Abstract

The secosteroid hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the natural ligand for the vitamin D receptor, a member of the nuclear receptor superfamily. Upon binding of the ligand, the vitamin D receptor heterodimerizes with the retinoid X receptor and binds to vitamin D response elements in the promoter region of target genes to induce/repress their expression. The target genes that have been identified so far are heterogeneous in nature and reflect the great spectrum of biological activities of 1,25(OH)2D3. Within the last two decades, the receptor has been shown to be present not only in classical target tissues such as bone, kidney, and intestine, but also in many other nonclassical tissues, for example, in the immune system (T and B cells, macrophages, and monocytes), in the reproductive system (uterus, testis, ovary, prostate, placenta, and mammary glands), in the endocrine system (pancreas, pituitary, thyroid, and adrenal cortex), in muscles (skeletal, smooth, and heart muscles), and in brain, skin, and liver. Besides the almost universal presence of vitamin D receptors, different cell types (for example, keratinocytes, monocytes, bone, placenta) are capable of metabolizing 25-hydroxyvitamin D3 to 1,25(OH)2D3 by the enzyme 25(OH)D3-1α-hydroxylase, encoded by CYP27B1. The combined presence of CYP27B1 and the specific receptor in several tissues introduced the idea of a paracrine/autocrine role for 1,25(OH)2D3. Moreover, it has been demonstrated that 1,25(OH)2D3 can induce differentiation and inhibit proliferation of normal and malignant cells. Moreover, vitamin D deficiency is associated with an increased risk for nearly all major human diseases such as cancer, autoimmune diseases, cardiovascular, and metabolic diseases. In addition to the treatment of bone disorders with 1,25(OH)2D3, these newly discovered functions open perspectives for the use of 1,25(OH)2D3 as an immune modulator (for example, for the treatment of autoimmune diseases or prevention of graft rejection), inhibitor of cell proliferation, and inducer of cell differentiation (cancer). The secosteroid hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the natural ligand for the vitamin D receptor, a member of the nuclear receptor superfamily. Upon binding of the ligand, the vitamin D receptor heterodimerizes with the retinoid X receptor and binds to vitamin D response elements in the promoter region of target genes to induce/repress their expression. The target genes that have been identified so far are heterogeneous in nature and reflect the great spectrum of biological activities of 1,25(OH)2D3. Within the last two decades, the receptor has been shown to be present not only in classical target tissues such as bone, kidney, and intestine, but also in many other nonclassical tissues, for example, in the immune system (T and B cells, macrophages, and monocytes), in the reproductive system (uterus, testis, ovary, prostate, placenta, and mammary glands), in the endocrine system (pancreas, pituitary, thyroid, and adrenal cortex), in muscles (skeletal, smooth, and heart muscles), and in brain, skin, and liver. Besides the almost universal presence of vitamin D receptors, different cell types (for example, keratinocytes, monocytes, bone, placenta) are capable of metabolizing 25-hydroxyvitamin D3 to 1,25(OH)2D3 by the enzyme 25(OH)D3-1α-hydroxylase, encoded by CYP27B1. The combined presence of CYP27B1 and the specific receptor in several tissues introduced the idea of a paracrine/autocrine role for 1,25(OH)2D3. Moreover, it has been demonstrated that 1,25(OH)2D3 can induce differentiation and inhibit proliferation of normal and malignant cells. Moreover, vitamin D deficiency is associated with an increased risk for nearly all major human diseases such as cancer, autoimmune diseases, cardiovascular, and metabolic diseases. In addition to the treatment of bone disorders with 1,25(OH)2D3, these newly discovered functions open perspectives for the use of 1,25(OH)2D3 as an immune modulator (for example, for the treatment of autoimmune diseases or prevention of graft rejection), inhibitor of cell proliferation, and inducer of cell differentiation (cancer). 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is a major calcitropic hormone with as primary purpose to keep the plasma Ca2+ concentration within narrow limits. The renal production of 1,25(OH)2D3 is therefore tightly regulated by two hormones. First, hypocalcemia induces the secretion of parathyroid hormone (PTH) by the parathyroid gland. PTH targets the kidney to decrease Ca2+ excretion, inhibit phosphate reabsorption, and stimulate 1,25(OH)2D3 production. 