The effect of vitamin D analogs and of vitamin D-binding protein on lymphocyte proliferation

In the absence of vitamin D-binding protein (DBP), 1,25-(OH)2D3 at 10(-12) M significantly inhibited the [3H]thymidine incorporation in human lymphocytes during mixed lymphocyte cultures (MLC) or after phyto-hemaglutinin (PHA) stimulation. In the presence of a physiological concentration of DBP (5 x 10(-6) M), the concentration of 1,25-(OH)2D3 required for inhibition was 10(-10) M (for PHA-cultures) and 10(-9) M (for MLC). Several vitamin D analogs were compared for their inhibitory action on PHA stimulation. In the absence of DBP, the concentration necessary for 50% inhibition of [3H]thymidine incorporation ranged from 10(-12) M [1,25-(OH)2D3 and 24,24-F2-1,25-(OH)2D3], over 10(-10) M [1,24R, 25-(OH)3D3; 1,25S, 26-(OH)3D3 and 26,27-F6-1,25-(OH)2D3] and 10(-8) M [25 OHD3 and 24,25-(OH)2D3] to 10(-6) M [calcitriol-lactone]. This rank order correlates with the binding affinity of the various analogs to the cytoplasmic 1,25-(OH)2D3-receptor. DBP counteracted the inhibitory effect of all analogs and the degree of counteraction was directly proportional to the binding affinity between DBP and the vitamin D analog. DBP thus decreased the in vitro inhibitory action of 1,25-(OH)2D3 and its analogs on lymphocyte proliferation. Of all analogs tested, only 1,25-(OH)2D3 had a significant effect at a physiological concentration.     

Probing the vitamin D sterol-binding pocket of human vitamin D-binding protein with bromoacetate affinity labeling reagents containing the affinity probe at C-3, C-6, C-11, and C-19 positions of parent vitamin D sterols

The multiple physiological properties of vitamin D-binding protein (DBP) include organ-specific transportation of vitamin D(3) and its metabolites, manifested by its ability to bind vitamin D sterols with high affinity. In the present investigation we probed the vitamin D sterol-binding pocket of human DBP with affinity labeling analogs of 25-hydroxyvitamin D(3) ?25-OH-D(3) and 1, 25-dihydroxyvitamin D(3) ?1,25(OH)(2)D(3) containing bromoacetate alkylating probe at C-3 (A-ring), C-6 (triene), C-11 (C-ring), and C-19 (exocyclic methylene) of the parent sterol. Competitive binding assays with DBP showed approximately 22-, 68-, and 2000-fold decrease in the binding of 1,25(OH)(2)-D(3)-11-BE, 25-OH-D(3)-3-BE, and 25-OH-D(3)-6-BE, respectively, compared to that seen with 25-OH-D(3), while there was no significant difference in the DBP-binding affinity of 25-OH-D(3)-19-BE and 25-OH-D(3). Surprisingly, ?(14)C25-OH-D(3)-11-BE and ?(14)C1, 25(OH)(2)-D(3)-19-BE failed to label DBP despite high-affinity DBP-binding, indicating the absence of any nucleophilic amino acid in the vicinity of their bromoacetate moiety to form a covalent bond, while these analogs are inside the binding pocket. In contrast, ?(14)C25-OH-D(3)-6-BE and ?(14)C25-OH-D(3)-3-BE specifically labeled DBP. BNPS-skatole digestion of DBP labeled with ?(14)C25-OH-D(3)-3-BE or ?(14)C25-OH-D(3)-6-BE produced two peptides (M(r) 17,400 and 33,840), with radioactivity associated with the N- and C-terminal peptides, respectively, raising the possibility that either different areas of the same vitamin D sterol-binding pocket, or different domains of DBP might be labeled by these analogs. Successful affinity labeling of recombinant domain I (1-203) of DBP with both reagents indicated that different areas of the same vitamin D-binding pocket (domain I) were labeled. These affinity analogs are potentially useful for "mapping" the vitamin D sterol-binding pocket and developing a functional model.     

