.1). The phospholipases that are activated by 1,25(OH)2D3 are PLC (e.g., PLCγ, Fig. 16.6) and PLA2 (Table 16.1). These two phospholipases control IP3 and arachidonic acid (AA) concentrations by hydrolyzing the membrane phosphatidylinositolphosphates (PIPs), PIP2 and PIP3. It is well documented that inositol-1,4,5-triphosphate (IP3) triggers the opening of the IP3R [77], which in turn facilitates an increase in cytosolic calcium by mobilizing it from the ER (Fig. 16.6). Alternately the liberation of AA from PIPs leads to the production of prostanoids and/or leukotrienes. These derivatives of AA have a wide range of functions that include, but are not limited to,

inflammatory signaling cascades [78]. Interestingly of the two PIPs, PIP3 is not found in yeast [78] and the enzyme that converts PIP2 to PIP3, PI3K, is a classic oncogene that, such as c-Src, is mutated in most cancers. Thus the known 1,25(OH)2D3/JN/ HL nongenomic functional effects on PI3K (Table 16.1) [79,80] may have coevolved by the interaction of the VDR with both vitamin D sterols and PIP3. It is noted that AA can also be obtained from diacylglycerol, the other product of PLC hydrolysis of PIP2. PLCγ was recently shown to be upregulated by 1,25(OH)2D3–VDR in antigen naive T-cells following the induction of VDR expression in response to T-cell antigen receptor (TCR) stimulation [81]. Upregulation of VDR mRNA by TCR is p38-MAPK–dependent, and PTEN, the enzyme that converts PIP3 to PIP2, is thought to be a VDR target gene [82–84]. Given the evidence that 1,25(OH)2D3 regulates PIP2/3 and p38-MAPK signaling in a nongenomic fashion (Table 16.1), it is possible that the innate and cognitive immune responses to 25(OH)D3 and 1,25(OH)2D3 [85,86] require cross-talk between 1,25(OH)2D3–VDR genomic and nongenomic signaling.

Cross-Talk There are two fundamental forms of cross-talk that exist between extranuclear and nuclear vitamin D sterol (VDS)

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16.  VITAMIN D STEROL/VITAMIN D RECEPTOR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS

signaling. The first type involves the cell-specific effect that 1,25(OH)2D3 nongenomic signaling has on the state of VDR phosphorylation. This type of cross-talk where 1,25(OH)2D3 activation of an extranuclear signaling factor can serve to regulate both VDR location within the cell and its potential to alter gene expression [87–90]. The second type of cross-talk involves 1,25(OH)2D3 regulation of cytoplasmic second messengers, which include, but are not limited to, cAMP, p53, NFκB, IκB, calcium, and ERK1/2 (e.g., c-fos) (Table 16.1). When activated, these second messengers can regulate genes that either do or do not contain a nuclear vitamin D response element. Perhaps the crucial importance of VDR-dependent nongenomic signaling and cross-talk in whole cell function is best embodied in the antiproliferative effect 1,25(OH)2D3 and JN have in human acute promyelocytic leukemia NB4 cells [4] (Table 16.1). In NB4 cells the prodifferentiative effect of 1,25(OH)2D3 and JN is blocked by coincubation with equimolar HL (Table 16.1) but not by addition of a tenfold excess of the human VDR genomic antagonist, analog MK (Fig. 16.5) [44,91]. In addition, the VDR genomic antagonist (ZK-series; Fig. 16.5) stimulates MEK–ERK signaling in psoriatic keratinocytes [92], and MK stimulates CLC-3 opening in TM4 Sertoli cells [63]. Other cellular functions shown and/or perceived to require nongenomic signaling and cross-talk include, but are not limited to, exocytosis (e.g., insulin [93,94] and ATP [61]), apoptosis (e.g., caspase-3 [95,96]), phagocytosis (e.g., soluble amyloid beta [97]), and protection against UVB skin damage and cyclobutane pyrimidine dimers ([98,99] and Table 16.1). Future work in the cross-talk subfield of vitamin D research is required to better define the interplay between 1,25(OH)2D3 nongenomic and genomic signaling in maintenance of good health and in therapeutic drug design.

THE PLASMA MEMBRANE VITAMIN D RECEPTOR Immunohistochemistry and confocal microscopy have convincingly shown that the classic nuclear VDR associates with a variety of cellular membranes and is observed in the cytosolic and nuclear compartments [11,61]. The Norman laboratory first discovered that the VDR localizes to the lipid-raft caveolae-enriched microdomain in many different tissues and cell types. In these caveolae-enriched membrane fractions 1,25(OH)2D3, JN, HL, and other VDS showed similar binding affinities when compared with tritiated 1,25(OH)2D3 competition assays performed with nuclear preps from the same cells [100]. In vitamin D-deficient chicks a significant reduction in the capacity of the caveolae fraction to bind physiological levels of tritiated-1,25(OH)2D3 was observed when compared with vitamin D3-replete chicks [2,11]. Huhtakangus et al. [11] also determined that [3H]-1,25(OH)2D3 localized to caveolae, as assessed by isolation of caveolae-enriched membrane fractions and HPLC. Finally, in VDR-KO mice there was a loss of [3H]-1,25(OH)2D3 binding to the caveolae-enriched membrane fraction and an associated loss in 1,25(OH)2D3-mediated rapid

responses of opening of chloride channels and stimulation of exocytosis [60]. Another 1,25(OH)2D3 membrane receptor has been proposed to play an important role in regulating calcium and phosphorous transport, namely the membrane-associated rapid response, steroid-binding protein (1,25(OH)2D3MARRS) [101]. MARRS is member of the group of a protein disulfide isomerases [101–103], which contribute, in some way, to the nongenomic effects of 1,25(OH)2D3 in chondrocytes [104], osteoblasts [105], and other cell types [106–109]. Most recently, 1,25(OH)2D3-MARRS was shown to regulate 1,25(OH)2D3 membrane effects in osteoblasts [105]. Because no structure–function studies have been carried out that demonstrate the shape specificity of 1,25(OH)2D3 for nongenomic responses mediated by MARRS are available, this protein will not be considered further here, although it is possible that both the VDR and 1,25(OH)2D3-MARRS are required for the regulation of some nongenomic signaling factors/functions.

