VDR, the Vitamin D Receptor
Almost from the time life began, vitamin D has been produced by plants and animals. As the structures of plants and animals became more complex, the sites of vitamin D production, its subsequent metabolism, as well as its sites of action separated. The ability to transport and metabolize vitamin D into more active forms therefore evolved. The metabolic active form 1,25(OH)2D3 of vitamin D exerts its actions through interaction with the vitamin D receptor, VDR (Bikle 2011). Although not as ancient, VDR has been highly conserved between species through evolution (Hochberg and Templeton 2010).VDR is found in almost all cells and tissues of higher-order animals, further emphasizing the importance of the receptor. Evidence for the existence of VDR was first provided in 1969 by Haussler and Norman (Feldman and Pike 2005), and since then a substantial amount of data on the structure and function of VDR has been accomplished.
The Source and Metabolism of the Vitamin D Receptor Ligand
Mechanism of Action, the Vitamin D Receptor
Besides regulation through VDREs of target genes, VDR can inhibit the expression of some genes by antagonizing the action of certain transcription factors, such as NFAT and NF-κΒ (Nagpal et al. 2005). An example is the VDR-dependent inhibition of the cytokines IL-2 and GM-CSF which appears more complex than the involvement of positive and negative VDREs. Here, VDR first competes with NFAT1 for binding to the enhancer motif of the NFAT1-activator protein 1 (AP1) and subsequently VDR binds to c-Jun. This cooccupancy of VDR-c-Jun to AP1 leads to inhibition of activated IL-2 and GM-CSF expression (Nagpal et al. 2005).
To fully accomplish its functions, the VDR protein is divided into an N-terminal domain including the DNA-binding region and a multifunctional C-terminal domain harboring the ligand-binding region, the RXR heterodimerization motif, and a ligand-dependent transactivation function (AF-2). The C-terminal domain consequently exerts absolute regulatory control over the DNA-binding properties of the VDR (Fig. 2) (Jones et al. 1998; Feldman and Pike 2005).
Regulation of Vitamin D Receptor Activity
Due to the influence of VDR on such a broad array of genes, its activity is ascribed strict control. Besides the obvious limitations of VDR activity due to availability of the VDR ligand, 1,25(OH)2D3 also directly influences the expression of VDR. Cells treated with 1,25(OH)2D3 induce VDR-mRNA and furthermore stabilize the mRNA, increasing the total amount of cellular VDR. VDR expression can also be modulated by numerous other physical stimuli such as dietary composition (calcium and phosphorus), steroid hormones and retinoids, growth factors, and peptide hormones (Feldman and Pike 2005).
The various physiological inputs that affect VDR levels involve complex interactions among the intracellular signaling transduction pathways. As an example, cellular response to the parathyroid hormone (PTH) activates protein kinase A (PKA) causing an increase in VDR level, whereas stimuli that induce protein kinase C (PKC) activity lead to a decrease in VDR expression. Given the central role of PKA and PKC in the integration of cellular responses to extracellular stimuli, these pathways are central in the mechanisms that regulate VDR levels in target cells (Feldman and Pike 2005). A signaling pathway to induce VDR expression not including PKA and PKC has recently been described in human naïve T cells of the immune system. Here, the cellular response to a foreign pathogen induces the expression of VDR through activation of the kinase p38, a process mandatory for the activation of the naive T cells (von Essen et al. 2010). In addition, cellular signaling motivating physical interaction of VDR-1,25(OH)2D3 with SUG1 of the proteasome complex targets VDR for ubiquitination and subsequent proteolysis (Dusso et al. 2005). Regulation of VDR expression and abundance is therefore an important mechanism for the modulation of cellular responsiveness to 1,25(OH)2D3 and hence for controlling a variety of cellular functions. It is also possible that phosphorylation of VDR, which induces a conformational change that releases corepressors as well as posttranslational VDR modifications, plays a role in regulating the activity of VDRs (Dusso et al. 2005).
Biological Functions of the Vitamin D Receptor
Within the last two decades, it has been clear that VDRs are not only present in tissues associated with calcium and phosphorus metabolism but also in “nonclassical” organs like immune cells, brain, eyes, heart, pancreatic islets (β cells), muscle, adipose tissue, thyroid, parathyroid, and adrenal glands, suggesting a broader role for VDR (Baeke et al. 2010; Feldman and Pike 2005). In particular, its role in the immune cells has received much attention due to a great potential as a preventive or therapeutic target in a variety of inflammatory and autoimmune diseases as well as in cancer. The immune system is divided into an innate immune system that makes up the critical first line of defense against invading pathogens and an adaptive immune system containing pathogen-specific cells to combat the foreign intruder (Abbas et al. 2007). VDR is widely expressed in most immune cell types and consequently 1,25(OH)2D3 has proven to have potent immunomodulatory effects on cells of both the innate and adaptive immune systems. Notably, several of these cells also express vitamin D-activating enzymes such as 1α-hydroxylase, allowing local activation of vitamin D (Bikle 2009; Baeke et al. 2010).
