Vitamin D-binding protein directs monocyte responses to 25-hydroxy- and 1,25-dihydroxyvitamin D.

BACKGROUND: Serum 25-hydroxyvitamin D (25OHD) is a key factor in determining monocyte induction of the antimicrobial protein cathelicidin, which requires intracrine conversion of 25OHD to 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. Both vitamin D metabolites circulate bound to vitamin D-binding protein (DBP), but the effect of this on induction of monocyte cathelicidin remains unclear. METHODS: Human monocytes were cultured in medium containing 1) serum from DBP knockout (DBP(-/-)) or DBP(+/-) mice, 2) serum-free defined supplement reconstituted with DBP or albumin (control), and 3) human serum with different DBP [group-specific component [Gc]] genotypes with varying affinities for vitamin D metabolites. In each case, response to added 1,25(OH)(2)D(3) or 25OHD(3) was determined by measuring expression of mRNA for cathelicidin and 24-hydroxylase. Monocyte internalization of DBP was assessed by fluorescent tagging followed by microscopic and flow cytometric analysis of tagged DBP. RESULTS: Monocytes cultured in DBP(-/-) serum showed more potent induction of cathelicidin by 25OHD(3) or 1,25(OH)(2)D(3) when compared with DBP(+/-) serum. Likewise, DBP added to serum-free medium attenuated 25OHD(3)/1,25(OH)(2)D(3) responses. Fluorescently tagged DBP showed low-level uptake by monocytes, but this did not appear to involve a megalin-mediated mechanism. Human serum containing low-affinity Gc2-1S or Gc2-2, respectively, supported 2.75-fold (P = 0.003) and 2.43-fold (P = 0.016) higher induction of cathelicidin by 25OHD relative to cells cultured with high affinity Gc1F-1F. CONCLUSION: These data indicate that DBP plays a pivotal role in regulating the bioavailablity of 25OHD to monocytes. Vitamin D-dependent antimicrobial responses are therefore likely to be strongly influenced by DBP polymorphisms.

Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.

The vitamin D receptor (VDR) mediates the endocrine actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and autoregulates the expression of its own gene in target cells. In studies herein, we used chromatin immunoprecipitation-chip analyses to examine further the activities of 1,25(OH)(2)D(3) and to assess the consequences of VDR/retinoid X receptor heterodimer binding at the VDR gene locus. We also explored mechanisms underlying the ability of retinoic acid, dexamethasone, and the protein kinase A activator forskolin to induce VDR up-regulation as well. We confirmed two previously identified intronic 1,25(OH)(2)D(3)-inducible enhancers and discovered two additional regions, one located 6 kb upstream of the VDR transcription start site. Although RNA polymerase II was present at the transcription start site in the absence of 1,25(OH)(2)D(3), it was strikingly up-regulated at both this site and at individual enhancers in its presence. 1,25(OH)(2)D(3) also increased basal levels of H4 acetylation at these enhancers as well. Surprisingly, many of these enhancers were targets for CCAAT enhancer-binding protein-beta and runt-related transcription factor 2; a subset also bound cAMP response element binding protein, retinoic acid receptor, and glucocorticoid receptor. Unexpectedly, many of these factors were resident at the Vdr gene locus in the absence of inducer, suggesting that they might contribute to basal Vdr gene expression. Indeed, small interfering RNA down-regulation of CCAAT enhancer-binding protein-beta suppressed basal VDR expression. These regulatory activities of 1,25(OH)(2)D(3), forskolin, and dexamethasone were recapitulated in MC3T3-E1 cells stably transfected with a full-length VDR bacterial artificial chromosome (BAC) clone-luciferase reporter gene. Finally, 1,25(OH)(2)D(3) also induced accumulation of VDR and up-regulated H4 acetylation at conserved regions in the human VDR gene. These data provide important new insights into VDR gene regulation in bone cells.

The enhanced hypercalcemic response to 20-epi-1,25-dihydroxyvitamin D3 results from a selective and prolonged induction of intestinal calcium-regulating genes.

