Class 3 semaphorins are transcriptionally regulated by 1,25(OH)2D3 in osteoblasts.

The vitamin D endocrine system is essential for calcium metabolism and skeletal integrity. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates bone mineral homeostasis and acts directly on osteoblasts. In the present study we characterized the transcriptional regulation of the class 3 semaphorin (Sema3) gene family by 1,25(OH)2D3 in osteoblastic cells. Class 3 semaphorins are secreted proteins that regulate cell growth, morphology and migration, and were recently shown to be involved in bone homeostasis. In ST2, MC3T3-E1 and primary calvarial osteoblast cell cultures we found that all members of the Sema3 gene family were expressed, and that Sema3e and Sema3f were the most strongly induced 1,25(OH)2D3 target genes among the studied cell types. In addition, transcription of Sema3b and Sema3c was upregulated, whereas Sema3d and Sema3g was downregulated by 1,25(OH)2D3 in different osteoblastic cells. Chromatin immunoprecipitation analysis linked to DNA sequencing (ChIP-seq analysis) revealed the presence of the vitamin D receptor at multiple genomic loci in the proximity of Sema3 genes, demonstrating that the genes are primary 1,25(OH)2D3 targets. Furthermore, we showed that recombinant SEMA3E and SEMA3F protein were able to inhibit osteoblast proliferation. However, recombinant SEMA3s did not affect ST2 cell migration. The expression of class 3 semaphorins in osteoblasts together with their regulation by 1,25(OH)2D3 suggests that these genes, involved in the regulation of bone homeostasis, are additional mediators for 1,25(OH)2D3 signaling in osteoblasts.

Dissecting high from low responders in a vitamin D3 intervention study.

Vitamin D3 is a pleiotropic signaling molecule that has via activation of the transcription factor vitamin D receptor (VDR) a direct effect on the expression of more than 100 genes. The aim of this study was to find transcriptomic and clinical biomarkers that are most suited to identify vitamin D3 responders within 71 pre-diabetic subjects during a 5-month intervention study (VitDmet). In hematopoietic cells, the genes ASAP2, CAMP, CD14, CD97, DUSP10, G0S2, IL8, LRRC8A, NINJ1, NRIP1, SLC37A2 and THBD are known as primary vitamin D targets. We demonstrate that each of these 12 genes carries a conserved VDR binding site within its genomic region and is expressed in human peripheral blood mononuclear cells (PBMCs). The changes in the expression of these genes in human PBMCs at the start and the end of the vitamin D-intervention were systematically correlated with the alteration in the circulating form of vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3). Only 39-44 (55-62%) of the study subjects showed a highly significant response to vitamin D3, i.e., we considered them as "responders". In comparison, we found for 37-53 (52-75%) of the participants that only 12 biochemical and clinical parameters, such as concentrations of parathyroid hormone (PTH) and insulin, or computed values, such as homeostatic model assessment and insulin sensitivity index, show a correlation with serum 25(OH)D3 levels that is as high as that of the selected VDR target genes. All 24 parameters together described the pleiotropic vitamin D response of the VitDmet study subjects. Interestingly, they demonstrated a number of additional correlations that define a network, in which PTH plays the central role. In conclusion, vitamin D3-induced changes in human PBMCs can be described by transcriptomic and serum biomarkers and allow a segregation into high and low responders. This article is part of a Special Issue entitled '17th Vitamin D Workshop' .

The transcriptional regulator BCL6 participates in the secondary gene regulatory response to vitamin D.

The vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the high affinity ligand of the transcription factor vitamin D receptor (VDR) and therefore a direct regulator of transcription. Transcriptome-wide analysis of THP-1 human monocytes had indicated more than 600 genes to be significantly (p<0.05) stimulated after 4h incubation with 1,25(OH)2D3, but only 67 of them where more than 1.5-fold up-regulated. These include the genes encoding for the transcription factors BCL6, NFE2, POU4F2 and ELF4, which are controlled by one or two VDR binding sites within their chromosomal domains. The latter are defined via DNA loop formation mediated by the transcription factor CTCF that is highly conserved in its genome-wide loci. We found BCL6 being most responsive to 1,25(OH)2D3 and selected it for further analysis. An incubation of THP-1 cells with 1,25(OH)2D3 for 24 h resulted in a significant (p<0.001) change in the mRNA expression of more than 1600 genes, of which 132 were at least 2-fold up-regulated. About half of the latter genes are secondary 1,25(OH)2D3 targets, since they do not carry any VDR binding site within their chromosomal domain. Chromatin immunoprecipitation sequencing datasets indicated that the majority of these domains contain a BCL6 binding site. We followed the secondary transcriptional response to 1,25(OH)2D3 for eight representative gene examples and confirmed the binding of CTCF and BCL6 to their respective chromosomal domains. In conclusion, our study indicated that in monocytes most of the physiological responses to 1,25(OH)2D3 involve the action of the transcription factor BCL6.

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

SCOPE: Vitamin D3, its biologically most active metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), 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.

The ASAP2 gene is a primary target of 1,25-dihydroxyvitamin D3 in human monocytes and macrophages.

