Nicholas J. Koszewski

Evidence of functional vitamin D receptors in rat hippocampus.

The steroid hormone vitamin D has important biological roles in calcium transport, cell growth, and cell differentiation. Its cellular activities are mediated by high affinity interaction with the vitamin D receptor. In brain, autoradiographic, immunohistologic, and messenger RNA expression studies implicate a number of neuronal systems, including the hippocampus, as potential targets of vitamin D. However, cellular distribution and protein expression, and binding of the receptor to vitamin D response elements have yet to be established in hippocampus. This investigation was undertaken to characterize the vitamin D receptor in rat hippocampus with western blot, immunocytochemistry, and gel shift analyses. The presence of the receptor protein in hippocampus extracts was revealed with western blotting using an anti-rat vitamin D receptor antiserum. In vivo and in vitro immunocytochemical results confirmed the presence of vitamin D receptor in neuronal and glial cells. In the hippocampus, the receptor was localized in pyramidal and granule cell layers, CA1, CA2, and CA3 subfields and in the dentate gyrus. Double labeling for the vitamin D receptor and glial fibrillary acidic protein revealed that glia also expressed the receptor protein. Gel shift analyses evaluated with the murine osteopontin vitamin D response element indicated a specific, bound receptor-containing complex from hippocampal extracts. Altogether, these findings clearly document the localization of vitamin D receptor in rat hippocampus and that hippocampus contains vitamin D receptors capable of specifically binding to DNA. In combination with reports of a neuroprotective role for vitamin D in hippocampal cell survival, these data suggest that the endogenous vitamin D receptor may mitigate processes related to cellular homeostasis, perhaps through a calcium buffering mechanism.

Vitamin D Receptor Interactions with the Rat Parathyroid Hormone Gene: Synergistic Effects Between Two Negative Vitamin D Response Elements

Vitamin D response elements (VDREs) that are required for negative regulation of rat parathyroid hormone (rPTH) gene expression have been characterized. Gel mobility shift assays using DNA restriction enzyme fragments and recombinant proteins for vitamin D and retinoic acid X receptors (VDR/RXR) revealed a sequence between −793 and −779 that bound a VDR/RXR heterodimer with high affinity (VDRE1). Furthermore, a lower affinity site (VDRE2) was detected that acted in combination with VDRE1 to bind a second VDR/RXR complex. As determined by ethylation interference analysis, the nucleotide sequence of VDRE1 consisted of GGTTCA GTG AGGTAC, which is remarkably similar to the sequence of the negative VDRE found in the chicken PTH (cPTH) gene. Using the same technique, VDRE2 was identified between positions −760 and −746 and contained the sequence AGGCTA GCC AGTTCA. Functional analysis was determined by transfection studies with plasmid constructs that expressed the gene for chloramphenicol acetyl transferase (CAT). The ability of the VDREs to regulate gene expression was tested in their native context with the rPTH promoter as well as when positioned immediately upstream from the cPTH promoter. With either plasmid construct, exposure to 10−8 M 1,25(OH)2D3 resulted in a 60–70% decrease in CAT gene expression when both VDRE1 and VDRE2 were present. Examination of the individual VDREs showed that inhibition by 10−8 M 1,25(OH)2D3 was only 35–40% when just VDRE1 was present. By itself, VDRE2 was even less effective, as significant inhibition of CAT activity (20%) was observed only in the presence of higher concentrations of 1,25(OH)2D3 (10−7 M) or when a plasmid vector that overexpressed the VDR protein was cotransfected. In conclusion, the rPTH gene contains two negative VDREs that act in concert to bind two RXR/VDR heterodimer complexes and that both VDREs are required for maximal inhibition by 1,25(OH)2D3.

Parathyroid hormone/parathyroid hormone-related peptide type 1 receptor in human bone.