1,25(OH)2D3 will then increase active Ca2+ and phosphate transport in the small intestine and stimulate Ca2+ reabsorption in the kidney. In concert with the effects of PTH on bone, 1,25(OH)2D3 action also enhances Ca2+ exchange from bone and the cooperation of these pathways restores serum Ca2+ levels to normal. Second, 1,25(OH)2D3 stimulates the secretion of FGF23 by the osteocytes. FGF23 is the major phosphaturic hormone inhibiting renal phosphate reabsorption (by the specific transporter NaPiT2a) and decreasing the renal production of 1,25(OH)2D3. In addition, 1,25(OH)2D3 and phosphate stimulate and FGF23 inhibits PTH production creating a true phosphate feedback loop. The analysis of transgenic mouse models permitted to elucidate the molecular pathway involved and to delineate direct versus indirect action of 1,25(OH)2D3 on the different target tissues. Genetic deficiencies in 1,25(OH)2D3 signaling by loss of CYP27B1 or vitamin D receptor (VDR) result in hypocalcemia, hyperparathyroidism, hypophoshatemia, rickets, and osteomalacia. These effects, however, are only observed after weaning, when dietary calcium content decreases. The phenotype can largely be corrected by administration of a high calcium diet that bypasses active, 1,25(OH)2D3-mediated, Ca2+ absorption.1.Bouillon R. Carmeliet G. Verlinden L. et al.Vitamin D and human health: lessons from vitamin D receptor null mice.Endocr Rev. 2008; 29: 726-776Crossref PubMed Scopus (1182) Google Scholar The action of 1,25(OH)2D3 to promote intestinal transcellular Ca2+ transport is, therefore, mainly required when dietary calcium content is low to normal. The importance of VDR signaling in the intestine is underscored by the observation that reintroducing VDR specifically in the intestine of VDR null mice rescued the hypocalcemia and the bone phenotype.2.Xue Y. Fleet J.C. Intestinal vitamin D receptor is required for normal calcium and bone metabolism in mice.Gastroenterology. 2009; 136: 1317-1327Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 3.Lieben L. Masuyama R. Moermans K. et al.Intestinal-Specific Vitamin D receptor null mice maintain normal calcemia but display severe bone loss.J Bone Miner Res. 2008; 23: S104 The proteins involved in this active intestinal Ca2+ transport remain, however, poorly defined. Gene expression analysis suggested that the Ca2+ channel TRPV6 (transient receptor potential vanilloid type 6), mediating Ca2+ entry across the luminal membrane, and calbindin D9k, facilitating cytoplasmic Ca2+ transfer toward the basolateral membrane, are the main mediators of 1,25(OH)2D3-induced intestinal Ca2+ absorption.4.Van Cromphaut S.J. Dewerchin M. Hoenderop J.G. et al.Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects.Proc Natl Acad Sci USA. 2001; 98: 13324-13329Crossref PubMed Scopus (441) Google Scholar Surprisingly, inactivation of TRPV6 and calbindin D9k does not impair intestinal Ca2+ absorption when dietary calcium intake is normal, indicating the contribution of compensatory proteins. Nevertheless, TRPV6 becomes critical for Ca2+ absorption when dietary calcium intake is low, as evidenced by the impaired intestinal Ca2+ absorption in TRPV6 null mice on a low calcium diet.5.Bianco S.D. Peng J.B. Takanaga H. et al.Marked disturbance of calcium homeostasis in mice with targeted disruption of the Trpv6 calcium channel gene.J Bone Miner Res. 2007; 22: 274-285Crossref PubMed Scopus (214) Google Scholar, 6.Benn B.S. Ajibade D. Porta A. et al.Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k.Endocrinology. 2008; 149: 3196-3205Crossref PubMed Scopus (167) Google Scholar A possible explanation for the lack of effect is that 1,25(OH)2D3 action not only affects transcellular but also paracellular Ca2+ transport. Indeed, 1,25(OH)2D3 may regulate tight junction permeability in the intestine by modifying the expression of several tight junction proteins, including claudin-2 and claudin-12 and hereby promoting Ca2+ absorption.7.Fujita H. Sugimoto K. Inatomi S. et al.Tight junction proteins claudin-2 and -12 are critical for vitamin D-dependent Ca2+ absorption between enterocytes.Mol Biol Cell. 2008; 19: 1912-1921Crossref PubMed Scopus (288) Google Scholar As mentioned, 1,25(OH)2D3 promotes renal Ca2+ reabsorption that correlates with increased expression of TRPV5 and calbindin D9k. In contrast to the limited contribution of TRPV6 to intestinal Ca2+ absorption, TRPV5 is definitely critical for renal Ca2+ reabsorption. Indeed, TRPV5 null mice display a profound Ca2+ wasting with compensatory increased intestinal Ca2+ absorption, likely due to high 1,25(OH)2D3 serum levels, and normocalcemia as a result.8.Hoenderop J.G. van Leeuwen J.P. van der Eerden B.C. et al.Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5.J Clin Invest. 2003; 112: 1906-1914Crossref PubMed Scopus (367) Google Scholar If the major action of 1,25(OH)2D3 is to keep serum Ca2+ levels within restricted limits, the question can be raised whether the sole action of 1,25(OH)2D3 on bone is to diminish changes in the serum Ca2+ concentrations by increased bone resorption or whether there is also a direct (positive) effect of 1,25(OH)2D3 on bone homeostasis. Indeed, 1,25(OH)2D3 can stimulate bone resorption by increasing the expression of osteoclastogenic factors (for example, receptor activator of nuclear factor κB ligand) in osteoblasts. There is, however, increasing evidence that 1,25(OH)2D3-mediated signaling has a defined, albeit nonessential function in regulating the activity of chondrocytes and osteoblasts during endochondral bone formation and bone homeostasis. A first indication is the observation that aberrant growth plate development in VDR-deficient mice appears before the onset of hypocalcemia and that a high calcium diet corrected all aspects of the phenotype of CYP27B1-deficient mice, except long bone growth.1.Bouillon R. Carmeliet G. Verlinden L. et al.Vitamin D and human health: lessons from vitamin D receptor null mice.Endocr Rev. 2008; 29: 726-776Crossref PubMed Scopus (1182) Google Scholar Genetic evidence is provided by the phenotype of mice lacking VDR expression in chondrocytes.9.Masuyama R. Stockmans I. Torrekens S. et al.Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts.J Clin Invest. 2006; 116: 3150-3159Crossref PubMed Scopus (235) Google Scholar These mice display a normal growth plate, but develop a transient phenotype of increased bone volume postnatally, likely due to decreased expression of the angiogenic factor vascular endothelial growth factor and the osteoclastogenic factor receptor activator of nuclear factor κB ligand in chondrocytes. In addition, VDR signaling in chondrocytes increases, by an unidentified mechanism, the expression of the phosphaturic factor FGF23 in osteoblasts. FGF23 signals to the kidney where it reduces phosphate reabsorption and CYP27B1 activity. Although not completely identical, most of these effects are also observed when CYP27B1 is deleted in chondrocytes, supporting a physiological function for an autocrine/paracrine vitamin D pathway in developing chondrocytes.10.Naja R.P. Dardenne O. Arabian A. et al.Chondrocyte-specific modulation of CYP27B1 expression supports a role for local synthesis of 1,25-dihydroxyvitamin D3 in growth plate development.Endocrinology. 2009; 150: 4024-4032Crossref PubMed Scopus (73) Google Scholar These data indicate that 1,25(OH)2D3-mediated signaling in chondrocytes is necessary for timely vascular invasion and osteoclastogenesis during bone growth and that this pathway is required for proper endocrine action of bone in phosphate homeostasis. The function of VDR in osteoblasts seems to be modulated as a function of the differentiation stage of the cells. Indeed, VDR activation in pre-osteoblasts suppresses bone formation as calvarial osteoblasts from VDR null mice displayed enhanced osteogenesis in vitro,11.Sooy K. Sabbagh Y. Demay M.B. Osteoblasts lacking the vitamin D receptor display enhanced osteogenic potential in vitro.J Cell Biochem. 2005; 94: 81-87Crossref PubMed Scopus (67) Google Scholar whereas the expression of a VDR transgene in mature osteoblasts results in increased bone mass.12.Gardiner E.M. Baldock P.A. Thomas G.P. et al.Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage.FASEB J. 2000; 14: 1908-1916Crossref PubMed Scopus (139) Google Scholar Selective deletion of VDR from the parathyroid glands causes a doubling of PTH levels and increased bone turnover without overall negative effects on bone.13.Meir T. Levi R. Lieben L. et al.Deletion of the vitamin D receptor specifically in the parathyroid demonstrates a limited role for the VDR in parathyroid physiology.Am J Physiol Renal Physiol. 