Longitudinal study of vitamin D metabolites after long bone fracture.

Animal models suggest a key role for dihydroxylated vitamin D metabolites in fracture healing, as evidenced by increases in serum concentration of 24R,25-dihydroxyvitamin D (24R,25[OH]2D) after long bone fracture. Human studies investigating the kinetics of serum concentrations of 24R,25[OH]2D, 1,25-dihydroxyvitamin D (1,25[OH]2D) and their parent metabolite 25-hydroxyvitamin D (25[OH]D) are lacking. We, therefore, conducted a longitudinal study to determine whether total, free, or bioavailable concentrations of these vitamin D metabolites fluctuate in humans after long bone fracture. Twenty-eight patients with cross-shaft (diaphyseal) long bone fracture presenting to an emergency department in London, UK, were studied. Serum concentrations of 25(OH)D, 24R,25(OH)2D, 1,25(OH)2D, vitamin D binding protein, albumin, and calcium were determined within 48 hours of fracture and again at 1 and 6 weeks postfracture. Concentrations of free and bioavailable vitamin D metabolites were calculated using standard equations. No changes in mean serum concentrations of 25(OH)D or 24R,25(OH)2D were seen at either follow-up time point versus baseline. In contrast, mean serum 1,25(OH)2 D concentration declined by 21% over the course of the study, from 68.5 pmol/L at baseline to 54.1 pmol/L at 6 weeks (p < 0.05). This decline was associated with an increase in mean serum corrected calcium concentration, from 2.32 mmol/L at baseline to 2.40 mmol/L at 1 week (p < 0.001) that was maintained at 6 weeks. No changes in free or bioavailable concentrations of any vitamin D metabolite investigated were seen over the course of the study. We conclude that serum 1,25(OH)2D concentration declines after long bone fracture in humans but that the serum 24R,25(OH)2D concentration does not fluctuate. The latter finding contrasts with those of animal models reporting increases in serum 24R,25(OH)2D concentration after long bone fracture.

Opening of chloride channels by 1α,25-dihydroxyvitamin D3 contributes to photoprotection against UVR-induced thymine dimers in keratinocytes.

UVR produces vitamin D in skin, which is hydroxylated locally to 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). 1,25(OH)(2)D(3) protects skin cells against UVR-induced DNA damage, including thymine dimers, but the mechanism is unknown. As DNA repair is inhibited by nitric oxide (NO) products but facilitated by p53, we examined whether 1,25(OH)(2)D(3) altered the expression of nitrotyrosine, a product of NO, or p53 after UVR in human keratinocytes. 1,25(OH)(2)D(3) and the nongenomic agonist 1α,25-dihydroxylumisterol(3) reduced nitrotyrosine 16 hours after UVR, detected by a sensitive whole-cell ELISA. p53 was enhanced after UVR, and this was further augmented in the presence of 1,25(OH)(2)D(3). DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a chloride channel blocker previously shown to prevent 1,25(OH)(2)D(3)-induced chloride currents in osteoblasts, had no effect on thymine dimers on its own but prevented the 1,25(OH)(2)D(3)-induced protection against thymine dimers. Independent treatment with DIDS, at concentrations that had no effect on thymine dimers, blocked UVR-induced upregulation of p53. In contrast, reduction of nitrotyrosine remained in keratinocytes treated with 1,25(OH)(2)D(3) and DIDS at concentrations shown to block decreases in post-UVR thymine dimers. These results suggest that 1,25(OH)(2)D(3)-induced chloride currents help protect from UVR-induced thymine dimers, but further increases in p53 or reductions of nitrotyrosine by 1,25(OH)(2)D(3) are unlikely to contribute substantially to this protection.

The history of the discovery of vitamin D and its daughter steroid hormone.

It is largely through historical accident in the interval of 1920-1940 that vitamin D(3) became classified as a vitamin rather than as a steroid hormone. The formal definition of a vitamin is that it is a trace dietary constituent required to produce the normal function of a physiological process or processes. The emphasis here is on trace and the fact that the vitamin must be supplied regularly in the diet; this implies that the body is unable to metabolically synthesize the vitamin in question. However, the ultraviolet exposure of 7-dehydrocholesterol present in the skin results in the photochemical production of vitamin D(3). Thus, vitamin D(3) becomes a true vitamin only when the animal or human does not have regular access to sunlight or ultraviolet light. Under normal physiological circumstances, all mammals, including humans, can generate, via ultraviolet exposure of 7-dehydrocholesterol present in the skin, adequate quantities of vitamin D(3) to meet their nutritionally defined requirements. There is a vibrant historical record beginning in 1650 and culminating in 1963 concerned with the determination of the chemical structures of vitamin D(3) and vitamin D(2). A surprising aspect concerning vitamin D(3) is that it is itself biologically inert. There are no known essential biological actions or contributions that rely specifically on the molecule vitamin D(3). While chemists had certainly appreciated the strong structural similarity between the vitamins D and other steroids, this correlation was never widely acknowledged in the biological, clinical, or nutritional sciences until 1965-1970. The biological role of vitamin D(3) is to serve as a substrate for the liver 25-hydroxylase which produces 25-hydroxyvitamin D(3) [25(OH)D(3)]. 25(OH)D(3) in turn serves as the substrate for the kidney proximal tubule 25(OH)D(3)-1α-hydroxylase enzyme which produces the steroid hormone 1α,25(OH)(2)-vitamin D(3) [1α,25(OH)(2)D(3)].