The parathyroid hormone/parathyroid hormone‐related peptide (PTH/PTHrP) receptor (denoted as PTH‐1R) is a key signaling factor through which calcium‐regulating hormones PTH and PTHrP exert their effects on bone. There are contradictory reports regarding the capability of osteoclasts to express PTH‐1R. To address this issue in humans, bone biopsy specimen samples from 9 normal controls and 16 patients with moderate to severe secondary renal hyperparathyroid bone disease (2°HPT) with elevated PTH levels were studied to determine whether osteoclasts in the bone microenvironment express PTH‐1R messenger RNA (mRNA) and protein. We report that osteoclasts express the PTH‐1R mRNA but the protein is detected only in patients with 2°HPT. The PTH‐1R mRNA and protein also were found in osteoblasts, osteocytes, and bone marrow cells. Receptor expression was higher in osteoclasts and osteoblasts of patients with 2°HPT than normal controls (98.0 ± 1.1% vs. 65.7 ± 14.3% and 65.8 ± 3.4% vs. 39.1 ± 6.2%; p < 0.01, respectively). Approximately half of osteoclasts found in bone of patients with 2°HPT have the PTH‐1R protein. In patients with 2°HPT, a positive relationship exists between erosion depth, a parameter of osteoclastic activity, and the percentage of osteoclasts with PTH‐1R protein (r = 0.58; p < 0.05). In normal controls, an inverse relationship exists between the percentage of osteoblasts with receptor mRNA, mRNA signals/cell, and serum PTH levels (r = −0.82 and p < 0.05 and r = −0.78 and p < 0.01, respectively). The results provide the novel evidence of PTH‐1R in human osteoclasts and suggest a functional role for the receptors in 2°HPT.

Characterization of vitamin D receptor immunoreactivity in human bone cells

The present study examined the expression of the vitamin D receptor (VDR) in adult human bone by immunohistochemical analysis. Antiserum from goats immunized with an N-terminal rat VDR peptide was purified by affinity chromatography. The purified antiserum recognized both endogenous rat and recombinant human VDR in Western blots. The purified antiserum was also able to specifically supershift the recombinant human VDR when analyzed in mobility shift assays. Immunohistochemical analysis of MG-63 cells, a human osteoblastic cell line known to express the VDR, revealed prominent staining over the nuclei of these cells. Immunostaining was greatly attenuated in the presence of an excess of the immunizing peptide. Analysis of bone biopsy samples from 16 normal human subjects immunostained for VDR protein showed strong, immunopositive staining over bone cells, particularly osteoblasts, in keeping with prior studies. In addition, there was significant immunoreactivity observed in nuclei of osteoclasts, lining cells and scattered bone marrow stromal cells of the adult human bone. Results showed that 298 osteoblasts out of 808 (36.9%) examined were immunopositive. It was also observed that 29 osteoclasts out of 125 (23%) contained VDR immunoreactivity. The ability to detect VDR in osteoclasts and stromal cell populations suggests that in addition to regulating osteoblast function, these other cell types are also direct targets of the hormones action. These results demonstrate the utility of this purified antiserum in detecting the VDR in a variety of molecular techniques and should prove useful in examining receptor expression in various pathological conditions.

Vitamin D receptor interactions with the murine osteopontin response element

The nature of the DNA binding interactions of the human vitamin D receptor (hVDR) with the murine osteopontin vitamin D response element (mOP VDRE) was examined. Both recombinant hVDR and human retinoid X receptor beta (hRXRbeta) proteins were obtained from baculovirus-infected Sf9 insect cells. Mixing extracts of the two recombinant proteins resulted in the strong, specific formation of a slower migrating complex in the electrophoretic mobility shift assay. Crude extracts of the expressed hVDR alone were also capable of binding with high affinity to the mOP sequence, and this binding was enhanced in the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Competition experiments confirmed the specificity of this interaction and revealed that the human osteocalcin VDRE was a poor competitor for this binding. Ethylation interference footprint analyses of hVDR/hRXRbeta and hVDR complexes revealed only subtle differences in how these two different VDR-containing complexes interacted with the mOP VDRE. The footprints displayed contact points in both halves of the direct repeat format, confirming the dimeric and major groove interactions of both types of complexes. DNA affinity chromatography of labelled hVDR extracts revealed a peak eluting at ca. 290 mM KC1 that was capable of rebinding to the mOP sequence in gel shift experiments. Ultraviolet (UV) light-crosslinking experiments of hVDR extracts alone to radiolabelled DNA were consistent with the existence of a homodimeric hVDR interaction. Additionally, these experiments confirmed the direct interaction of a hVDR/hRXRbeta heterodimer when mixed extracts were utilized. From these results we infer that homodimers of the hVDR which respond with enhanced DNA binding to particular vitamin D response elements when exposed to 1,25-(OH)2D3 are possible. This may be of functional significance when RXR proteins are limiting or RXR ligand is present within a cell.