2009; 297: F1192-F1198Crossref PubMed Scopus (64) Google Scholar This indicates that the calcium-sensing parathyroid gland is a direct target for vitamin D action and thus may contribute to the secondary hyperparathyroidism in chronic renal failure. Taken together, the major function of 1,25(OH)2D3/VDR signaling is to guarantee normal serum Ca2+ levels, but this pathway also directly affects the homeostasis and endocrine function of bone. Several large-scale epidemiological studies have shown an inverse correlation between exposure to sunlight (the major source of vitamin D) or vitamin D intake and risk of colorectal, breast, and prostate cancer. However, the fact that people in sunny Southern Europe have a lower vitamin D status than those in Northern Europe indicates that sun exposure and diet are not such good indicators for the vitamin D status.14.van der Wielen R.P. Lowik M.R. van den B.H. et al.Serum vitamin D concentrations among elderly people in Europe.Lancet. 1995; 346: 207-210Abstract PubMed Google Scholar Moreover, the vitamin D content in food is highly variable. Therefore, prospective studies using serum 25(OH)D3 levels to assess vitamin D status are more accurate to study the relationship between 25(OH)D3 status and cancer incidence. A recent meta-analysis of the association between serum 25(OH)D3 and colorectal cancer risk15.Yin L. Grandi N. Raum E. et al.Meta-analysis: longitudinal studies of serum vitamin D and colorectal cancer risk.Aliment Pharmacol Ther. 2009; 30: 113-125Crossref PubMed Scopus (163) Google Scholar supports the conclusion that lower serum 25(OH)D3 levels are inversely associated with colorectal cancer and confirmed another meta-analysis,16.Gorham E.D. Garland C.F. Garland F.C. et al.Optimal vitamin D status for colorectal cancer prevention: a quantitative meta analysis.Am J Prev Med. 2007; 32: 210-216Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar which reported that a 51% lower incidence of colorectal cancer was associated with the highest serum 25(OH)D3 quintile (82 nmol/l) compared to the lowest quintile (≤30 nmol/l). The association between breast cancer risk and 25(OH)D3 status has been studied among others in the United States17.Bertone-Johnson E.R. Chen W.Y. Holick M.F. et al.Plasma 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D and risk of breast cancer.Cancer Epidemiol Biomarkers Prev. 2005; 14: 1991-1997Crossref PubMed Scopus (306) Google Scholar as well as in Europe.18.Colston K.W. Vitamin D and breast cancer risk.Best Pract Res Clin Endocrinol Metab. 2008; 22: 587-599Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar The UK study showed that women with 25(OH)D3 levels 50 nmol/l. However, other epidemiological studies failed to show an inverse correlation between 25(OH)D3 circulating levels and breast cancer incidence. Similarly the association between 25(OH)D3 status and prostate cancer19.Travis R.C. Crowe F.L. Allen N.E. et al.Serum vitamin D and risk of prostate cancer in a case-control analysis nested within the European Prospective Investigation into Cancer and Nutrition (EPIC).Am J Epidemiol. 2009; 169: 1223-1232Crossref PubMed Scopus (70) Google Scholar is less clear than for colorectal cancer. Moreover, the association between overall cancer outcome and 25(OH)D3 remains ambiguous. In the large WHI, combined treatment of vitamin D3 (400 IU per day) and calcium did not decrease colon cancer risk,20.Wactawski-Wende J. Kotchen J.M. Anderson G.L. et al.Calcium plus vitamin D supplementation and the risk of colorectal cancer.N Engl J Med. 2006; 354: 684-696Crossref PubMed Scopus (790) Google Scholar whereas in a much smaller randomized control trial 1100 IU per day of vitamin D3 along with calcium decreased the overall cancer risk.21.Lappe J.M. Travers-Gustafson D. Davies K.M. et al.Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial.Am J Clin Nutr. 2007; 85: 1586-1591PubMed Google Scholar Therefore, additional intervention studies are needed to investigate the effect of vitamin D supplementation alone on cancer risk. The conversion from 25(OH)D3 to 1,25(OH)2D3 by CYP27B1 is not restricted to the kidney but can also take place in other cell types such as keratinocytes, immune, or bone cells but also in breast, prostate, or colon cancer cells, raising the possibility that locally synthesized 1,25(OH)2D3 behaves in an autocrine or paracrine way. Indeed, the kidney tubular cell is the only normal cell that can export its 1,25(OH)2D3. The same CYP27B1 enzyme can, however, be activated by totally different stimuli (for example, cytokines stimulating monocyte CYP27B1) and generate 1,25(OH)2D3 with mainly autocrine and paracrine activity. Therefore, much like T4-T3 for the thyroid system, 25(OH)D3 can be a precursor for local action. However, the relative function of local versus systemic production of 1,25(OH)2D3 is yet poorly defined. 