Structural/Scaffolding Proteins (e.g., Caveolins) In this section we present an overview of how the VDR is thought to be localized to the plasma membrane caveolae microdomain and the signaling factors and extranuclear scaffolds the VDR has been shown to interact with. We also highlight how the VDR is distinct when compared with other NRs known to serve as membrane receptors for their endogenous steroid hormones. The main structural component of the plasma membrane caveolae microdomain is the group of membrane proteins termed caveolins (e.g., Cav-1 and/or Cav-3) [110]. VDR colocalizes with each of these molecules in either a liganddependent (e.g., osteoblasts) [61] or ligand-independent (e.g., cardiac myocytes) [111] fashion. The important protein domain(s) of the VDR/Cav proteins required for their direct interaction is not known. Nor is it understood what specific role the interaction between the VDR and Cavs plays in known nongenomic signaling by 1,25(OH)2D3. Nevertheless, the caveolae microdomain is home to an enhanced concentration of membrane PIP molecules and cholesterol [110,112,113], and it is also a cell loci enriched with signaling factors known to be regulated by 1,25(OH)2D3 (Table 16.1) [113–115].

Other Signaling Factor/Scaffolding Proteins (e.g., PP1c and Src) In addition to Cav-1 and Cav-3, the VDR has been reported to bind directly to c-Src [116] and PP1c [72,73]. Initial findings indicate that like the Cav-3–VDR interaction, the PP1c–VDR interaction is attenuated by 1,25(OH)2D3, demonstrating that in some cases the VDR itself can function as a negative inhibitor of enzyme catalytic function (Fig. 16.6). Boland’s group provided evidence that the VDR mediates rapid changes in muscle protein tyrosine phosphorylation induced by 1,25(OH)2D3 [117]. This leads to the activation of the Src/ MAP kinase pathway [116] (Fig. 16.6), presumably via the

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The Vitamin D Receptor Conformational Ensemble Model

VDR interacting with the SH2-domain of Src. The interaction of VDR with the SH2-domain of Src is believed to occur via phosphorylation of a Tyr-residue located in the hinge domain of the VDR (e.g., Y143 or Y147) [116]. Src tyrosine kinases, Gα-subunits, and H-Ras also bind to the common membraneanchored scaffolding protein, caveolin. According to the report by Li et al. [118], caveolin binding negatively regulates the autoactivation of Src tyrosine kinases. The VDR has also been reported to bind to the scaffolding protein tuberin [119]; like the VDR interaction with Src, the molecular details of this interaction are unknown. The functional relevance of the VDR interaction with tuberin has yet to be elucidated; however, the VDR may regulate different nongenomic signaling cassette(s) [120–122] by physically interacting with the tuberin/hamartin signaling scaffold [123]. Thus it appears that the VDR can interact with nongenomic signaling factors via its reversible interaction with intracellular protein scaffolds in both a 1,25(OH)2D3-dependent and -independent manner. Certainly, future work in this area of vitamin D research is of paramount importance in providing a molecular basis [3] for how 1,25(OH)2D3 nongenomic, extranuclear, rapid responses contribute to cell physiology and ultimately the therapeutic effects associated with vitamin D supplementation and/or analogs of VDR ligands (i.e., 1,25(OH)2D3).

Covalent Modification (e.g., Palmitoylation) Another way that the VDR could localize to the plasma membrane is through palmitoylation or myristoylation. This hypothesis is in large part based on the recent evidence published by the Levin laboratory indicating some NRs such as the sex hormone NRs for estrogen (ER), androgen, and progesterone contain a consensus sequence for palmitoylation and subsequent membrane anchoring [124,125]. Evaluation of the VDR primary sequence produced no such consensus sequence in the VDR ligand-binding domain. Thus it would seem that the VDR either localizes through the various cellular loci protein scaffolds, and/or the VDR could bind directly to membrane lipids, such as the PIPs (e.g., PIP2, Fig. 16.6).

Membrane Binding (e.g., PIP2/3) We and others have shown over the past 10 years that the VDR does not “selectively” bind just 1,25(OH)2D3. Rather, it can sample a very broad set of ligands that are capable of competing off specifically bound, tritiated 1,25(OH)2D3 [32,63,97,126,127]. It has also been recently demonstrated that the orphan NRs SF-1 and LRH-1 (NR5 family members) are capable of binding to membrane lipids [128] (e.g., phosphatidylinositol-3,4,5-triphosphate (PIP3)). Molecular mechanics calculations indicate that both PIP2 and PIP3 can bind to the VDR such that the inositol phosphate polar head group of the PIP is bound to the A-ring domain, the glycerol moiety is located in the region of the VDR-AP toward the surface of the VDR-AP (see below), and the hydrophobic tails are left free to suspend in the hydrophobic

