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DOI 10.1002/mnfr.201400291

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RESEARCH ARTICLE

Changes in vitamin D target gene expression in adipose tissue monitor the vitamin D response of human individuals 1 ¨ Jussi Ryynanen , Antonio Neme1 , Tomi-Pekka Tuomainen2 , Jyrki K. Virtanen2 , 2 Sari Voutilainen , Tarja Nurmi2 , Vanessa D. F. de Mello2 , Matti Uusitupa2 and Carsten Carlberg1 1 2

School of Medicine, Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio, Finland

Scope: Vitamin D3 , its biologically most active metabolite 1␣,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), and the vitamin D receptor (VDR) are important for adipose tissue biology. Methods and results: We extrapolated genomic VDR association loci in adipocytes from 55 conserved genome-wide VDR-binding sites in nonfat tissues. Taking the genes DUSP10, TRAK1, NRIP1, and THBD as examples, we confirmed the predicted VDR binding sites upstream of their transcription start sites and showed rapid mRNA up-regulation of all four genes in SGBS human pre-adipocytes. Using adipose tissue biopsy samples from 47 participants of a 5-month vitamin D3 intervention study, we demonstrated that all four primary VDR target genes can serve as biomarkers for the vitamin D3 responsiveness of human individuals. Changes in DUSP10 gene expression appear to be the most comprehensive marker, while THBD mRNA changes characterized a rather different group of study participants. Conclusion: We present a new approach to predict vitamin D target genes based on conserved genomic VDR-binding sites. Using human adipocytes as examples, we show that such ubiquitous VDR target genes can be used as markers for the individual’s response to a supplementation with vitamin D3 .

Received: April 30, 2014 Revised: April 30, 2014 Accepted: June 4, 2014

Keywords: Adipose tissue / Chromatin immunoprecipitation / Vitamin D / Vitamin D receptor / Vitamin D target genes



Additional supporting information may be found in the online version of this article at the publisher’s web-site

1

Correspondence: Professor Carsten Carlberg, School of Medicine, Institute of Biomedicine, University of Eastern Finland, POB 1627, FIN-70211 Kuopio, Finland E-mail: [email protected] Abbreviations: 1,25(OH)2 D3 or 1,25D, 1␣,25-dihydroxyvitamin D3 ; 25(OH)D3 , 25-dihydroxyvitamin D3 ; ChIP, chromatin immunoprecipitation; ChIP-seq, ChIP sequencing; CYP24A1, cytochrome P450, family 24, subfamily A, polypeptide 1; DR3, direct repeat spaced by three nucleotides; DUSP10, dual specificity phosphatase 10; GAPDH, glycerinaldehyde-3-phosphate dehydrogenase; MB, myoglobin; NRIP1, nuclear receptor interacting protein 1; qPCR, real-time quantitative PCR; RPLP0, ribosomal protein, large, P0; SGBS, Simpson–Golabi–Behmel syndrome; THBD, thrombomodulin; TRAK1, trafficking protein, kinesin binding 1; TSS, transcription start site; VDR, vitamin D receptor  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Introduction

Vitamin D3 is a micronutrient but can also be synthesized in the skin using energy provided by UV-B radiation [1]. The concentration of the vitamin D3 metabolite 25-dihydroxyvitamin D3 (25(OH)D3 ) in blood serum is considered as the indicator for the vitamin D3 status of human individuals [2]. Based on a summary of the Institute of Medicine [3], a serum 25(OH)D3 concentration of below 50 nM (20 ng/mL) can be considered as vitamin D3 deficiency, although other reports suggested that the 25(OH)D3 levels should be at least 75 nM [4]. The biologically most active metabolite of vitamin D3 — 1␣,25-dihydroxyvitamin D3 (1,25(OH)2 D3 )—acts as a key regulator of calcium homeostasis, and therefore is critical for skeletal health [5]. In addition, 1,25(OH)2 D3 has immunemodulatory functions and is involved in the regulation of differentiation and proliferation of a number of tissues and www.mnf-journal.com

