Annemieke Verstuyf

Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice

The vitamin D endocrine system is essential for calcium and bone homeostasis. The precise mode of action and the full spectrum of activities of the vitamin D hormone, 1,25-dihydroxyvitamin D [1,25-(OH)(2)D], can now be better evaluated by critical analysis of mice with engineered deletion of the vitamin D receptor (VDR). Absence of a functional VDR or the key activating enzyme, 25-OHD-1alpha-hydroxylase (CYP27B1), in mice creates a bone and growth plate phenotype that mimics humans with the same congenital disease or severe vitamin D deficiency. The intestine is the key target for the VDR because high calcium intake, or selective VDR rescue in the intestine, restores a normal bone and growth plate phenotype. The VDR is nearly ubiquitously expressed, and almost all cells respond to 1,25-(OH)(2)D exposure; about 3% of the mouse or human genome is regulated, directly and/or indirectly, by the vitamin D endocrine system, suggesting a more widespread function. VDR-deficient mice, but not vitamin D- or 1alpha-hydroxylase-deficient mice, and man develop total alopecia, indicating that the function of the VDR and its ligand is not fully overlapping. The immune system of VDR- or vitamin D-deficient mice is grossly normal but shows increased sensitivity to autoimmune diseases such as inflammatory bowel disease or type 1 diabetes after exposure to predisposing factors. VDR-deficient mice do not have a spontaneous increase in cancer but are more prone to oncogene- or chemocarcinogen-induced tumors. They also develop high renin hypertension, cardiac hypertrophy, and increased thrombogenicity. Vitamin D deficiency in humans is associated with increased prevalence of diseases, as predicted by the VDR null phenotype. Prospective vitamin D supplementation studies with multiple noncalcemic endpoints are needed to define the benefits of an optimal vitamin D status.

Vitamin D: Metabolism, Molecular Mechanism of Action, and Pleiotropic Effects

1,25-Dihydroxvitamin D3 [1,25(OH)2D3] is the hormonally active form of vitamin D. The genomic mechanism of 1,25(OH)2D3 action involves the direct binding of the 1,25(OH)2D3 activated vitamin D receptor/retinoic X receptor (VDR/RXR) heterodimeric complex to specific DNA sequences. Numerous VDR co-regulatory proteins have been identified, and genome-wide studies have shown that the actions of 1,25(OH)2D3 involve regulation of gene activity at a range of locations many kilobases from the transcription start site. The structure of the liganded VDR/RXR complex was recently characterized using cryoelectron microscopy, X-ray scattering, and hydrogen deuterium exchange. These recent technological advances will result in a more complete understanding of VDR coactivator interactions, thus facilitating cell and gene specific clinical applications. Although the identification of mechanisms mediating VDR-regulated transcription has been one focus of recent research in the field, other topics of fundamental importance include the identification and functional significance of proteins involved in the metabolism of vitamin D. CYP2R1 has been identified as the most important 25-hydroxylase, and a critical role for CYP24A1 in humans was noted in studies showing that inactivating mutations in CYP24A1 are a probable cause of idiopathic infantile hypercalcemia. In addition, studies using knockout and transgenic mice have provided new insight on the physiological role of vitamin D in classical target tissues as well as evidence of extraskeletal effects of 1,25(OH)2D3 including inhibition of cancer progression, effects on the cardiovascular system, and immunomodulatory effects in certain autoimmune diseases. Some of the mechanistic findings in mouse models have also been observed in humans. The identification of similar pathways in humans could lead to the development of new therapies to prevent and treat disease.

Identification and immune regulation of 25-hydroxyvitamin D-1-alpha-hydroxylase in murine macrophages

