Vitamin D and Lumisterol Hydroxyderivatives Can Act on Liver X Receptors (LXRs)

Abstract New pathways of vitamin D3 (D3) activation initiated by CYP11A1 and involving other CYPs have been discovered. At least 15 hydroxyderivatives, including 20(OH)D3 as the major product, are generated by these pathways (1,2) with some being present in human serum, epidermis, and pig adrenals. CYP11A1 can also metabolize 7-dehydrocholesterol to produce 7-dehydropregnenolone, which can be further modified by steroidogenic enzymes generating Δ7-steroids (1,2). Lastly, CYP11A1 and CYP27A1 act on lumisterol (L3) producing at least 9 biologically active derivatives (1,2). Thus, new pathways generating a large number of biologically active secosteroids and lumisterol-derivatives have now been described. These compounds interact with the vitamin D receptor (VDR), retinoic acid receptors (RORs) α and γ, and the aryl hydrocarbon receptor (AhR)(1). These findings challenge dogmas that lumisterol is biologically inactive and that 1,25(OH)2D3 is the only active form of D3 exerting its effects exclusively through interaction with the VDR. In view of the above and since liver X receptors (LXRs) can be activated by oxysterols, we investigated the interactions of novel products of L3 and D3 metabolism with LXRs. Molecular docking, using crystal structures of the ligand binding domains (LBDs) of LXRα and β, revealed high docking scores for L3 and D3 hydroxymetabolites, like those of the natural ligands, predicting good receptor binding. RNA sequencing of murine dermal fibroblasts stimulated with D3-hydroxyderivatives revealed LXR as the second major nuclear receptor signaling pathway for several D3-hydroxyderivatives, including 1,25(OH)2D3. The involvement of LXRs was validated by the induction of several genes downstream of LXR. Furthermore, L3 and D3-hydroxyderivatives activated an LXR-response element (LXRE)-driven reporter in CHO cells and human keratinocytes. For keratinocytes, enhanced expression of LXR target genes was also observed supporting the involvement of LXR. Importantly, L3 and D3 derivatives showed high affinity binding to the LBD of the LXRα and β in LanthaScreen TR-FRET LXRα and β coactivator assays. The majority of metabolites functioned as LXRα/β agonists; however, 1,20,25(OH)3D3, 1,25(OH)2D3, 1,20(OH)2D3 and 25(OH)D3 acted as inverse agonists of LXRα, but as agonists of LXRβ. Molecular dynamics simulations performed for selected compounds, including 1,25(OH)2D3, 1,20(OH)2D3, 25(OH)D3, 20(OH)D3, 20(OH)L3 and 20,22(OH)2L3, showed overlapping and different interactions with LXRs. Identification of D3 and L3 derivatives as ligands for LXRs changes the accepted paradigms on their biological role and mechanism of action. 1. Cell Biochem Biophys. 2020;78(2):165-180. 2. J Steroid Biochem Mol Biol. 2019;186:4-21.

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Vitamin D and lumisterol derivatives can act on liver X receptors (LXRs)

Scientific Reports ◽

10.1038/s41598-021-87061-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Andrzej T. Slominski ◽

Tae-Kang Kim ◽

Shariq Qayyum ◽

Yuwei Song ◽

Zorica Janjetovic ◽

...

Keyword(s):

Target Genes ◽

Human Keratinocytes ◽

Dermal Fibroblasts ◽

Liver X Receptors ◽

Binding Domains ◽

Natural Ligands ◽

Enhanced Expression ◽

X Receptors ◽

Dynamics Simulations ◽

Derivatives Of

AbstractThe interactions of derivatives of lumisterol (L3) and vitamin D3 (D3) with liver X receptors (LXRs) were investigated. Molecular docking using crystal structures of the ligand binding domains (LBDs) of LXRα and β revealed high docking scores for L3 and D3 hydroxymetabolites, similar to those of the natural ligands, predicting good binding to the receptor. RNA sequencing of murine dermal fibroblasts stimulated with D3-hydroxyderivatives revealed LXR as the second nuclear receptor pathway for several D3-hydroxyderivatives, including 1,25(OH)2D3. This was validated by their induction of genes downstream of LXR. L3 and D3-derivatives activated an LXR-response element (LXRE)-driven reporter in CHO cells and human keratinocytes, and by enhanced expression of LXR target genes. L3 and D3 derivatives showed high affinity binding to the LBD of the LXRα and β in LanthaScreen TR-FRET LXRα and β coactivator assays. The majority of metabolites functioned as LXRα/β agonists; however, 1,20,25(OH)3D3, 1,25(OH)2D3, 1,20(OH)2D3 and 25(OH)D3 acted as inverse agonists of LXRα, but as agonists of LXRβ. Molecular dynamics simulations for the selected compounds, including 1,25(OH)2D3, 1,20(OH)2D3, 25(OH)D3, 20(OH)D3, 20(OH)L3 and 20,22(OH)2L3, showed different but overlapping interactions with LXRs. Identification of D3 and L3 derivatives as ligands for LXRs suggests a new mechanism of action for these compounds.