1,25(OH)2D3 exerts its biological effects by binding to the VDR and after dimerization with retinoid X receptor, the complex binds to vitamin D responsive elements in the promoter region of target genes, recruits coregulators and the transcriptional preinitiation complex to initiate target gene transcription. During the last decades it became clear that supraphysiological doses of 1,25(OH)2D3 have potent growth-inhibitory and pro-differentiating effects on a large variety of cell types. Moreover, a physiological function for 1,25(OH)2D3 in the regulation of normal cell growth is suggested by the finding that in VDR−/− mice hyperproliferation in the colon and mammary gland is present. Although these mice do not develop cancer spontaneously, they are predisposed to cancer when challenged with carcinogenic agents.1.Bouillon R. Carmeliet G. Verlinden L. et al.Vitamin D and human health: lessons from vitamin D receptor null mice.Endocr Rev. 2008; 29: 726-776Crossref PubMed Scopus (1182) Google Scholar 1,25(OH)2D3 inhibits cell growth by hampering the transition from the G1 to the S phase of the cell cycle (Figure 1). 1,25(OH)2D3 accumulates cells in the G1 phase by targeting cell-cycle regulators in a direct or indirect way. As such the cyclin-dependent kinase inhibitor p21(waf1/cip1) is directly activated by 1,25(OH)2D3 through a vitamin D responsive element in its promoter region (Figure 1). In addition to an increase of cyclin-dependent kinase inhibitor p27(kip1) protein levels due to posttranslational modifications, it has been recently shown that 1,25(OH)2D3-induced repression of the miR181 family contributes to the accumulation of p27(kip1)22.Wang X. Gocek E. Liu C.G. et al.MicroRNAs181 regulate the expression of p27Kip1 in human myeloid leukemia cells induced to differentiate by 1,25-dihydroxyvitamin D3.Cell Cycle. 2009; 8: 736-741Crossref PubMed Scopus (117) Google Scholar by 1,25(OH)2D3. 1,25(OH)2D3 also decreases cyclin D1 and cyclin E expression and the activity of complexes between cyclins and cyclin-dependent kinases (Figure 1). As a result 1,25(OH)2D3 influences the phosphorylation status of the retinoblastoma pocket protein family, preventing the release of the E2F transcription factors 1, 2, 3 and keeps the inhibitory complexes of E2F4 and E2F5 with p107 and p130 pocket proteins on the promoter of cell-cycle target genes (Figure 1).23.Verlinden L. Eelen G. Van H.R. et al.1alpha,25-Dihydroxyvitamin D3-induced down-regulation of the checkpoint proteins, Chk1 and Claspin, is mediated by the pocket proteins p107 and p130.J Steroid Biochem Mol Biol. 2007; 103: 411-415Crossref PubMed Scopus (16) Google Scholar To identify new key genes that are regulated by 1,25(OH)2D3, a large number of microarray studies have been performed on different types of cancer cells. From these studies, it became clear that not one single gene or one single pathway mediates the antiproliferative effects of 1,25(OH)2D3 but that multiple pathways are involved, among which epidermal growth factor, insulin-like growth factor, transforming growth factor, prostaglandins, and Wnt-β-catenin signaling cascades.1.Bouillon R. Carmeliet G. Verlinden L. et al.Vitamin D and human health: lessons from vitamin D receptor null mice.Endocr Rev. 2008; 29: 726-776Crossref PubMed Scopus (1182) Google Scholar, 24.Deeb K.K. Trump D.L. Johnson C.S. Vitamin D signalling pathways in cancer: potential for anticancer therapeutics.Nat Rev Cancer. 2007; 7: 684-700Crossref PubMed Scopus (1039) Google Scholar The variety of target genes identified through these studies reflect the pleiotropic action of 1,25(OH)2D3 in different cellular processes next to cell-cycle progression, such as apoptosis, cellular adhesion, and oxidative stress. The presence of the VDR in tumors together with the growth-regulatory effect of 1,25(OH)2D3 opens perspectives for the use of this compound in cancer prevention and/or treatment. 1,25(OH)2D3 has been evaluated in different dose settings and in different combinations (dexamethasone, docetaxel, estramustine, mitoxantrone, and prednisone) in patients with prostate cancer. These small trials indicated that a high weekly dose of 1,25(OH)2D3 instead of daily administration was safe and combination therapies were well tolerated. In the Ascent phase III clinical trial (Novacea, South San Francisco, CA, USA) a large cohort of patients (950) with androgen-independent prostate cancer was treated either with Taxotere administered every 3 weeks (control arm) or with a weekly high dose of 1,25(OH)2D3 (DN-101) in combination with once-weekly Taxotere. Unfortunately, this trial was terminated earlier than the design of 30 weeks of treatment due to an unexplained imbalance of deaths between the treatment and control group of the trial. Worldwide research has focused on