Mammary adipocytes bioactivate 25‐hydroxyvitamin D3 and signal via vitamin D3 receptor,modulating mammary epithelial cell growth

The vitamin D₃ receptor (VDR) is present in all microenvironments of the breast, yet it is hypothesized to signal through the epithelium to regulate hormone induced growth and differentiation. However, the influence or contribution of the other microenvironments within the breast that express VDR, like the breast adipose tissue, are yet to be investigated. We hypothesized that the breast adipocytes express the signaling components necessary to participate in vitamin D₃ synthesis and signaling via VDR, modulating ductal epithelial cell growth and differentiation. We utilized human primary breast adipocytes and VDR wild type (WT) and knockout (KO) mice to address whether breast adipocytes participate in vitamin D₃‐induced growth regulation of the ductal epithelium. We report in this study that breast primary adipocytes express VDR, CYP27B1 (1α‐hydroxylase, 1α‐OHase), the enzyme that generates the biologically active VDR ligand, 1α,25‐dihydroxyvitamin D₃ (1,25D₃), and CYP24 (24‐hydroxylase, 24‐OHase), a VDR‐1,25D₃ induced target gene. Furthermore, the breast adipocytes participate in bioactivating 25‐hydroxyvitamin D₃ (25D₃) to the active ligand, 1,25D₃, and secreting it to the surrounding microenvironment. In support of this concept, we report that purified mammary ductal epithelial fragments (organoids) from VDR KO mice, co‐cultured with WT breast adipocytes, were growth inhibited upon treatment with 25D₃ or 1,25D₃ compared to vehicle alone. Collectively, these results demonstrate that breast adipocytes bioactivate 25D₃ to 1,25D₃, signal via VDR within the adipocytes, and release an inhibitory factor that regulates ductal epithelial cell growth, suggesting that breast adipose tissue contributes to vitamin D₃‐induced growth regulation of ductal epithelium. J. Cell. Biochem. 112: 3393–3405, 2011. © 2011 Wiley Periodicals, Inc.     

Enhanced effects of guggulsterone plus 1,25(OH)2D3 on 3T3-L1 adipocytes

Guggulsterone (GS) and 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] have been shown to influence adipogenesis in 3T3-L1 cells. We investigated the ability of GS and 1,25(OH)₂D₃, alone and in combination to inhibit adipogenesis and induce apoptosis in 3T3-L1 adipocytes. Maturing preadipocytes were treated with 1,25(OH)₂D₃ in combination with GS for 6 days during differentiation. GS and 1,25(OH)₂D₃ each inhibited lipid accumulation, but the combination potentiated the inhibition of lipid accumulation. Apoptosis was increased by 1,25(OH)₂D₃ while GS had no effect, but GS + 1,25(OH)₂D₃ increased apoptosis more than either compound alone. Furthermore, GS + 1,25(OH)₂D₃ caused a potentiated decrease in the expression of aP2 and farnesoid X receptor expression more than either compound alone. In addition, 1,25(OH)₂D₃ increased vitamin D receptor expression after 6 days, while GS had no effect. GS + 1,25(OH)₂D₃, however, caused a potentiated increase in the expression of VDR. These findings show that GS potentiates 1,25(OH)₂D₃’s anti-adipogenic and pro-apoptotic effects in maturing 3T3-L1 preadipocytes.     

Enhanced effects of 1,25(OH)(2)D(3) plus genistein on adipogenesis and apoptosis in 3T3-L1 adipocytes