For adaptive immunity to be encouraged, naïve T lymphocytes (T cells) must receive an activation signal from antigen-presenting cells like macrophages and in particular dendritic cells (DCs). Depending on the nature of the signal provided and the local environment, the lymphocytes can pursue different developmental programs (Abbas et al. 2007). Both DCs and T cells express the VDR and 1α-hydroxylase and have been identified as direct targets of 1,25(OH)2D3 (Baeke et al. 2010). As for innate immunity, 1,25(OH)2D3 has been shown to promote adaptive responses; 1,25(OH) 2D 3-VDR is required for the early phase of T-cell priming as VDR-signaling induces expression of PLC-γ1, which is mandatory for differentiation of the naïve T cells (von Essen et al. 2010). Still, the most well-known described effect of 1,25(OH)2D3 on adaptive immunity is that of inhibition (Bikle 2009; Baeke et al. 2010; van Etten and Mathieu 2005). DCs under the influence of 1,25(OH)2D3 alter their phenotype to favor differentiation of a regulatory subset of T cells (Treg) with immune suppressive functions, instead of priming the T-cell subset with inflammatory effects (Th1 and Th17). Both Th1 and Th17 cells are commonly accepted to be main players in the development and progression of various inflammatory and autoimmune diseases. Furthermore, 1,25(OH)2D3 also directly modulates the function of the different T-cell subsets. Treg cells increase their ability to suppress immunity and the effector T cells decreases their ability to proliferate and exert their effector functions, for example, through secretion of cytokines. The net result is downregulation of the effector T-cell response (Fig. 4) (van Etten and Mathieu 2005). The apparent discrepancies of the influence of 1,25(OH)2D3 on adaptive immunity might be explained by the availability of 1,25(OH)2D3 at the different stages of an immune response. Early studies have shown a proproliferative effect of low concentrations of 1,25(OH)2D3 on T cells (Lacey et al. 1987), whereas more recent studies using higher concentrations have shown the exact opposite. Although it has to be investigated, one can speculate that the modest quantity of 1,25(OH)2D3 present at the beginning of an immune response is sufficient to induce T-cell priming, whereas the increasing amount supposedly generated during the response exerts a negative feedback to ensure that the immune system does not overreact.
VDR-Mediated Signaling in Health and Disease
Allelic variants (polymorphisms) of the VDR gene which occurs naturally in the human population are associated with various medical conditions such as decreased bone density, tendency to hyperparathyroidism (excess PTH production), and susceptibility to infections, autoimmune diseases, and cancer. This may in part be due to the effect of VDR polymorphism on VDR-mRNA stability and reduced expression of VDR (Feldman and Pike 2005). The importance of such changes in VDR expression for disease development is strengthened by the observation that low VDR expression is associated with more invasive and lethal tumors (Hendrickson et al. 2011; Lopez et al. 2010) perhaps due to less proliferative control and apoptosis induction, which are two major processes modulated by VDR-signaling.
In addition to allelic variations of the VDR gene, two rare genetic disorders have been described in which either the CYP27B1 gene or the VDR gene itself contain mutations that render the gene products nonfunctional. In patients with a nonfunctional 1α-hydroxylase (due to a mutation in CYP27B1) 1,25(OH)2D3 is no longer synthesized. Individuals with a nonfunctional VDR suffer from absence of VDR-signaling giving rise to the disease hereditary vitamin D resistant rickets (HVDRR). Both diseases are characterized by hypocalcemia (low blood calcium), hyperparathyroidism, and early onset of rickets (bone deformities in children). Even though the two diseases appear alike, a crucial difference is the blood level of 1,25(OH)2D3: It is almost absent in patients with 1α-hydroxylase deficiency, whereas it is exceedingly high in HVDRR. The apparent consequence is that patients with a defect CYP27B1 gene respond to treatment with 1,25(OH)2D3 and can expect complete remission of the disease, whereas most HVDRR-affected individuals are resistant to vitamin D therapy. In this case, intensive calcium therapy is used, surprisingly reversing all aspects of the disease. As there are only very few cases of HVDRR and as the abnormalities are corrected relatively early in life, long-term effects of defective VDR-signaling such as development of autoimmune diseases and cancer have not been observed (Malloy and Feldman 2010). A promising model system regarding these issues is a mouse model in which the VDR gene has been deleted, or in which severe vitamin D deficiency has been introduced through a vitamin D depleted diet. These mice do show increased sensitivity to certain autoimmune diseases and are also more prone to oncogene- and chemocarcinogen-induced tumors (Bouillon et al. 2008). Furthermore, it has been demonstrated in a variety of animal disease models that pretreatment with 1,25(OH)2D3 is effective in preventing the onset of both multiple sclerosis, type I diabetes, rheumatoid arthritis, and inflammatory bowel disease (Nagpal et al. 2005; Guillot et al. 2010).