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cell types [6, 7]. One of these is adipose tissue, which is an important metabolic organ having a central role in energy balance and glucose homeostasis [8]. Adipose tissue is not only the major organ for vitamin D storage [9], but also expresses the vitamin D receptor (VDR) [10] and enzymes for vitamin D metabolism [11]. In the mouse preadipocyte model 3T3-L1, 1,25(OH)2 D3 inhibits adipogenesis [12, 13], and also in the human Simpson–Golabi–Behmel syndrome (SGBS) preadipocyte model, vitamin D3 metabolites modulate the differentiation process [10, 14]. In addition, evidence for an in vivo role of VDR in adiposity was demonstrated by studies with transgenic and knockout mice [15–17]. VDR is a transcription factor [18] and member of the superfamily of nuclear receptors, many of which can be activated by small lipophilic ligands [19]. The receptor is expressed in many human tissues, in which between 200 and 600 primary 1,25(OH)2 D3 target genes are described [20–23]. However, most of these genes are regulated in a tissue-specific fashion, i.e. a simple extrapolation of the transcriptome profile from one tissue or cell type to another is not very reliable, as exemplified for monocytes and B cells [24]. Nevertheless, all primary 1,25(OH)2 D3 target genes should have at least one VDR-binding site in relative vicinity to their transcription start sites (TSSs). This suggests that a comparison of vitamin D target tissues on the level of the genome-wide VDR-binding profile may provide a better overview on the tissue-specific and conserved actions of 1,25(OH)2 D3 . The method chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq) is widely used to determine the genome-wide location of transcription factors [25]. For VDR six of these datasets are available from human cellular models, of which we recently performed a harmonized reanalysis [26]. In total, the VDR ChIP-seq datasets from THP-1 human monocytic leukemia cells [21], macrophage-like LPS-polarized THP-1 cells [26], the lymphoblastoid cells GM10855 and GM10861 [20], LX2 hepatic stellate cells [27], and LS180 colon cancer cells [28] indicated more than 20 000 nonoverlapping VDR-binding loci within the human genome. However, when allowing a maximal distance of 250 bp between overlapping VDR peaks some 68% of these sites are unique for only one cell type, while only 55 genomic loci are found throughout all datasets [26]. VDR preferentially associates as a heterodimer with the retinoid X receptor on genomic DNA motifs that are formed by two directly repeated binding motifs spaced by three nucleotides (DR3) [29, 30]. However, only one in six of the more than 20 000 genomic VDR-binding sites carry such a DR3type sequence [26]. In contrast, for the majority of the VDRbinding sites, which have been demonstrated to be involved in gene activation [31, 32], a DR3-type motif was found. This means that the presence of a DR3-type sequence is a strong indication of a functional VDR-binding site. In line with this, 48 of the 55 conserved VDR-binding loci (87.3%) are associated with a DR3-type sequence [26]. Thus, the list of the conserved VDR-binding sites highlights those genomic loci, at which the receptor may be found also in tissues and cell  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

types, such as adipocytes, that have not yet been investigated genome wide. Moreover, genes that are in vicinity to these conserved VDR-binding sites, may act in many tissues as primary vitamin D targets, i.e. the use of VDR’s genome-wide binding profile should allow more reliable extrapolations to other tissues than transcriptome-wide data on their own. In order to confirm our assumption, we selected in this study the genes dual-specific phosphatase 10 (DUSP10), trafficking protein, kinesin binding 1 (TRAK1), nuclear receptor interacting protein 1 (NRIP1), and thrombomodulin (THBD) as representative examples. In SGBS cells we confirmed VDR-binding to these loci and demonstrated that the genes’ mRNA expression are rapidly upregulated after exposure to 1,25(OH)2 D3 . In adipose tissue samples from 47 human individuals, who participated in a 5-month vitamin D3 intervention study, we showed that all four primary VDR target genes can serve as biomarkers for the vitamin D3 response of fat tissue.