3,3

1XFOHDU +

&RUHJXODWRU

5;5

6XUIDFH ++ ++

+

6XUIDFH

+

FIGURE 16.7  The speculative vitamin D receptor–phosphatidylinositolphosphate (VDR–PIP) membrane model. In the figure, the theoretical (i.e., computed) complex formed between the VDR alternative ligandbinding pocket (Fig. 16.9A) and PIP2 is shown. The PIP2 is rendered in space filling with hydrogen atoms included. In the complex, the phosphate at C4 of the inositol ring forms a strong electrostatic interaction with R274. The R274 residue is central in the figure and is shown in space filling with hydrogen atoms absent. In this theoretical complex, the vitamin D receptor–retinoid X receptor (VDR–RXR) heterodimerization surface (H7 and H10) and the nuclear coregulator surface (portions of H3/H4/H5 and H12) are oriented in such a manner that they are theoretically accessible when the VDR is anchored noncovalently and reversibly to the underside of the plasma membrane. These surfaces are shaded in the figure and labeled.

membrane (Fig. 16.7). This VDR membrane model has not yet been experimentally studied, but it does provide a direct link between 1,25(OH)2D3 and PLC hydrolysis of PIP2 and the subsequent rapid (∼5 s) increase in cytosolic calcium from ER stores observed following hormone addition to cells [129] (Fig. 16.6). According to the molecular models, PIP2 is envisioned to be displaced from the VDR by 1,25(OH)2D3, allowing it to be exposed and subjected to hydrolysis by PLC (Fig. 16.6).

THE VITAMIN D RECEPTOR CONFORMATIONAL ENSEMBLE MODEL The first model describing ligand activation of a NR was presented by the Moras laboratory over 15 years ago [130–132]. The model, termed the “mouse-trap,” was based on comparison of the apo- and holo-retinoic acid receptors and posited that, in the absence of the high-affinity hormone ligand, the C-terminus of helix-11 occupies the NR ligandbinding pocket (Fig. 16.8A, center left ribbon structure). Helix-11 therefore physically blocks the ability of hormone to bind the classical and highly conserved X-ray ligand-binding pocket [133] and orients helix-12 away from the remainder of the NR ligand-binding domain. Based on the tenets of the mouse-trap model, the ligand induces a conformational

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282

16.  VITAMIN D STEROL/VITAMIN D RECEPTOR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS

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FIGURE 16.8  Aspects of the vitamin D receptor (VDR) conformational ensemble model. (A) Molecular dynamics: the ribbon diagrams in the upper left portion of the figure illustrate a putative thermodynamic cycle for the VDR molecule and more specifically helix-12. For example, different helix-12 VDR conformations predicted from X-ray crystallization of various apo- and holo-NR complexes are illustrated in the upper, middle panel by coloring different conformations of the C-terminal activation helix of the VDR, helix-12 (H12, c1, c2, and c3). The numbers 1 and 2 highlight a theoretical inducedfit, where an apo-VDR of favored conformation (i.e., high population) binds to 1,25(OH)2D3 (1) producing a transitory complex (VDR–VDS*). This complex relaxes to prefer a specific distribution of VDR conformations. For the process of vitamin D receptor–retinoid X receptor (VDR–RXR) transactivation, the major body of structural evidence suggests that helix-12 must be closed to potently upregulate gene products that contain a positive vitamin D response element. The VDR conformational ensemble (2) or mutually induced-fit model [191] (3 and 4 in the cycle) differs from the induced-fit, mousetrap model in that the unliganded VDR is considered to be a continuum of conformational isomers with a number of different sampled and populated states. According to this model, the VDR ligand pool and cell signaling state modulate the population distribution of VDR ensemble members [1,32]. In theory the VDR ensemble can be perturbed in vivo by differential and dynamic socialization with ligand and coregulatory/scaffolding protein pools. For example, nutritional intake, pro- and hormone metabolism, posttranslational modifications, intracellular pH, isoelectric environment, intracellular localization, mutagenesis, content, and concentration of coregulatory molecules that either form the scaffold of a quaternary complex containing the VDR or form direct intermolecular interactions with the VDR surface, all can influence the VDR ensemble state (i.e., distribution of conformers) and/or the presence/movement of the VDR to different cellular loci. (B) The bottom panels in the figure are representative SDS-PAGE gels obtained following a limited proteolytic (i.e., trypsin) digest of radiolabeled apo- or holo-VDR coding for the full-length hVDRwt, I268Y/V300Y, and H305F/H397F constructs. These two mutations significantly reduce the free volume of the VDR genomic pocket (VDR-GP) and introduce intramolecular hydrophobic interaction with H12 residues (see Fig. 16.9B). The ligands used at 10 μM concentration were 1,25(OH)2D3 (C, control), BS, and KH (structures shown and labeled). These vitamin D sterols stabilize unique distribution of three VDR solution state conformations (c1, c2, and c3). The detailed structure–function studies carried out with BS, a natural, terminal metabolite of 1,25(OH)2D3 side-chain metabolism (Fig. 16.3), and KH, a synthetic two side-chain analog of 1,25(OH)2D3, are compared in detail in Mizwicki et al. [32] but not elaborated on extensively herein. In both the I268Y/V300Y and H305F/H397F, no ligand (NL = no exogenous vitamin D sterol added) showed significant protection of the VDR against trypsin cleavage, demonstrating that the population distribution of VDR conformational isomers can be largely influenced by changes in the chemistry of the ligand-binding surface and not just ligand itself.

