Abstract
The central role of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) is to regulate calcium and phosphorus homeostasis through actions in the intestine, kidney, and bone. These actions are mediated by the vitamin D receptor (VDR), which acts in all target tissues to regulate the expression of genes that are integral to cellular response. Studies over the past decade or more have identified a number of target genes in a myriad of tissues, including the intestine and colon. Recent studies on a genome-wide scale have confirmed and extended old ideas and lead to new concepts as well. In the intestine, the actions of 1,25(OH) 2 D 3 and its receptor are focused upon calcium and phosphate transport, xenobiotic metabolism, and growth regulation. This latter activity may be relevant to the antitumor effects of the hormone in colorectal cancer. Future studies are designed to further understand the actions of the vitamin D hormone in not only the intestine but also other tissues.
Keywords
Vitamin D, Mineral homeostasis, Hormone activation, Gene regulation, Calcium transport, Lithocholic acid ligand, C-Myc, Transporters, Cyps
51.1
Introduction
Studies of Mellanby, McCollum, Steenbock, Windaus, and others earlier in the 20th century resulted in the discovery and characterization of an important new bioactive substance termed vitamin D. Although this vitamin was found to be produced in the skin following exposure to sunlight, studies that rapidly followed indicated that the compound undergoes sequential hydroxylations in the liver to 25-hydroxyvitamin D 3 and then in the kidney to 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), the biologically active form of the original vitamin. The discovery that its mode of action involves the regulation of gene expression led to the suggestion that 1,25(OH) 2 D 3 might represent a novel steroid hormone rather that a true vitamin. This hypothesis proved to be correct as a result of extensive mechanistic studies that were carried out during the ensuing decades.
It is now known that 1,25(OH) 2 D 3 operates in target tissues such as the intestinal tract, kidney, and bone through its capacity to activate a nuclear receptor/transcription factor termed the vitamin D receptor (VDR). This regulatory protein functions to modulate the expression of specific genes whose products are responsible for maintaining calcium and phosphorus homeostasis. This factor is also known to regulate many additional biological processes. Regulation is enabled through direct interaction of the 1,25(OH) 2 D 3 -bound VDR with specific DNA sequence elements (VDREs) located within individual, cell-specific genetic loci. The localization of the VDR at these sites provides a nucleation center for the recruitment of additional sets of coregulatory complexes that are essential for the diverse genetic and epigenetic processes that are ultimately required to modify gene expression. Common features of this mechanism are utilized by all the steroidal and lipophilic hormones.
1,25(OH) 2 D 3 functions to integrate the calcium and phosphorus regulating actions of intestine, kidney, and bone such that blood calcium and to a lesser extent blood phosphorus levels are maintained within tight limits. An elevation in circulating 1,25(OH) 2 D 3 levels accelerates the absorption of calcium and phosphorus by the intestine, the reabsorption of filtered calcium at the kidney, and under certain conditions mobilization of both calcium and phosphorus from the skeleton. 1,25(OH) 2 D 3 is produced in response to parathyroid hormone (PTH), a peptide whose elaboration from the parathyroid gland is exquisitely sensitive to blood calcium levels. Decreasing blood calcium triggers increased parathyroid gland production of PTH, which causes increased synthesis and elaboration of 1,25(OH) 2 D 3 and its subsequent actions on the three tissues responsible for elevating blood calcium and phosphorus levels. Importantly, elevated phosphate levels prompt the secretion of a second recently discovered hormone termed fibroblast growth factor 23 (FGF23), a bone cell-derived phosphaturic hormone, which acts in the kidney to increase phosphate diuresis. Other hormones are also involved in mineral metabolism, although to a lesser extent.
In this chapter, we provide an historical overview of the vitamin D endocrine system. We then provide a contemporary view of how the vitamin D hormone functions to modulate gene expression in the intestine, kidney, and bone. We also provide a focus on genes that are directly involved in intestinal calcium transport across the intestinal epithelium and describe the molecular mechanisms through which these genes are regulated by 1,25(OH) 2 D 3 . As will be evident, our understanding of these mechanisms has been greatly enhanced through the discovery and implementation of powerful new approaches for the study of gene expression which have also enabled an examination of epigenetic contributions linked to the expression of genes. In the final section, we discuss briefly newer discoveries of the actions of vitamin D and/or its receptor in the intestinal tract, including the novel activation of the VDR by lithocholic acid (LC) in the colon, the unexpected role of the VDR as an inducer of enzymes responsible for the detoxification of secondary fecal bile acids, and the potential impact of the vitamin D hormone on the microbiome.