2

Materials and methods

2.1 Cell culture The human SGBS preadipocyte cell line was kindly provided by Prof. M. Wabitsch (University of Ulm, Germany) [33]. The cells had been obtained from adipose tissue of a patient with SGBS, which is a rare X chromosome linked multiple congenital disorder causing general overgrowth [34]. SGBS cells are neither transformed nor immortalized and have retained capacity to differentiate, i.e. they may represent at present the best cellular model of human adipocytes. However, we observed previously [10] that the cells show in their undifferentiated form a more pronounced response to 1,25(OH)2 D3 . The cells were grown in a DMEM/F12 (1:1) mixture supplemented with 8 ␮g/mL biotin, 4 ␮g/mL pantothenate, 2 mM L-glutamine, 0.1 mg/mL streptomycin, 100 U/mL penicillin, and 10% fetal calf serum on cell culture flasks or plates, which had been coated with a solution of 10 ␮g/mL fibronectin and 500 ␮g/mL gelatin in PBS. Prior to chromatin or mRNA extraction, cells were grown overnight in medium containing 5% charcoal-stripped fetal calf serum and were treated with solvent (0.01% ethanol) or 100 nM 1,25(OH)2 D3 (SigmaAldrich, St. Louis, MO, USA) for the indicated time periods.

2.2 RNA extraction, cDNA synthesis, and qPCR Total RNA from SGBS cells was extracted using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland). During the RNA extraction remains of genomic DNA were cleared through DNase treatment. cDNA synthesis was performed with the Transcriptor First Strand cDNA Synthesis Kit (Roche), where 500 ng total RNA and oligo(dT)18 primers were denatured at 65⬚C, and reverse transcription was carried out for 30 min at 55⬚C. Real-time quantitative PCR (qPCR) www.mnf-journal.com

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reactions were performed using 280 nM of reverse and forward primers (Supporting Information Table 1) and the LightCycler 480 SYBRGreen I Master mix (Roche). The hotstart Taq polymerase was activated for 10 min at 95⬚C, followed by 43 amplification cycles of 20 s denaturation at 95⬚C, 15 s annealing at 60⬚C, and 15 s elongation at 72⬚C, and a final elongation for 10 min at 72⬚C. PCR product specificity was monitored using post-PCR melt curve analysis. Relative mRNA expression levels were determined for each sample separately using internal reference genes ribosomal protein, large, P0 (RPLP0) and glycerinaldehyde-3-phosphate dehydrogenase (GAPDH) with the formula 2−(⌬Ct) , where ⌬Ct is Ct(target gene) − mean of Ct(reference genes) [35]. The relative mRNA expression levels in ligand-treated samples were compared against untreated control samples, in order to obtain fold change values.

2.3 Samples of the VitDmet study The 73 participants of the VitDmet study (NCT01479933, ClinicalTrials.gov) came from the region of Kuopio, Finland (63 N) and were supplemented during 5 winter months with placebo, 40 or 80 ␮g vitamin D3 (Tuomainen, T. P. et al., Glucose metabolism effects of vitamin D supplementation in prediabetes—the VitDmet study; submitted for publication) and [36]. The individuals were selected to be ࣙ60 years of age, showed evidence of disturbed glucose homeostasis, i.e. impaired fasting glucose or impaired glucose tolerance, but no type 2 diabetes, and had a BMI between 25 and 35. The research ethics committee of the Northern Savo Hospital District had approved the study protocol (#37/2011). All participants gave a written informed consent to participate in the study. From subcutaneous abdominal adipose tissue of 47 individuals needle biopsies (0.5–1 g) were taken from under local anesthesia (10 mg/mL lidocaine without adrenalin) at the begin and the end of the study. The samples were washed twice with PBS, frozen quickly in liquid nitrogen, and stored at −80⬚C until used for RNA extraction. Total RNA was extracted by dissolving the homogenized samples in TRIzol followed by purification with miRNeasy Mini Kit columns (Qiagen, Hilden, Germany). The purified RNA was then reverse transcribed into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Carlsbad, CA, USA). qPCR was performed as described in Section 2.2. Serum 25(OH)D3 concentrations were measured from venous blood samples using a HPLC with coulometric electrode array as described previously [37]. The baseline serum 25(OH)D3 concentrations of the 47 investigated study participants ranged between 35.9 and 73.6 nM at the start of the intervention and raised in average by 20.8 nM (Supporting Information Table 2). The measurement of other basic clinical and biochemical variables showed that neither the BMI nor the serum calcium concentrations changed during the intervention (for further details see (Tuomainen, T. P. et al., Glucose metabolism effects of vitamin D supplementation in  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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prediabetes—the VitDmet study; submitted for publication) and [36]).