Effects of orchidectomy and different modes of high dose estrogen treatment on circulating "free" and total 1,25-dihydroxyvitamin D in patients with prostatic cancer

Serum levels of total 1,25-dihydroxyvitamin D (1,25(OH)2D), vitamin D binding protein (DBP), sex hormone binding globulin (SHBG), testosterone, estradiol 17 beta (E2) and the "free" 1,25(OH)2D index were measured before and during treatment in prostatic cancer patients treated by orchidectomy (n = 15), with combined i.m. polyestradiol phosphate (PEP) + oral ethinyl estradiol (EE) (n = 10) and with i.m. PEP only for 3 months, followed by addition of oral EE (n = 9). Total concentrations of 1,25(OH)2D and DBP were unaffected by orchidectomy and treatment with i.m. PEP only, but were significantly elevated during treatment including oral EE. SHBG levels were unaffected by orchidectomy, slightly increased by i.m. PEP only and greatly increased by oral EE. The free 1,25(OH)2D index was slightly elevated by treatment including oral EE. Evidence was obtained that the increase in 1,25(OH)2D levels observed during oral estrogen treatment was secondary to the estrogen-augmented increase in DBP and not a result of an estrogen-stimulated synthesis of 1,25(OH)2D. Furthermore, the stimulatory effect of estrogen on DBP concentrations seemed to be dependent on the route of administration of the hormone.     

A structural basis for the unique binding features of the human vitamin D-binding protein.

The human serum vitamin D-binding protein (DBP) has many physiologically important functions, ranging from transporting vitamin D3 metabolites, binding and sequestering globular actin and binding fatty acids to functioning in the immune system. Here we report the 2.3 A crystal structure of DBP in complex with 25-hydroxyvitamin D3, a vitamin D3 metabolite, which reveals the vitamin D-binding site in the N-terminal part of domain I. To more explicitly explore this, we also studied the structure of DBP in complex with a vitamin D3 analog. Comparisons with the structure of human serum albumin, another family member, reveal a similar topology but also significant differences in overall, as well as local, folding. These observed structural differences explain the unique vitamin D3-binding property of DBP.     

Polyunsaturated fatty acids decrease the apparent affinity of vitamin D metabolites for human vitamin D-binding protein

The affinity of purified human vitamin D-binding protein from serum (DBP) for 25-hydroxyvitamin D₃ (25-OHD₃) and 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] was measured in the presence of free fatty acids (FFA), cholesterol, prostaglandins and several drugs. Mono- and polyunsaturated fatty acids markedly decreased the affinity of both 25-OHD₃ and 1,25-(OH)₂D₃ for DBP, whereas saturated fatty acids (stearic and arachidic acid), cholesterol, cholesterol esters, retinol, retinoic acid and prostaglandins (A₁ and E₁) did not affect the apparent affinity. Several chemicals known to decrease the binding of thyroxine to its plasma-binding protein did not affect the affinity of DBP.

The apparent affinity of DBP for both 25-OHD₃ and 1,25-(OH)₂D₃ decreased 2.4- to 4.6-fold in the presence of 36 μM of linoleic or arachidonic acid, respectively. Only a molar ratio of FFA:DBP higher than 10,000 was able to decrease the binding of 25-OHD₃ to DBP by 20%. Much smaller ratio's of FFA:DBP (25 for arachidonic and 45 for oleic acid), however, decreased the binding of 1,25-(OH)₂D₃ to DBP. These latter ratio's are well within the physiological range. The addition of human albumin in a physiological albumin:DBP molar ratio did not impair the inhibitory effect of linoleic acid on the binding of [³H]25-OHD₃ to DBP. The binding and bioavailability of vitamin D metabolites thus might be altered by mono- and polyunsaturated but not by saturated fatty acids.     

Association of Single Nucleotide Polymorphisms in VDR and DBP Genes with HBV-Related Hepatocellular Carcinoma Risk in a Chinese Population

Background

Polymorphisms of genes encoding components of the vitamin D pathway including vitamin D receptor (VDR) and vitamin D binding protein (DBP) have been widely investigated because of the complex role played by vitamin D in cancer tumorogenesis. In this study, we investigated the association between VDR and DBP gene polymorphisms and HBV-related HCC risk in a Chinese population.