change in the NR by replacing the C-terminal end of helix-11 in the ligand-binding pocket and causing helix-12 to swing into a closed position (Fig. 16.8A steps 1 and 2). In this position, helix-12 can form extensive intramolecular contacts with the ligand-binding domain and contact the ligand as well; this is discussed in detail below. Closure of helix-12 completes the NR surface, termed the nuclear coregulatory binding site (Fig. 16.9A). Thus closure constitutes a switch from the transcriptionally off-state to the on-state (Fig. 16.8A steps 1 and 2), presumably by displacing nuclear corepressor and recruiting nuclear coactivators to the coregulatory binding site [134]. In 2004 we proposed that the VDR molecule, like its native high-affinity hormone ligand, 1,25(OH)2D3, is a flexible body

whose conformation is not static in the in vitro unliganded apostate (Fig. 16.8A). This hypothesis was largely based on the inability to form an apo-VDR crystal and solve its structure. The concept helix-12 mobility has been validated in solution by limited proteolytic digest [32] (Fig. 16.8B) and by VDR hydrogen/ deuterium exchange mass spectrometry [135]. The results from these studies showed that no favored apo-VDR conformation [2] exists, which correlates with one predicted by the mousetrap model to be of high population. Recently we showed that the conformational distribution of helix-12 of the apo-VDR could be significantly altered by changes in either ligand chemistry or mutation of the VDR [32,44] (Fig. 16.8B). The ensemble concept also provides a sound, rationale, structural explanation for how the VDR antagonist, MK (Fig. 16.5), can be converted to

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283

The Vitamin D Receptor Conformational Ensemble Model

(A)

(B)

(C)

V418

H1

L414 H305

H5

S237

H2 R402

C288

C288

H12

L227

Y143 H3

H397

W286

W286 S278

S274

K413

FIGURE 16.9  The vitamin D receptor (VDR) two-pocket model. (A) The solvent exposed surface of the VDR (aa118–427; Δ165–215) is shown as a

transparent clear surface. In the diagram two amino acids are rendered in space filling and labeled R402 and K413. These two cationic residues serve as the residues recognized by the trypsin active site in the production of the c3 and c2 limited trypsin digest (i.e., protease sensitivity assay) fragments (see Fig. 16.8 and [32]). The VDR alternative ligand-binding pocket (VDR-AP) is represented by a colored sky blue solid surface and the VDR genomic pocket (VDR-GP) by a faint yellow transparent surface. The region where H1/H2, H3, H5, and the β-sheet meet forms the region where the VDR-AP and VDR-GP ligand-binding pockets share three-dimensional, steric space. This region is termed the VDR A-ring domain, highlighted in the figure by a dashed circle. (B) The 25-OH hydrogen bonds, the C26 and C27 hydrophobic, Van der Waals (vdW) interactions, and the seco-B-ring intermolecular stabilization when 1,25(OH)2D3 (ball and stick) is flexibly docked to the VDR-GP are illustrated. Note that the 1,25(OH)2D3 pose shown here is in strong agreement with the VDR X-ray pose of 1,25(OH)2D3 [40]. That same pose was produced blindly in the PC_Model conformational search calculation (see Figs. 16.2 and 16.4 and the text). The residues forming favorable contacts with C5, C6, C7, C8, and C19 are W286 and C288. Hydrophobic, vdW contacts are made between the terminal side-chain methyl groups of 1,25(OH)2D3 (i.e., C26 and C27) and the hydrophobic crown residues L227, L414, and V418. These hydrophobic interactions form the buried foundation of the nuclear coregulatory surface (labeled, shaded regions of H3 and H12 in panel A and Fig. 16.7). H305 and H397 are required for 1,25(OH)2D3 for genomic transactivation. This is because the dynamics of the 1,25(OH)2D3 side-chain must be energetically augmented so as to allow for it to interact with the hydrophobic crown residues in a manner that shifts the Boltzmann distribution of VDR conformations to favor the agonist conformation, where H12 is protected from trypsin and therefore ordered when compared with 1,25(OH)2D3 (Fig. 16.8) [32]. (C) The polar or unsaturated amino acid R-groups that form the VDR A-ring domain: Y143, S237, R274, S278, W286, and C288. The β-chair, 6-s-cis, Pop. A conformation of 1,25(OH)2D3 observed following the 1,25(OH)2D3/VDR-AP flexible docking simulation [37] is shown in the panel as a ball and stick structure. The nearest-neighbor contacts made between polar or unsaturated carbon atoms with the A-ring domain residues are indicated by solid lines and the distances in angstroms are provided in the figure.

a superagonist by a single-point mutation and why removal of both hydrogen bonds to the 25-OH group of 1,25(OH)2D3 does not attenuate the ability of 1,25(OH)2D3 to transactivate [44]. Based on the evidence that the VDR is a highly flexible molecule and the results of structure–function studies indicate that the VDR is, without question, involved in 1,25(OH)2D3 extranuclear, nongenomic, rapid cellular responses; we proposed the VDR could function as both a membrane receptor and transcription factor modulating the nongenomic and genomic functions of VDS [1,37]. Although attempts have been made, X-ray crystallography has not yet provided a structural explanation for the mechanistic nuances (i.e., shape specificity, see above) of VDR nongenomic specific ligands (Fig. 16.5). Also X-ray crystallography has been unable to provide a unique structural understanding induced by genomic superagonist ligands [40,41,136], discussed in greater detail above. Alternatively, through the use of molecular mechanics modeling techniques, we obtained evidence that ligands could in theory regulate the population of different VDR ensemble conformations and thereby genomic and nongenomic signaling by their ability to bind to different, yet overlapping, regions of the VDR ligand-binding and hinge domains [1,37,44,63] (Fig. 16.9A). Thus we posit that the innate VDR conformational flexibility and the presence

of two overlapping ligand-binding pockets allow the VDR to send signals dynamically [137].