2.4 ChIP-qPCR ChIP was carried out following the protocol described earlier [38]. After treatment of SGBS cells, nuclear proteins were cross-linked to DNA by 10-min formaldehyde treatment. Cross-linking was stopped with glycine and cells were washed three times with ice-cold PBS and scraped into Farnham Lysis buffer (0.5% NP-40, 85 mM KCl, protease inhibitors, 5 mM PIPES, pH 8.0). Immunoprecipitation was carried out using 1 ␮g of anti-VDR antibody (sc-1008X, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Final DNA concentrations R dsDNA Assay Kit (Invitwere determined by PicoGreen rogen, Carlsbad, CA, USA) and selected genomic regions containing VDR peaks were analyzed by qPCR using equal DNA amounts of chromatin fragments, 280 nM of reverse and forward primers (Supporting Information Table 3) and the LightCycler 480 SYBRGreen I Master mix (Roche). As negative control showing background VDR binding, a genomic region of exon 2 of the myoglobin (MB) gene was chosen, which is only expressed in muscle cells and represents in other tissues constitutive heterochromatin. Accordingly, based on ChIP-seq data in six cell types [26], there is no VDR binding to this genomic region.

2.5 Correlation analysis Linear regression analysis was performed using Microsoft Excel, version 2011 and StatPlus, version 5.8.2. The ranking of vitamin D3 responsiveness was done under the assumption of a linear positive correlation between changes of the mRNA expression of the VDR target genes and alterations in the serum 25(OH)D3 concentrations. The study participants were sorted, in ascending order, according to their ratios of gene expression and serum 25(OH)D3 concentration changes (for each gene separately). Starting from the mean, individuals were ranked both in ascending and descending order for each VDR target gene. The ranking was performed based on the absolute difference between the mean and the ratio of gene expression and serum 25(OH)D3 concentration changes of the individual. For values of equal rank the distance to the mean determined the final ranking (see Supporting Information Table 2).

3

Results

3.1 Conserved VDR-binding sites The list of conserved VDR-binding loci within the human genome (Supporting Information Table 4), which we recently obtained [26], contains 55 genomic loci. These sites have the www.mnf-journal.com

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Figure 1. Conserved VDR peak locations. The Integrative Genomics Viewer [42] was used to visualize normalized VDR ChIP-seq signals from unstimulated (−) and ligandstimulated (+) undifferentiated THP-1 cells (THP-1 [21], orange), LPS-differentiated THP-1 cells (THP, LPS [26], red), the lymphoblastoid cell lines GM10855 ([20], dark blue) and GM10861 ([20], light blue), LX2 cells ([27], purple), and LS180 cells ([28], gray) for the loci of the genes DUSP10 (A), TRAK1 (B), NRIP1 (C), and THBD (D). Conserved VDR peak loci are shaded in gray and the sequence of the respective DR3-type VDR-binding motifs is provided below. Gene structures are indicated in blue and 1,25(OH)2 D3 target gene names are highlighted in red. The VDR ChIP-seq datasets are available at GEO (www.ncbi.nlm.nih.gov/geo) under the accession number GSE53041.

highest likelihood to be used by VDR also in other tissues and cell types, for which so far no VDR ChIP-seq datasets are available, such as adipocytes. In order to identify the most appropriate candidate VDR sites and target genes for studies in adipocytes, we used the selection criteria that (i) the sequence  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

below the summit of the VDR peak should contain a DR3-type binding site, (ii) the VDR peak should be located in an enhancer region distant from the gene’s TSS, and (iii) the gene has been already described as a 1,25(OH)2 D3 target in at least one of the six cellular system for which VDR ChIP-seq data www.mnf-journal.com