Methods

Study subjects were divided into three groups: 184 HBV patients with HCC, 296 HBV patients without HCC, and 180 healthy controls. The VDR rs2228570, and rs3782905 and the DBP rs7041 polymorphisms were genotyped using PCR-RFLP and the VDR rs11568820 polymorphism was genotyped by PCR-SSP, respectively. DNA sequencing was performed to validate the genotype results.

Results

We found that there were significant differences in the genotype and allele frequencies of the VDR rs2228570 and DBP rs7041 polymorphisms between HBV patients with HCC and healthy controls. The rs2228570 T allele was associated with a significant increased HBV-related HCC risk as compared with the C allele. The rs2228570 TT and TT/TC genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type CC homozygote. Similarly, the rs7041 G allele was associated with a significant increased HBV-related HCC risk as compared with the T allele. The rs7041 GG and GG/TG genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type TT homozygote. However, we did not observe any significant effect of VDR rs11568820, and rs3782905 polymorphisms on HBV-related HCC risk in this population. In haplotype analysis, we also did not find any significant differences in haplotype frequencies of the VDR gene between HBV patients with HCC and the healthy controls.

Conclusions

We conclude that the VDR rs2228570 and DBP rs7041 polymorphisms may contribute to increased susceptibility to HBV-related HCC in the Chinese population. Due to the marginal significance, further large and well-designed studies in diverse ethnic populations are needed to confirm our results.     

Photoaffinity labeling of the rat plasma vitamin D binding protein with [26,27-3H]-25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate]

It is well recognized that the vitamin D binding protein (DBP) is important for the transport of vitamin D, 25-hydroxyvitamin D (25-OH-D), and its metabolites. In an attempt to better understand the molecular-binding properties of this ubiquitous protein, we designed and synthesized a photoaffinity analogue of 25-OH-D3 and its radiolabeled counterpart. This analogue, 25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (25-OH-D3-ANG), was recognized by the rat DBP and was about 10 times less active than 25-OH-D3 in terms of binding. Incubation of [3H]25-OH-D3 or [3H]25-OH-D3-ANG with rat DBP revealed that both compounds were specifically bound to a protein with a sedimentation coefficient of 4.1 S. Each was displaced with a 500-fold excess of 25-OH-D3. When [3H]25-OH-D3-ANG was exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3, there was no displacement of tritium from the 4.1S peak. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of [3H]25-OH-D3-ANG exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3 revealed one major band with a molecular weight of 52 000. These data provide strong evidence that [3H]25-OH-D3-ANG was covalently linked to the rat DBP. This photoaffinity probe should provide a valuable tool for the analysis of the binding site on this transport protein.     

Vitamin D binding protein and monocyte response to 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D: analysis by mathematical modeling

Vitamin D binding protein (DBP) plays a key role in the bioavailability of active 1,25-dihydroxyvitamin D (1,25(OH)(2)D) and its precursor 25-hydroxyvitamin D (25OHD), but accurate analysis of DBP-bound and free 25OHD and 1,25(OH)(2)D is difficult. To address this, two new mathematical models were developed to estimate: 1) serum levels of free 25OHD/1,25(OH)(2)D based on DBP concentration and genotype; 2) the impact of DBP on the biological activity of 25OHD/1,25(OH)(2)D in vivo. The initial extracellular steady state (eSS) model predicted that 50 nM 25OHD and 100 pM 1,25(OH)(2)D), <0.1% 25OHD and <1.5% 1,25(OH)(2)D are 'free' in vivo. However, for any given concentration of total 25OHD, levels of free 25OHD are higher for low affinity versus high affinity forms of DBP. The eSS model was then combined with an intracellular (iSS) model that incorporated conversion of 25OHD to 1,25(OH)(2)D via the enzyme CYP27B1, as well as binding of 1,25(OH)(2)D to the vitamin D receptor (VDR). The iSS model was optimized to 25OHD/1,25(OH)(2)D-mediated in vitro dose-responsive induction of the vitamin D target gene cathelicidin (CAMP) in human monocytes. The iSS model was then used to predict vitamin D activity in vivo (100% serum). The predicted induction of CAMP in vivo was minimal at basal settings but increased with enhanced expression of VDR (5-fold) and CYP27B1 (10-fold). Consistent with the eSS model, the iSS model predicted stronger responses to 25OHD for low affinity forms of DBP. Finally, the iSS model was used to compare the efficiency of endogenously synthesized versus exogenously added 1,25(OH)(2)D. Data strongly support the endogenous model as the most viable mode for CAMP induction by vitamin D in vivo. These novel mathematical models underline the importance of DBP as a determinant of vitamin D 'status' in vivo, with future implications for clinical studies of vitamin D status and supplementation.     