Receptors for 1,25(OH)2vitaminD3 are found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to examine the presence and nature of the enzyme responsible for final activation of the molecule, 1‐α‐hydroxylase, in murine macrophages and to analyse its regulation and possible role in the immune system. Peritoneal macrophages from C57Bl/6 mice were incubated with lipopolysaccharide (LPS; 100 μg/ml), interferon‐gamma (IFN‐γ; 500 U/ml) or a combination of both. By quantitative reverse transcriptase‐polymerase chain reaction, using primers based on the murine renal cDNA sequence, low levels of 1‐α‐hydroxylase mRNA were detected in freshly isolated cells (18 ± 7 × 10−6 copies/β‐actin copies). Analysis of the cDNA sequence of the gene revealed identical coding sequences for the macrophage and renal enzymes. mRNA levels rose three‐fold with LPS (NS), but a six‐fold increase was seen after IFN‐γ stimulation (P < 0·05). Combining LPS and IFN‐γ did not result in a major additional increase, but addition of cyclosporin A further increased levels 2·5‐fold both in IFN‐γ‐ and combination‐stimulated cells (P < 0·05). Time course analysis revealed that up‐regulation of 1‐α‐hydroxylase was a late phenomenon, preceded by the up‐regulation of activating macrophage products such as IL‐1 and tumour necrosis factor‐alpha. Finally, a defect in 1‐α‐hydroxylase up‐regulation by immune stimuli was found in autoimmune non‐obese diabetic mice. In conclusion, we propose that the up‐regulation of 1‐α‐hydroxylase in activated macrophages, resulting in the synthesis of 1,25(OH)2D3, might be a negative feedback loop in inflammation. A defect in this system might be an additional element in tipping the balance towards autoimmunity.

Vitamin D: a pleiotropic hormone

The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) 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)(2)D(3). 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 D(3) to 1,25(OH)(2)D(3) by the enzyme 25(OH)D(3)-1alpha-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)(2)D(3). Moreover, it has been demonstrated that 1,25(OH)(2)D(3) 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)(2)D(3), these newly discovered functions open perspectives for the use of 1,25(OH)(2)D(3) 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).

ACTION OF 1,25(OH)2D3 ON THE CELL CYCLE GENES, CYCLIN D1, P21 AND P27 IN MCF-7 CELLS

1,25(OH)2D3 is a known growth inhibitor and differentiation inducer of several cancer cell lines. To establish the molecular mechanism of 1,25(OH)2D3 as an antiproliferating agent, its effect on proliferation and gene regulation was studied in human breast cancer MCF-7 cells. 1,25(OH)2D3 inhibited cell proliferation dose dependently through G1 arrest. Cyclin D1 transcription levels decreased rapidly in 1,25(OH)2D3-treated cells while protein levels only decreased after 72 h of treatment. Transcription levels of p21 and p27 were upregulated with chronologically consistent changes in cell cycle distribution. Experiments with TGF-beta neutralising antibodies revealed that the largest effect of 1,25(OH)2D3 on cell proliferation is likely due to a TGF-beta independent mechanism of action. The cell cycle regulatory genes, cyclin D1 and p27, are probably involved herein as their expression was not affected by the presence of neutralising antibodies. However, upregulation of p21 was completely abrogated. Therefore, the TGF-beta signalling pathway is thought to be responsible for p21 upregulation.