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are available. Since presumably all conserved VDR sites have an important function, the restriction to DR3-type containing VDR peaks excluded only seven of the 55 candidates. However, only 50% (24) of the remaining 48 VDR-binding sites are located more than 10 kb in distance from the closest protein coding gene, i.e. by distance definition they are clearly located within enhancer and not in promoter regions. As representatives for further investigations, we selected the VDR-binding sites (i) 216 kb upstream of the TSS of the DUSP10 gene (Fig. 1A), (ii) 78 kb upstream of the TRAK1 gene (Fig. 1B), (iii) 156 kb upstream of the NRIP1 gene (Fig. 1C), and (iv) 285 kb upstream of the THBD gene (Fig. 1D). For all four genes close to these VDR loci we knew from previous studies that they are primary 1,25(OH)2 D3 targets, at least in THP-1 cells [39, 40]. The visualization of the harmonized VDR ChIP-seq datasets (Fig. 1) indicated that macrophage-like LPS-polarized THP-1 cells, which from the six available cellular models are closest related to adipocytes [41], demonstrate prominent VDR binding at all four genomic loci. In summary, a harmonized analysis of six VDR ChIP-seq datasets has identified an overlap of only 55 genomic loci for the receptor. From these, the VDR-binding sites upstream of the genes DUSP10, TRAK1, NRIP1, and THBD showed to be most appropriate for more detailed investigations in human adipocytes.

3.2 Genomic VDR association in adipocytes The immortalized human preadipocyte cell line SGBS is an established human adipocyte model [10]. In this cellular system, we performed ChIP-qPCR, in order to confirm our prediction that also in adipocytes VDR binds to the indicated genomic loci upstream of the genes DUSP10, TRAK1, NRIP1, and THBD (Fig. 2). At the sites of DUSP10, NRIP1, and THBD a treatment of the cells for 1 and 2 h with 1,25(OH)2 D3 led to a statistically significant increase of VDR binding between 2.3- and 3.4-fold. In contrast, the basal VDR association to the site of the TRAK1 gene was already in the absence of 1,25(OH)2 D3 approximately threefold higher than that of the three other VDR-binding sites and, after ligand stimulation, did not show any further significant increase. This means that VDR binds already in absence of ligand to the genomic site close to the TRAK1 gene, which is in accordance with the ChIP-seq data from monocytes and macrophages (Fig. 1B). Taken together, ChIP-qPCR in SGBS cells could confirm our prediction on VDR binding to genomic loci upstream of the genes DUSP10, TRAK1, NRIP1, and THBD.

3.3 1,25(OH)2 D3 -dependent mRNA expression in adipocytes The observed VDR occupancy of predicted binding loci upstream of the genes DUSP10, TRAK1, NRIP1, and THBD in adipocytes (Fig. 2) suggested that the genes may be primary  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 2. VDR association with genomic regions of target genes. SGBS cells were stimulated for 0, 1, and 2 h with 100 nM 1,25(OH)2 D3 and chromatin was extracted. ChIP-qPCR was performed to determine VDR association at the indicated loci (see Fig. 1) of the genes DUSP10, TRAK1, NRIP1, and THBD relative to the negative control region of the MB gene. Columns represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of the VDR association at each time point separately (*p < 0.05; **p < 0.01; ***p < 0.001).

1,25(OH)2 D3 targets. We tested this assumption by performing qPCR with RNA from SGBS cells, which had been treated for 1, 2, 3, 4, 5, and 6 h with 1,25(OH)2 D3 (Fig. 3). A statistically significant upregulation of mRNA expression was observed already after 2 h in case of the THBD gene, after 3 h for the genes DUSP10 and NRIP1, and after 4 h for the TRAK1 gene. The apparent downregulation of NRIP1 at time point 5 h is in the range of the normal fluctuation of the expression of the gene. During the 6-h time course the maximal induction of the genes’ mRNA ranged from 1.5-fold for TRAK1, 1.6fold for NRIP1, 1.8-fold for DUSP10 to 3.7-fold for THBD. We used as a negative control the VDR gene, which did not show any induction within 6-h 1,25(OH)2 D3 treatment (Supporting Information Fig. 1A). The expression changes of DUSP10, TRAK1, and NRIP1 are rather low compared with that of the cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1) gene, which is in SGBS cells (Supporting Information Fig. 1B), like in other adipocytes [14], the most inducible primary 1,25(OH)2 D3 target. However, in the absence of VDR ligand no CYP24A1 expression could be www.mnf-journal.com