the development of structural analogs of 1,25(OH)2D3 with a clear dissociation between antiproliferative and calcemic effects. One of these analogs, TX 522 (Inecalcitol; Hybrigenics, Paris, France) is currently in phase II in patients with hormone-refractory prostate cancer. Preliminary results showed that 27 of the 31 patients treated with Inecalcitol (at different doses up to 600 μg per day) and Taxotere during 18 weeks showed a decrease in prostate-specific antigen levels of more than 30% within 3 months of initiation of treatment without any changes in calcium parameters.25.Medioni J. Deplanque G. Maurina T. et al.Dose finding and safety analysis of inecalcitol in combination with a docetaxel-prednisone regimen in hormone-refractory prostate cancer (HRPC) patients.J Clin Oncol. 2009; 155: 5151Google Scholar Exposure of skin to UVB results in immunosuppression and part of this effect is mediated through synthesis of vitamin D and activation to its active form 1,25(OH)2D3 under the influence of UVB.26.Lehmann B. The vitamin D3 pathway in human skin and its role for regulation of biological processes.Photochem Photobiol. 2005; 81: 1246-1251Crossref PubMed Scopus (85) Google Scholar This phenomenon hints toward a physiological function for vitamin D in immune regulation and in recent years, an increasing amount of data have become available to support such a physiological function for the vitamin D system in immune function, both in defense and in maintenance of self-tolerance. Receptors for vitamin D have been described in most cells of the immune system and many immune cells express the hydroxylase enzymes that can provide hydroxylation of vitamin D on the 25 and the 1α site (CYP27A1/CYP2R1 and CYP27B1, respectively).27.Baeke F. van Etten E. Gysemans C. et al.Vitamin D signaling in immune-mediated disorders: evolving insights and therapeutic opportunities.Mol Aspects Med. 2008; 29: 376-387Crossref PubMed Scopus (78) Google Scholar Immune cells can thus activate 25(OH)D3 to 1,25(OH)2D3, providing within the immune system a paracrine presence of 1,25(OH)2D328.Sigmundsdottir H. Pan J. Debes G.F. et al.DCs metabolize sunlight-induced vitamin D3 to ‘program’ T cell attraction to the epidermal chemokine CCL27.Nat Immunol. 2007; 8: 285-293Crossref PubMed Scopus (475) Google Scholar (Figure 2). Secretion of 1,25(OH)2D3 by immune cells is regulated differently from its secretion by kidney cells, with immune stimuli, rather than calcemic stimuli regulating levels of 1,25(OH)2D3 in inflammatory foci. A crucial immune stimulus in the regulation of the final hydroxylation step (1α-hydroxylation by CYP27B1) is interferon-γ. However, although important, interferon-γ is not sufficient by its own, because other macrophage activators/differentiators, such as lipopolysaccharide, phorbol myristate acetate, or tumor necrosis factor-α, need to be present simultaneously to activate a complex network of signaling pathways, necessary for CYP27B1 induction. Adding to this, not only TLR4 activation, but also TLR2/1 triggering is able to induce 1α-hydroxylase in human macrophages, paralleled by induction of an antimicrobial response. Finally, also the 1,25(OH)2D3-metabolizing enzyme, CYP24A1, responsible for 24-hydroxylation, is expressed by antigen-presenting cells. 1,25(OH)2D3 affects the immune system in many different ways, but the overall picture is one of enhanced capacity of innate antibacterial defense and a more tolerogenic profile toward autoimmune phenomena. The dendritic cell (DC) and other antigen-presenting cells, such as the Langerhans cells in skin, are central to the effect of vitamin D, affecting dramatically the direction in which the immune system is skewed. Human and animal data indicate that maturation, differentiation, and function of DCs is affected, with downregulation of major histocompatibility complex class II and costimulatory molecules, such as CD40, CD80, and CD86, resulting in a tolerance-promoting phenotype. The effect of 1,25(OH)2D3 on DC phenotype and function is an integrated effect, not fully explained by prevention of differentiation as indicated by both transcriptional and translational data.29.Szeles L. Keresztes G. Torocsik D. et al.1,25-Dihydroxyvitamin D3 is an autonomous regulator of the transcriptional changes leading to a tolerogenic dendritic cell phenotype.J Immunol. 2009; 182: 2074-2083Crossref PubMed Scopus (156) Google Scholar Through these DC alterations and effects on cytokine secretion by the DC (favoring IL10 and inhibiting IL12), T-cell function is fundamentally affected, promoting development of Th2 lymphocytes within the CD4+ subset, but also affecting CD8+ CTL development.30.Adorini L. Penna G. Dendritic cell tolerogenicity: a key mechanism in immunomodulation by vitamin D receptor agonists.Hum Immunol. 