Vitamin D Binding Protein (VDBP) and Its Gene Polymorphisms-The Risk of Malignant Tumors and Other Diseases.

Vitamin D is an important component of the endocrine system that controls calcium homeostasis and bone mineralization. Because of the very short half-life of free serum vitamin D it is stabilized and transported to target tissues by being bound to the vitamin D binding protein (VDBP). The most common polymorphisms: rs4588 and rs7041 in the vitamin D binding protein gene may correlate with differences in vitamin D status in the serum. This review presents data that relate to the presence of genetic variants in the VDBP gene in correlation with certain diseases, mostly concerning cancers (breast, prostate, pancreatic, lung, colorectal, basal cell carcinoma cancer and cutaneous melanoma) or other related diseases (thyroid autoimmunity disorders, obesity, diabetes mellitus, bone metabolism, rheumatoid arthritis, ankylosing spondylitis, asthma, chronic obstructive pulmonary disease, tuberculosis and coronary artery diseases).

Identification of HLA-A2-restricted immunogenic peptides derived from Vitamin D-Binding Protein.

T-cell-mediated destruction of pancreatic ß cells leads to Type 1 diabetes (TID). Vitamin D-Binding Protein (VDBP) has been identified as an autoantigen and T cell reactivity against VDBP increases in the development of T1D. Autoreactive cytotoxic T lymphocytes (CTLs) recognize ß-cell-derived peptides in the context of major histocompatibility complex class I molecules. However, little is known about the VDBP-derived immunogenic peptides that are presented in the context of human HLA molecules. Here, we predicted and identified VDBP derived immunogenic peptides that were presented in association with human HLA-A2 molecule. The VDBP derived peptides binding to HLA-A∗0201 were predicted by using a computer-assisted algorithm. The candidate peptides were synthesized, then affinity between peptides and HLA-A∗0201 were analyzed. In addition, the CTL activity of the peptides was detected by cytotoxicity assay and ELISPOT assay in vitro. Furthermore, HLA-A∗0201-transgenic mice were immunized with peptides to induce the CTL activity in vivo. The results demonstrated that peptides of VDBP containing residues 211-219 and 235-243 had high affinity with HLA-A∗0201. In addition, these peptides elicited potent CTL responses in vitro, and induced T1D in vivo. Therefore, this experiment identified immunogenic HLA-A∗0201-restricted epitopes derived from VDBP, and provided pathogenesis theory of T1D.

Placental amino acid transport may be regulated by maternal vitamin D and vitamin D-binding protein: results from the Southampton Women's Survey.

Both maternal 25-hydroxyvitamin D (25(OH)D) concentrations during pregnancy and placental amino acid transporter gene expression have been associated with development of the offspring in terms of body composition and bone structure. Several amino acid transporter genes have vitamin D response elements in their promoters suggesting the possible linkage of these two mechanisms. We aimed to establish whether maternal 25(OH)D and vitamin D-binding protein (VDBP) levels relate to expression of placental amino acid transporters. RNA was extracted from 102 placental samples collected in the Southampton Women's Survey, and gene expression was analysed using quantitative real-time PCR. Gene expression data were normalised to the geometric mean of three housekeeping genes, and related to maternal factors and childhood body composition. Maternal serum 25(OH)D and VDBP levels were measured by radioimmunoassay. Maternal 25(OH)D and VDBP levels were positively associated with placental expression of specific genes involved in amino acid transport. Maternal 25(OH)D and VDBP concentrations were correlated with the expression of specific placental amino acid transporters, and thus may be involved in the regulation of amino acid transfer to the fetus. The positive correlation of VDBP levels and placental transporter expression suggests that delivery of vitamin D to the placenta may be important. This exploratory study identifies placental amino acid transporters which may be altered in response to modifiable maternal factors and provides a basis for further studies.

Vitamin D-binding protein contributes to COPD by activation of alveolar macrophages.