51.2
Overview of Vitamin D Production and Physiology
51.2.1
Synthesis, Activation, and Degradation of Vitamin D
Following the initial discovery of vitamin D, extensive work over the ensuing half century has revealed that vitamin D is produced, activated through hydroxylation, and then degraded by very specific metabolic processes. Indeed, vitamin D is synthesized in the skin following exposure to sunlight through a process that involves initial photolysis of cutaneous 7-dehydrocholesterol (provitamin D) to previtamin D followed by rapid isomerization to authentic vitamin D. Importantly, vitamin D was then discovered to undergo further metabolism, first in the liver to 25-hydroxyvitamin D 3 (25OHD 3 ) and then in the kidney to 1,25(OH) 2 D 3 . The enzymes responsible for these conversions are cytochrome p 450-containing mixed-function oxidases. Several hydroxylases have been identified that can carry out 25-hydroxylation of vitamin D, including mitochondrial CYP2R1, as well as microsomal CYP27A1, CYP2D11, CYP2D25, CYP2J2/3, and CYP3A4. CYP2R1, a 25-hydroxylase (25-OHase) recently discovered in the liver by Cheng et al. demonstrates highest affinity for vitamin D and is likely to be the most important of the 25-OHases. By far, the most critical hydroxylation of vitamin D that results in the synthesis of 1,25(OH) 2 D 3 , however, occurs in the kidney through the actions of mitochondrial CYP27B1 ( Fig. 51.1 ). First discovered by Fraser and Kodicek in 1970, this enzyme was cloned by St. Arnaud and coworkers as well as others and shown to be solely responsible for the synthesis of 1,25(OH) 2 D 3 . Importantly, genetic mutations in both CYP2R1 and CYP27B1 are associated with human disease, the former with a unique and perhaps rare form of hereditary rickets, the latter with the syndrome of vitamin D resistant rickets, type 1 (VDDR-1). The biochemical and skeletal phenotype of this latter syndrome has been recapitulated recently through genetic deletion of the Cyp27b1 gene in mice using homologous recombination. Recent studies of the deletion of Cyp2r1 in mice led to a significant reduction in circulating 25(OH)D 3 levels, but leave open the likelihood that other 25-hydroxylases may also contribute.
The activity of renal CYP27B1 is critical to the production and maintenance of physiologic levels of circulating 1,25(OH) 2 D 3 . As a consequence, the synthesis and activity of CYP27B1 are tightly regulated through factors that are elaborated in response to changes in blood calcium and/or phosphorus ( Fig. 51.1 ). Most notable is PTH, which is produced in the parathyroid glands in response to hypocalcemia, and which acts directly on the kidney to stimulate CYP27B1 gene expression. CYP27B1 is also modulated independently through phosphate signaling, although the mechanism through which this regulation is carried out has yet to be fully understood. Recent studies suggest that FGF23 also plays a key role in the modulation of CYP27B1 expression. FGF23 represents the long sought after hormone phosphatonin (or a member thereof) that is induced by 1,25(OH) 2 D 3 and is the major mediator of phosphorus homeostasis. Linkage of FGF23 to the regulation of phosphate was initially derived from phenotypes associated with tumor-induced osteomalacia (TIO), autosomal dominant hypophosphatemic rickets (ADHRs), and X-linked hypophosphatemic (XLH) syndromes. The FGF23 gene has been deleted and overexpressed and the emerging phenotypes strongly support this linkage. The molecular mechanisms and signaling pathways whereby PTH and FGF23 suppresses the expression of renal CYP27B1 also remain obscure, although new approaches are seeking to delineate details of this regulation. Importantly, completion of the vitamin D endocrine circuit involves a potent feedback mechanism through which 1,25(OH) 2 D 3 acts to suppress CYP27B1 expression in the kidney, to downregulate PTH expression and production by the parathyroid gland and, as stated earlier, to upregulate FGF23. A number of additional factors also regulate CYP27B1 including the sex and adrenal hormones, prolactin, and growth hormone.