2009; 70: 345-352Crossref PubMed Scopus (153) Google Scholar T lymphocytes can also be direct targets for 1,25(OH)2D3, resulting in inhibition of several cytokines, such as interferon-γ and IL-17, or stimulation of others such as IL-4.1.Bouillon R. Carmeliet G. Verlinden L. et al.Vitamin D and human health: lessons from vitamin D receptor null mice.Endocr Rev. 2008; 29: 726-776Crossref PubMed Scopus (1182) Google Scholar Importantly, 1,25(OH)2D3 affects the expression of chemokine receptors on the surface of T lymphocytes and will thus determine the target tissue where any primed T lymphocytes will migrate to.31.Yu S. Bruce D. Froicu M. et al.Failure of T cell homing, reduced CD4/CD8alphaalpha intraepithelial lymphocytes, and inflammation in the gut of vitamin D receptor KO mice.Proc Natl Acad Sci USA. 2008; 105: 20834-20839Crossref PubMed Scopus (147) Google Scholar The integrated effect of 1,25(OH)2D3 on DC and T lymphocytes provides a unique potential of this substance to generate regulator cells (Tregs). In different model systems, 1,25(OH)2D3 has been shown to induce Tregs. The nature of these Tregs differs from system to system, but their presence is clear and they appear to be stable.32.Adorini L. Giarratana N. Penna G. Pharmacological induction of tolerogenic dendritic cells and regulatory T cells.Semin Immunol. 2004; 16: 127-134Crossref PubMed Scopus (166) Google Scholar Finally, another important target cell of 1,25(OH)2D3 is the monocyte/macrophage, the cell responsible for antibacterial and antiviral defenses. Here 1,25(OH)2D3 is a crucial factor in promoting the antibacterial defense system of macrophages, by promoting secretion of antibacterial products such as cathelicidin.33.Liu P.T. Stenger S. Li H. et al.Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response.Science. 2006; 311: 1770-1773Crossref PubMed Scopus (2739) Google Scholar These immunomodulatory effects of 1,25(OH)2D3 can clearly be observed in vivo in circumstances of vitamin D deficiency both in humans and in animal models of human disease. Decreased antibacterial and antiviral defenses are obvious in vitamin D deficiency and restoration of the vitamin D-sufficient state restores immune responses. Correlations between the existence of vitamin D deficiency and higher prevalence of autoimmunity have been described for many diseases and in different populations. Indeed, many epidemiological studies have shown a clear association between low vitamin D status and autoimmune diseases, such as type 1 diabetes,34.Mathieu C. Gysemans C. Giulietti A. et al.Vitamin D and diabetes.Diabetologia. 2005; 48: 1247-1257Crossref PubMed Scopus (470) Google Scholar multiple sclerosis,35.Cantorna M.T. Vitamin D and multiple sclerosis: an update.Nutr Rev. 2008; 66: S135-S138Crossref PubMed Scopus (72) Google Scholar, 36.Munger K.L. Levin L.I. Hollis B.W. et al.Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis.JAMA. 2006; 296: 2832-2838Crossref PubMed Scopus (1326) Google Scholar and inflammatory bowel disease. Preclinical animal models have confirmed that vitamin D deficiency especially early in life enhances such autoimmune diseases. Prospective intervention studies with either vitamin D or selective VDR modulators are, however, needed to evaluate their therapeutic potential. Vitamin D-deficient rats or mice with deletion of VDR or 1α-hydroxylase develop high renin hypertension with ultimately cardiac hypertrophy. Selective VDR deficiency in cardiac myocytes can reproduce the effects.37.Gardner D.G. Glenn D. Nei W. et al.Cardiomyocyte-specific vitamin D receptor gene knockout causes cardiac hypertrophy.in: Abstract at the 14th workshop on vitamin D4–8 October 2009Google Scholar In normotensive and hypertensive Caucasian, Hispanic, or African Americans lower serum 25(OH)D levels are associated with higher blood pressure.38.Bouillon R. Vitamin D as potential baseline therapy for blood pressure control.Am J Hypertens. 