BACKGROUND: Vitamin D-binding protein (DBP) genetic polymorphisms have been associated with chronic obstructive pulmonary disease (COPD). DBP has an indirect role in macrophage activation; thus it was hypothesised that DBP is present in the airway and contributes to lung disease by this mechanism. METHODS: 471 PiZZ subjects with α1-antitrypsin deficiency (AATD) were genotyped for tag single nucleotide polymorphisms (SNPs) covering the DBP gene (GC), together with known functional variants, prior to seeking association with COPD phenotypes. 140 subjects with usual COPD and 480 controls were available for replication. Vitamin D and DBP levels were measured by tandem mass spectrometry and ELISA, respectively, in serum and DBP in the sol phase of sputum in a subset of 60 patients. Concentrations were related to phenotype and to alveolar macrophage activation. RESULTS: rs2070741 was associated with airway bacterial colonisation (p=0.04) and bronchiectasis (p=0.01), as was rs7041 (p=0.03) which also influenced vitamin D concentrations (p=0.01). The GC2 variant predisposed to bronchiectasis in AATD (p=0.04) and protected against COPD (p=0.05); the latter association was replicated in usual COPD versus controls (p=0.04). Circulating DBP related inversely to forced expiratory volume in 1 s (FEV(1)) (p=0.02), in direct contrast to vitamin D, where deficiency related to low FEV(1) (p=0.04). Sol DBP related directly to alveolar macrophage activation (p=0.004). CONCLUSIONS: The genetic association of DBP with COPD may be mediated by effects on macrophage activation, since DBP relates to FEV(1), and affects macrophage activation. Vitamin D effects may be independent of this, relating more strongly to innate immunity.

Trophoblast uptake of DBP regulates intracellular actin and promotes matrix invasion.

Early pregnancy is characterised by elevated circulating levels of vitamin D binding protein (DBP). The impact of this on maternal and fetal health is unclear but DBP is present in the placenta, and DBP gene variants have been linked to malplacentation disorders such as preeclampsia. The functional role of DBP in the placenta was investigated using trophoblastic JEG3, BeWo and HTR8 cells. All three cell lines showed intracellular DBP with increased expression and nuclear localisation of DBP in cells treated with the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). When cultured in the serum of mice lacking DBP (DBP-/-), JEG3 cells showed no intracellular DBP indicating uptake of exogenous DBP. Inhibition of the membrane receptor for DBP, megalin, also suppressed intracellular DBP. Elimination of intracellular DBP with DBP-/- serum or megalin inhibitor suppressed matrix invasion by trophoblast cells and was associated with increased nuclear accumulation of G-actin. Conversely, treatment with 1,25D enhanced matrix invasion. This was independent of the nuclear vitamin D receptor but was associated with enhanced ERK phosphorylation, and inhibition of ERK kinase suppressed trophoblast matrix invasion. When cultured with serum from pregnant women, trophoblast matrix invasion correlated with DBP concentration, and DBP was lower in first-trimester serum from women who later developed preeclampsia. These data show that the trophoblast matrix invasion involves uptake of serum DBP and associated intracellular actin-binding and homeostasis. DBP is a potential marker of placentation disorders such as preeclampsia and may also provide a therapeutic option for improved placenta and pregnancy health.

The vitamin D axis in the lung: a key role for vitamin D-binding protein.

There has been much recent interest in the role of the vitamin D axis in lung disease, which includes vitamin D, vitamin D receptor (VDR) and vitamin D-binding protein (VDBP; also known as Gc-globulin). VDBP is a serum protein which has immunomodulatory functions relevant in the lung, predominantly relating to macrophage activation and neutrophil chemotaxis. Variations within its gene are also associated with airways disease, implying a role for the protein product in pathogenesis. Thus far the majority of evidence relates to chronic obstructive pulmonary disease (COPD), but is scant in other airways diseases, such as asthma and bronchiectasis. VDBP also acts as a scavenger protein to clear extracellular G-actin released from necrotic cells, which may be of relevance in severe lung infections and acute lung injury. Vitamin D protects against the development of cancer and tuberculosis, although optimal levels are unknown. The majority of circulating vitamin D is bound to VDBP, and its uptake into cells occurs in both bound and unbound forms, which suggests the role of VDBP warrants further study in these conditions as well. This article reviews the evidence of the role VDBP and its gene (GC) in a range of lung diseases, including asthma, COPD and tuberculosis.