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Key Vitamin D Target Genes with Functions in the Immune System

Nutrients ◽

10.3390/nu12041140 ◽

2020 ◽

Vol 12 (4) ◽

pp. 1140 ◽

Cited By ~ 6

Author(s):

Oona Koivisto ◽

Andrea Hanel ◽

Carsten Carlberg

Keyword(s):

Vitamin D ◽

Adaptive Immunity ◽

Vitamin D3 ◽

Target Genes ◽

Mononuclear Cells ◽

Active Form ◽

Biologically Active ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Innate And Adaptive Immunity

The biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), modulates innate and adaptive immunity via genes regulated by the transcription factor vitamin D receptor (VDR). In order to identify the key vitamin D target genes involved in these processes, transcriptome-wide datasets were compared, which were obtained from a human monocytic cell line (THP-1) and peripheral blood mononuclear cells (PBMCs) treated in vitro by 1,25(OH)2D3, filtered using different approaches, as well as from PBMCs of individuals supplemented with a vitamin D3 bolus. The led to the genes ACVRL1, CAMP, CD14, CD93, CEBPB, FN1, MAPK13, NINJ1, LILRB4, LRRC25, SEMA6B, SRGN, THBD, THEMIS2 and TREM1. Public epigenome- and transcriptome-wide data from THP-1 cells were used to characterize these genes based on the level of their VDR-driven enhancers as well as the level of the dynamics of their mRNA production. Both types of datasets allowed the categorization of the vitamin D target genes into three groups according to their role in (i) acute response to infection, (ii) infection in general and (iii) autoimmunity. In conclusion, 15 genes were identified as major mediators of the action of vitamin D in innate and adaptive immunity and their individual functions are explained based on different gene regulatory scenarios.

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Common and personal target genes of the micronutrient vitamin D in primary immune cells from human peripheral blood

Scientific Reports ◽

10.1038/s41598-020-78288-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Andrea Hanel ◽

Antonio Neme ◽

Marjo Malinen ◽

Emmi Hämäläinen ◽

Henna-Riikka Malmberg ◽

...

Keyword(s):

Vitamin D ◽

Immune System ◽

Immune Cells ◽

Peripheral Blood ◽

Target Genes ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Active Form ◽

Biologically Active ◽

Expression Change

AbstractVitamin D is essential for the function of the immune system. In this study, we treated peripheral blood mononuclear cells (PBMCs) of healthy adults with the biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) using two different approaches: single repeats with PBMCs obtained from a cohort of 12 individuals and personalized analysis based on triplicates of five study participants. This identified 877 (cohort approach) and 3951 (personalized approach) genes that significantly (p < 0.05) changed their expression 24 h after 1,25(OH)2D3 stimulation. From these, 333 and 1232 were classified as supertargets, a third of which were identified as novel. Individuals differed largely in their vitamin D response not only by the magnitude of expression change but also by their personal selection of (super)target genes. Functional analysis of the target genes suggested the overarching role of vitamin D in the regulation of metabolism, proliferation and differentiation, but in particular in the control of functions mediated by the innate and adaptive immune system, such as responses to infectious diseases and chronic inflammatory disorders. In conclusion, immune cells are an important target of vitamin D and common genes may serve as biomarkers for personal responses to the micronutrient.

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The Biological Activities of Vitamin D and Its Receptor in Relation to Calcium and Bone Homeostasis, Cancer, Immune and Cardiovascular Systems, Skin Biology, and Oral Health

BioMed Research International ◽

10.1155/2018/9276380 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 19

Author(s):

R. A. G. Khammissa ◽

J. Fourie ◽

M. H. Motswaledi ◽

R. Ballyram ◽

J. Lemmer ◽

...