20-Epi-1,25-dihydroxyvitamin D(3) (20-epi-1,25(OH)(2)D(3)) is a vitamin D analog that exhibits unique biologic properties. The mechanism(s) responsible for these activities remains unclear. Here we explore the ability of 20-epi-1,25(OH)(2)D(3) to induce calcemic responses in mice in vivo and identify a potential mechanism. Surprisingly, the levels of calcemia induced at 24 h after single injections of equivalent doses of 1,25(OH)(2)D(3) or 20-epi-1,25(OH)(2)D(3) were similar, suggesting that both compounds were equal in both potency and efficacy. This similarity was also observed at genes involved in calcium homeostasis including, S100g (calbindin D9K), Trpv6, Cldn2 (claudin 2), Trpv5, and Tnfsf11 (Rankl) as well as Cyp24a1. Despite this, the activities of the two compounds at 48 h were strikingly different. Thus, whereas the activity of 1,25-dihydroxyvitamin D(3) declined at this time point, the response to 20-epi-1,25(OH)(2)D(3) was increased. This unique profile was not due to an exaggerated induction of calcium regulating genes in the intestine, kidney, or bone but to a sustained action on these genes in the intestine. This conclusion was supported by studies using in vivo chromatin immunoprecipitation analysis, which revealed a prolonged presence of vitamin D receptor and RNA polymerase II at the Trpv6 and Cyp24a1 promoters and a sustained increase in histone 4 acetylation in these gene regions as well. We conclude that 20-epi-1,25(OH)(2)D(3) displays superagonist properties largely as a result of its duration of action in the intestine. This action is likely due to a decrease in the rate of intestinal-specific degradation of the ligand rather than to an increase in the functional stability of the vitamin D receptor.

Vitamin D-binding protein influences total circulating levels of 1,25-dihydroxyvitamin D3 but does not directly modulate the bioactive levels of the hormone in vivo.

Mice deficient in the expression of vitamin D-binding protein (DBP) are normocalcemic despite undetectable levels of circulating 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. We used this in vivo mouse model together with cells in culture to explore the impact of DBP on the biological activity of 1,25(OH)(2)D(3). Modest changes in the basal expression of genes involved in 1,25(OH)(2)D(3) metabolism and calcium homeostasis were observed in vivo; however, these changes seemed unlikely to explain the normal calcium balance seen in DBP-null mice. Further investigation revealed that despite the reduced blood levels of 1,25(OH)(2)D(3) in these mice, tissue concentrations were equivalent to those measured in wild-type counterparts. Thus, the presence of DBP has limited impact on the extracellular pool of 1,25(OH)(2)D(3) that is biologically active and that accumulates within target tissues. In cell culture, in contrast, the biological activity of 1,25(OH)(2)D(3) is significantly impacted by DBP. Here, although DBP deficiency had no effect on the activation profile itself, the absence of DBP strongly reduced the concentration of exogenous 1,25(OH)(2)D(3) necessary for transactivation. Surprisingly, analogous studies in wild-type and DBP-null mice, wherein we explored the activity of exogenous 1,25(OH)(2)D(3), produced strikingly different results as compared with those in vitro. Here, the carrier protein had virtually no impact on the distribution, uptake, activation profile, or biological potency of the hormone. Collectively, these experiments suggest that whereas DBP is important to total circulating 1,25(OH)(2)D(3) and sequesters extracellular levels of this hormone both in vivo and in vitro, the binding protein does not influence the hormone's biologically active pool.

Targeted deletion of a distant transcriptional enhancer of the receptor activator of nuclear factor-kappaB ligand gene reduces bone remodeling and increases bone mass.

Receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for osteoclast differentiation, and hormones and cytokines that stimulate bone resorption increase RANKL expression in stromal/osteoblastic cells. We have previously shown that PTH and 1,25-dihydroxyvitamin D(3) control murine RANKL gene expression in vitro, in part, via an evolutionarily conserved transcriptional enhancer, designated the distal control region (DCR), located 76 kb upstream from the transcription start site. Herein we describe the phenotype of mice lacking this enhancer. Deletion of the DCR reduced PTH and 1,25-dihydroxyvitamin D(3) stimulation of RANKL mRNA and osteoclast formation in primary bone marrow cultures as well as stimulation of RANKL mRNA in bone. DCR deletion also reduced basal RANKL mRNA levels in bone, thymus, and spleen. Moreover, mice lacking the DCR exhibited increased bone mass and strength. The increase in bone mass was due to reduced osteoclast and osteoblast formation leading to a low rate of bone remodeling similar to that observed in humans and mice with hypoparathyroidism. These findings demonstrate that hormonal control of RANKL expression via the DCR is a critical determinant of the rate of bone remodeling.

Characterizing early events associated with the activation of target genes by 1,25-dihydroxyvitamin D3 in mouse kidney and intestine in vivo.