Vitamin D binding protein is related to cardiac autonomic function and metabolic status in prediabetes.

A putative causal relationship between vitamin D status and glucose metabolism and a direct effect of vitamin D on cardiac autonomic function (CAF) have been suggested. We hypothesized that vitamin D binding protein (DBP), as a transporter of vitamin D, might also influence CAF and the overall metabolic risk. The present study aims to assess the relationship between DBP and CAF and metabolic status in a high-risk population with prediabetes. A total of 174 subjects (mean age 49.1±12.9 years, mean body mass index 30.2±6.2 kg/m2) were divided into 2 groups according to glucose tolerance: 48 with normal glucose tolerance and 126 with prediabetes. Glucose tolerance was assessed during oral glucose tolerance test, applying 2006 World Health Organization criteria. Fasting and postload glucose and immunoreactive insulin were measured (homeostatic model assessment of insulin resistance and homeostatic model assessment of ß-cell function were calculated). Anthropometric indexes, blood pressure, hemoglobin A1c, creatinine, lipids, high-sensitivity C-reactive protein, total 25-hydroxyvitamin D, DBP (free 25-hydroxyvitamin D was calculated), and intact parathormone were measured. Body composition was estimated by impedance analysis (InBody 720), whereas tissue advanced glycation end products were assessed by skin autofluorescence (AGE Reader, DiagnOptics, the Netherlands). CAF was evaluated by АNX-3.0 system, applying standard autonomic tests. DBP was found to be elevated in women, as well as in the presence of cardiac autonomic dysfunction and metabolic syndrome. DBP was related to parasympathetic activity in both sexes and in prediabetes; to body fat in women and in prediabetes; and to age and high-density lipoprotein cholesterol in men. Vitamin D deficiency was established in 40.8% of the studied cohort. These results support the hypothesis that DBP is associated with CAF and some metabolic parameters in prediabetes.

Vitamin D and cardiovascular disease.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide. Recently vitamin D deficiency has been identified as a potential risk factor for many diseases not traditionally associated with vitamin D, such as cancer and CVD. This review discusses the evidence suggesting an association between low 25-hydroxyvitamin D levels and CVD and the possible mechanisms mediating it. Vitamin D deficiency has been associated with CVD risk factors such as hypertension and diabetes mellitus, with markers of subclinical atherosclerosis such as intima-media thickness and coronary calcification as well as with cardiovascular events such as myocardial infarction and stroke as well as congestive heart failure. It could be suggested that vitamin D deficiency contributes to the development of CVD through its association with risk factors, such as diabetes and hypertension. However, direct effects of vitamin D on the cardiovascular system may also be involved. Vitamin D receptors are expressed in a variety of tissues, including cardiomyocytes, vascular smooth muscle cells and endothelial cells and vitamin D has been shown to affect inflammation and cell proliferation and differentiation. While much evidence supports a potential antiatherosclerotic effect of vitamin D, prospective, placebo-controlled randomized as well as mechanistic studies are needed to confirm this association. Since vitamin D deficiency is easy to screen for and treat, the confirmation of such an association could have important implications for both, patient care and health policy.

Vitamin D: physiology and pathophysiology.

Although vitamin D was initially considered a nutrient, it has been recognized that the molecules derived from vitamin D metabolism are best considered as a complex endocrine system. In this review article we summarize the basic concepts regarding vitamin D metabolism, transport, and genomic activity through the vitamin D receptor, facilitating activation or suppression of target genes. We also examine non-genomic actions, biological responses to vitamin D in classic target organs (intestine, bone, kidneys, and parathyroid glands), and in organs and tissues not related to mineral homeostasis.

Vitamin D-binding protein influences total circulating levels of 1,25-dihydroxyvitamin D3 but does not directly modulate the bioactive levels of the hormone in vivo.