The Overlapping Two-Pocket Model The ability of the VDR to function as a membrane receptor for 1,25(OH)2D3, JN, and 25(OH)D3-initiated rapid responses was for the first time structurally understood by the discovery that a hydrogen bond donor–acceptor flip-flop between His229 and Tyr295 opened additional steric space in the VDR molecule forming what is termed the VDR-AP (Fig. 16.9A) [37,38]. The VDR-AP shares a significant amount of steric (i.e., 3D) space, termed the A-ring domain (Fig. 16.9A, dashed circle), with the VDR ligandbinding pocket defined by X-ray crystallography, termed the VDR-GP (Fig. 16.9A). When bound to the VDR-GP, the 1,25(OH)2D3 molecule is bowllike in shape (Figs. 16.4 and 16.8B). According to many lines of structure–function evidence, obtained both before [14] and after [44] the 1,25(OH)2D3–VDR X-ray structure, when bound to the VDR-GP, the 1,25(OH)2D3 A-ring and seco-Bring are rigidly held in a β-chair (Fig. 16.2B), 6-s-trans (Fig. 16.2A) conformation. The rigidity results from the hydrogen bonds formed between the equatorial 1α-OH group and

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284

16.  VITAMIN D STEROL/VITAMIN D RECEPTOR CONFORMATIONAL DYNAMICS AND NONGENOMIC ACTIONS

S237 and R274 and the axial 3β-OH group and Y143 and/or S278 (Fig. 16.9B) and the buried nature of the VDR-GP (Fig. 16.9A). In contrast, the side-chain atoms of 1,25(OH)2D3 show enhanced heterogeneity but are somewhat conformationally restricted by hydrogen bonds formed with His305 and His397 (Fig. 16.9B and see pdb code: 1DB1 heavy atom B-factors). Finally, the H-bonds between the 25-OH group (Fig. 16.1) and H305/H397 are intermolecular interactions that allow the C26 and C27 terminal methyl groups of 1,25(OH)2D3 to contact crucial hydrophobic residues in the VDR-GP (Fig. 16.9B), which are essential for potent 1,25(OH)2D3–VDR genomic agonism [44,138]. When bound to the VDR-AP, the A-ring of the 1,25(OH)2D3 molecule can exist in either the α- or β-chair conformation (Fig. 16.2B). According to the molecular models, the seco-Bring can exist in the 6-s-cis or 6-s-trans conformations (Fig. 16.2C), and the side chain prefers the lowest energy and highest populated Pop. A configuration (Fig. 16.2C) [37]. All of these 1,25(OH)2D3 conformational rotomers that are accepted by the VDR-AP have a more planar-like molecular shape (Fig. 16.2A). In the VDR-AP, the 3β-OH group forms hydrogen bonds with S237 and/or R274 and when in the β-chair, 6-s-cis conformation the 1α-OH group forms H-bonds with Y143 and/or S278 (Fig. 16.8C). The triene atoms of 1,25(OH)2D3 make a significant number of contacts with Cys288 and also make intermolecular contacts with S278 and W286 (Fig. 16.9C). Based on the computational results that show the VDR-AP accepts more conformational isomers of 1,25(OH)2D3 when compared with the VDR-GP [37,45] and

the evidence that the potential interaction energy and Gibbs free energy of binding (ΔGbinding) are better for the VDR-GP for nearly all VDS (Table 16.2), the VDR-AP has been described as being kinetically favored, whereas the VDR-GP is the thermodynamically favored ligand-binding pocket for 1,25(OH)2D3, if not all 1-OH vitamin D sterols (Table 16.2 and Fig. 16.9A legend).

Experimental Support for the Vitamin D Receptor Alternative Ligand-Binding Pocket To test the validity of the VDR-AP model we designed single and double S237A/R274A or Y142A/S278A point mutations and measured and compared the binding affinity and genomic EC50 of 1,25(OH)2D3, 25(OH)D3, and 3-deoxy1,25(OH)2D3 (analog CF) [30,32,37]. Based on the modeling results it was predicted that either single or double alanine mutants against S237/R274 would enhance the VDR affinity and genomic agonist potential of 25(OH)D3. This was because the hydrogen bonds formed between the 3-OH group and these residues in the VDR-AP would be lost in the Ala mutants (Fig. 16.9C), but those hydrogen bonds formed between the 3-OH group of 25(OH)D3 and Y143/S278 in the VDR-GP would still exist (Fig. 16.9B). These changes in the intermolecular interactions between the vitamin D sterol and the VDR A-ring domain would then have the consequence of increasing the fractional occupancy of the VDR-GP in the mutants as compared with VDRwt. Alternatively the VDR affinity and genomic

TABLE 16.2  Relative Competitive Index, Genomic EC50 Values, and Simulated Binding Properties of 1,25(OH)2D3 (1,25D3), JN, HL, 25(OH) D3 (25D3), CF, MK, and IE in the VDR Genomic Pocket (VDR-GP) and VDR Alternative Ligand-Binding Pocket (VDR-AP) VDR Ligand

VDR-GP ΔGbinding (kcal/mol)

VDR-AP ΔGbinding (kcal/mol)

Relative Competitive Index (RCI), Chick Intestinal Mucosa

Genomic Effective Concentration (EC50, nM)

1,25D3*

−47.3 (−63.5)

−37.0

100%

1.4 ± 0.15

JN*

−37.3 (−59.2)

−34.4

1.8% ± 0.5%

178 ± 37

HL*

−45.6 (−57.6)

−41.7

1.0% ± 1.5%

160 ± 1.8

25D3**

−36.7 (−43.7)

−37.2

0.15% ± 0.020%

280 ± 73

CF**

−44.7 (−50.8)

−29.7

5.7% ± 0.73%

5.4 ± 1.3

MK

−37.1 (−55.2)

−36.2

10.6% ± 5.3%

>1000

IE*

−41.8 (−62.0)