Keyword(s):

Vitamin D ◽

Calcium Homeostasis ◽

Dna Sequences ◽

Target Genes ◽

Biological Activities ◽

Biological Effects ◽

Active Form ◽

Vitamin D Supplementation ◽

Biologically Active ◽

Bone Homeostasis

Vitamin D plays an important role in calcium homeostasis and bone metabolism, with the capacity to modulate innate and adaptive immune function, cardiovascular function, and proliferation and differentiation of both normal and malignant keratinocytes. 1,25(OH)2D, the biologically active form of vitamin D, exerts most of its functions through the almost universally distributed nuclear vitamin D receptor (VDR). Upon stimulation by 1,25(OH)2D, VDR forms a heterodimer with the retinoid X receptor (RXR). In turn, VDR/RXR binds to DNA sequences termed vitamin D response elements in target genes, regulating gene transcription. In order to exert its biological effects, VDR signalling interacts with other intracellular signalling pathways. In some cases 1,25(OH)2D exerts its biological effects without regulating either gene expression or protein synthesis. Although the regulatory role of vitamin D in many biological processes is well documented, there is not enough evidence to support the therapeutic use of vitamin D supplementation in the prevention or treatment of infectious, immunoinflammatory, or hyperproliferative disorders. In this review we highlight the effects of 1,25(OH)2D on bone and calcium homeostasis, on cancer, and refer to its effects on the cardiovascular and immune systems.

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A hierarchical regulatory network analysis of the vitamin D induced transcriptome reveals novel regulators and complete VDR dependency in monocytes

Scientific Reports ◽

10.1038/s41598-021-86032-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Timothy Warwick ◽

Marcel H. Schulz ◽

Stefan Günther ◽

Ralf Gilsbach ◽

Antonio Neme ◽

...

Keyword(s):

Vitamin D ◽

Transcription Factor ◽

Transcription Factors ◽

Target Genes ◽

Active Form ◽

Transcriptional Response ◽

Biologically Active ◽

Open Chromatin ◽

Monocytic Cell ◽

Monocytic Cell Line

AbstractThe transcription factor vitamin D receptor (VDR) is the high affinity nuclear target of the biologically active form of vitamin D3 (1,25(OH)2D3). In order to identify pure genomic transcriptional effects of 1,25(OH)2D3, we used VDR cistrome, transcriptome and open chromatin data, obtained from the human monocytic cell line THP-1, for a novel hierarchical analysis applying three bioinformatics approaches. We predicted 75.6% of all early 1,25(OH)2D3-responding (2.5 or 4 h) and 57.4% of the late differentially expressed genes (24 h) to be primary VDR target genes. VDR knockout led to a complete loss of 1,25(OH)2D3–induced genome-wide gene regulation. Thus, there was no indication of any VDR-independent non-genomic actions of 1,25(OH)2D3 modulating its transcriptional response. Among the predicted primary VDR target genes, 47 were coding for transcription factors and thus may mediate secondary 1,25(OH)2D3 responses. CEBPA and ETS1 ChIP-seq data and RNA-seq following CEBPA knockdown were used to validate the predicted regulation of secondary vitamin D target genes by both transcription factors. In conclusion, a directional network containing 47 partly novel primary VDR target transcription factors describes secondary responses in a highly complex vitamin D signaling cascade. The central transcription factor VDR is indispensable for all transcriptome-wide effects of the nuclear hormone.

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Altered regulation of cytosolic Ca2+ concentration in dendritic cells from klotho hypomorphic mice

AJP Cell Physiology ◽

10.1152/ajpcell.00355.2012 ◽

2013 ◽

Vol 305 (1) ◽

pp. C70-C77 ◽

Cited By ~ 10

Author(s):

Ekaterina Shumilina ◽

Meerim K. Nurbaeva ◽

Wenting Yang ◽

Evi Schmid ◽

Kalina Szteyn ◽

...

Keyword(s):