2009; 22: 816Crossref PubMed Scopus (18) Google Scholar Moreover, in normotensive and hypertensive patients blood pressure is inversely correlated with plasma 1,25(OH)2D and renin concentrations.1.Bouillon R. Carmeliet G. Verlinden L. et al.Vitamin D and human health: lessons from vitamin D receptor null mice.Endocr Rev. 2008; 29: 726-776Crossref PubMed Scopus (1182) Google Scholar However, low 25(OH)D levels are also associated with nearly all aspects of the metabolic syndrome, such as obesity, insulin resistance, type 2 diabetes. The fat–vitamin D axis is further underlined by lower fat mass and resistance to diet-induced obesity in VDR null mice and a negative effect of the major fat cell hormone, leptin, on renal 1α-hydroxylase activity. However, there are only very small scale intervention studies using vitamin D or its active hormone on blood pressure or other aspects of the metabolic syndrome.39.Schmitz K.J. Skinner H.G. Bautista L.E. et al.Association of 25-hydroxyvitamin D with blood pressure in predominantly 25-hydroxyvitamin D deficient Hispanic and African Americans.Am J Hypertens. 2009; 22: 867-870Crossref PubMed Scopus (51) Google Scholar, 40.Margolis K.L. Ray R.M. Van H.L. et al.Effect of calcium and vitamin D supplementation on blood pressure: the Women's Health Initiative Randomized Trial.Hypertension. 2008; 52: 847-855Crossref PubMed Scopus (203) Google Scholar The major function of 1,25(OH)2D3/VDR signaling is to control the calcium and bone homeostasis. Data obtained in animal models of vitamin D deficiency and resistance are generally perfectly in line with observations in rare patients with such human diseases. The combined presence of 25(OH)D3-1α-hydroxylase as well as VDR in several nonclassical tissues introduced the concept of a paracrine/autocrine function for 1,25(OH)2D3 outside the calcium and bone metabolism. Moreover, the fact that 1,25(OH)2D3 was found to be capable of regulating cell differentiation and proliferation of normal (immune) cells and malignant cells created new perspectives for this molecule. The extensive studies in cells, tissues, and whole animals with modified vitamin D action have generated clear hypothesis to evaluate the function of vitamin D (deficiency) on a whole spectrum of human diseases. A poor vitamin D status is linked with all major diseases causing the majority of human morbidity, such as cancer, immune diseases, metabolic syndrome, hypertension, and cardiovascular risk factors. Correction of vitamin D deficiency or insufficiency usually requires vitamin D supplementation as higher exposure to UVB light carries a risk for skin damage and carcinogenesis and as naturally vitamin D-rich food is scarce. In Europe, the vitamin D3 is preferred whereas vitamin D2 is widely added to food or used as supplement in North America. The natural D3 compound is probably slightly more potent than vitamin D2 especially when given intermittently. The optimal daily dosage is highly debated but most randomized controlled trials used 600–1000 IU of vitamin D3 per day and such therapy was found to decrease the risk of fractures and falls in the elderly. The serum 25(OH)D concentration should certainly exceed 20 ng/ml (or 50 nmol/l) and probably even 30 ng/ml (80 nmol/l) in patients with impaired renal function. As the mean level of 25(OH)D around the world is only about 20 ng/ml41.Hagenau T. Vest R. Gissel T.N. et al.Global vitamin D levels in relation to age, gender, skin pigmentation and latitude: an ecologic meta-regression analysis.Osteoporos Int. 2009; 20: 133-140Crossref PubMed Scopus (297) Google Scholar and not higher than 30 ng/ml in the US NHANES IV participants,42.Ginde A.A. Liu M.C. Camargo Jr, C.A. Demographic differences and trends of vitamin D insufficiency in the US population, 1988–2004.Arch Intern Med. 2009; 169: 626-632Crossref PubMed Scopus (840) Google Scholar it can be easily assumed that a very large part of the human population and thus even billions of people is at least mildly vitamin D deficient. Patients with chronic renal failure have a greater than normal risk for bone diseases, cancer, immune disorders, as well as increased cardiovascular mortality. They also have impaired production of 1,25(OH)2D3 and frequently combine this with 25(OH)D3 deficiency. A number of observational studies have shown that treatment of hemodialysis patients with active vitamin D analogs decreases (cardiovascular) mortality. The overall contribution of combined impaired 25(OH)D3–1,25(OH)2D3 status on morbidity and mortality in patients with chronic renal failure may be substantial but needs further randomized control trials. We thank Femke Baeke and Lieve Verlinden for the creation of the figures and Evelyne van Etten for editorial assitance. CM is a clinical researcher for the Fund for Scientific Research (FWO-Vlaanderen). The work in the author's laboratories was supported by FWO Vlaanderen (FWO G.0587.09, G.0553.06, G.0552.06), the University of Leuven (GOA/04/10, EF/05/007), and Belgium Program on Interuniversity Poles of Attraction (P6/34).