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Figure 3. Expression profiling of primary VDR target genes. SGBS cells were stimulated for 1, 2, 3, 4, 5, and 6 h with 100 nM 1,25(OH)2 D3 or solvent and RNA was isolated. qPCR was performed to determine the relative changes of mRNA expression of the genes DUSP10, TRAK1, NRIP1, and THBD normalized by the reference genes GAPDH and RPLP0. Data points represent the means of three independent experiments (each performed in triplicate) and the bars indicate standard deviations. Two-tailed Student’s ttests were performed to determine the significance of the mRNA induction by 1,25(OH)2 D3 at each time point separately (*p < 0.05; **p < 0.01).

detected, and therefore actual inducibility cannot be calculated. Similarly, the rather high inducibility of the THBD gene is related to its 500- to 1000-fold lower basal expression than that of the three other genes (Supporting Information Fig. 1C). For example, the stimulation with 1,25(OH)2 D3 led to the synthesis of an approximately 220-fold higher number of new DUSP10 mRNA molecules than of THBD RNAs. This means that a high fold induction of a low expressed gene has lower impact than a modest induction of a gene with far higher basal expression. In summary, the genes DUSP10, TRAK1, NRIP1, and THBD are also in adipocytes targets of 1,25(OH)2 D3 with first responses between 2 and 4 h and maximal inductions between 1.5- and 3.7-fold. THBD is the most inducible gene, but it is also lower expressed than the three other 1,25(OH)2 D3 responding genes.

3.4 Response of VDR target genes in primary human adipose tissue samples In order to improve the understanding of the role of vitamin D3 in human health and disease, the 1,25(OH)2 D3 responsiveness of the genes DUSP10, TRAK1, NRIP1, and THBD in SGBS cells has to be translated to primary human adipocytes. Therefore, we studied the expression of the four genes in adipose tissue samples, which were obtained by peripheral subcutaneous needle biopsies of 47 participants of the VitDmet study. The 5-month intervention resulted in changes of serum 25(OH)D3 concentrations between a 2.1-fold decrease (maximal reduction 30.2 nM) and a 2.4-fold increase (maximal addition 64.4 nM, Supporting Information Table 2).  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

In the adipose tissue samples, the changes of relative DUSP10 mRNA expression ranged from a 1.62-fold decrease to a 2.33-fold increase, for TRAK1 from a 1.91-fold decrease to a 2.15-fold increase, for NRIP1 from a 2.02-fold decrease to a 3.15-fold increase, and for THBD from a 4.71-fold decrease to a 2.91-fold increase (Supporting Information Table 2). As a reference, at the begin of the intervention the relative mean mRNA expression of NRIP1 was 19.6, 10.6, and 2.5 times higher than that of THBD, DUSP10, and TRAK1, respectively (Supporting Information Fig. 2A). This confirms that also in primary adipocytes THBD shows the lowest mRNA levels of all tested genes. For a ranking of the study participants according to their responsiveness to vitamin D3 , we assumed for each of the four primary VDR target genes a positive correlation between their expression changes and the respective alterations of serum 25(OH)D3 concentrations during the intervention (Supporting Information Table 2), and calculated the respective correlation coefficient (r). For each gene separately, we then omitted in the reverse order of the ranking one participant after the other from the group and determined again the r-value. When plotting the r-values in relation to the number of the remaining study participants, they increased for all four genes with the decreasing number of considered participants (Fig. 4). However, in order to obtain an r-value of at least 0.71 (which equals to a r2 -value of more than 0.5) for the ranking based on DUSP10 expression changes, only 17 of the 47 study participants had to be removed, while based on TRAK1 24 individuals and for NRIP1 and THBD, even 25 persons were not taken into the calculations, as illustrated by the respective correlation graphs (Fig. 5). Interestingly, with the exception of one subject, the ranking by DUSP10 described the same www.mnf-journal.com