In this report, we explore the interaction of the vitamin D receptor (VDR) at regulatory sites within both the Cyp24a1 and the Trpv6 genes using chromatin immunoprecipitation techniques in a mouse model in vivo. We show that exogenous 1,25(OH)(2)D(3) induces rapid VDR and RXR (retinoid X receptor) binding to the Cyp24a1 gene in both the kidney and the intestine and to the Trpv6 gene in the intestine. Separate studies of Trpv6 in vitro suggest that VDR binding occurs directly to VDR response elements located -2 and -4 kb upstream of the TSS. VDR binding is dose-dependent, demonstrating EC(50) values that are comparable with those for the induction of both Cyp24a1 and Trpv6 mRNA. Importantly, interaction of the VDR with these targets results in rapid changes in histone 4 acetylation as well as the recruitment of RNA polymerase II. The presence of both VDR and RNA polymerase II at these sites declines between 3-6 h, whereas the changes observed in acetylation decrease more slowly. Finally, we show that whereas mediator protein 1 is recruited to the Cyp24a1 promoter in the intestine, this coactivator is apparently not required for Trpv6 activation. These studies provide the first evidence for 1,25(OH)(2)D(3)-induced VDR interaction at key target genes in vivo, revealing the consequences of that interaction on the Cyp24a1 and Trpv6 genes.

Multiple enhancer regions located at significant distances upstream of the transcriptional start site mediate RANKL gene expression in response to 1,25-dihydroxyvitamin D3.

One of the primary regulators of receptor activator of NF-kappaB ligand (RANKL) is 1,25-dihydoxyvitamin D(3) (1,25(OH)(2)D(3)). To elucidate the mechanism whereby 1,25(OH)(2)D(3) activates RANKL expression we screened some 300kb of the RANKL gene locus using a ChIP on chip analysis and identified five potential regulatory regions lying significant distances upstream of the transcription start site (TSS), the farthest over 70kb from the TSS. A direct ChIP analysis confirmed the presence of the VDR/RXR heterodimer at these sites. The binding of the VDR was associated with histone modification and enhanced entry of RNA polymerase II, indicating an important functional consequence to the localization of these transcription factors in response to 1,25(OH)(2)D(3). The region -76kb upstream from the TSS, termed D5, was capable of mediating VDR-dependent transcriptional output in response to 1,25(OH)(2)D(3) in luciferase assays. The identified VDRE in this region was able to confer dramatic 1,25(OH)(2)D(3) sensitivity to heterologous promoters. This region was highly evolutionarily conserved and functionally active in the human RANKL gene as well. We propose that the RANKL gene is regulated via multiple enhancers that while located at significant distances from the TSS, likely form a chromatin hub centered on the RankL promoter.

Perspectives on mechanisms of gene regulation by 1,25-dihydroxyvitamin D3 and its receptor.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) functions as a systemic signal in vertebrate organisms to control the expression of genes whose products are vital to the maintenance of calcium and phosphorus homeostasis. This regulatory capability is mediated by the vitamin D receptor (VDR) which localizes at DNA sites adjacent to the promoter regions of target genes and initiates the complex events necessary for transcriptional modulation. Recent investigations using chromatin immunoprecipitation techniques combined with various gene scanning methodologies have revealed new insights into the location, structure and function of these regulatory regions. In the studies reported here, we utilized the above techniques to identify key enhancer regions that mediate the actions of vitamin D on the calcium ion channel gene TRPV6, the catabolic bone calcium-mobilizing factor gene RankL and the bone anabolic Wnt signaling pathway co-receptor gene LRP5. We also resolve the mechanism whereby 1,25(OH)(2)D(3) autoregulates the expression of its own receptor. The results identify new features of vitamin D-regulated enhancers, including their locations at gene loci, the structure of the VDR binding sites located within, their modular nature and their functional activity. Our studies suggest that vitamin D enhancers regulate the expression of key target genes by facilitating the recruitment of both the basal transcriptional machinery as well as the protein complexes necessary for altered gene expression.

Enhancers located in the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.

The regulatory actions of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on target genes are mediated by the vitamin D receptor (VDR). Interestingly, one of the genomic targets of 1,25(OH)(2)D(3) action is the VDR gene itself; however, the mechanism underlying this regulation is unknown. We investigated VDR autoregulation by screening the mouse VDR locus from 20kb upstream of the transcriptional start site (TSS) to 10kb downstream of the last exon using chromatin immunoprecipitation (ChIP)-DNA microarray analysis (ChIP/chip). Three potential VDR binding sites were located within introns lying downstream of the TSS and their activities confirmed through direct ChIP analysis. Further exploration revealed that one of these intronic regions was capable of conferring 1,25(OH)(2)D(3) response to both a downstream heterologous promoter and the minimal VDR promoter. Importantly, this regulatory region contained a classic vitamin D response element and was highly conserved within the human gene. We also demonstrated using ChIP analysis that the binding of VDR is associated with co-localization of RXR and the enhanced entry of RNA polymerase II. Thus, each of these sites appears likely to contribute to VDR autoregulation. Our studies using ChIP/chip analysis coupled to more traditional approaches define a direct mechanism whereby the VDR gene is upregulated by 1,25(OH)(2)D(3).

