Development of a Biologically Active Fluorescent-Labeled Calcitriol and Its Use to Study Hormone Binding to the Vitamin D Receptor

Bársony, Julia; Renyi, Istvan; McKoy, Wilma M.; Kang, Hee Choi; Haugland, Richard P.; Smith, Catharine L.

doi:10.1006/abio.1995.1380

Cited by 10 publications

(12 citation statements)

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“…Binding studies with BP-calcitriol and with radiolabeled calcitriol gave comparable results on VDR content in various cell lines. Binding studies with BP-calcitriol also showed the same VDR content whether we used spectrofluorimetry or microscopy (26). Thus, this new reagent opens the possibility of studying the subcellular distribution of VDR in living cells by microscopy.…”

mentioning

confidence: 61%

“…We have shown the utility of BP-calcitriol to measure hormone binding to VDR, and the specificity of BPcalcitriol binding has been established (26). The best feature of this reagent is that its fluorescence emission increases when bound to VDR but does not increase when bound to other proteins.…”

Section: Discussionmentioning

confidence: 99%

“…To obtain BP-calcitriol, the fluorescent dye BODIPY was attached at the A-ring of calcitriol by an ester link at the 3␤-position (26). We have shown the utility of BP-calcitriol to measure hormone binding to VDR, and the specificity of BPcalcitriol binding has been established (26).…”

Section: Discussionmentioning

confidence: 99%

“…Software for automated measurements, calibration, morphometry, and statistical analysis was developed in our laboratory, incorporating functions from a vendor-supplied library (G. W. Hannaway & Associates). Image aquisition and calibration were done as described earlier (26). Brightness values were calculated from unprocessed images and were expressed in brightness units (B).…”

Section: Methodsmentioning

confidence: 99%

“…The use of fluorescent ligands could overcome these obstacles. Recently, we succeeded in developing a pharmacologically relevant fluorescently labeled steroid hormone, 3␤-BODIPY-calcitriol (BP-calcitriol) (26). This reagent is stable, is resistant to endogenous esterases, freely enters living cells, and retains most of the biologic activity of the parent hormone and its affinity to the receptor.…”

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confidence: 99%

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Subcellular Distribution of Normal and Mutant Vitamin D Receptors in Living Cells

Bársony

Renyi

McKoy

1997

Journal of Biological Chemistry

116

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To understand the subcellular localization of the vitamin D receptor (VDR) and to measure VDR content in single cells, we recently developed a fluorescent labeled ligand, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-calcitriol. This tagged hormone has intact biological activity, high affinity and specific binding to the receptor, and enhanced fluorescent emission upon receptor binding. Using BODIPY-calcitriol, here we monitored the subcellular distribution of VDR in living cultured cells by microscopy. Time course studies showed that an equilibrium between the cytoplasmic and nuclear hormone binding developed within 5 min and was maintained thereafter. We found a substantial proportion of VDR residing in the cytoplasm, colocalized with endoplasmic reticulum, the Golgi complex, and microtubules. Confocal microscopy clarified the presence of VDR within discrete regions of the nucleus and along the nuclear envelope. There was no VDR in the plasma membrane. Low affinity BODIPY-calcitriol binding sites were in the mitochondria. Mutations in the VDR gene selectively and specifically altered BODIPYcalcitriol distribution. Defects in the hormone binding region of VDR prevented both nuclear and cytoplasmic hormone binding. Defects in the DNA binding region decreased the nuclear retention of VDR and prevented localization to nuclear foci. These results with BODIPYcalcitriol reveal cytoplasmic VDR localization in living cells and open the possibility of studying the three-dimensional architecture of intranuclear target sites. The vitamin D receptor (VDR)1 belongs to the v-erb-A superfamily of ligand activated transcription factors. The hormonal forms of vitamin D and other steroid hormones act through their receptors to regulate the transcription of target genes and thus modulate a variety of cell functions. These hormones also exert rapid, so-called "nongenomic" actions that take place outside the nucleus. Over the last 30 years we learned many aspects of steroid receptor activation, but the subcellular distribution of the receptors remained controversial. The use of radioligands led to the classical model for steroid receptor activation, which placed the receptors in the cytoplasm (1). Later, antibodies were raised against steroid receptors, and immunocytology suggested that unactivated steroid receptors reside exclusively in the nucleus (2). In the past few years, evidence has accumulated showing that glucocorticoid, mineralocorticoid, and androgen receptors reside both in the cytoplasm and in the nucleus. Nevertheless, the consensus has remained unchanged for the nuclear localization of estrogen receptors, thyroid hormone receptors, and VDR (3). Several studies with immunocytology on aldehyde-fixed cells showed that VDR resides exclusively in the nucleus (4). However, using microwave fixation, we found a significant portion of VDR in the cytoplasm (5). Since then, cytoplasmic VDR has been found by others (6 -10), but the existence of cytoplasmic VDR is still not generally accepted (11,12).While the role of nucle...