−38.6

147% ± 72%

0.019 ± 0.0019

The table summarizes the theoretical and empirical binding affinities for the vitamin D receptor (VDR) ligands highlighted in this article. The (*) next to 1,25(OH)2D3, JN, HL, and IE indicates that the molecular formula of the sterol is identical, but the atom connectivity or stereochemistry at C1, C9/C10, or C20 differs when compared to 1,25(OH)2D3 (see Fig. 16.5 for the chemical structures). The (**) next to 25D3 and CF indicates that these two compounds have the same molecular formulas but differ in the position of the A-ring hydroxyl group (C1 for CF and C3 for 25D3, see Fig. 16.2B). So long as the molecular formulas (i.e., the number and type of atoms in the ligand are identical) direct comparisons in computational affinities (i.e., Gibbs free energy of binding (ΔGbinding)) can be made. The theoretical affinities for the VDR-GP and VDR-AP are listed in the table. These values were obtained using the Discovery Studio 2.0 software package and a flexible docking simulation protocol, where vitamin D sterol conformations were generated using a PC_Model conformational search calculation (see Figs. 16.2 and 16.4 and text). The first values listed in the VDR-GP column represent the average of the top seven VDR-GP complexes for each ligand. The top seven were averaged because it was observed that the averaged value for 1,25(OH)2D3 was identical to that computed using the ligand pose extracted from the 1,25(OH)2D3–VDR X-ray complex in the Gibbs free energy calculation [44]. Consistent with our previous VDR-AP and VDR-GP molecular mechanics calculations [30,32,37,38], a mixture of 6-s-cis and 6-s-trans, α/β-chair poses was observed in the VDR-AP flexible docking studies. Alternatively, a 6-s-trans, β-chair conformation was the only seco-B-ring, A-ring rotomer observed in the top 7 1,25(OH)2D3–VDR-GP flexible docking results [45]. The value in parentheses represents the highest affinity complex for each vitamin D sterol. This value is presented because it correlates better to the empirical affinity of the VDS for the VDR and their function where IE ≥ 1,25(OH)2D3 > JN ≥ HL (see text). This is based on the RCI value for each VDS measured by a steroid competition assay where increasing concentrations of the cold ligand are measured for their ability to compete off [3H]-1,25(OH)2D3 (0.2 pmoles) and the genomic effective concentration (EC50) measured for each VDS in CV1 cells transiently transfected with VDRwt and a SEAP-reporter construct [138].

II.  MECHANISM OF ACTION

The Vitamin D Receptor Conformational Ensemble Model

TABLE 16.3 1,25(OH)2D3 (1,25D3), 25(OH)D3 (25D3), and 3-Deoxy-1,25D3 Structure–Function Results in the Vitamin D Receptor (VDR) A-Ring Domain Mutant Constructs in Transfected COS and CV1 Cells

VDR Construct

Analog Code

Relative Competitive Index, %

Genomic Transactivation EC50 (nM)

VDRwt

1,25D3

100

1.4 ± 0.15 (n = 21)

VDRwt

3-deoxy-1,25D3

13 ± 5.3

5.4 ± 1.3 (n = 3)

VDRwt

25D3

0.08 ± 0.03

280 ± 73 (n = 3)

S278A

1,25D3

100

0.88 ± 0.29 (n = 3)

S278A

3-deoxy-1,25D3

20 ± 2.4

1.9 ± 0.30 (n = 5)

Y143F/S278A

1,25D3

100

94 ± 34 (n = 3)

Y143F/S278A

3-deoxy-1,25D3

130 ± 41

1.3 ± 0.30 (n = 3)

S237A

1,25D3

100

93 ± 24 (n = 2)

S237A

25D3

11.0 ± 4.5

230 ± 54 (n = 2)

R274A

1,25D3

ND

6700 ± 3700 (n = 3)

R274A

25D3

ND

860 ± 374 (n = 2)

S237A/R274A

1,25D3

ND

21,000 ± 64,000 (n = 3)

S237A/R274A

25D3

ND

510 ± 7.6 (n = 3)

The table summarizes the relative competitive index (RCI) values and effective concentrations (EC50) of 1,25D3, 25D3 (1-deoxy-1,25D3), and 3-deoxy-1,25D3 in COS-1 cells transfected with hVDRwt, S278A, Y143F/S278A, S237A, R274A, and S237A/R274A VDR constructs [30,37,38]. RCI values were obtained using the cell lysate from the transfected cells in a steroid competition assay [178]. ND indicates that the ability of [3H]-1,25(OH)2D3 to bind the construct was too weak for an RCI to be determined. The EC50 value was obtained by cotransfecting CV1 cells with a secreted alkaline phosphatase reporter construct driven by an osteocalcin vitamin D response element [32].

agonist potential of 3-deoxy-1,25(OH)2D3 were posited to be increased in the Y143F and/or S278A mutants because the mutation would, in theory, reduce the energetic stability of 3-deoxy-1,25(OH)2D3 in the VDR-AP and thereby increase its fractional occupancy of the VDR-GP. The actual structure–function results were quite consistent with the predicted changes in the measured VDR affinity and genomic EC50 (Table 16.3 and Refs. [30,32,37]).