The vitamin D receptor interacts preferentially with DRIP205-like LxxLL motifs.

The vitamin D receptor (VDR) mediates the biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) through its capacity to recruit coregulatory proteins. This interaction is mediated via a coregulatory LxxLL motif. We screened a combinatorial (x)7LxxLL(x)7 phage library with purified VDR to identify peptides that displayed high affinity and selectivity for VDR. These peptides contained the consensus sequence Lx E/H x H/F P L/M/I LxxLL and exhibited significant sequence similarity to the active LxxLL box found in DRIP205. Nearly all LxxLL peptides interacted in a ligand-dependent manner directly with human VDR. However, a pattern of selectivity of the peptides for other members of the nuclear receptor family was also observed. Interestingly, the interaction between the VDR and many of the peptides was differentially sensitive to a broad assortment of VDR ligands. Finally, several of these peptides were shown to inhibit activation of a 1,25(OH)2D3-sensitive reporter gene. These studies suggest that the LxxLL motif can interact directly with the VDR and that this interaction is regulated by chemically diverse vitamin D ligands.

1,25-Dihydroxyvitamin D3 induces expression of the Wnt signaling co-regulator LRP5 via regulatory elements located significantly downstream of the gene's transcriptional start site.

Canonical Wnt signaling is essential for bone formation. Activation involves binding of secreted members of the Wnt family of proteins with a membrane receptor Frizzled on osteoblasts, an interaction that is facilitated by LRP5/LRP6 co-receptors. LRP5 is known to play a particularly important role in bone formation such that the loss of this protein results in a reduction in osteoblast number, a delay in mineralization and a reduction in peak BMD. During the course of a VDR ChIP-chip analysis we found that 1,25(OH)(2)D(3) could induce binding of the VDR to sites within the Lrp5 gene locus. Importantly, this interaction between 1,25(OH)(2)D(3)-activated VDR and the Lrp5 gene led to both a modification in chromatin structure within the Lrp5 locus and the induction of LRP5 mRNA transcripts in vivo as well as in vitro. One site within Lrp5 was discovered to confer 1,25(OH)(2)D(3) response to a heterologous promoter in osteoblastic cells, permitting both the identification and characterization of the component VDRE. While the regulatory region in Lrp5 was highly conserved in the human genome, the VDRE was not. Our studies show that 1,25(OH)(2)D(3) can enhance the expression of a critical component of the Wnt signaling pathway which is known to impact osteogenesis.

Molecular actions of 1,25-dihydroxyvitamin D3 on genes involved in calcium homeostasis.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] functions in vertebrate organisms as a primary regulator of calcium and phosphorus homeostasis, an activity that is achieved through direct actions on gene expression in intestine, kidney, and bone. Recent studies have identified novel genes such as TRPV5, TRPV6, and RANKL whose products are integral to the maintenance of extracellular calcium. The objective of this progress report/review is to describe our recent results that identify the mechanisms of 1,25(OH)(2)D(3) action on the expression of TRPV6 and RANKL. A series of molecular, cellular, and in vivo studies have been conducted to define the molecular mechanisms that control the expression of TRPV6 and RANKL. Cell culture-based assays, chromatin immunoprecipitation (ChIP) and ChIP-DNA microarray (ChIP-chip) methods, and a series of molecular techniques were used to identify and characterize upstream regions of mouse and human TRPV6 and RANKL genes. We discovered that these genes were regulated by at least five separate enhancer regions. In the TRPV6 gene, these enhancers were all located within 5 kb of the transcriptional start site (TSS), and each contained one or more vitamin D regulatory elements (VDREs). In the RANKL gene, these regulatory regions span over 80 kb of upstream sequence, the most distal 76 kb from the TSS. This regulatory region is central to the regulation of RANKL expression in vitro and in vivo. Our studies identified key regulatory regions within the TRPV6 and RANKL genes that are essential for their individual expression in the intestine and bone, respectively.

Activation of receptor activator of NF-kappaB ligand gene expression by 1,25-dihydroxyvitamin D3 is mediated through multiple long-range enhancers.

