Mathew T. Mizwicki

Steroid-hormone rapid actions, membrane receptors and a conformational ensemble model

Steroid hormones can act as chemical messengers in a wide range of species and target tissues to produce both slow genomic responses, and rapid non-genomic responses. Although it is clear that genomic responses to steroid hormones are mediated by the formation of a complex of the hormone and its cognate steroid-hormone nuclear receptor, new evidence indicates that rapid responses are mediated by a variety of receptor types associated with the plasma membrane or its caveolae components, potentially including a membrane-associated nuclear receptor. This review summarizes our current knowledge of membrane-associated steroid receptors, as well as details of structure–function relationships between steroid hormones and the ligand-binding domains of their nuclear and membrane-associated receptors. Furthermore, a new receptor conformational ensemble model is presented that suggests how the same receptor could produce both rapid and genomic responses. It is apparent that there is a cornucopia of new drug development opportunities in these areas.

Vitamin D receptor (VDR)-mediated actions of 1α,25(OH)2vitamin D3: Genomic and non-genomic mechanisms

The conformationally flexible secosteroid, 1α,25(OH)₂vitamin D₃ (1α,25(OH)₂D₃) initiates biological responses via binding to the vitamin D receptor (VDR). The VDR contains two overlapping ligand binding sites, a genomic pocket (VDR-GP) and an alternative pocket (VDR-AP), that respectively bind a bowl-like ligand configuration (gene transcription) or a planar-like ligand shape (rapid responses). When occupied by 1α,25(OH)₂D₃, the VDR-GP interacts with the retinoid X receptor to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1α,25(OH)₂D₃. By recruiting complexes of either coactivators or corepressors, activated VDR modulates the transcription of genes encoding proteins that promulgate the traditional genomic functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. 1α,25(OH)₂D₃/VDR control of gene expression and rapid responses also delays chronic diseases of aging such as osteoporosis, cancer, type-1 and -2 diabetes, arteriosclerosis, vascular disease, and infection.

The Vitamin D Sterol–Vitamin D Receptor Ensemble Model Offers Unique Insights into Both Genomic and Rapid-Response Signaling

Different Vitamin D conformations binding to different pockets of a flexibile receptor could elicit distinct responses. Vitamin D3 was discovered 90 years ago as a dietary agent that prevented the bone disease rickets. Subsequently, it was learned that vitamin D3 itself is biologically inert and only generates biological effects through participation in a two-step metabolic process that generates the steroid hormone 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3 or 1,25D]. Binding of 1,25D to its vitamin D receptor (VDR) results in various biological responses. The VDR is present in 37 tissues of the body, where it has been shown to function in five physiological systems in addition to the intestine and bone (where it plays a critical role in calcium homeostasis). In these target tissues, the VDR functions both in the cell nucleus (as a transcriptional factor to regulate genes containing a vitamin D response element) and at or near the plasma membrane (as a mediator of rapid signal transduction pathways). Here, we discuss the evidence supporting a vitamin D sterol–VDR conformational ensemble model, whereby different shapes of the 1,25D hormone bind to two different ligand-binding pockets of a flexible VDR to selectively modulate either genomic or rapid responses. Steroid hormones serve as chemical messengers in a wide number of species and target tissues by transmitting signals that result in both genomic and nongenomic responses. Genomic responses are mediated by the formation of a ligand-receptor complex with its cognate steroid hormone nuclear receptor (NR). Nongenomic responses can be mediated at the plasma membrane by a membrane-localized NR. The focus of this Review is on the structural attributes and molecular mechanisms underlying vitamin D sterol (VDS)–vitamin D receptor (VDR) selective and stereospecific regulation of nongenomic and genomic signaling. The VDS-VDR conformational ensemble model describes how VDSs can selectively initiate or block either nongenomic or genomic biological responses by interacting with two VDR ligand-binding pockets, one kinetically favored by 1α,25(OH)2D3 (1,25D) and the other thermodynamically favored. We describe the variables that affect the three major elements of the model: the conformational flexibility of the unliganded (apo) protein, the flexibility of the VDS, and the physicochemical selectivity of the VDR genomic pocket (VDR-GP) and alternative pocket (VDR-AP). We also discuss how these three factors collectively provide a rational explanation for the complexities of VDS regulation of cell biology and highlight the current limitations of the model.

Photoprotection by 1α,25-dihydroxyvitamin D and analogs: Further studies on mechanisms and implications for UV-damage

Ultraviolet (UV) irradiation causes DNA damage in skin cells, immunosuppression and photocarcinogenesis. 1alpha,25-dihydroxyvitamin D3 (1,25D) reduces UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in human keratinocytes in culture and in mouse and human skin. UV-induced immunosuppression is also reduced in mice by 1,25D, in part due to the reduction in CPD and a reduction in interleukin (IL-6. The cis-locked analog, 1alpha,25-dihydroxylumisterol3 (JN), which has almost no transactivating activity, reduces UV-induced DNA damage, apoptosis and immunosuppression with similar potency to 1,25D, consistent with a non-genomic signalling mechanism. The mechanism of the reduction in DNA damage in the form of CPD is unclear. 1,25D doubles nuclear expression of p53 compared to UV alone, which suggests that 1,25D facilitates DNA repair. Yet expression of a key DNA repair gene, XPG is not affected by 1,25D. Chemical production of CPD has been described. Incubation of keratinocytes with a nitric oxide donor, SNP, induces CPD in the dark. We previously reported that 1,25D reduced UV-induced nitrite in keratinocytes, similar to aminoguanidine, an inhibitor of nitric oxide synthase. A reduction in reactive nitrogen species has been shown to facilitate DNA repair, but in view of these findings may also reduce CPD formation via a novel mechanism.

