1,25-Dihydroxvitamin D₃[1,25(OH)₂D₃] is the hormonally active form of vitamin D. The genomic mechanism of 1,25(OH)₂D₃action involves the direct binding of the 1,25(OH)₂D₃activated 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)₂D₃involve 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)₂D₃including 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.

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)₂D], 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-1α-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)₂D 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 1α-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.

Metabolic switches in various immune cell subsets enforce phenotype and function. In the present study, we demonstrate that the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), induces human monocyte-derived tolerogenic dendritic cells (DC) by metabolic reprogramming. Microarray analysis demonstrated that 1,25(OH)2D3 upregulated several genes directly related to glucose metabolism, tricarboxylic acid cycle (TCA), and oxidative phosphorylation (OXPHOS). Although OXPHOS was promoted by 1,25(OH)2D3, hypoxia did not change the tolerogenic function of 1,25(OH)2D3-treated DCs. Instead, glucose availability and glycolysis, controlled by the PI3K/Akt/mTOR pathway, dictate the induction and maintenance of the 1,25(OH)2D3-conditioned tolerogenic DC phenotype and function. This metabolic reprogramming is unique for 1,25(OH)2D3, because the tolerogenic DC phenotype induced by other immune modulators did not depend on similar metabolic changes. We put forward that these metabolic insights in tolerogenic DC biology can be used to advance DC-based immunotherapies, influencing DC longevity and their resistance to environmental metabolic stress.

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.

Monocytes express 1α‐hydroxylase, the enzyme responsible for final hydroxylation of vitamin D₃, in response to IFNγ and CD14/TLR4 activation. Cross‐talk between the JAK‐STAT, the NF‐κB, and the p38 MAPK pathways is necessary, and direct binding of C/EBPβ to its recognition sites in the promoter of the 1α‐hydroxylase gene is a prerequisite. Introduction: The activated form of vitamin D₃, 1,25(OH)₂D₃, known for its action in bone and mineral homeostasis, has important immunomodulatory effects. 1,25(OH)₂D₃ modulates the immune system through specific nuclear receptors, whereas macrophages produce 1,25(OH)₂D₃. In monocytes, the expression of 1α‐hydroxylase, the enzyme responsible for final hydroxylation of vitamin D₃, is regulated by immune stimuli. The aim of this study was to elucidate the intracellular pathways through which interferon (IFN)γ and Toll‐like receptor (TLR) modulation regulate expression of 1α‐hydroxylase in monocytes/macrophages. Materials and Methods: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated with IFNγ (12.5 U/ml) and/or lipopolysaccharide (LPS; 100 ng/ml) for 48 h. The following inhibitors were used: janus kinase (JAK) inhibitor AG490 (50 μM), NF‐κB inhibitor sulfasalazine (0.25 mM), p38 mitogen‐activated protein kinase (MAPK) inhibitor SB203580 (5 μM). 1α‐hydroxylase mRNA expression was monitored by qRT‐PCR. Phosphorylation of transcription factors was studied by Western blotting. Transfection of mutated or deletion promoter constructs, cloned in the pGL3‐luciferase reporter plasmid, were performed in the RAW264.7 cell line. Cells were stimulated with IFNγ (100 U/ml) and LPS (100 μg/ml), and promoter activity was studied. Binding of signal transducer and activator of transcription (STAT)1α, NF‐κB, and C/EBPβ to their respective binding sites in the promoter was analyzed by gel shift assays. Results: 1α‐hydroxylase mRNA expression in monocytes is synergistically induced by IFNγ and CD14/TLR4 ligation and paralleled by 1,25(OH)₂D₃ production. This induction requires the JAK‐STAT, NF‐κB, and p38 MAPK pathways. Each of them is essential, because blocking individual pathways is sufficient to block 1α‐hydroxylase expression (JAK inhibitor, 60% inhibition, p < 0.01; NF‐κB inhibitor, 70% inhibition, p < 0.05; p38 MAPK inhibitor, 95% inhibition, p < 0.005). In addition, we show the involvement of the p38 MAPK pathway in phosphorylation of C/EBPβ. Direct binding of C/EBPβ to its recognition sites in the 1α‐hydroxylase promoter is necessary to enable its immune‐stimulated upregulation. Conclusion: IFNγ and CD14/TLR4 binding regulate expression of 1α‐hydroxylase in monocytes in a synergistic way. Combined activation of the JAK‐STAT, p38 MAPK, and NF‐κB pathways is necessary, with C/EBPβ most probably being the essential transcription factor controlling immune‐mediated transcription.