A genome-wide data set on vitamin D receptor (VDR) binding sites in human THP-1 cells (monocytes) led us to the genomic region around the ASAP2 (Arf-GAP with SH3 domain, ankyrin repeat and PH domain 2) gene, whose product is involved in the regulation of vesicular transport, cellular migration and autophagy. Using ENCODE data, we demonstrated that the ASAP2 gene is flanked by conserved binding sites of the insulating transcription factor CTCF. These sites define different chromosomal domains containing the ASAP2 gene, up to six additional genes and three VDR binding sites. In human monocytes (THP-1 cells) the ASAP2 gene is more weakly expressed but more and faster inducible by the biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), than in M2-type macrophages (phorbol ester-differentiated THP-1 cells). Within the investigated genomic region, the basal mRNA expressions of the neighboring genes are comparably high in both monocytes and macrophages, but the ASAP2 gene is the only primary 1,25(OH)2D3 target. The three VDR binding sites located 54, 436 and 574kb downstream of the ASAP2 transcription start site each carry a sequence formed by a direct repeat with three intervening nucleotides (DR3). Ligand-inducible VDR binding was confirmed to all three genomic sites in monocytes and macrophages. Taken together, the region around the ASAP2 gene is genome-wide highlighted as a special attraction point for the VDR, but the presently sole known functional consequence of the binding of VDR to three sites within this chromosomal region is that ASAP2 is a primary 1,25(OH)2D3 target gene in monocytes and macrophages. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Gene regulatory scenarios of primary 1,25-dihydroxyvitamin d3 target genes in a human myeloid leukemia cell line.

Genome- and transcriptome-wide data has significantly increased the amount of available information about primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes in cancer cell models, such as human THP-1 myelomonocytic leukemia cells. In this study, we investigated the genes G0S2, CDKN1A and MYC as master examples of primary vitamin D receptor (VDR) targets being involved in the control of cellular proliferation. The chromosomal domains of G0S2 and CDKN1A are 140-170 kb in size and contain one and three VDR binding sites, respectively. This is rather compact compared to the MYC locus that is 15 times larger and accommodates four VDR binding sites. All eight VDR binding sites were studied by chromatin immunoprecipitation in THP-1 cells. Interestingly, the site closest to the transcription start site of the down-regulated MYC gene showed 1,25(OH)2D3-dependent reduction of VDR binding and is not associated with open chromatin. Four of the other seven VDR binding regions contain a typical DR3-type VDR binding sequence, three of which are also occupied with VDR in macrophage-like cells. In conclusion, the three examples suggest that each VDR target gene has an individual regulatory scenario. However, some general components of these scenarios may be useful for the development of new therapy regimens.

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.

Genome-wide landscape of liver X receptor chromatin binding and gene regulation in human macrophages.

BACKGROUND: The liver X receptors (LXRs) are oxysterol sensing nuclear receptors with multiple effects on metabolism and immune cells. However, the complete genome-wide cistrome of LXR in cells of human origin has not yet been provided. RESULTS: We performed ChIP-seq in phorbol myristate acetate-differentiated THP-1 cells (macrophage-type) after stimulation with the potent synthetic LXR ligand T0901317 (T09). Microarray gene expression analysis was performed in the same cellular model. We identified 1357 genome-wide LXR locations (FDR < 1%), of which 526 were observed after T09 treatment. De novo analysis of LXR binding sequences identified a DR4-type element as the major motif. On mRNA level T09 up-regulated 1258 genes and repressed 455 genes. Our results show that LXR actions are focused on 112 genomic regions that contain up to 11 T09 target genes per region under the control of highly stringent LXR binding sites with individual constellations for each region. We could confirm that LXR controls lipid metabolism and transport and observed a strong association with apoptosis-related functions. CONCLUSIONS: This first report on genome-wide binding of LXR in a human cell line provides new insights into the transcriptional network of LXR and its target genes with their link to physiological processes, such as apoptosis.The gene expression microarray and sequence data have been submitted collectively to the NCBI Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo under accession number GSE28319.

Cyclical regulation of the insulin-like growth factor binding protein 3 gene in response to 1alpha,25-dihydroxyvitamin D3.

The nuclear receptor vitamin D receptor (VDR) is known to associate with two vitamin D response element (VDRE) containing chromatin regions of the insulin-like growth factor binding protein 3 (IGFBP3) gene. In non-malignant MCF-10A human mammary cells, we show that the natural VDR ligand 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) causes cyclical IGFBP3 mRNA accumulation with a periodicity of 60 min, while in the presence of the potent VDR agonist Gemini the mRNA is continuously accumulated. Accordingly, VDR also showed cyclical ligand-dependent association with the chromatin regions of both VDREs. Histone deacetylases (HDACs) play an important role both in VDR signalling and in transcriptional cycling. From the 11 HDAC gene family members, only HDAC4 and HDAC6 are up-regulated in a cyclical fashion in response to 1α,25(OH)(2)D(3), while even these two genes do not respond to Gemini. Interestingly, HDAC4 and HDAC6 proteins show cyclical VDR ligand-induced association with both VDRE regions of the IGFBP3 gene, which coincides with histone H4 deacetylation on these regions. Moreover, combined silencing of HDAC4 and HDAC6 abolishes the cycling of the IGFBP3 gene. We assume that due to more efficient VDR interaction, Gemini induces longer lasting chromatin activation and therefore no transcriptional cycling but monotonically increasing IGFBP3 mRNA. In conclusion, 1α,25(OH)(2)D(3) regulates IGFBP3 transcription through short-term cyclical association of VDR, HDAC4 and HDAC6 to both VDRE-containing chromatin regions.