1α,25(OH)2-Vitamin D3 stimulation of secretion via chloride channel activation in Sertoli cells

Sertoli cell secretory activities are highly dependent on ion channel functions and critical to spermatogenesis. The steroid hormone 1alpha,25(OH)2-vitamin D3 (1,25(OH)2-D3) stimulates exocytosis in different cell systems by activating a nongenotropic vitamin D receptor (VDR). Here, we described 1,25(OH)2-D3 stimulation of secretion via Cl(-) channel activation in the mouse immature Sertoli cell line TM4. 1,25(OH)2-D3 potentiation of chloride currents was dependent on hormone concentration, and correlated with a significant increase in whole-cell capacitance within 20-40 min. In addition, Cl(-) currents were potentiated by the nongenomic VDR agonist 1alpha,25(OH)2 lumisterol D3 (JN), while 1,25(OH)2-D3 potentiation of channels was suppressed by nongenomic VDR antagonist 1beta,25(OH)2-vitamin D3 (HL). Treatment of TM4 cells with PKC and PKA activators PMA and forskolin respectively, increased Cl(-) currents significantly, while PKC and PKA inhibitors Go6983 and H-89, respectively, abolished 1,25(OH)2-D3 stimulation of Cl(-) currents, suggesting phosphorylation pathways in 1,25(OH))2-D3 mediated channel responses. RT-PCR demonstrated the expression of outwardly rectifying ClC-3 channels in TM4 cells. Taken together, our results demonstrate a PKA/PKC-dependent 1,25(OH)2-D3/VDR nongenotropic pathway leading to Cl(-) channel and exocytosis activation in Sertoli cells. We conclude that 1,25(OH)2-D3 appears to be a modulator of male reproductive functions at least in part by stimulating Sertoli cell secretory functions.

A molecular description of ligand binding to the two overlapping binding pockets of the nuclear vitamin D receptor (VDR): structure-function implications

Molecular modeling results indicate that the VDR contains two overlapping ligand binding pockets (LBP). Differential ligand stability and fractional occupancy of the two LBP has been physiochemically linked to the regulation of VDR-dependent genomic and non-genomic cellular responses. The purpose of this report is to develop an unbiased molecular modeling protocol that serves as a good starting point in simulating the dynamic interaction between 1alpha,25(OH)2-vitamin D3 (1,25D3) and the VDR LBP. To accomplish this goal, the flexible docking protocol developed allowed for flexibility in the VDR ligand and the VDR atoms that form the surfaces of the VDR LBP. This approach blindly replicated the 1,25D3 conformation and side-chain dynamics observed in the VDR X-ray structure. The results are also consistent with the previously published tenants of the vitamin D sterol (VDS)-VDR conformational ensemble model. Furthermore, we used flexible docking in combination with whole-cell patch-clamp electrophysiology and steroid competition assays to demonstrate that (a) new non-vitamin D VDR ligands show a different pocket selectivity when compared to 1,25D3 that is qualitatively consistent with their ability to stimulate chloride channels and (b) a new route of ligand binding provides a novel hypothesis describing the structural nuances that underlie hypercalceamia.

1α,25-Dihydroxyvitamin D3 and Resolvin D1 Retune the Balance between Amyloid-β Phagocytosis and Inflammation in Alzheimer's Disease Patients

As immune defects in amyloid-β (Aβ) phagocytosis and degradation underlie Aβ deposition and inflammation in Alzheimers disease (AD) brain, better understanding of the relation between Aβ phagocytosis and inflammation could lead to promising preventive strategies. We tested two immune modulators in peripheral blood mononuclear cells (PBMCs) of AD patients and controls: 1α,25(OH)2-vitamin D3 (1,25D3) and resolvin D1 (RvD1). Both 1,25D3 and RvD1 improved phagocytosis of FAM-Aβ by AD macrophages and inhibited fibrillar Aβ-induced apoptosis. The action of 1,25D3 depended on the nuclear vitamin D and the protein disulfide isomerase A3 receptors, whereas RvD1 required the chemokine receptor, GPR32. The activities of 1,25D3 and RvD1 commonly required intracellular calcium, MEK1/2, PKA, and PI3K signaling; however, the effect of RvD1 was more sensitive to pertussis toxin. In this case study, the AD patients: a) showed significant transcriptional up regulation of IL1RN, ITGB2, and NFκB; and b) revealed two distinct groups when compared to controls: group 1 decreased and group 2 increased transcription of TLRs, IL-1, IL1R1 and chemokines. In the PBMCs/macrophages of both groups, soluble Aβ (sAβ) increased the transcription/secretion of cytokines (e.g., IL1 and IL6) and chemokines (e.g., CCLs and CXCLs) and 1,25D3/RvD1 reversed most of the sAβ effects. However, they both further increased the expression of IL1 in the group 1, sβ-treated cells. We conclude that in vitro, 1,25D3 and RvD1 rebalance inflammation to promote Aβ phagocytosis, and suggest that low vitamin D3 and docosahexaenoic acid intake and/or poor anabolic production of 1,25D3/RvD1 in PBMCs could contribute to AD onset/pathology.