Understanding the Nongenomic Agonist/ Antagonist Affinity/Function Conundrum The vitamin D sterols, JN, 25(OH)D3, and HL, all modulate nongenomic rapid responses when 1.0 nM of each VDS is added alone (i.e., agonist ligands, JN, and 25(OH)D3) or in the presence of 1,25(OH)2D3 (i.e., antagonist HL). However, JN, 25(OH)D3, and HL bind to the VDR with an approximately 50–500-fold reduced affinity, in a steroid competition assay, when compared with 1,25(OH)2D3 (Table 16.2). Thus when rapid, nongenomic responses were first identified it was unclear how 1 nM JN could stimulate and how HL could block nongenomic, rapid responses. These VDR affinity–function

285

conundrums formed the basis for this laboratory’s original hypothesis that a distinct, novel, membrane VDR for rapid responses must exist, which differs from the classic nuclear VDR [139]. Our most recent 1,25(OH)2D3, JN, 25(OH)D3, and HL molecular mechanics simulations [45] demonstrate that based on the overlay of the bound ligand poses and the potential interaction energy and ΔGbinding calculations (Table 16.2), 1,25(OH)2D3, JN, and 25(OH)D3 share similar VDR-AP physicochemical binding characteristics [30,37]. Alternately, HL is observed to form hydrogen bonds with only R274 and, when bound to the VDR-AP it is positioned differently with respect to the agonist ligands [1]. Lastly, 1,25(OH)2D3 is a significantly more stable VDR-GP ligand when compared with JN, 25(OH)D3, and HL (Table 16.2). Thus the models suggest that the enhanced VDR-AP selectivity of JN and 25(OH)D3 (Table 16.2), when compared with 1,25(OH)2D3, is not a result of a significant increase in their VDR-AP affinity, but rather a reduced capacity to interact favorably with the VDR-GP. This is consistent with the hypothesis that the VDR-AP is kinetically favored for all VDR ligands [1]. In closing, the enhanced VDR-AP affinity of HL when compared with 1,25(OH)2D3 (Table 16.2) and its stabilization of a unique VDR-AP geometry supports it functioning as a competitive antagonist of 1,25(OH)2D3, JN, and 25(OH)D3 nongenomic responses (Table 16.1).   

CA S E S TUD Y I : VO LTA G E -G AT E D CHL O RI D E CHA NNE L S From the perspective of small-molecule specificity and nongenomic, rapid response structure–function studies, the system studied in the greatest detail is VDS potentiation of ORCCs of the CLC type (see above). Most recently we have extended our screening of vitamin D sterols for their ability to potentiate ORCC currents in TM4 Sertoli cells. Specifically, we have shown that 1,25(OH)2D3, JN, MK, IE (Fig. 16.5), and 25(OH)D3 all function as agonist ligands in this system (Table 16.1). The side chain analogs MK and IE are most well known as an antagonist and superagonist of VDR gene transcription, respectively [14,140,141]. When flexibly docked to the VDR-AP and VDR-GP, MK displayed a good affinity in the VDR-AP (Table 16.2). In the VDR-GP, MK shows increased conformational heterogeneity when compared with 1,25(OH)2D3 [44], a result that supports the inability to form a cocrystal of the human VDRwt bound to MK [142]. The same was observed for IE, with the exception that its VDR-GP affinity was more similar to 1,25(OH)2D3 (Table 16.2). This evidence in conjunction with the results showing that 1,25(OH)2D3, JN, and 25(OH)D3 currents are blocked by coincubation with HL clearly demonstrates that the changes in 1,25(OH)2D3 chemistry known to alter VDR genomic function do not necessarily alter VDR-dependent nongenomic potentiation of ORCC. This may also be true for other nongenomic signaling factors.   

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TABLE 16.4  Theoretical 1,25(OH)2D3 (1,25D3) and Phosphatidylinositolphosphate (PIP) VDRwt and W286R Complex Scores

C A SE STU D Y II: W286R AND C A LC I U M SI GNALING Individuals with the W286R VDR mutation are not able to stimulate VDRwt genomic responses; however, they do respond rapidly to 1,25(OH)2D3, as manifested by an increase in intracellular calcium. Thus the W286R mutation is a natural mutation that causes vitamin D-resistant rickets, but unlike other natural mutations that inhibit binding of the VDR to DNA, the mutation does not cause alopecia, considered a VDR genomic effect [129,143]. The conundrum here lies in the fact that 1,25(OH)2D3 binds very poorly to the W286R mutant (Fig. 16.10) yet can stimulate an increase in intracellular calcium at subnanomolar concentrations [129,143]. Thus, the change in VDR function induced by the W286R mutation is somewhat similar to that induced by the absence of the 1α-OH group or fusion of the 1,25(OH)2D3 B-ring as discussed above. Flexible docking molecular mechanics simulations show that 1,25(OH)2D3 is not a good VDR-GP ligand in the W286R mutant; however, in the W286R mutant, there is no observed change in the affinity of 1,25(OH)2D3 for the VDR-AP (Table 16.4). According to the VDR–PIP membrane model, when bound to a PIP, all of the known surface regions of the VDR molecule shown to interact with coregulator proteins would remain solvent exposed (Fig. 16.7). This may allow nuclear VDR–PIP to form a repressive complex with DNA and provide a basis for why patients with the W286R mutant lack alopecia [129,143]. Thus our empirical evidence that [3H]-1,25(OH)2D3 can bind weakly to the W286R mutant (Fig. 16.10) confirms the results of Nguyen

Ligand

VDR Construct/Binding Pocket

ΔGbinding (kcal/mole)