With respect to catabolism, 1,25(OH) 2 D 3 is degraded through the primary actions of CYP24A1, a mitochondrial enzyme also found in the kidney and in virtually all vitamin D target tissues. As perhaps expected, its expression is strongly induced by 1,25(OH) 2 D 3 itself. The degradative pathway involves a third hydroxylation of 1,25(OH) 2 D 3 at carbon 24 to 1,24,25-trihydroxyvitamin D 3 (1,24,25(OH) 3 D 3 ) followed by multistep destruction of the vitamin D side chain to calcitroic acid. Hydroxylation at C-23 also occurs as well. 25OHD 3 is also hydroxylated by CYP24A1 leading to high circulating levels of 24,25-dihydroxyvitamin D 3 (24,25(OH) 2 D 3 ). Thus, CYP24A1 regulates not only 1,25(OH) 2 D 3 catabolism in all tissues but also controls the renal production of other metabolites including 25OHD 3 , 24,25(OH) 2 D 3 , and 1,25(OH) 2 D 3 , and their concentrations in the blood. A biological role for 24,25(OH) 2 D 3 has been hypothesized for many years, although little in vivo evidence exists to support this view. St. Arnaud and coworkers deleted the Cyp24a1 gene in the mouse, an action that resulted in hypercalcemia, hypercalciuria, renal calcification, and skeletal abnormalities; all of these effects were subsequently attributed to toxic circulating levels of 1,25(OH) 2 D 3 , which coexist as a result of Cyp24a1 deletion. As indicated above, one of the fundamental actions of 1,25(OH) 2 D 3 in all target cells is to stimulate the expression of CYP24A1 , thus initiating the means to its own self destruction. Interestingly, while the mechanism through which 1,25(OH) 2 D 3 induces CYP24A1 transcription was believed to be well understood at the molecular level, more recent studies using unbiased approaches have revealed a much more complex mode of activation by the vitamin D hormone through coregulatory regions located distal to the gene’s transcriptional start site (TSS). This regulatory mechanism represents a paradigm for the current view of how most genes are believed to be regulated by transcriptional activators.
51.2.2
Regulation of Calcium and Phosphorus Homeostasis by 1,25(OH) 2 D 3
Calcium and phosphorus homeostasis is maintained through the activity of the intestinal tract, kidney, and bone ( Fig. 51.1 ). These tissues serve to acquire mineral from the diet, to conserve mineral from glomerular filtrate, and to provide an immediately available source of skeletal mineral when the diet is deficient in calcium and/or phosphorus. Integrating the actions of these tissues so that serum calcium and phosphorus levels are maintained within tight limits and at supersaturating concentrations relative to mineralized bone is the central function of 1,25(OH) 2 D 3 . The actions of 1,25(OH) 2 D 3 in the intestine are therefore focused upon stimulating the production of proteins essential to the processes of dietary calcium and phosphorus absorption. 1,25(OH) 2 D 3 , particularly at aberrantly high concentrations, can also provoke calcium and phosphorus mobilization from the skeleton through a process involving both stimulation of bone-resorbing osteoclast activity as well as the induction of new osteoclast formation from cellular precursors. Interestingly, this mechanism involves the ability of 1,25(OH) 2 D 3 to induce expression of the autocrine TNF-like factor receptor activator of NF-κB Ligand (RANKL) from osteoblasts and osteocytes. This factor acts in turn on both osteoclast precursors and fully mature osteoclasts. The mechanism has a particularly profound consequence when dietary levels of calcium are insufficient. Accordingly, a homeostatic attempt in mice and in humans is made to maintain serum calcium and phosphorus levels at the expense of bone, which leads to bone demineralization, a weakening of the structure of the skeleton and increased risk of bone fracture. Interestingly, recent studies suggest that osteocytes, mature osteoblasts that have become fully encased in bone mineral, may also function to resorb bone and could represent critical targets of vitamin D action as well. Indeed, it is the osteocyte that elaborates FGF23 in response to a variety of stimulants including the vitamin D hormone. Finally, 1,25(OH) 2 D 3 also acts on the kidney to increase the resorption of calcium. Although this action is modest, the actual amount of calcium recovered through reabsorption can be highly significant due to the large daily load of calcium that is filtered by the kidney.