Mice deficient in the expression of vitamin D-binding protein (DBP) are normocalcemic despite undetectable levels of circulating 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. We used this in vivo mouse model together with cells in culture to explore the impact of DBP on the biological activity of 1,25(OH)(2)D(3). Modest changes in the basal expression of genes involved in 1,25(OH)(2)D(3) metabolism and calcium homeostasis were observed in vivo; however, these changes seemed unlikely to explain the normal calcium balance seen in DBP-null mice. Further investigation revealed that despite the reduced blood levels of 1,25(OH)(2)D(3) in these mice, tissue concentrations were equivalent to those measured in wild-type counterparts. Thus, the presence of DBP has limited impact on the extracellular pool of 1,25(OH)(2)D(3) that is biologically active and that accumulates within target tissues. In cell culture, in contrast, the biological activity of 1,25(OH)(2)D(3) is significantly impacted by DBP. Here, although DBP deficiency had no effect on the activation profile itself, the absence of DBP strongly reduced the concentration of exogenous 1,25(OH)(2)D(3) necessary for transactivation. Surprisingly, analogous studies in wild-type and DBP-null mice, wherein we explored the activity of exogenous 1,25(OH)(2)D(3), produced strikingly different results as compared with those in vitro. Here, the carrier protein had virtually no impact on the distribution, uptake, activation profile, or biological potency of the hormone. Collectively, these experiments suggest that whereas DBP is important to total circulating 1,25(OH)(2)D(3) and sequesters extracellular levels of this hormone both in vivo and in vitro, the binding protein does not influence the hormone's biologically active pool.

Therapeutic potential of vitamin D for multiple sclerosis.

Multiple sclerosis (MS) is a major inflammatory and demyelinating disease of the central nervous system and has an increasing prevalence in populations residing at higher latitudes. This observation may indicate a protective effect of sunlight exposure, which is reduced at higher latitudes and may contribute to insufficient levels of vitamin D in the MS population. The vitamin D hormone is important for bone metabolism and can regulate cell proliferation and differentiation as well as apoptosis and immune regulation in immune cells such as T helper cells and dendritic cells. Evidence from experimental autoimmune encephalomyelitis and prospective studies on MS suggests an important role of vitamin D as a modifiable environmental factor in MS. These provide guidance for future studies with regard to the potential role of vitamin D in the prevention and/or treatment of MS. Here, we first review the metabolism and immune functions of vitamin D. Then, we describe the current thinking on the etiology of vitamin D in MS and the accumulating evidence pointing to a link between vitamin D and MS. Further, we describe how genetic susceptibility interacts with environmental risk factors at the population level, MS-associated risk factors, and genetic studies related to the vitamin D receptor. This review also discusses the therapeutic potential of vitamin D for treating MS.

Gc-globulin in liver disease.

This doctoral thesis is based on seven previously published papers and reports on the role of the actin-scavenger Gc-globulin in acute and chronic liver diseases. Gc-globulin is synthesized in the liver and is a multifunctional protein; however, its main physiologic function is presumably actin binding and actin scavenging. Actin is a major cellular protein released during cell necrosis that may cause fatal formation of actin-containing thrombi in the circulation if the actin scavenging capacity of Gc-globulin is exceeded. In my studies, I found serum Gc-globulin levels to be reduced in liver disease, most so in patients with acute liver failure (ALF). In patients admitted with acetaminophen (paracetamol) overdose, Gc-globulin concentrations were lower in patients with hepatic encephalopathy than in those without and the levels nadired at approximately 60-72 hours after acetaminophen ingestion, corresponding with the peak in aminotransferese levels (and thus, hepatic necrosis). In patients with ALF, admission Gc-globulin was significantly lower in 47 nonsurvivors than in 30 survivors, 26% and 46% of normal, respectively (P<0.001). The predictive value of outcome using a Gc-globulin cutoff level of 100 mg/L equaled that of the internationally accepted King's College Hospital criteria. The prognostic value of Gc-globulin was confirmed in a separate study including 106 patients from the United States with nonacetaminophen-induced ALF now using an automated nephelometric assay whereas the prognostic value seemed less obvious for acetaminophen-induced ALF. Multiple organ failure (MOF) is a frequent complication of ALF. In ALF patients with deep coma (hepatic encephalopathy grade III or IV) Gc-globulin levels correlated inversely with the number of failing organs. Levels were lower in patients who later developed MOF than in those who did not. Surprisingly, kinetic studies in patients with ALF and acute on chronic liver disease showed Gc-globulin production to be 7-fold increased in these conditions. Despite this increase Gc-globulin levels were severely reduced and the reduction must therefore be due to a highly increased consumption of Gc-globulin - probably because of hepatocyte necrosis and removal from the circulation of Gc-globulin:actin complexes or because of its role in immune-related functions. Patients with chronic liver disease had reduced Gc-globulin levels, but the reduction was less pronounced than in ALF. After liver transplantation, Gc-globulin concentrations normalized within two weeks, in contrast to the continuous decrease in albumin levels suggesting a very different regulation of these two phylogenetically related proteins. It remains to be studied if lack of Gc-globulin contributes to the pathogenesis of patients with ALF or chronic liver disease. Future studies should focus on the potential value of Gc-globulin substitution in these patients.