The role of the vitamin D receptor and ERp57 in photoprotection by 1α,25-dihydroxyvitamin D3.

UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.

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.

1α,25(OH)2-vitamin D and a nongenomic vitamin D analogue inhibit ultraviolet radiation-induced skin carcinogenesis.

Exposure to ultraviolet radiation (UVR) can lead to a range of deleterious responses in the skin. An important form of damage is the DNA photolesion cyclobutane pyrimidine dimer (CPD). CPDs can be highly mutagenic if not repaired prior to cell division and can lead to UV-induced immunosuppression, making them potentially carcinogenic. UVR exposure also produces vitamin D, a prehormone. Different shapes of the steroid hormone 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] can produce biological responses through binding either to its cognate nuclear receptor (VDR) to regulate gene transcription or to the VDR associated with plasma membrane caveolae to produce, via signal transduction, nongenomic physiologic responses. Here, we show that both 1,25(OH)2D3 and 1α,25(OH)2-lumisterol (JN), a conformationally restricted analogue that can generate only nongenomic responses, are effective inhibitors of UV damage in an immunocompetent mouse (Skh:hr1) model susceptible to UV-induced tumors. Both 1,25(OH)2D3 and JN significantly reduced UVR-induced CPD, apoptotic sunburn cells, and immunosuppression. Furthermore, these compounds inhibited skin tumor development, both papillomas and squamous cell carcinomas, in these mice. The observed reduction of these UV-induced effects by 1,25(OH)2D3 and JN suggests a role for these compounds in prevention against skin carcinogenesis. To the best of our knowledge, this is the first comprehensive report of an in vivo long-term biological response generated by chronic dosing with a nongenomic-selective vitamin D steroid.

Vitamin D receptor (VDR) regulation of voltage-gated chloride channels by ligands preferring a VDR-alternative pocket (VDR-AP).

We have postulated that the vitamin D receptor (VDR) contains two overlapping ligand binding sites, a genomic pocket and an alternative pocket (AP), that mediate regulation of gene transcription and rapid responses, respectively. Flexible VDR + ligand docking calculations predict that the major blood metabolite, 25(OH)-vitamin D(3) (25D3), and curcumin (CM) bind more selectively to the VDR-AP when compared with the seco-steroid hormone 1α,25(OH)(2)-vitamin D(3) (1,25D3). In VDR wild-type-transfected COS-1 cells and TM4 Sertoli cells, 1,25D3, 25D3, and CM each trigger voltage-gated, outwardly rectifying chloride channel (ORCC) currents that can be blocked by the VDR antagonist 1ß,25(OH)(2)-vitamin D(3) and the chloride channel antagonist (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid). VDR mutational analysis in transfected COS-1 cells demonstrate the DNA-binding domain is not, but the ligand binding and hinge domains of the VDR are, required for 1,25D3 and 25D3 to activate the ORCC. Dose-response studies demonstrate that 25D3 and 1,25D3 are approximately equipotent in stimulating ORCC rapid responses, whereas 1 nm 1,25D3 was 1000-fold more potent than 25D3 and CM in stimulating gene expression. The VDR-AP agonist effects of 1,25D3, 25D3, and low-dose CM are lost after pretreatment of TM4 cells with VDR small interfering RNA. Collectively, these results are consistent with an essential role for the VDR-AP in initiating the signaling required for rapid opening of ORCC. The fact that 25D3 is equipotent to 1,25D3 in opening ORCC suggests that reconsideration of the ability of 25D3 to generate biological responses in vivo may be in order.

Structural basis of the histidine-mediated vitamin D receptor agonistic and antagonistic mechanisms of (23S)-25-dehydro-1alpha-hydroxyvitamin D3-26,23-lactone.