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Figure 4. VDR target gene-specific ranking of vitamin D3 intervention study participants. RNA was isolated from peripheral subcutaneous adipose tissue biopsies obtained from 47 participants before and after a 5-month vitamin D3 intervention trial. qPCR was performed to determine the relative changes of the expression of the genes DUSP10, TRAK1, NRIP1, and THBD normalized by the reference genes RPLP0 and GAPDH. The study participants were ranked for the changes of the four VDR target genes in relation to changes in their 25(OH)D3 serum levels (see Supporting Information Table 2). Starting from the lowest ranking participants, the number of persons considered for correlation analysis was stepwise reduced and the respective r-values were plotted over the number of remaining subjects. The numbers of study participants that resulted r-value higher than 0.71 (r2 = 0.5) are indicated in red. The respective correlation graphs are shown in Fig. 5.

group of persons (but seven and eight additional study participants) than those of TRAK1 and NRIP1. In contrast, the ranking by the previously published biomarker THBD [36,43] described a different group of individuals. Moreover, when the relative mean mRNA expression of DUSP10, TRAK1, NRIP1, and THBD at the beginning and end of the 5-month intervention was calculated only based on the respective responsive individuals (Supporting Information Fig. 2B, for selected individuals see Supporting Information Table 2), the four genes showed some induction compared to the average of all 47 individuals (Supporting Information Fig. 2A). In summary, in primary human adipocytes the primary VDR target genes DUSP10, TRAK1, NRIP1, and THBD can be used as markers for the vitamin D3 response of human individuals. DUSP10 showed the best suited, while THBD described a differently composed subgroup of the study participants.

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Discussion

In this study, we took advantage of a harmonized analysis of six VDR ChIP-seq datasets [26] and selected from a  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

shortlist of 55 conserved VDR-binding loci the genomic positions upstream of the genes DUSP10, TRAK1, NRIP1, and THBD. We confirmed our prediction that these sites are also functional in human adipocytes and could show in SGBS cells that (i) each of the four genomic loci binds VDR and (ii) all four genes in the vicinity of the VDR loci are primary 1,25(OH)2 D3 targets. In the candidate selection process, we used information from our previous studies on the identification and characterization of 1,25(OH)2 D3 target genes in THP-1 monocytic leukemia cells [21,39]. The observation that all four genes respond both in monocytes and in adipocytes to 1,25(OH)2 D3 suggests that for genes, which use the same active VDR-binding sites, also their transcriptome profile may be transferred from one cell type to another. This concept indicates that DUSP10, TRAK1, NRIP1, and THBD may also be 1,25(OH)2 D3 targets in other tissues and cell types that express the VDR. VDR is known to have targets with rather divergent functions [44]. Accordingly, the four genes, which were investigated in this study, encode for proteins that range from the transmembrane anticoagulant glycoprotein THBD (also known as CD141) [45], via the intracellular adaptor molecule TRAK1 [46] and the mitogen-activated protein kinase-specific

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Figure 5. Changes in VDR target gene expression in adipose tissue samples correlate with alterations in serum 25(OH)D3 concentrations. The relative changes of the expression of the genes DUSP10, TRAK1, NRIP1, and THBD were correlated with relative changes of serum 25(OH)D3 concentrations. Data from study participants leading to r-values higher than 0.71 are shown (see Fig. 4). Grey dots indicate the 30 study participants that were top-ranked based on DUSP10 mRNA expression, while black dots represent individuals outside of this group.