Bone Marrow ◽

Vitamin D ◽

Dendritic Cells ◽

Active Form ◽

Biologically Active ◽

Deficient Diet ◽

Dihydroxyvitamin D3 ◽

Mhc Ii ◽

Macrophage Colony ◽

Antigen Presenting

The function of dendritic cells (DCs), antigen-presenting cells regulating naïve T-cells, is regulated by cytosolic Ca2+ concentration ([Ca2+]i). [Ca2+]i is increased by store-operated Ca2+ entry and decreased by K+-independent (NCX) and K+-dependent (NCKX) Na+/Ca2+ exchangers. NCKX exchangers are stimulated by immunosuppressive 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active form of vitamin D. Formation of 1,25(OH)2D3 is inhibited by the antiaging protein Klotho. Thus 1,25(OH)2D3 plasma levels are excessive in Klotho-deficient mice ( klotho hm). The present study explored whether Klotho deficiency modifies [Ca2+]i regulation in DCs. DCs were isolated from the bone marrow of klotho hm mice and wild-type mice ( klotho+/+) and cultured for 7–9 days with granulocyte-macrophage colony-stimulating factor. According to major histocompatibility complex II (MHC II) and CD86 expression, differentiation and lipopolysaccharide (LPS)-induced maturation were similar in klotho hm DCs and klotho+/+ DCs. However, NCKX1 membrane abundance and NCX/NCKX-activity were significantly enhanced in klotho hm DCs. The [Ca2+]i increase upon acute application of LPS (1 μg/ml) was significantly lower in klotho hm DCs than in klotho+/+ DCs, a difference reversed by the NCKX blocker 3′,4′-dichlorobenzamyl (DBZ; 10 μM). CCL21-dependent migration was significantly less in klotho hm DCs than in klotho+/+ DCs but could be restored by DBZ. NCKX activity was enhanced by pretreatment of klotho+/+ DC precursors with 1,25(OH)2D3 the first 2 days after isolation from bone marrow. Feeding klotho hm mice a vitamin D-deficient diet decreased NCKX activity, augmented LPS-induced increase of [Ca2+]i, and enhanced migration of klotho hm DCs, thus dissipating the differences between klotho hm DCs and klotho+/+ DCs. In conclusion, Klotho deficiency upregulates NCKX1 membrane abundance and Na+/Ca2+-exchange activity, thus blunting the increase of [Ca2+]i following LPS exposure and CCL21-mediated migration. The effects are in large part due to excessive 1,25(OH)2D3 formation.

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Vitamin D Treatment Sequence Is Critical for Transcriptome Modulation of Immune Challenged Primary Human Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.754056 ◽

2021 ◽

Vol 12 ◽

Author(s):

Henna-Riikka Malmberg ◽

Andrea Hanel ◽

Mari Taipale ◽

Sami Heikkinen ◽

Carsten Carlberg

Keyword(s):

Vitamin D ◽

Mononuclear Cells ◽

Fungal Infections ◽

Human Peripheral Blood ◽

Active Form ◽

Biologically Active ◽

Treatment Sequence ◽

Peripheral Blood Mononuclear ◽

Lps Challenge ◽

Immune Challenges

Microbe-associated molecular patterns, such as lipopolysaccharide (LPS) and β-glucan (BG), are surrogates of immune challenges like bacterial and fungal infections, respectively. The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), supports the immune system in its fight against infections. This study investigated significant and prominent changes of the transcriptome of human peripheral blood mononuclear cells that immediately after isolation are exposed to 1,25(OH)2D3-modulated immune challenges over a time frame of 24-48 h. In this in vitro study design, most LPS and BG responsive genes are downregulated and their counts are drastically reduced when cells are treated 24 h after, 24 h before or in parallel with 1,25(OH)2D3. Interestingly, only a 1,25(OH)2D3 pre-treatment of the LPS challenge results in a majority of upregulated genes. Based on transcriptome-wide data both immune challenges display characteristic differences in responsive genes and their associated pathways, to which the actions of 1,25(OH)2D3 often oppose. The joined BG/1,25(OH)2D3 response is less sensitive to treatment sequence than that of LPS/1,25(OH)2D3. In conclusion, the functional consequences of immune challenges are significantly modulated by 1,25(OH)2D3 but largely depend on treatment sequence. This may suggest that a sufficient vitamin D status before an infection is more important than vitamin D supplementation afterwards.

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Vitamin D Enhances Radiosensitivity of Colorectal Cancer by Reversing Epithelial-Mesenchymal Transition

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.684855 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xinyue Yu ◽

Qian Wang ◽

Baocai Liu ◽

Ning Zhang ◽

Guanghui Cheng

Keyword(s):

Colorectal Cancer ◽

Vitamin D ◽

Epithelial Mesenchymal Transition ◽

Active Form ◽

Plasminogen Activator Inhibitor ◽

Biologically Active ◽

Plasminogen Activator Inhibitor 1 ◽

Mesenchymal Transition

Colorectal cancer (CRC) is often resistant to conventional therapies. Previous studies have reported the anticancer effects of vitamin D in several cancers, its role in radiotherapy (RT) remains unknown. We found that 1α, 25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, had antitumor effect on CRC and sensitized CRC cells to ionizing radiation (IR). VD3 demonstrated synergistic effect in combination with IR, which were detected by colony formation and cell proliferation assay. Radiosensitivity restoration induced by VD3 was associated with a series of phenotypes, including apoptosis, autophagy, and epithelial-mesenchymal transition (EMT). Using proteomics, “regulation of cell migration” and “cadherin” were found to be obviously enriched GO terms. Moreover, cystatin D and plasminogen activator inhibitor-1 (PAI-1), the differentially expressed proteins, were associated with EMT. Next, we confirmed the contributions of these two genes in enhancing IR sensitivity of CRC cells upon inhibition of EMT. As determined by proteomics, the mechanism underlying such sensitivity involved partially block of JAK/STAT3 signaling pathway. Furthermore, VD3 also elicited sensitization to RT in xenograft CRC models without additional toxicity. Our study revealed that VD3 was able to act in synergy with IR both in vitro and in vivo and could also confer radiosensitivity by regulating EMT, thereby providing a novel insight for elevating the efficacy of therapeutic regimens.

