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Keywords:

  • ion channels;
  • osteoclast-inducing factor;
  • 1,25-dihydroxyvitamin D3;
  • chromatin immunoprecipitation analysis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL OBSERVATIONS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] 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)2D3 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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL OBSERVATIONS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES

The fundamental actions of the steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are to maintain calcium and phosphorus homeostasis in vertebrate organisms.[1] This activity is achieved through direct actions of the hormone on the intestine, kidney, and bone and through feedback inhibition of PTH production at the parathyroid glands. In the intestine and kidney, the transepithelial transport of calcium is now known to involve the apical calcium ion channels TRPV5 and TRPV6,[2, 3] soluble components such as the calbindins (D9K and D28K), the basolateral ATPase-driven calcium pump PMCA1b, and the sodium/calcium exchanger NCX1.[4, 5] Of these protein components, however, it is the calcium ion channels in the intestine and kidney that have emerged most recently as key to the control of calcium translocation. Indeed, TRPV5 and TRPV6 have been termed the “gatekeepers” of epithelial calcium transport.[6] Activity at the skeleton, on the other hand, is driven primarily by RANKL, a TNF-like factor produced by stromal cells and osteoblasts that is both necessary and sufficient for the formation, activation, and survival of bone-resorbing osteoclasts.[7] Perhaps most important is the observation that a primary regulator of TRPV5, TRPV6, and RANKL expression is 1,25(OH)2D3. Thus, both renal TRPV5 and intestinal TRPV5 and TRPV6 expression are substantially reduced in both 25-hydroxyvitamin D3-1α-hydroxylase (Cyp27b1)-null[8] and vitamin D receptor (VDR)-null[9] mice. Mice containing a deleted VDR gene cannot produce RANKL in response to 1,25(OH)D3, and therefore cannot support osteoclastogenesis.[10] Collectively, these recent experiments highlight the importance of TRPV5, TRPV6, and RANKL in the processes that are both central to calcium homeostasis in intestine, kidney, and bone and the focus of a key activity of 1,25(OH)2D3. We therefore explored the molecular mechanism whereby 1,25(OH)2D3 mediates the upregulation of these genes both in vivo and in vitro.

EXPERIMENTAL OBSERVATIONS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL OBSERVATIONS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES

Mechanism of action of 1,25(OH)2D3

Figure 1 depicts a model that outlines the mechanism of action of 1,25(OH)2D3 on target genes within a generic cell.[11] In this model, the hormone diffuses into the cell and interacts directly with either a cytoplasmic or nuclear form of the VDR or both. This interaction leads to the formation of a VDR-retinoid X receptor (RXR) heterodimer that localizes subsequently to sequence-specific DNA sites on cell-specific target genes. VDR/RXR localization is followed by the recruitment of a series of co-regulatory complexes or molecular machines that act in turn to modify nucleosome occupancy, alter the chemical state of histones that participate in the organization of chromatin structure, and facilitate the entry of basal transcriptional machinery that includes RNA polymerase II. Thus, these actions lead directly to changes in the transcriptional output of genes that are targeted for modification. The output of these genes encode proteins responsible for mediating the actions of 1,25(OH)2D3 in a cell- and tissue-specific manner.

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Figure FIG. 1.. Model for the molecular actions of 1,25(OH)2D3 on gene expression in both generic and specific target cells. Regulatory responses to 1,25(OH)2D3 can be both ubiquitous and cell specific. Cyp24a1 upregulation by 1,25(OH)2D3 represents a ubiquitous response. VDR upregulation and Cyp27b1 suppression by 1,25(OH)2D3 are cell type–specific responses. C, cytoplasm; N, nucleus; V, VDR; R, RXR.

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Several target genes play a direct and perhaps mechanistic role in modulating the biological activity of 1,25(OH)2D3. As seen in Fig. 1, these include the VDR itself, Cyp24a1, and Cyp27b1. Accordingly, the VDR gene is a direct target of its own receptor, thus facilitating an upregulation of the VDR protein in certain target tissues.[12] This autoregulatory mechanism likely functions to accommodate the altered “consumption” of activated VDR. Additional target genes include Cyp24a1,[13] which functions to modify the incoming ligand signal through degradation, and Cyp27b1,[14] which functions to produce the ligand from its proximal precursor 25-hydroxyvitamin D3. In the latter case, the actions of 1,25(OH)2D3 and its receptor are to suppress the synthesis of the enzyme responsible for 1,25(OH)2D3 production. It seems reasonable that the regulation of these specific target genes are integral to the overall biological actions of 1,25(OH)2D3 in all tissues and cell types.