phosphatase DUSP10 [47] to the nuclear coactivator NRIP1 [48]. The biological function of these proteins indicates that vitamin D3 is on all levels—membrane receptors, adaptor molecules, phosphatases, and nuclear coregulator proteins— involved in the control of cellular signaling. Moreover, as explained above, this most likely applies to all vitamin D3 responsive human tissues. This pleiotropy emphasizes again the need for sufficient synthesis of the vitamin D3 in the skin or adequate intake from diet or supplements in order to keep the VDR ligand activated. However, the optimal serum 25(OH)D3 concentration may vary within individuals. This means that some persons may need to be supplemented with more vitamin D3 than others. Therefore, molecular biomarkers, such as the ubiquitous 1,25(OH)2 D3 target genes DUSP10, TRAK1, NRIP1, and THBD, are well suited for a molecular monitoring of the vitamin D status of human individuals. We suggest that in particular the change of the expression of these genes is important. This assumption we tested with adipose tissue samples of 47 participants of the vitamin D3 intervention trial VitDmet. We found that the DUSP10 gene is the most comprehensive biomarker, since it identified with high significance (p = 1.38 × 10−5 ) in 30 of 47 individuals (63.8%) the relation between changes in the serum 25(OH)D3 concentration and gene expression. The genes TRAK1 and NRIP1  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

monitored the same subgroup of individuals as DUSP10, but included, at comparable significance levels, only 23 (48.9%) or 22 (45.8%) of the 47 study participants. In contrast, THBD gene expression changes described a subgroup, from which only 13 of 22 (59%) individuals overlapped with the group described by the three other genes. The THBD gene is more responsive to vitamin D3 metabolites than the three other investigated genes. This was the main reasons that, based on a comparison of 1,25(OH)2 D3 modulated transcriptome datasets, the genes THBD and CD14 were suggested as indicators of the vitamin D status of human individuals [43]. In a previous study [36], we used the combined expression changes of both genes in two human tissues, peripheral blood mononuclear cells and adipocytes, to monitor human individuals for benefits of vitamin D3 supplementation. However, while CD14 shows high basal expression, THBD is far lower expressed. The latter could lead to some instable measurement and may discourage from using THBD as a biomarker in the clinical practice. In this study, we used far higher threshold levels for the required significance (p < 2.5 × 10−4 ) of the linear regression than in our previous report [36]. This was only possible by focusing on one tissue and using genes, such as DUSP10, TRAK1, and NRIP1, which show higher basal expression than THBD. Therefore, in contrast to most other approaches, not www.mnf-journal.com

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the most inducible genes, such as CYP24A1, which also show very rapid changes, are the most suitable biomarkers, but rather the modestly induced genes with high basal expression. The 30 individuals that were selected by DUSP10 expression changes in their adipocytes are considered as being full responsive to vitamin D3 . Interestingly, this property is not related to the basal 25(OH)D3 levels at the start of the invention, which ranged from 36.9 to 73.6 nM. This suggests that all 30 individuals would benefit from vitamin D3 supplementation, although at the start of the intervention, only two of them had serum 25(OH)D3 levels below 50 nM. The latter concentration is the value, below which the Institute of Medicine recommends vitamin D3 supplementation [3]. Our observation suggests that rather the individual’s responsiveness to vitamin D3 than a fixed serum 25(OH)D3 level should be considered, when recommending vitamin D3 supplementation. Moreover, it should be noted that the changes in gene expression have been measured over a time span of a 5-month intervention, i.e. far longer than the response of primary vitamin D target genes normally takes under cell culture conditions. Therefore, our measurements may reflect the slow natural changes in the vitamin D response of human individuals. In conclusion, we demonstrated a new approach how to predict 1,25(OH)D3 target genes based on conservation of genomic VDR-binding sites in the genes DUSP10, TRAK1, NRIP1, and THBD. We suggest that gene expression changes of such ubiquitous 1,25(OH)D3 targets can be used as molecular biomarkers for the vitamin D3 responsiveness not only of human adipocytes but also of many other tissues and cell types. J.R. acknowledges Ingrid Felicidade for help in handling SGBS samples. This work was supported by the Academy of Finland (grant no. 267067 to C.C. and 137826 for the VitDmet study), the Juselius Foundation, and the Diabetes Research Foundation. The authors have declared no conflict of interest.

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Changes in vitamin D target gene expression in adipose tissue monitor the vitamin D response of human individuals.

Vitamin D₃, its biologically most active metabolite 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and the vitamin D receptor (VDR) are important for adipos...
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