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Dystrobrevin alpha gene is a direct target of the vitamin D receptor in muscle

Journal of Molecular Endocrinology ◽

10.1530/jme-19-0229 ◽

2020 ◽

Vol 64 (3) ◽

pp. 195-208 ◽

Cited By ~ 1

Author(s):

Maria K Tsoumpra ◽

Shun Sawatsubashi ◽

Michihiro Imamura ◽

Seiji Fukumoto ◽

Shin’ichi Takeda ◽

...

Keyword(s):

Vitamin D ◽

Vitamin D Receptor ◽

Target Genes ◽

Musculoskeletal Diseases ◽

Myogenic Differentiation ◽

Biologically Active ◽

Site Directed Mutagenesis ◽

C2c12 Cells ◽

Expression Vectors ◽

Gene And Protein Expression

The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (VD3), exerts its tissue-specific actions through binding to its intracellular vitamin D receptor (VDR) which functions as a heterodimer with retinoid X receptor (RXR) to recognize vitamin D response elements (VDRE) and activate target genes. Upregulation of VDR in murine skeletal muscle cells occurs concomitantly with transcriptional regulation of key myogenic factors upon VD3 administration, reinforcing the notion that VD3 exerts beneficial effects on muscle. Herein we elucidated the regulatory role of VD3/VDR axis on the expression of dystrobrevin alpha (DTNA), a member of dystrophin-associated protein complex (DAPC). In C2C12 cells, Dtna and VDR gene and protein expression were upregulated by 1–50 nM of VD3 during all stages of myogenic differentiation. In the dystrophic-derived H2K-mdx52 cells, upregulation of DTNA by VD3 occurred upon co-transfection of VDR and RXR expression vectors. Silencing of MyoD1, an E-box binding myogenic transcription factor, did not alter the VD3-mediated Dtna induction, but Vdr silencing abolished this effect. We also demonstrated that VD3 administration enhanced the muscle-specific Dtna promoter activity in presence of VDR/RXR only. Through site-directed mutagenesis and chromatin immunoprecipitation assays, we have validated a VDRE site in Dtna promoter in myogenic cells. We have thus proved that the positive regulation of Dtna by VD3 observed during in vitro murine myogenic differentiation is VDR mediated and specific. The current study reveals a novel mechanism of VDR-mediated regulation for Dtna, which may be positively explored in treatments aiming to stabilize the DAPC in musculoskeletal diseases.

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Impact of Epigenetics on Complications of Fanconi Anemia: The Role of Vitamin D-Modulated Immunity

Nutrients ◽

10.3390/nu12051355 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1355

Author(s):

Eunike Velleuer ◽

Carsten Carlberg

Keyword(s):

Vitamin D ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Active Form ◽

Cellular Growth ◽

Biologically Active ◽

Fine Tuning ◽

Dihydroxyvitamin D3 ◽

Increased Risk ◽

The Individual

Fanconi anemia (FA) is a rare disorder with the clinical characteristics of (i) specific malformations at birth, (ii) progressive bone marrow failure already during early childhood and (iii) dramatically increased risk of developing cancer in early age, such as acute myeloid leukemia and squamous cell carcinoma. Patients with FA show DNA fragility due to a defect in the DNA repair machinery based on predominately recessive mutations in 23 genes. Interestingly, patients originating from the same family and sharing an identical mutation, frequently show significant differences in their clinical presentation. This implies that epigenetics plays an important role in the manifestation of the disease. The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 controls cellular growth, differentiation and apoptosis via the modulation of the immune system. The nuclear hormone activates the transcription factor vitamin D receptor that affects, via fine-tuning of the epigenome, the transcription of >1000 human genes. In this review, we discuss that changes in the epigenome, in particular in immune cells, may be central for the clinical manifestation of FA. These epigenetic changes can be modulated by vitamin D suggesting that the individual FA patient’s vitamin D status and responsiveness are of critical importance for disease progression.

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