On the Mechanism Underlying (23S)-25-Dehydro-1α(OH)-vitamin D3-26,23-lactone Antagonism of hVDRwt Gene Activation and Its Switch to a Superagonist

(23S)-25-Dehydro-1α(OH)-vitamin D3-26,23-lactone (MK) is an antagonist of the 1α,25(OH)2-vitamin D3 (1,25D)/human nuclear vitamin D receptor (hVDR) transcription initiation complex, where the activation helix (i.e. helix-12) is closed. To study the mode of antagonism of MK an hVDR mutant library was designed to alter the free molecular volume in the region of the hVDR ligand binding pocket occupied by the ligand side-chain atoms (i.e. proximal to helix-12). The 1,25D-hVDR structure-function studies demonstrate that 1) van der Waals contacts between helix-12 residues Leu-414 and Val-418 and 1,25D enhance the stability of the closed helix-12 conformer and 2) removal of the side-chain H-bonds to His-305(F) and/or His-397(F) have no effect on 1,25D transactivation, even though they reduce the binding affinity of 1,25D. The MK structure-function results demonstrate that the His-305, Leu-404, Leu-414, and Val-418 mutations, which increase the free volume of the hVDR ligand binding pocket, significantly enhance MK antagonist potency. Surprisingly, the H305F and H305F/H397F mutations turn MK into a VDR superagonist (EC50 ∼ 0.05 nm) but do not concomitantly alter MK binding affinity. Molecular modeling studies demonstrate that MK antagonism stems from its side chain energetically preferring a pose in the VDR ligand binding pocket where its terminal C26-methylene atom is far removed from helix-12. MK superagonism results from an energetically favored increase in interaction between Leu-404/Val-418 and C26, resulting in an increase in the stability and population of the closed, helix-12 conformer. Finally, the results/model generated, coupled with application of a VDR ensemble allosterics model, provide an understanding for the species specificity of MK.

Evidence that annexin II is not a putative membrane receptor for 1α, 25(OH)2-Vitamin D3

The seco‐steroid hormone 1α,25(OH)2‐vitamin D3 (1,25‐D3) is known to generate biological responses via both genomic and non‐genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII2/p112] was proposed by Baran and co‐authors to be the membrane receptor responsible for mediating non‐genomic, rapid actions of 1,25‐D3, based on ligand affinity labeling, competition, and saturation analysis experiments. Given the cytosolic presence of both the monomeric and heterotetrameric form of AII and their functional regulation by intracellular calcium concentrations, which are known to be affected by 1,25‐D3 rapid, non‐genomic activities, we investigated in vitro the affinity of [3H]1,25‐D3 for the AII monomer and AII2/p112 in the absence and presence of calcium using saturation analysis and gel‐filtration chromatography. Using two different techniques for separating bound from free ligand (perchlorate and hydroxylapatite (HAP)) over a series of 30 experiments, no evidence for specific binding of [3H]1,25‐D3 was obtained with or without the presence of 700 nM exogenous calcium, using either the AII monomer or AII2/p112. However saturable binding of [3H]1,25‐D3 to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (Kd = 3.0 nM; Bmax = 45 fmols/mg total protein). AII was detected in lipid raft/caveolae enriched fractions from rat and mouse intestine and ROS 17/2.8 and NB4 cells by Western blot, but incubation in the presence of exogenous calcium did not ablate 1,25‐D3 binding as reported by Baran et al. Our results suggest that AII does not bind 1,25‐D3 in a physiologically relevant manner; however, recent studies linking AII2/p112 phosphorylation to vesicle fusion and its calcium regulated localization may make AII a possible down‐stream substrate for 1,25‐D3 induced rapid cellular effects.

Two Key Proteins of the Vitamin D Endocrine System Come Into Crystal Clear Focus: Comparison of the X-ray Structures of the Nuclear Receptor for 1α,25(OH)2 Vitamin D3, the Plasma Vitamin D Binding Protein, and Their Ligands†

RECENTLY TWO BREAKTHROUGHS have been achieved with respect to understanding the three-dimensional protein structures of both the vitamin D binding protein (DBP) and the nuclear receptor (VDR) for the steroid hormone 1 ,25(OH)2-vitamin D3 [1 ,25(OH)2D3] and the detailed shape of their respective bound ligands. The determination of the crystal structure of the ligand binding domain (LBD) of the VDR bound to its natural ligand, 1 ,25(OH)2D3, (1)