Vitamin D metabolism and action

Vitamin D3 is a secosteroid derived either from photosynthesis, whereby 7-dehydrocholesterol is transformed into previtamin D by short-wave ultraviolet (UVB) light, or from nutritional origin. Nutritional vitamin D3 (from most natural sources) or D 2 (mostly from pharmaceutical origin) in fact also originates from photosynthesis in living organisms. The vitamin D content of most food products is rather low (Table 1), with the exception of some fatty fish. Moreover, its stated content has not always been verified by accurate up-to-date measurements. Even food supplemented with vitamin D does not always contain the stated concentrations, as gross errors have been detected even in critical baby milk products [1]. The skin synthesis of vitamin D is therefore considered the common source of vitamin D in man and probably also in most vertebrates (from reptiles to mammals; see below). The photosynthesis requires the presence of 7-dehydrocholesterol, which requires de novo synthesis of cholesterol (Fig. 1) in the epidermis. If the enzyme 7-dehydrocholesterolD7-reductase is overactive, as seems to be the case in cats, UV light cannot activate vitamin D synthesis so that vitamin D 3 is a true vitamin for this species [2]. The congenital absence of a functional 7-dehydrocholesterolD7-reductase enzyme is responsible for a serious disease called Smith–Lemli– Opitz syndrome, in which deficiency of cholesterol and/ or excess 7-dehydrocholesterol causes abnormal brain development and other soft and hard tissue malformations (e.g. syndactyly and cleft palate) [3,4]. Previtamin D3 slowly equilibrates with vitamin D3, a process thought to be non-enzymatic and highly temperaturedependent. However, more recent indirect data support the idea that its thermal isomerization might be accelerated in vivo by entrapment in the lipid bilayers of the keratinocytes [5]. Additional or excess UV light can degrade (pre)vitamin D into lumisterol or tachysterol and other sterols that are largely devoid of vitamin D activity. This phenomenon is largely responsible for avoiding ‘natural’ vitamin D intoxication. Vitamin D from nutritional origin is transported via chylomicrons from the intestine via lymph veins to the liver, whereas skin-produced vitamin D is probably transported mainly via DBP, an albumin-like plasma carrier protein. The liver but also some other tissues metabolise vitamin D3 into 25-OHD, probably by the enzyme CYP27. This sterol-27-hydroxylase (EC 1.14.13.15) is a multifunctional enzyme responsible for 27-hydroxylation of cholesterol and bile acid precursors (e.g. 5b-cholestane-3 a,7a12a-triol) as main function (rate of metabolism) and 25or 24-hydroxylation of vitamin D3 or D2, respectively, as minor function [6]. The enzyme is encoded by a gene located on the human chromosome 2, but its regulation and promoter structure have not been studied in detail. The enzyme is, however, found in many tissues outside the liver (e.g. duodenum, adrenal gland, lung, macrophages). A mouse KO model is not yet available, but a human disease, cerebrotendinous xanthomatosis, is due to a genetic abnormality or deficiency of CYP27 [7]. The disease is mainly characterized by neurological abnormalities, believed to be due to cholestanol overloading, premature atherosclerosis and osteoporotic bone fractures (with Osteoporos Int (1998) Suppl. 8:S13–S19 ß 1998 European Foundation for Osteoporosis and National Osteoporosis Foundation Osteoporosis International

The vitamin D receptor gene FokI polymorphism: Functional impact on the immune system

1α,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) has important effects on the growth and function of multiple cell types. These pleiotropic effects of 1,25(OH)2D3 are mediated through binding to the vitamin D receptor (VDR). Several polymorphisms of the human VDR gene have been identified, with the FokI polymorphism resulting in VDR proteins with different structures, a long f‐VDR or a shorter F‐VDR. The aim of this study was to investigate the functional consequences of the FokI polymorphism in immune cells. In transfection experiments, the presence of the shorter F‐VDR resulted in higher NF‐κB‐ and NFAT‐driven transcription as well as higher IL‐12p40 promoter‐driven transcription. Marginal differences were observed for AP‐1‐driven transcription, and no differential effects were observed for transactivation of a classical vitamin D‐responsive element. Concordantly, in human monocytes and dendritic cells with a homozygous short FF VDR genotype, expression of IL‐12 (mRNA and protein) was higher than in cells with a long ff VDR genotype. Additionally, lymphocytes with a short FF VDR genotype proliferated more strongly in response to phytohemagglutinin. Together, these data provide the first evidence that the VDR FokI polymorphism affects immune cell behavior, with a more active immune system for the short F‐VDR, thus possibly playing a role in immune‐mediated diseases.

In Vitro and In Vivo Analysis of the Immune System of Vitamin D Receptor Knockout Mice

Immune cells carry receptors for 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3; vitamin D receptor (VDR)] and individuals with severe vitamin D deficiency have immune abnormalities. The aim of this study was to investigate the role of vitamin D in the immune system by studying VDR‐knockout (VDR‐KO) mice. VDR‐KO mice had the same metabolic phenotype as rachitic animals with severe hypocalcemia. Leukocytosis, lymphocyte subset composition in different immune organs, and splenocyte proliferation to several stimuli were normal, except for a lower response to anti‐CD3 stimulation (simulation index [SI] of 13 ± 4 vs. 24 ± 9 in wild‐type mice; p < 0.01). Macrophage chemotaxis was impaired (41 ± 19% vs. 60 ± 18% in wild‐type mice; p < 0.01) but phagocytosis and killing were normal. In vivo rejection of allogeneic (31 ± 12 days vs. 45 ± 26 days of survival in wild‐type mice, NS) or xenogeneic (10 ± 2 days vs. 16 ± 9 days of survival in wild‐type mice, NS) islet grafts was comparable with wild‐type mice. Surprisingly, VDR‐KO mice were protected from low‐dose streptozotocin‐induced diabetes mellitus (LDSDM; 5% vs. 65% in wild‐type mice; p < 0.001). Correcting hypocalcemia by use of lactose‐rich or polyunsaturated fat‐rich diets fully restored the immune abnormalities in vitro and the sensitivity to diabetes in vivo. On the other hand, treatment with 1,25(OH)2D3 protected wild‐type mice against diabetes but did not protect normocalcemic VDR‐KO mice. We conclude that immune defects observed in VDR‐KO mice are an indirect consequence of VDR disruption because they can be restored by calcium homeostasis normalization. This study proves that although 1,25(OH)2D3 is a pharmacologic and probably a physiological immunomodulator, its immune function is redundant. Moreover, we confirm the essential role of calcium in the immune system.