1,25D3

VDRwt/VDR-GP

−47.3

1,25D3

VDRwt/VDR-AP

−37.0

1,25D3

W286R/VDR-GP

−8.3

1,25D3

W286R/VDR-AP

−33.7

PIP2

VDRwt/VDR-AP

−178

PIP3

VDRwt/VDR-AP

−233

PIP2

W286R/VDR-AP

−192

PIP3

W286R/VDR-AP

−92

The table summarizes the computational results obtained when 1,25(OH)2D3 and the phosphatidylinositolphosphates, PIP2 and PIP3, were docked to the vitamin D receptor (VDR) using molecular mechanics. For docking 1,25(OH)2D3, the published flexible docking protocol was used [44,45], wherein 1,25(OH)2D3 conformational isomers generated using a PC_Model conformational search calculation (Figs. 16.2 and 16.4) were docked to a 10.0 Å3 VDR genomic pocket (VDR-GP) or VDR alternative ligand-binding pocket (VDR-AP) site spheres (see [45]). For the PIP-VDR complexes, the PIP2 and PIP3 inositol head groups (i.e., inositol-1,4,5-triphosphate and inositol-1,3,4,5-tetraphosphate) where flexibly docked to the VDR A-ring domain (Discovery Studio 2.0, DS2.0), the phosphotriglyceride was then manually built off the highest scored complex. The resulting PIP2 or PIP3 complex was energy optimized to a convergence derivative of 0.01. As in Table 16.2, the 1,25(OH)2D3 Gibbs free energy of binding (ΔGbinding) values represent the average of the top seven complexes ranked using the cDOCKER scoring function of DS2.0. Alternatively the PIP ΔGbinding value is calculated from one complex. Based on the theoretical results, the W286R mutation hinders the binding of 1,25(OH)2D3 to the VDR-GP and PIP3 to the VDR-AP as deduced from the more positive (i.e., weaker affinity) ΔGbinding values when compared with their VDRwt results.

et al. [129,143]. The structure–function and molecular modeling results provide the first model explaining why W286 patients lack alopecia. Also they provide evidence that alopecia may still occur when the VDR is occupied with a ligand and therefore not strictly a ligand-independent effect of the VDR [144,145].   

VITAMIN D RECEPTOR LIGAND SPECIFICITY: DOES AN UNLIGANDED VITAMIN D RECEPTOR EVER EXIST IN VIVO? FIGURE 16.10 [3H]-1,25(OH)2D3 binding to the VDR W286R natural mutant. The graph shows the percent (%) of [3H]-1,25(OH)2D3 bound to the W286R construct. The value was obtained following incubation of expressed hVDRwt and W286R with 0.04 and 1.0 nM of tritiated 1,25(OH)2D3, in COS-1 cell lysate (Table 16.3). The percentage for hVDRwt was set to 100% and the amount specifically bound to W286R reported.

In the past few years it has emerged that botanical ligands isolated from the roots of plants (e.g., curcuminoids and withanolides, Fig. 16.5) can bind to the VDR [45]. Surprisingly, even tocopherols (i.e., vitamin E) have been shown to bind to the VDR [127]. In addition, the nonsteroidal synthetic analogs [146], DIDS [45], LCA [147] and 25(OH)D3, and 1,25(OH)2D3 metabolites [32] (Fig. 16.3) all bind specifically

II.  MECHANISM OF ACTION

References

to the VDR. Finally, computational results suggest the VDR binds to PIPs (Fig. 16.7). Taken together, these observations allow us to envision that the VDR is seldom, if ever, in a situation where it is unliganded in vivo. As a consequence, each ligand can, under given circumstances, either mimic or differ from 1,25(OH)2D3 in the VDR functions it is able to modulate [1,32]. For example, 1,25(OH)2D3 [148], curcumin [149], and withaferin A [150] all inhibit NFκB; however, 1,25(OH)2D3 stimulates MAPK activity, whereas curcumin blocks it. Perhaps these data will incline more researchers to consider the fact that when flexibly docked to the VDR-AP, VDR-GP, and/or the overlapping A-ring domain, all of the small molecules highlighted in this work show differences in the intermolecular contacts made and pocket selectivity observed [30,32,37,38,45].

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[184] Ishizuka S, Ishimoto S, Norman AW. Biological activity assessment of 1α,25-dihydroxyvitamin D3-26, 23-lactone in the rat. J Steroid Biochem 1984;20:611–5. [185] Ishizuka S, Kiyoki M, Orimo H, Norman AW. Biological activity and characteristics of 1α,25-(OH)2D3-26,23-lactone. In: Norman AW, Schaefer K, Grigoleit H-G, von Herrath D, editors. Vitamin D: chemical, biochemistry and clinical update. Berlin: Walter de Gruyter; 1985. p. 402–3. [186] Ando H, Mizutani A, Kiefer H, Tsuzurugi D, Michikawa T, Mikoshiba K. IRBIT suppresses IP3 receptor activity by competing with IP3 for the common binding site on the IP3 receptor. Mol Cell 2006;22:795–806. [187] Jirmanova L, Ashwell JD. T cell priming: let there be light. Cell Res 2010;20:608–10. [188] Deeb KK, Trump DL, Johnson CS. Vitamin D signalling pathways in cancer: potential for anticancer therapeutics. Nat Rev Cancer 2007;7:684–700. [189] Barletta F, Wong CW, McNally C, Komm BS, Katzenellenbogen B, Cheskis BJ. Characterization of the interactions of estrogen receptor and MNAR in the activation of cSrc. Mol Endocrinol 2004;18:1096–108. [190] Biggins JB, Koh JT. Chemical biology of steroid and nuclear hormone receptors. Curr Opin Chem Biol 2007;11:99–110. [191] Okamura WH, Midland MM, Hill DK, Ringe K, Takeuchi JA, Vassar VC, Vu TH, Zhu G-D, Norman AW, Bouillon R, Farach-Carson MC. Vitamin D drug design and synthesis: towards understanding the “mutually induced fit” of vitamin D ligands and various proteins which bind metabolites and analogs. In: Norman AW, Bouillon R, Thomasset M, editors. Vitamin D, chemistry, biology and clinical applications of the steroid hormone. Riverside (CA): University of California; 1997. p. 11–8.

II.  MECHANISM OF ACTION