RANKL is a tumor necrosis factor (TNF)-like factor secreted by mesenchymal cells, osteoblast derivatives, and T cells that is essential for osteoclastogenesis. In osteoblasts, RANKL expression is regulated by two major calcemic hormones, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and parathyroid hormone (PTH), as well as by several inflammatory/osteoclastogenic cytokines; the molecular mechanisms for this regulation are unclear. To identify such mechanisms, we screened a DNA microarray which tiled across the entire mouse RankL gene locus at a 50-bp resolution using chromatin immunoprecipitation (ChIP)-derived DNA precipitated with antibodies to the vitamin D receptor (VDR) and the retinoid X receptor (RXR). Five sites of dimer interaction were observed on the RankL gene centered at 16, 22, 60, 69, and 76 kb upstream of the TSS. These regions contained binding sites for not only VDR and RXR, but also the glucocorticoid receptor (GR). The most distant of these regions, termed the distal control region (RL-DCR), conferred both VDR-dependent 1,25(OH)(2)D(3) and GR-dependent glucocorticoid (GC) responses. We mapped these activities to an unusual but functionally active vitamin D response element and to several potential GC response elements located over a more extensive region within the RL-DCR. An evolutionarily conserved region within the human RANKL gene contained a similar vitamin D response element and exhibited an equivalent behavior. Importantly, hormonal activation of the RankL gene was also associated with chromatin modification and RNA polymerase II recruitment. Our studies demonstrate that regulation of RankL gene expression by 1,25(OH)(2)D(3) is complex and mediated by at least five distal regions, one of which contains a specific element capable of mediating direct transcriptional activation.

1,25-Dihydroxyvitamin D3 regulates the expression of low-density lipoprotein receptor-related protein 5 via deoxyribonucleic acid sequence elements located downstream of the start site of transcription.

The skeleton is a direct target of vitamin D action, where the hormone modulates the proliferation of osteoblast precursors, their differentiation into mature osteoblasts, and their functional activity. Some of these effects of vitamin D are reminiscent of those orchestrated by the Wnt signaling pathway wherein stimulation of the membrane receptor Frizzled and its coreceptor LRP5 leads to activation of beta-catenin and subsequent transcription-mediated changes in osteoblast biology. Indeed, LRP5 is now known to play a particularly important role in bone formation such that the loss of this component results in a reduction in osteoblast number, a delay in mineralization, and a reduction in peak bone mineral density. Interestingly, we discovered during the course of a vitamin D receptor (VDR) chromatin immunoprecipitation/DNA microarray analysis that 1,25-(OH)2D3 could induce binding of the VDR to sites within the Lrp5 gene locus. VDR and retinoid X receptor binding was evident both in primary osteoblasts as well as in osteoblasts of cell line origin. Importantly, this interaction between 1,25-(OH)2D3-activated VDR and the Lrp5 gene led to both a modification in chromatin structure within the Lrp5 locus and the induction of Lrp5 mRNA transcripts in vivo as well as in vitro. One of these sites within the Lrp5 locus was discovered to confer vitamin D response to a heterologous promoter when introduced into osteoblastic cells, permitting both the identification and characterization of the vitamin D response element located within. Interestingly, additional studies revealed that whereas the regulatory region in the mouse Lrp5 gene was highly conserved in the human genome, the vitamin D response element was not. Our studies show that 1,25-(OH)2D3 can enhance the expression of a critical component of the Wnt signaling pathway that is known to impact osteogenesis.

Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.

The biological actions of 1,25-(OH)2D3 are mediated by the vitamin D receptor (VDR), a protein that binds to target genes and alters their expression. 1,25-(OH)2D3 is also capable of inducing transcription of the VDR gene itself. In the present study, we explored both the capacity of 1,25-(OH)2D3 to induce VDR gene expression in bone cells and the mechanism instrumental to this up-regulation. After establishing the ability of 1,25-(OH)2D3 to stimulate VDR mRNA up-regulation both in bone in vivo and in osteoblastic cells, we screened the mouse VDR gene locus from 20 kb upstream of the gene's transcriptional start site (TSS) to 10 kb downstream of the final exon to identify VDR binding sites using chromatin immunoprecipitation-DNA microarray (ChIP-chip) analysis. Three conserved regions were identified 20, 27, and 29 kb downstream of the TSS. VDR binding to these sites in response to 1,25-(OH)2D3 was confirmed by ChIP analysis and was accompanied by differential localization of retinoid X receptor, histone acetylation, and RNA polymerase II recruitment. One of these regions was able to confer 1,25-(OH)2D3 regulation to downstream promoters, thereby permitting identification and characterization of the regulatory element located within. Importantly, a highly conserved region within the human VDR gene analogous to that discovered in the mouse was also capable of mediating 1,25-(OH)2D3 response. Our results demonstrate that 1,25-(OH)2D3 and its receptor autoregulate the expression of the VDR gene. The location of these regulatory regions and their apparent distances from the TSS are consistent with new findings suggesting the emerging relevance of distant enhancers.