Targeted delivery of vitamin D to the colon using β-glucuronides of vitamin D: therapeutic effects in a murine model of inflammatory bowel disease

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D] has been shown to inhibit development of dextran sodium sulfate (DSS)-induced colitis in mice but can also cause hypercalcemia. The aim of this study was to evaluate whether β-glucuronides of vitamin D could deliver 1,25(OH)(2)D to the colon to ameliorate colitis while reducing the risk of hypercalcemia. Initial studies demonstrated that bacteria residing in the lower intestinal tract were capable of liberating 1,25(OH)(2)D from 1,25-dihydroxyvitamin D(3)-25-β-glucuronide [β-gluc-1,25(OH)(2)D]. We also determined that a much greater upregulation of the vitamin D-dependent 24-hydroxylase gene (Cyp24) was induced in the colon by treatment of mice with an oral dose of β-gluc-1,25(OH)(2)D than 1,25(OH)(2)D, demonstrating targeted delivery of 1,25(OH)(2)D to the colon. We then tested β-glucuronides of vitamin D in the mouse DSS colitis model in two studies. In mice receiving DSS dissolved in distilled water and treated with 1,25(OH)(2)D or β-gluc-1,25(OH)(2)D, severity of colitis was reduced. Combination of β-gluc-1,25(OH)(2)D with 25-hydroxyvitamin D(3)-25-β-glucuronide [β-gluc-25(OH)D] resulted in the greatest reduction of colitis lesions and symptoms in DSS-treated mice. Plasma calcium concentrations were lower in mice treated with β-gluc-1,25(OH)(2)D alone or in combination with β-gluc-25(OH)D than in mice treated with 1,25(OH)(2)D, which were hypercalcemic at the time of death. β-Glucuronides of vitamin D compounds can deliver 1,25(OH)(2)D to the lower intestine and can reduce symptoms and lesions of acute colitis in this model.

Vitamin D4 in Mushrooms

An unknown vitamin D compound was observed in the HPLC-UV chromatogram of edible mushrooms in the course of analyzing vitamin D2 as part of a food composition study and confirmed by liquid chromatography-mass spectrometry to be vitamin D4 (22-dihydroergocalciferol). Vitamin D4 was quantified by HPLC with UV detection, with vitamin [3H] itamin D3 as an internal standard. White button, crimini, portabella, enoki, shiitake, maitake, oyster, morel, chanterelle, and UV-treated portabella mushrooms were analyzed, as four composites each of a total of 71 samples from U.S. retail suppliers and producers. Vitamin D4 was present (>0.1 µg/100 g) in a total of 18 composites and in at least one composite of each mushroom type except white button. The level was highest in samples with known UV exposure: vitamin D enhanced portabella, and maitake mushrooms from one supplier (0.2–7.0 and 22.5–35.4 µg/100 g, respectively). Other mushrooms had detectable vitamin D4 in some but not all samples. In one composite of oyster mushrooms the vitamin D4 content was more than twice that of D2 (6.29 vs. 2.59 µg/100 g). Vitamin D4 exceeded 2 µg/100 g in the morel and chanterelle mushroom samples that contained D4, but was undetectable in two morel samples. The vitamin D4 precursor 22,23-dihydroergosterol was found in all composites (4.49–16.5 mg/100 g). Vitamin D4 should be expected to occur in mushrooms exposed to UV light, such as commercially produced vitamin D enhanced products, wild grown mushrooms or other mushrooms receiving incidental exposure. Because vitamin D4 coeluted with D3 in the routine HPLC analysis of vitamin D2 and an alternate mobile phase was necessary for resolution, researchers analyzing vitamin D2 in mushrooms and using D3 as an internal standard should verify that the system will resolve vitamins D3 and D4.