Objective: To investigate the ability of 1,25(OH)₂D₃ (D) and genistein (G), alone and in combination, to inhibit adipogenesis and induce apoptosis in 3T3‐L1 adipocytes. Methods and Procedures: 3T3‐L1 preadipocytes and mature adipocytes were incubated with various concentrations of D and G, alone and in combination, for 48 h. Viability was determined using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay. Post‐confluent preadipocytes were incubated with D and G for up to 6 days during adipogenesis and lipid content was quantified by Nile Red dye; apoptosis was quantified by measurement of single‐stranded DNA. Expression of adipocyte‐specific proteins and VDR was analyzed by western blotting. Results: Combining D and G did not cause an enhanced effect on cell viability in either preadipocytes or mature adipocytes. In maturing preadipocytes, D at 0.5 nmol/l (D0.5) increased apoptosis by 47 ± 10.25% (P < 0.05) and inhibited lipid accumulation by 28 ± 10% (P < 0.001), while G at 25 μmol/l (G25) had no significant effect. However, D+G caused an enhanced apoptosis by 136 ± 12.6% (P < 0.001) and enhanced inhibition of lipid accumulation by 82.46 ± 2.95% (P < 0.001). Similarly, D0.5 alone decreased adipose‐specific gene 422 (aP2) expression to 34.2 ± 2.3% and increased VDR expression levels by 41.8 ± 11% (P < 0.001), but G25 showed no effect. However, D0.5+G25 decreased aP2 expression to 52 ± 4.2% (P < 0.05) and increased VDR expression levels by 131 ± 14.5% (P < 0.0001). Discussion: These findings suggest that combining 1,25(OH)₂D₃ with genistein results in an enhanced inhibition of lipid accumulation and induction of apoptosis in maturing 3T3‐L1 preadipocytes.     

Increased nuclear expression and transactivation of vitamin D receptor by the cardiotonic steroid bufalin in human myeloid leukemia cells

The active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], is a potent ligand for the nuclear receptor vitamin D receptor (VDR) and induces myeloid leukemia cell differentiation. The cardiotonic steroid bufalin enhances vitamin D-induced differentiation of leukemia cells and VDR transactivation activity. In this study, we examined the combined effects of 1,25(OH)₂D₃ and bufalin on differentiation and VDR target gene expression in human leukemia cells. Bufalin in combination with 1,25(OH)₂D₃ enhanced the expression of VDR target genes, such as CYP24A1 and cathelicidin antimicrobial peptide, and effectively induced differentiation phenotypes. An inhibitor of the Erk mitogen-activated protein (MAP) kinase pathway partially inhibited bufalin induction of VDR target gene expression. 1,25(OH)₂D₃ treatment induced transient nuclear expression of VDR in HL60 cells. Interestingly, bufalin enhanced 1,25(OH)₂D₃-induced nuclear VDR expression. The MAP kinase pathway inhibitor increased nuclear VDR expression induced by 1,25(OH)₂D₃ and did not change that by 1,25(OH)₂D₃ plus bufalin. A proteasome inhibitor also enhanced 1,25(OH)₂D₃-induced CYP24A1 expression and nuclear VDR expression. Bufalin-induced nuclear VDR expression was associated with histone acetylation and VDR recruitment to the CYP24A1 promoter in HL60 cells. Thus, the Na⁺,K⁺-ATPase inhibitor bufalin modulates VDR function through several mechanisms, including Erk MAP kinase activation and increased nuclear VDR expression.     

Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,Differentiation, and Myotube Hypertrophy

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)₂D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)₂D₃ and 25(OH)D₃ affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. We show that myoblasts not only responded to 1,25(OH)₂D₃, but also to the precursor 25(OH)D₃ by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)₂D₃ as well as 25(OH)D₃ stimulated VDR mRNA expression and in myotubes 1,25(OH)₂D₃ also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D₃ metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)₂D₃ or 25(OH)D₃ myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D₃ to 24R,25(OH)₂D₃ which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D₃ metabolites, but is also able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. J. Cell. Physiol. 231: 2517–2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3

The most biologically active metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D₃ (25(OH)D₃) can affect osteoblast function via conversion to 1,25(OH)₂D₃, however, it is largely unknown whether 25(OH)D₃ can affect primary osteoblast function on its own. Furthermore, 25(OH)D₃ is not only converted to 1,25(OH)₂D₃, but also to 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D₃ itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)₂D₃ can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D₃ and 1,25(OH)₂D₃ by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)₂D₃ synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D₃. We demonstrated that 24R,25(OH)₂D₃ increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)₂D₃ strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D₃, 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)₂D₃, but also to 24R,25(OH)₂D₃ by enhancing osteoblast differentiation. This suggests that 25(OH)D₃ can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)₂D₃, but also via conversion to 24R,25(OH)₂D₃. Whether 25(OH)D₃ has direct actions on osteoblast function needs further investigation.     