Role of group-specific component (vitamin D binding protein) in clearance of actin from the circulation in the rabbit.

The possible role of group specific component (Gc) (vitamin D-binding protein) in the clearance of cellular actin entering the circulation was examined with 125I-labeled Gc and actin injected into a rabbit model. Although filamentous F-actin is depolymerized primarily by plasma gelsolin, greater than or equal to 90% 125I-actin injected in either monomeric G- or F-form became complexed eventually with Gc (1:1 molar ratio). Clearance of Gc complexes was much faster (greater than 90% within 5 h) than that of native Gc (t1/2 = 17.2 h). Nephrectomy did not significantly alter the clearance of either Gc or actin. Since Gc complexes are dramatically increased in situations of tissue necrosis such as in fulminant hepatic failure, the current results suggest a crucial role for Gc in sequestration and clearance of released cellular actin.

Gc-globulin (vitamin D-binding protein) enhances the neutrophil chemotactic activity of C5a and C5a des Arg.

Several serum proteins have been shown to be important in modulating leukocyte chemotaxis and inflammation. We investigated the possibility that the multifunctional serum protein Gc-globulin (vitamin D-binding protein) may also enhance the neutrophil chemotactic activity of complement-derived peptides. Purified Gc-globulin by itself did not induce chemotaxis of human neutrophils. However, as little as 0.01 nM Gc-globulin greatly enhanced the neutrophil chemotactic activity of C5a and its derivative, C5a des Arg over a wide concentration range. The effect was most pronounced at nonchemotactic doses of C5a (0.01 nM) and C5a des Arg (1 nM). Gc-globulin was unable to augment the neutrophil chemotactic activity of FMLP and leukotriene B4. This enhancing activity was not due to a nonspecific effect of anionic proteins since other purified serum proteins, of similar size and charge as Gc-globulin (alpha 1 acid glycoprotein, alpha 2 HS glycoprotein, alpha 2 histidine-rich glycoprotein), could not increase the chemotactic activity of C5a des Arg. Serum depleted of Gc-globulin by immunoaffinity chromatography totally lacked chemotactic enhancing activity for C5a des Arg. Gc-globulin-depleted serum activated with zymosan also had significantly less chemotactic activity than control- (sham-depleted) activated serum. Finally, radioiodinated C5a or C5a des Arg formed a 1:1 complex with purified Gc-globulin when analyzed by gel filtration chromatography. These results indicate that Gc-globulin is the major chemotactic enhancing factor in serum and may function as an up-regulator of the chemotactic activity of C5-derived peptides.

Biological and clinical aspects of the vitamin D binding protein (Gc-globulin) and its polymorphism.