TRPV5 and TRPV6 genes

As indicated in the Introduction, the TRPV5 and TRPV6 genes encode calcium ion channels that are essential for the transepithelial uptake of calcium by the kidney and the absorption of calcium from the intestinal lumen.[2, 3, 6] TRPV5 is expressed primarily in the kidney, whereas TRPV6 is produced in the intestine. In the human genome, TRPV5 and TRPV6 lie proximal to each other at chromosome 7q35, with TRPV5 located upstream. In view of the sensitivity of these genes to 1,25(OH)2D3,[8, 9] we embarked on a series of studies to determine the mechanisms whereby 1,25(OH)2D3 promotes the upregulation of these genes both in vivo and in vitro. Figure 2 shows, in part, the results of those studies. We used a chromatin immunoprecipitation (ChIP) PCR scanning method together with an in silico assessment of the first 7 kb of the human TRPV6 gene to identify potential binding sites or vitamin D response elements (VDREs) for ligand-activated VDR and its heterodimer partner on the upstream portion of the gene.[15] As shown in Fig. 2, major sites were located at approximately −1.2, −2.1, −3.5, −4.3, and −5.5 kb upstream of the transcriptional start site (TSS). Of these, the VDREs that contribute most significantly to the activity of 1,25(OH)2D3 at the TRPV6 promoter include the elements at −1.2, −2.1, and −4.3 kb. Indeed, the VDREs located at the −2.1- and −4.3-kb sites are made up of two independent regulatory elements. Similar sites have been identified and are being characterized in the mouse TRPV6 gene.[16] These regulatory regions of the TRPV6 gene function to recruit co-activator complexes that in turn alter the chemical composition of chromatin at histone 4. Our studies provide preliminary support for a direct action of 1,25(OH)2D3 at the TRPV6 locus. Ongoing studies are now focused on similar mechanisms at the TRPV5 gene in the kidney.

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Figure FIG. 2.. Position and sequence of VDREs located in the upstream region of the human TRPV6 gene. Black bars represent positions of putative VDREs. Experimental evaluation of these regulatory elements provides strong support for the five regulatory elements located in the TRPV6 promoter at −1.2, −2.1, and −4.3 kb. Uppercase letters represent binding half-sites; lowercase letters represent spacing sequences between the half-sites.

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RANKL gene

RANKL represents a long sought after factor that induces the formation, activation, and survival of bone-resorbing osteoclasts.[7] RANKL is expressed in a variety of supportive cell types including stromal cells, osteoblasts, fibroblasts, and chondrocytes, and acts on both osteoclast precursors and mature cells largely through direct contact.[17, 18] The role of RANKL in bone resorption is well established. Indeed, its upregulation is responsible for the osteoporosis that is associated with many disease states, making it a striking therapeutic target. The human gene is located on chromosome 13q14 in what seems to be a nominal gene desert. Thus, its proximal neighbors are located over 200 kb upstream or downstream of the RANKL gene itself. The calcemic hormones 1,25(OH)2D3 and PTH are both primary regulators of RANKL expression from stromal cells and osteoblasts.[7] We therefore set out to determine the mechanisms whereby each of these hormones induce an upregulation of RANKL and therefore an increase in bone resorption. Figure 3 shows the upstream organization of the mouse RANKL gene; the human gene is similarly organized. Initial studies suggested that 1,25(OH)2D3 modulates the expression of RANKL through a regulatory element located within the proximal promoter.[19] The overall contribution of this element to RANKL induction by 1,25(OH)2D3 is unclear, however. We therefore used an unbiased ChIP-DNA microarray (ChIP-chip) approach that enabled us to scan broad regions of the RANKL locus for binding sites for the VDR and its RXR partner.[20] As can be seen in Fig. 3, five sites of VDR/RXR interaction were identified and characterized. All of these sites were located significant distances from the TSS, the most distal at −76 kb in the mouse genome and −99 kb in the human genome. In the context of this experiment,[20] it is noteworthy that the putative regulatory element located at the proximal region of the RANKL promoter[19] was not observed. The upstream regions were shown to mediate the actions of 1,25(OH)2D3 to induce RANKL gene expression. Interestingly, these enhancer regions were also discovered to mediate the transcriptional activity of PTH and its surrogate activator forskolin on RANKL gene expression through stimulation and phosphorylation of the transcription factor CREB.[21, 22] Importantly, the removal of the most distal of these regulatory regions, termed the RANKL distal control region (RL-DCR), from the mouse genome leads to a failure of full osteoclast activation and an increase in BMD.[22, 23] More importantly, neither PTH nor 1,25(OH)2D3 are fully effective in inducing RANKL expression. Our studies show, therefore, that the calcemic hormones 1,25(OH)2D3 and PTH mediate the upregulation of RANKL through unusual regulatory enhancers that are located at significant distances upstream of the RANKL TSS. Interestingly, a subset of these enhancer regions in the RANKL gene also mediates the activity of osteoclastogenic cytokines in osteoblastic cells.[21] Future studies will be needed to determine whether these enhancers mediate the actions of these cytokine or other known regulators of RANKL expression in T cells and other cell types where RANKL is known to be expressed.[18]

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Figure FIG. 3.. Organization of the mouse RANKL gene upstream region. Black bars represent the locations of five distal regulatory enhancers (D1–D5). The sequences of regulatory elements that have been functionally characterized for RUNX2 (OSE), CREB (CRE1 and 2), and VDR/RXR (VDRE1/VDRE2) are shown. Uppercase and lowercase letters are as indicated in Fig. 2.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL OBSERVATIONS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES

This work was supported by National Institutes of Health Grants AR-45173, DK-72881, and DK-74993.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL OBSERVATIONS AND DISCUSSION
  5. Acknowledgements
  6. REFERENCES
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