Vitamin D Receptor Gene Expression and Function in a South African Population: Ethnicity,Vitamin D and FokI

Polymorphisms of the vitamin D receptor gene (VDR) have been associated inconsistently with various diseases, across populations of diverse origin. The T(f) allele of the functional SNP FokI, in exon 2 of VDR, results in a longer vitamin D receptor protein (VDR) isoform, proposed to be less active. Genetic association of VDR with disease is likely confounded by ethnicity and environmental factors such as plasma 25(OH)D₃ status. We hypothesized that VDR expression, VDR level and transactivation of target genes, CAMP and CYP24A1, depend on vitamin D, ethnicity and FokI genotype. Healthy volunteers participated in the study (African, n = 40 and White, n = 20). Plasma 25(OH)D₃ levels were quantified by LC-MS and monocytes cultured, with or without 1,25(OH)₂D₃. Gene expression and protein level was quantified using qRT-PCR and flow cytometry, respectively. Mean plasma 25(OH)D₃ status was normal and not significantly different between ethnicities. Neither 25(OH)D₃ status nor 1,25(OH)₂D₃ supplementation significantly influenced expression or level of VDR. Africans had significantly higher mean VDR protein levels (P<0.050), nonetheless transactivated less CAMP expression than Whites. Genotyping the FokI polymorphism by pyrosequencing together with HapMap data, showed a significantly higher (P<0.050) frequency of the CC genotype in Africans than in Whites. FokI genotype, however, did not influence VDR expression or VDR level, but influenced overall transactivation of CAMP and 1,25(OH)₂D₃-elicited CYP24A1 induction; the latter, interacting with ethnicity. In conclusion, differential VDR expression relates to ethnicity, rather than 25(OH)D₃ status and FokI genotype. Instead, VDR transactivation of CAMP is influenced by FokI genotype and, together with ethnicity, influence 1,25(OH)₂D₃-elicited CYP24A1 expression. Thus, the expression and role of VDR to transactivate target genes is determined not only by genetics, but also by ethnicity and environment involving complex interactions which may confound disease association.     

Clonal Differences in Expression of 25-Hydroxyvitamin D3-1α-hydroxylase, of 25-Hydroxyvitamin D3-24-hydroxylase, and of the Vitamin D Receptor in Human Colon Carcinoma Cells: Effects of Epidermal Growth Factor and 1α,25-Dihydroxyvitamin D3

Human colon carcinoma cells express 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D₃ (1,25-D3), which can be metabolized by 25-hydroxyvitamin D₃-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

Vitamin D Up-Regulates the Vitamin D Receptor by Protecting It from Proteasomal Degradation in Human CD4+ T Cells

The active form of vitamin D₃, 1,25(OH)₂D₃, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4⁺ effector T cells. The biological actions of 1,25(OH)₂D₃ are mediated by the vitamin D receptor (VDR) and are believed to correlate with the VDR protein expression level in a given cell. The aim of this study was to determine if and how 1,25(OH)₂D₃ by itself regulates VDR expression in human CD4⁺ T cells. We found that activated CD4⁺ T cells have the capacity to convert the inactive 25(OH)D₃ to the active 1,25(OH)₂D₃ that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)₂D₃ does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4⁺ T cells. Furthermore, 1,25(OH)₂D₃ induces a significant intracellular redistribution of the VDR. We show that 1,25(OH)₂D₃ stabilizes the VDR by protecting it from proteasomal degradation. Finally, we demonstrate that proteasome inhibition leads to up-regulation of VDR protein expression and increases 1,25(OH)₂D₃-induced gene activation. In conclusion, our study shows that activated CD4⁺ T cells can produce 1,25(OH)₂D₃, and that 1,25(OH)₂D₃ induces a 2-fold up-regulation of the VDR protein expression in activated CD4⁺ T cells by protecting the VDR against proteasomal degradation.     