Central to the orchestration of intestinal, kidney, and bone actions by 1,25(OH) 2 D 3 are the additional systems that monitor the content of calcium and phosphorus in the blood. In the case of calcium, the level of this ion is continually monitored by the calcium-sensing receptor (CsR) in the parathyroid gland, which responds when calcium levels drop by increasing parathyroid gland secretion of PTH. As noted earlier, increased PTH levels enhance the synthesis of 1,25(OH) 2 D 3 , stimulating, in turn, both calcium uptake at the intestine and calcium reabsorption by the kidney. The actions of 1,25(OH) 2 D 3 in conjunction with increased PTH on bone serve to promote bone mobilization, which also leads to an increase in blood levels of mineral. While 1,25(OH) 2 D 3 also acts to increase phosphate levels in the blood via actions on the intestine, both PTH and FGF23 act collectively on the kidney to modulate phosphate reabsorption. With regard to FGF23, a novel mechanism is now known whereby FGF23 promotes a redistribution of the phosphate transporters NaPi2a ( SLC34A1 ) and NaPi2c ( SLC34A3 ) such that proximal tubular reabsorption of phosphate is reduced. FGF23’s ability to reduce circulating levels of 1,25(OH) 2 D 3 through downregulation of the renal CYP27B1 gene also results in a reduction in intestinal phosphate uptake. The molecular mechanisms whereby 1,25(OH) 2 D 3 induces the uptake of calcium as well as phosphorus across the intestinal epithelium as well as its molecular actions in kidney and bone will be considered in subsequent sections.
51.3
The Molecular Mechanism of Action of 1,25(OH) 2 D 3
51.3.1
Discovery, Properties, and Cloning of the Vitamin D Receptor (VDR)
The VDR was discovered first in the chick intestine in the early 1970s, and rapidly determined to be expressed in other tissues such as kidney, bone, and parathyroid glands shortly thereafter. It is now clear that this factor is expressed in selected cell types in almost all vertebrate tissues, the majority of which do not play roles in calcium and phosphorus homeostasis. The VDR is a protein of approximately 50 kDa that is able to bind 1,25(OH) 2 D 3 with very high affinity and selectivity, an activation process that leads to a complex set of downstream events. This largely nuclear-localized protein was discovered subsequently to bind directly to DNA through a domain separate from that which binds ligand. These functional properties of the VDR provided further support for the hypothesis that the actions of 1,25(OH) 2 D 3 occur at the level of transcription. Details of the overall process of ligand-activated, VDR-mediated gene regulation are depicted in the model in Fig. 51.2 and will be considered in detail in subsequent sections. In this model, the VDR is activated by 1,25(OH) 2 D 3 , although the secondary bile acid LCA appears able to modulate receptor activity in the colon as well ( Section 51.7 ). While much was accomplished following the discovery of the VDR, it is clear that the most important advance following its identification was the cloning of the chicken VDR gene in 1987 and the subsequent cloning of the human and rat homologues as well. As the overall structural similarities of the VDR with that of several other steroid receptors cloned during that period of time were strikingly evident, the cloning of the VDR provided the final evidence that 1,25(OH) 2 D 3 operated in a manner mechanistically similar to that of other steroidal ligands. These latter achievements opened the door for a myriad of additional studies that have revealed not only the molecular domain structure of the protein but also precise details of its functional capabilities.