The C(19) position of 25-hydroxyvitamin D(3) faces outward in the vitamin D sterol-binding pocket of vitamin D-binding protein

The radiolabeled affinity and photoaffinity analogues of 25-hydroxyvitamin D(3) (25-OH-D(3)) with probes at the C-19 position failed to specifically label the 25-OH-D(3)-binding pocket of vitamin D-binding protein (DBP). However, a hybrid analogue, with a bromoacetate affinity probe and a photoaffinity probe at C(3)-OH and C(19) positions, respectively, specifically labeled the ligand-binding pocket, suggesting that C(3)-OH points towards the 'inside' of the binding cavity while the C(19) position faces away from it.     

Endothelial stress induces the release of vitamin D-binding protein, a novel growth factor

Endothelial cells (EC) under stress release paracrine mediators that facilitate accumulation of vascular smooth muscle cells (VSCM) at sites of vascular injury. We found that medium conditioned by serum-starved EC increase proliferation and migration of VSCM in vitro. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as vitamin D-binding protein (DBP). DBP induced both proliferation and migration of VSMC in vitro in association with increased phosphorylation of ERK 1/2. PD 98059, a biochemical inhibitor of ERK 1/2, abrogated these proliferative and migratory responses in VSMC. DBP is an important carrier for the vitamin-D sterols, 25-hydroxyvitamin-D, and 1alpha,25-dihydroxyvitamin-D. Both sterols inhibited the activity of DBP on VSMC, suggesting that vitamin D binding sites are important for initiating the activities of DBP on VSMC. Release of DBP at sites of endothelial injury represents a novel pathway favoring accumulation of VSMC at sites of vascular injury.     

The rat vitamin D binding protein (Gc-globulin) gene is syntenic with the rat albumin and alpha-fetoprotein genes on chromosome 14

Using a panel of mouse X rat somatic cell hybrids and a characterized rat vitamin D binding protein (DBP) cDNA probe, we assigned the gene coding for rat DBP to chromosome 14. We conclude that the genes for DBP, albumin, and alpha-fetoprotein, all of which derive from a common precursor, are syntenic in the rat, as they are in man.     

Initial characterization of the vitamin D binding protein (Gc-globulin) binding site on the neutrophil plasma membrane: evidence for a chondroitin sulfate proteoglycan.

The vitamin D binding protein (DBP) is a multifunctional plasma protein that can modulate certain immune and inflammatory responses. The diverse cellular functions of DBP appear to require cell surface binding to mediate these processes. Numerous reports have detected DBP bound to the surface of several cell types and would support the concept of a cell surface binding site for DBP. However, direct evidence for such a molecule has been lacking and essentially nothing is known about its basic biochemical properties. In the present study, radioiodinated DBP was used as a probe to characterize biochemically the neutrophil DBP binding site. Radiolabeled DBP binds to and remains associated with the plasma membrane and is not degraded. Quantitation of DBP binding to either intact cells or purified plasma membranes showed nonsaturable (linear) binding with positive cooperativity, possibly suggesting DBP oligomer formation. Solubilization of cell bound 125I-DBP with various nonionic and zwitterionic detergents demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fraction. Moreover, this molecule does not associate with the cytoskeleton. Cross-linking of radiolabeled DBP bound to plasma membranes increased the amount of protein that partitioned to the insoluble fraction, and analysis of these complexes by SDS-PAGE revealed that they may be very large since they did not enter the gel. Finally, treatment of plasma membranes with either proteases or chondroitinase ABC completely abrogated membrane binding of DBP, suggesting that the protein binds to a chondroitin sulfate proteoglycan.     

Crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution

A high-affinity complex formed between G-actin and plasma vitamin D-binding protein (DBP) is believed to form part of a scavenging system in the plasma for removing actin released from damaged cells. In the study presented here, we describe the crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution. The complex contains one molecule of each protein bound together by extensive ionic, polar, and hydrophobic interactions. It includes an ATP and a calcium ion bound to actin, but no evidence of vitamin D metabolites bound to the DBP. Both actin and DBP are multidomain molecules, two major domains in actin and three in DBP. All of these domains contribute to the interaction between the molecules. DBP enfolds the end of the actin molecule, principally in actin subdomain 3 but with additional interactions in actin subdomain 1. This orientation is similar to the binding of profilin to actin, as predicted from previous studies. The more extensive interactions of DBP give an affinity for actin some 3 orders of magnitude higher than that for profilin. The larger "footprint" of DBP on actin also leads to an overlap with the actin-binding site of gelsolin domain I.     

Aspirin inhibits phospholipase C

We have shown previously that aspirin (ASA) ingestion by normal human volunteers inhibits peripheral blood monocyte phospholipase C (PLC) activities ex vivo. In order to explore further the mechanism of action of ASA, normal human monocytes and differentiated human U937 cells were treated with ASA and other salicylates. Cells preincubated with ASA were found to have decreased PLC activities. Phospholipase A2 activities were not affected by salicylates. Sodium salicylate and salicylic acid, nonacetylated relatives of ASA also inhibited PLC activity. This effect was dose and time dependent and addition of cycloheximide or actinomycin D to the preincubation mixture abrogated the inhibitory effect of salicylates on PLC. This PLC inhibitory protein induced by ASA appears distinct from lipocortin, a phospholipase A2 inhibitory protein inducible by corticosteroids.     

Glycosylation status of vitamin D binding protein in cancer patients

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase‐derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O‐linked trisaccharide glycosylation of serum‐derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization‐based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.     

The vitamin D system in iguanian lizards

The apparent plasma concentration of vitamin D binding protein (DBP) in an iguanian lizard, Pogona barbata, and the affinity of this protein for 25-hydroxyvitamin D₃ (25(OH)D₃), 25-hydroxyvitamin D₂ (25(OH)D₂), and 1,25-dihydroxyvitamin D₃ (1,25(OH)D₃) was found to resemble more closely that of the domestic hen than that of the human. The characteristics of Pogona DBP, the pattern of vitamin D metabolites derived from injected radioactive vitamin D₃ and the plasma concentrations of endogenous 25-hydroxyvitamin D (25(OH)D) in a range of iguanian lizards have been examined. The findings suggest that 25-hydroxyvitamin D (25(OH)D) is the major metabolite of vitamin D, and that it may represent the storage form of vitamin D in these species in the same way as in mammals. High concentrations of vitamin D within iguanian embryos and egg yolks suggest a role for this compound in embryogenesis in these species, and perhaps indicates that there is a mechanism for vitamin D delivery to eggs comparable to that found in the domestic chicken.     

Direct regional assignment of the gene for vitamin D binding protein (Gc-globulin) to human chromosome 4q11-q13 and identification of an associated DNA polymorphism

Summary Using a characterized human vitamin D binding protein (DBP) cDNA probe and a panel of rodent X human somatic cell hybrids, we established the chromosomal location of the structural gene for DBP on human chromosome 4. In situ hybridization of ³H-labeled DBP cDNA to human metaphase chromosomes confirmed this assignment and allowed regional localization to bands 4q11–4q13. A restriction fragment length polymorphism associated with the DBP gene that should prove useful in future linkage studies was identified with the enzyme BamHI.