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

The Paracrine Feedback Loop Between Vitamin D3 (1,25(OH)2D3) and PTHrP in Prehypertrophic Chondrocytes

The endocrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH‐related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)₂D₃ and PTH. This was investigated in ATDC5 cells treated with 10^?8 M 1,25(OH)₂D₃ or PTHrP, Col2‐pd2EGFP transgenic mice, and primary Col2‐pd2EGFP growth plate chondrocytes isolated by FACS, using RT‐qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)₂D₃ receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)₂D₃ decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α‐ and 24‐hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)₂D₃ treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate. 1,25(OH)₂D₃ decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)₂D₃ by increasing VDR production. In light of the role of 1,25(OH)₂D₃ and PTHrP in modulating chondrocyte differentiation, 1,25(OH)₂D₃ in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration. J. Cell. Physiol. 229: 1999–2014, 2014. © 2014 Wiley Periodicals, Inc.

     

Membrane localization,Caveolin-3 association and rapid actions of vitamin D receptor in cardiac myocytes

The active form of vitamin D, 1alpha, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), mediates both genomic and rapid non-genomic actions in heart cells. We have previously shown that the vitamin D receptor (VDR) is located in the t-tubular structure of cardiomyocytes. Here we show that VDR specifically interacts with Caveolin-3 in the t-tubules and sarcolemma of adult rat cardiac myocytes. Co-immunoprecipitation studies using VDR antibodies revealed that Caveolin-3 specifically co-precipitates with the VDR and similarly the VDR is co-precipitated with Caveolin-3 antibody. Confocal immuno-fluorescence microscopy analysis also showed co-localization of VDR and Caveolin-3 in t-tubules and sarcolemma. The non-genomic effects of the functional VDR were studied in electrically stimulated myocytes isolated from adult rat hearts. Sarcomere shortening and re-lengthening were measured in 1,25(OH)₂D₃ treated cardiac myocytes. A 1 nM treatment decreased peak shortening within minutes, suggesting a rapid effect through the membrane-bound VDR. This novel finding of the interaction between VDR and Caveolin-3 is fundamentally important in understanding 1,25(OH)₂D₃ signal transduction in heart cells and provides further evidence that VDR plays a role in regulation of heart structure and function.     

Primary 1,25-Dihydroxyvitamin D3 Response of the Interleukin 8 Gene Cluster in Human Monocyte- and Macrophage-Like Cells

Genome-wide analysis of vitamin D receptor (VDR) binding sites in THP-1 human monocyte-like cells highlighted the interleukin 8 gene, also known as chemokine CXC motif ligand 8 (CXCL8). CXCL8 is a chemotactic cytokine with important functions during acute inflammation as well as in the context of various cancers. The nine genes of the CXCL cluster and the strong VDR binding site close to the CXCL8 gene are insulated from neighboring genes by CCCTC-binding factor (CTCF) binding sites. Only CXCL8, CXCL6 and CXCL1 are expressed in THP-1 cells, but all three are up-regulated primary 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) target genes. Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)₂D₃-dependent chromatin opening exclusively for the VDR binding site. In differentiated THP-1 cells the CXCL8 gene showed a 33-fold higher basal expression, but is together with CXCL6 and CXCL1 still a primary 1,25(OH)₂D₃ target under the control of the same genomic VDR binding site. In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)₂D₃ and its receptor VDR. Our observation provides further evidence for the immune-related functions of vitamin D.