Suppression of the Human Parathyroid Hormone Promoter by Vitamin D Involves Displacement of NF-Y Binding to the Vitamin D Response Element

An earlier report in the literature indicated the vitamin D response element (VDRE) in the human parathyroid hormone (hPTH) promoter could be specifically bound by an unidentified transcription factor in addition to the vitamin D receptor (VDR) complex. We confirmed that OK and HeLa cell nuclear extracts formed a specific complex with the hPTH VDRE that was insensitive to competition with other VDRE sequences. However, this factor could be competed for by a consensus NF-Y DNA-binding site, and an anti-NF-Y antibody was able to supershift the bound band. Mutational analysis indicated that the NF-Y-binding site partially overlapped the 3′ portion of the VDRE. Transfection studies using an hPTH promoter construct in Drosophila SL2 cells demonstrated strong synergistic transactivation by NF-Y interactions with both the VDRE site and a previously described distal NF-Y-binding site. Finally, mobility shift studies indicated that the VDR heterodimer competed with NF-Y for binding to the VDRE sequence, and NF-Y-stimulated activity of the hPTH promoter could be suppressed in a hormone-dependent manner when the VDR heterodimer complex was coexpressed in SL2 cells. In summary, these findings establish the presence of a proximal NF-Y-binding site in the hPTH promoter and highlight the potential for synergism between distal and proximal NF-Y DNA elements to strongly enhance transcription. Furthermore, findings suggest that the repressive effects of vitamin D on hPTH gene transcription may involve displacement of NF-Y binding to the proximal site by the VDR heterodimer, which subsequently attenuates synergistic transactivation.

Use of a modified yeast one-hybrid screen to identify BAF60a interactions with the Vitamin D receptor heterodimer.

A modified yeast one-hybrid screen was used to isolate proteins capable of interacting with the Vitamin D receptor (VDR) heterodimer complex while driving expression from a repressor Vitamin D response element (VDRE). Four of nine independent colonies recovered in the screen coded for full-length BAF60a, a component of the mammalian SWI/SNF complex. Deletion studies in yeast were unable to localize a unique region of BAF60a responsible for interaction with the heterodimer complex, as only the full-length protein would support reporter gene expression. Pull-down analyses revealed that BAF60a displayed strong interactions with either the unliganded or liganded heterodimer complex, but neither individual receptor component alone. Transient transfection analysis in opossum kidney (OK) cells indicated that BAF60a decreased basal transcriptional activity from the negative VDRE, but had no effect on hormone-induced repression. Transcriptional activity from an enhancer VDRE also exhibited decreased basal transcriptional activity, but also augmented hormone-dependent enhancer activity, resulting in an overall increased sensitivity to hormone. In summary, BAF60a has been identified as a factor that specifically interacts with the VDR heterodimer complex using a modified yeast one-hybrid selection strategy. This suggests that BAF60a may be a link between mammalian SWI/SNF-like chromatin remodeling complexes and the VDR heterodimer.

Differential Effects of 20-Epi Vitamin D Analogs on the Vitamin D Receptor Homodimer

Vitamin D analogs have received increased attention because of their possible therapeutic benefits in treating osteoporosis and various proliferative disorders. Several analogs were examined for their effects on DNA binding of the vitamin D receptor (VDR) homodimer complex with the murine osteopontin vitamin D response element. All of the tested analogs increased complex binding by recombinant human VDR in the electrophoretic mobility shift assay and notable differences in mobility of these complexes were observed. A panel of C‐terminal anti‐VDR antisera were screened for their ability to interact with analog‐bound VDR homodimer complexes or as a heterodimer complex with recombinant human retinoid X receptor α (rhRXRα). Like calcitriol, analog‐bound heterodimer complexes were largely resistant to interaction with these antisera; however, striking differences were observed with the various antisera in an analogous homodimer binding experiment. KH1060 and CB1093, analogs with 20‐epi conformations, produced homodimer complexes that were 3‐ to 6‐fold more resistant to supershifting with Ab180 compared with the hormone or EB1089. Chymotrypsin digestion in combination with Western blotting using a C‐terminal anti‐VDR antiserum revealed similar digestion patterns for all ligands. However, KH1060‐ and CB1093‐bound VDR complexes were more resistant to digestion than either calcitriol or EB1089. Finally, the ability of these compounds to yield stable homodimer complexes was assessed by challenging preformed homodimer with the exogenous addition of rhRXRα extracts. Although new heterodimer complexes appeared in a time‐dependent fashion, the preformed homodimer complexes exhibited stable binding throughout the time course of the experiment. The results indicate that VDR homodimers are targets of vitamin D analogs with differential effects on C‐terminal protein conformation that may partially explain the varied biological responses of these compounds.