Consistent with the observations from mice models, an inverse association between the vitamin D status (blood level of 25(OH)D3) and development of a variety of autoimmune and inflammatory diseases as well as an increased risk for development of certain types of cancers has been described in epidemiological studies in humans (Nagpal et al. 2005; van Etten and Mathieu 2005; Guillot et al. 2010; Luong and Nguyen 2010). Even so, many of the epidemiological studies performed measured the vitamin D status at the time of disease diagnosis. It therefore has to be considered that the low vitamin D status could be a consequence of disease development rather than a direct cause, as daily routines and habits are often changed upon development of these diseases (e.g., more indoor activities). Thus, the usage of the vitamin D status as a “probability marker” for disease development awaits clarification through future studies.
Summary and Future Directions
VDR is a nuclear, ligand-induced transcription factor that in complex with hormonally active vitamin D, 1,25(OH)2D3, regulates the expression of more than 900 genes involved in a wide array of physiological functions, for example, calcium homeostasis, growth control, differentiation, and immune responses.
The ligand for VDR, 1,25(OH)2D3 is attained mainly from 7-DHC in the skin. 7-DHC is transformed to a vitamin D precursor through UVβ irradiation from the sun. This vitamin D precursor can also be obtained in minor amounts through diet. Either way, two hydroxylation steps involving 25-hydrolylase and 1α-hydroxylase transform the precursor into its active metabolite. Once formed 1,25(OH)2D3 binds to VDR in the target cell and enables the receptor to bind to RXR and translocate into the nucleus where it participates in the regulation of the expression of a great variety of genes. In the target cell, 1,25(OH)2D3 limits its own activity by inducing the enzyme 24-hydroxylase that transforms 1,25(OH)2D3 to a less active metabolite. Other mechanisms important for the regulation of VDR activity include control of VDR expression and possibly phosphorylation and posttranslational VDR modifications. The biological functions of VDR are numerous, with its role as a regulator of blood calcium and phosphorus level as the most commonly known. However, lately the role of VDR in modulating immune responses has been the focus of many studies.
It is generally accepted that vitamin D deficiency is highly prevalent in many populations around the world (Baeke et al. 2010). Therefore, vitamin D supplementation represents an attractive strategy to ensure sufficient 25(OH)D3 levels for adequate bone metabolism and immune functions thereby eliminating one of the proposed risk factors that may underlie inflammatory or autoimmune diseases as well as certain types of cancer. In addition to a potential disease-preventive role of 1,25(OH)2D3, the immunomodulating effects of vitamin D suggest that vitamin D holds therapeutic promises in inflammatory and autoimmune diseases. In a variety of animal studies, 1,25(OH)2D3 has been shown either to halt progression or reduce disease severity, counting a mouse model for multiple sclerosis, type I diabetes, rheumatoid arthritis, and inflammatory bowel disease (Nagpal et al. 2005; Plum and DeLuca 2010; Guillot et al. 2010). Unfortunately, the therapeutic application of 1,25(OH)2D3 is obstructed by toxicity issues as the supraphysiological doses needed to exert an immune inhibitory effect elicit unfortunate calcemic side effects. To overcome this limitation, structural analogues of 1,25(OH)2D3 are being designed that have reduced calcemic effects with similar immunoregulatory activity (Yee et al. 2005). Today, such analogs have become the first-line therapy for the treatment of psoriasis. The vitamin D analogue is applied topically onto the psoriasis plaques where it inhibits the massive keratinocyte proliferation and the underlying inflammation. Due to its short half-life after it enters circulation, it is destroyed before it has an opportunity to affect calcium homeostasis (Yee et al. 2005). A similar “soft-drug-approach” cannot be employed for other inflammatory, autoimmune, and cancer diseases as a systemic effect (not including calcemic effects) would be the goal of therapy. However, the development of orally available noncalcemic and even tissue-specific vitamin D analogs is a working field in progress, and the prospect of vitamin D therapeutics for treating diseases beyond metabolic bone disorders seems promising.
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