The vitamin D binding protein (DBP) is the major plasma carrier protein of vitamin D and its metabolites. Unlike other hydrophobic hormone-binding systems, it circulates in a considerably higher titer compared to its ligands. Apart from its specific sterol binding capacity, DBP exerts several other important biological functions such as actin scavenging, fatty acid transport, macrophage activation and chemotaxis. The DBP-gene is a member of a multigene cluster that includes albumin, alpha-fetoprotein, and alpha-albumin/afamin. All four genes are expressed predominantly in the liver with overlapping developmental profiles. DBP is a highly polymorphic serum protein with three common alleles (Gc1F, Gc1S and Gc2) and more than 120 rare variants. The presence of unique alleles is a useful tool for anthropological studies to discriminate and to reveal ancestral links between populations. Many studies have discussed the link between DBP-phenotypes and susceptibility or resistance to osteoporosis, Graves' disease, Hashimoto's thyroiditis, diabetes, COPD, AIDS, multiple sclerosis, sarcoidosis and rheumatic fever. This article reviews the general characteristics, functions and clinical aspects of DBP.

Tandem arrangement of the human serum albumin multigene family in the sub-centromeric region of 4q: evolution and chromosomal direction of transcription.

The albumin gene family is comprised of four genes encoding: serum albumin (ALB), alpha-fetoprotein (AFP), alpha-albumin (ALF), and vitamin D-binding protein (DBP; also known as GC). The genes are regulated developmentally, expressed in the liver, and the proteins are secreted into the bloodstream. The GC gene, and the tandemly linked ALB and AFP genes, have been previously localized to human chromosome 4q11-13. Using techniques of fluorescence in situ hybridization to chromatin fibres, chromosome walking and DNA sequencing of genomic clones, we now report on the chromosomal location of the ALF gene and the organization of the entire gene family. The four genes are tandemly linked in the 4q sub-centromeric region: 5'ALB-5'AFP-5'ALF-5'GC3'-centromere, and hence are transcribed in the same, centromere-bound, direction. The linear arrangement of the four genes along the chromosome is not correlated with their temporal expression in the human ontogeny. It appears that GC is very close (and may be the gene proximal) to the centromere. The linear chromosomal arrangement of the four genes and the structural differences between them are congruent with the following evolutionary divergence of the gene family. Starting with the first duplication of an ancestral progenitor gene, a single evolutionary line led to the contemporary GC, leaving ALB/AFP/ALF on the other line of descent. The second duplication occurred in this ALB lineage, giving rise to ALB and the AFP/ALF progenitor, and the third, most recent one, gave rise to the AFP-ALF pair.

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Human monocyte chemotaxis to complement-derived chemotaxins is enhanced by Gc-globulin.

Gc-globulin has been found in bronchoalveolar lavage fluid in patients with chronic obstructive pulmonary disease (COPD) and adult respiratory distress syndrome (ARDS) and has been shown to enhance neutrophil chemotaxis to C5-derived peptides in vitro. We proposed that Gc-globulin may enhance the inflammatory response in lungs by influencing monocyte chemotaxis to C5-derived peptides as it does with neutrophils. Monocyte chemotaxis was measured in blind well chambers by a leading-front technique. Purified human Gc-globulin had no intrinsic chemotactic activity for monocytes at concentrations ranging from 1 fM to 1 microM. However, Gc-globulin, at concentrations as low as 10 pM, increased monocyte chemotaxis over 10-fold in a concentration-dependent fashion when added to non-chemotactic doses of C5a (0.1 nM) and C5a des Arg (0.5 nM). The chemotaxis-enhancing effect of Gc-globulin was specific for C5-derived peptides, as Gc-globulin did not enhance monocyte chemotaxis to other chemoattractants such as leukotriene B4 or formyl-Met-Leu-Phe. The enhancement of monocyte chemotaxis to C5-derived peptides by Gc-globulin was not a nonspecific effect of anionic proteins, as other serum proteins of similar size and charge did not enhance monocyte chemotaxis to C5a des Arg. These results indicate that Gc-globulin enhances the monocyte response to C5-derived peptides and, together with previous work, indicates that its presence in the airways of patients with COPD and ARDS may up-regulate the monocyte inflammatory response in the lungs.