TEI-9647 antagonizes vitamin D receptor (VDR) mediated genomic actions of 1alpha,25(OH)2D3 in human cells but is agonistic in rodent cells. The presence of Cys403, Cys410 or of both residues in the C-terminal region of human VDR (hVDR) results in antagonistic action of this compound. In the complexes of TEI-9647 with wild-type hVDR (hVDRwt) and H397F hVDR, TEI-9647 functions as an antagonist and forms a covalent adduct with hVDR according to MALDI-TOF MS. The crystal structures of complexes of TEI-9647 with rat VDR (rVDR), H305F hVDR and H305F/H397F hVDR showed that the agonistic activity of TEI-9647 is caused by a hydrogen-bond interaction with His397 or Phe397 located in helix 11. Both biological activity assays and the crystal structure of H305F hVDR complexed with TEI-9647 showed that the interaction between His305 and TEI-9647 is crucial for antagonist activity. This study indicates the following stepwise mechanism for TEI-9647 antagonism. Firstly, TEI-9647 forms hydrogen bonds to His305, which promote conformational changes in hVDR and draw Cys403 or Cys410 towards the ligand. This is followed by the formation of a 1,4-Michael addition adduct between the thiol (-SH) group of Cys403 or Cys410 and the exo-methylene group of TEI-9647.

Vitamin D nutritional policy needs a vision for the future.

Historically vitamin D is known to be essential for normal bone growth and quality, and thus appropriate dietary vitamin D supplementation can eliminate vitamin D deficiency childhood rickets and adult osteomalacia. In spite of many government and medical associations' worldwide guidelines for the reference daily intake (RDI) of vitamin D, scientists and nutritionists from many countries agree that at present about half of elderly North Americans and Western Europeans and probably also of the rest of the world are not receiving enough vitamin D to maintain healthy bone. In addition, over the past decade there has been a dramatic increase in our understanding of the many biological actions that result from vitamin D acting through its daughter steroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] in collaboration with its cognate vitamin D receptor (VDR). Consequently, evidence has accumulated that beside intestine and bone, there are five additional physiological systems where the VDR with 1alpha,25(OH)(2)D generates biological responses. These include the immune system (both the innate and adaptive), pancreas and metabolic homeostasis, heart-cardiovascular, muscle and brain systems as well as the control of the cell cycle, and thus of the disease process of cancer. Acting through the VDR, 1alpha,25(OH)(2)D(3) can produce a wide array of favorable biological effects that collectively are projected to contribute to the improvement of human health. Responsible medicine demands that worldwide vitamin D nutritional guidelines reflect current scientific knowledge about vitamin D's spectrum of activities. Thus, worldwide vitamin D nutritional policy is now at a crossroads. This paper presents several proposed policy changes with regard to the amount of vitamin D daily intake that if implemented will maximize vitamin D's contribution to reducing the frequency of many diseases, which would then increase the quality and longevity of life and significantly reduce the cost of medical care worldwide.

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.

1alpha,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.

On the mechanism underlying (23S)-25-dehydro-1alpha(OH)-vitamin D3-26,23-lactone antagonism of hVDRwt gene activation and its switch to a superagonist.

(23S)-25-Dehydro-1alpha(OH)-vitamin D(3)-26,23-lactone (MK) is an antagonist of the 1alpha,25(OH)(2)-vitamin D(3) (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 (EC(50) approximately 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.

The vitamin D sterol-vitamin D receptor ensemble model offers unique insights into both genomic and rapid-response signaling.

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 1alpha,25(OH)(2)D(3) (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.

Prolactin blocks nuclear translocation of VDR by regulating its interaction with BRCA1 in osteosarcoma cells.

Based on their content of prolactin receptors, osteosarcoma cells were predicted to be responsive to prolactin (PRL), but whether PRL would be beneficial or contribute to pathogenesis was unclear. 1,25(OH)(2) vitamin D(3) [1alpha,25(OH)(2)D(3)] has antiproliferative effects on osteosarcoma cells, and a complex interregulatory situation exists between PRL and 1alpha,25(OH)(2)D(3). Using osteosarcoma cells, Western blot, real time RT-PCR, and promoter-luciferase assays, we have examined the interaction between PRL and 1alpha,25(OH)(2)D(3) and demonstrated that physiological concentrations of PRL block increased osteocalcin and vitamin D receptor (VDR) expression in response to 1alpha,25(OH)(2)D(3.) This blockade was shown to be the result of lack of nuclear accumulation of the VDR in response to 1alpha,25(OH)(2)D(3). Although inhibition of proteasomic degradation with MG132 had no effect on the VDR itself in a 30-min time frame, it relieved the blockade by PRL. Analysis of ubiquitinated proteins brought down by immunoprecipitation with anti-VDR showed PRL regulation of a 250-kDa protein-VDR complex. P250 was identified as the breast cancer tumor suppressor gene product, BRCA1, by Western blot of the VDR immunoprecipitate and confirmed by immunoprecipitation with anti-BRCA1 and blotting for the VDR in the absence and presence of PRL. Knockdown of BRCA1 inhibited nuclear translocation of the VDR and the ability of 1alpha,25(OH)(2)D(3) to induce the VDR. This, to our knowledge, is the first demonstration of a role for BRCA1 in nuclear accumulation of a steroid hormone and the first demonstration that PRL has the potential to affect the cell cycle through effects on BRCA1.