Analogs of 1,25-dihydroxyvitamin D3 as dose-reducing agents for classical immunosuppressants.

BACKGROUND Most immunosuppressants have a narrow margin between efficacy and side effects. A major goal in the development of immunomodulatory strategies is the discovery of combinations of drugs exerting synergistic immunomodulatory effects. The active form of vitamin D, 1,25(OH)2D3, is an immunomodulator that interacts with T cells but mainly targets antigen-presenting cells. We have demonstrated synergism between 1,25(OH)2D3 and cyclosporine, rapamycin, and FK506. The aim of this study was to investigate whether this synergism could be observed with other immunosuppressants (mycophenolate mofetil, leflunomide, and the methylxanthine A802715) and whether analogs of 1,25(OH)2D3 share this synergistic capacity in vivo. METHODS In vitro, the median effect analysis was applied to the inhibition of phytohemagglutinin A-induced lymphocyte proliferation. In vivo, synergism between analogs of 1,25(OH)2D3 and cyclosporine or mycophenolate mofetil was evaluated in experimental autoimmune encephalomyelitis. RESULTS In vitro, all combinations with 1,25(OH)2D3 were synergistic. The strongest synergism was seen with the inhibitors of interleukin 2 secretion, cyclosporine and FK506 (indexes 0.16 and 0.27, respectively). The weakest synergism was observed in combinations using A802715, a second-signal inhibitor (index 0.52), or the nucleotide synthesis inhibitor mycophenolate mofetil (index 0.43). In vivo, analogs of 1,25(OH)2D3 share the in vitro-observed synergism with 1,25(OH)2D3. Moreover, the differences in synergism with different immunomodulators were also present in vivo, where the best synergism was again seen in combination with cyclosporine (up to 100% paralysis protection). CONCLUSIONS These data confirm that 1,25(OH)2D3 and its analogs are potent dose-reducing drugs for other immunomodulators, making them potentially interesting for clinical use in autoimmunity and transplantation.

Human T lymphocytes are direct targets of 1,25-dihydroxyvitamin D3 in the immune system

Besides its actions on minerals and bone, the bioactive vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has important immunomodulatory properties. Within the immune system, dendritic cells represent key targets for this hormone and 1,25(OH)2D3-induced changes in their phenotype and function ultimately affects T lymphocytes. However, the presence of vitamin D receptors (VDR) in activated T cells proposes additional mechanisms for 1,25(OH)2D3 to directly regulate T cell responses. Here, we investigated the expression and kinetics of vitamin D-related genes in human activated T lymphocytes. Different activation stimuli elicited increased VDR- and 1-alpha-hydroxylase expression, with a highly similar kinetic pattern. Addition of 1,25(OH)2D3 effectively triggered VDR signaling, as evidenced by 24-hydroxylase induction, but only when introduced to T lymphocytes expressing high levels of VDR. This enhanced degree of VDR signaling correlated with a stronger inhibition of cytokines (IFN-gamma, IL-10) and modulation of homing receptor expression (CCR10, CLA) in long-term T cell cultures. Importantly, chronic 1,25(OH)2D3-exposure further amplified VDR signaling and the concomitant T cell modulating effects. In conclusion, we validate T cells as direct targets for 1,25(OH)2D3 and provide this optimized in vitro model to improve our understanding of the role of vitamin D as a direct regulator of T cell responses.