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mentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Subcellular Distribution of Normal and Mutant Vitamin D Receptors in Living Cells

Bársony

Renyi

McKoy

1997

Journal of Biological Chemistry

116

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Cell biology of paget's disease

Reddy¹,

Menaa²,

Singer³

et al. 1999

Journal of Bone and Mineral Research

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Paget's disease is characterized by markedly increased osteoclast formation and bone resorption followed by excessive new bone formation. Osteoclasts in Paget's disease are increased both in number and size, contain paramyxoviral-like nuclear inclusions, and can have up to 100 nuclei per cell. Marrow culture studies have identified several abnormalities in osteoclast formation in Paget's disease. Osteoclast-like multinucleated cells formed more rapidly in marrow cultures from patients with Paget's disease, produced increased levels of interleukin-6 (IL-6), and expressed high levels of IL-6 receptors compared to normals. IL-6 levels were also increased in bone marrow and peripheral blood of patients with Paget's disease. In addition, osteoclast precursors from patients with Paget's disease are hyperresponsive to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and calcitonin. The increased sensitivity of osteoclast precursors to 1,25(OH)2D3 is mediated through the vitamin D receptor (VDR), since 24-hydroxylase activity is also up-regulated at concentrations of 1,25(OH)2D3 that are one log less than that needed to induce 24-hydroxylase activity in osteoclast precursors from normals. However, VDR numbers and affinity for 1,25(OH)2D3 do not differ in osteoclast precursors from Paget's patients compared to those from normals. Synergistic interactions between cytokines such as IL-6 and 1,25(OH)2D3 also cannot explain the enhanced sensitivity of osteoclast precursors from patients with Paget's disease to 1,25(OH)2D3. Interestingly, coculture studies of osteoclast precursors and cells from the marrow microenvironment of patients with Paget's disease and normals have demonstrated that the marrow microenvironment is more osteoclastogenic than normal. Thus, studies of the cell biology of osteoclasts in Paget's disease have demonstrated an increased rate of osteoclast formation and abnormalities in both osteoclast precursors and the marrow microenvironment. Enhanced IL-6 production by osteoclasts in Paget's disease may further amplify the increased osteoclast formation already ongoing in the pagetic lesion, and may explain the increased bone turnover at uninvolved sites distant from the pagetic lesion.

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Evidence that annexin II Is not a putative membrane receptor for 1α,25(OH)₂‐vitamin D₃

Mizwicki

Bishop

Olivera

et al. 2004

J of Cellular Biochemistry

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The seco-steroid hormone 1alpha,25(OH)(2)-vitamin D(3) (1,25-D(3)) is known to generate biological responses via both genomic and non-genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII(2)/p11(2)] was proposed by Baran and co-authors to be the membrane receptor responsible for mediating non-genomic, rapid actions of 1,25-D(3), 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-D(3) rapid, non-genomic activities, we investigated in vitro the affinity of [(3)H]1,25-D(3) for the AII monomer and AII(2)/p11(2) 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 [(3)H]1,25-D(3) was obtained with or without the presence of 700 nM exogenous calcium, using either the AII monomer or AII(2)/p11(2). However saturable binding of [(3)H]1,25-D(3) to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (K(d) = 3.0 nM; B(max) = 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-D(3) binding as reported by Baran et al. Our results suggest that AII does not bind 1,25-D(3) in a physiologically relevant manner; however, recent studies linking AII(2)/p11(2) phosphorylation to vesicle fusion and its calcium regulated localization may make AII a possible down-stream substrate for 1,25-D(3) induced rapid cellular effects.

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Development of a Biologically Active Fluorescent-Labeled Calcitriol and Its Use to Study Hormone Binding to the Vitamin D Receptor

Cited by 10 publications

References 0 publications

Subcellular Distribution of Normal and Mutant Vitamin D Receptors in Living Cells

Subcellular Distribution of Normal and Mutant Vitamin D Receptors in Living Cells

Cell biology of paget's disease

Evidence that annexin II Is not a putative membrane receptor for 1α,25(OH)₂‐vitamin D₃

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Development of a Biologically Active Fluorescent-Labeled Calcitriol and Its Use to Study Hormone Binding to the Vitamin D Receptor

Cited by 10 publications

References 0 publications

Subcellular Distribution of Normal and Mutant Vitamin D Receptors in Living Cells

Subcellular Distribution of Normal and Mutant Vitamin D Receptors in Living Cells

Cell biology of paget's disease

Evidence that annexin II Is not a putative membrane receptor for 1α,25(OH)2‐vitamin D3

Contact Info

Product

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Evidence that annexin II Is not a putative membrane receptor for 1α,25(OH)₂‐vitamin D₃