51.3.2
Interaction of the VDR With Target Genes
1,25(OH) 2 D 3 is known to regulate the expression of a number of genes, as summarized in the model in Fig. 51.2 , not only in intestine, kidney, and bone but also in other tissues. Perhaps its most ubiquitous regulatory target is CYP24A1 , which as indicated earlier functions negatively to downregulate cellular levels of 1,25(OH) 2 D 3 . In the intestine, 1,25(OH) 2 D 3 regulates the transcriptional output of the calcium-binding proteins (calbindins D9K and D28K), basolateral calcium-stimulated ATPases such as PMCA1b and TRPV6 (considered in more detail in a subsequent section). Proteins with analogous functions are also regulated by 1,25(OH) 2 D 3 in the kidney. In bone, however, 1,25(OH) 2 D 3 regulates a variety of additional genes whose products include the matrix proteins osteocalcin, osteopontin, and bone sialoprotein that are involved in bone osteoid formation and which control mineralization. 1,25(OH) 2 D 3 also regulates RANKL and OPG, two factors that as previously indicated participate in the bone resorbing activities of osteoclasts that are central to bone calcium and phosphorus mobilization. Investigation of each of these transcriptional targets at the molecular level has revealed the presence of specific DNA sequences or VDREs that represent high-affinity-binding sites for the VDR. In initial studies using traditional methodological approaches, many VDR-binding sites were found within several kilobases of the genes’ promoters. Further exploration of these VDREs has revealed adjacent binding sites for many additional regulatory proteins as well, suggesting that these regions are modular and likely mediate and integrate the actions of a variety of signaling pathways. VDREs are comprised largely, but not exclusively, of two directly repeated hexanucleotide half-sites of the consensus sequence AGGTCA that are separated by an identity-irrelevant three base pairs. Several additional configurations have been defined as well, although these sequences have yet to be fully validated via contemporary techniques. The response elements that mediate the actions of 1,25(OH) 2 D 3 in cis are similar, but not identical, to those for other nuclear receptors that utilize RXR as partner. Most striking is that while the arrangement of the half-sites and their sequences are retained, the degree of nucleotide spacing that exists between the two half-sites diverges. Thus, the diagnostic feature for a potential VDRE is not the sequence of the half-sites, but rather their three base pair separation. As will be seen in a subsequent section, evaluation of both the placement and structural organization of VDREs across thousands of VDR-binding sites identified through recent genome-wide analyses has confirmed the structural organization of these VDREs but not their most frequent locations relative to the genes they regulate.
1,25(OH) 2 D 3 is also known to suppress the expression of genes, among others, those that are targets of negative feedback regulation such as PTH and CYP27B1 . Early studies suggested that the sequences of the VDREs that mediate repression by 1,25(OH) 2 D 3 might be slightly different from those that mediate transactivation. Thus, small differences in the sequence of one or both of the half-sites might be capable of dictating suppression, suggesting that DNA sequence can act as a ligand and regulates VDR activity allosterically. A key example is that of the PTH gene. Additional studies, however, have revealed that repression likely occurs most frequently as a result of the ability of the VDR to displace or to prevent the binding of key transcription factors that are necessary for the maintenance of basal expression . Importantly, recent genome-wide studies suggest that the mechanisms of repression are not only cell type-specific but also highly diverse.
51.3.3
VDR Functions as a Heterodimer With Retinoid X Receptor (RXR)
Accompanying the discovery of the first VDRE was the important finding that VDR binding to these specific DNA sequences was dependent upon an unknown nuclear factor. The identity of this protein was subsequently revealed when it was discovered that specific members of the steroid receptor family termed retinoid X receptors (RXRs) were capable of forming heterodimeric complexes with the VDR and other members of this class of steroid receptors. Indeed, one of the roles of 1,25(OH) 2 D 3 upon ligand activation of VDR is to increase this receptor’s affinity for its RXR partner such that cooperative, high-affinity DNA binding can be accomplished, as illustrated in Figs. 51.2 and 51.3 . The requirement of a VDR/RXR heterodimer for DNA binding differs from that of the true steroid receptors, which bind DNA as homodimers, but explains the structural need for two directly repeated DNA half-sites. Interestingly, it is known from both molecular as well as structural studies that the VDR binds to the upstream 5′ half-site of a typical VDRE, whereas RXR binds to the downstream 3′ half-site. This orientation is determined by unique domains within the two proteins that facilitate the formation of a dimer during the process of DNA binding. Whether this orientation is consistently maintained across many thousands of regulatory elements for the VDR/RXR heterodimer remains unknown, although protein-interaction studies of the complexes on DNA at the 3D level suggest that this structural orientation may be an essential requirement for high-affinity DNA binding.
Three different genes encode the RXRs, including RXRα, RXRβ, and RXRɤ. While all interact with the VDR in vitro, the precise hierarchy of interaction of the VDR with these related proteins that may occur in the same cell in vivo is not known. As indicated earlier, the RXRs also represent heterodimeric-binding partners for many other nuclear receptors, including those for retinoic acid, thyroid hormone, and peroxisome proliferators. Interestingly, RXR can be activated independently through several of its own ligands, which include 9- cis -retinoic acid and certain fatty acids. This feature of the RXRs both as an independent transactivator and as a common interacting component of heterologous nuclear receptors potentially links specific ligand-activated signaling systems together in cells where they are coexpressed depending upon the intracellular abundance of the RXRs. Early studies suggested that the role of RXR in VDR action was to enhance VDR-binding affinity at target VDREs. Indeed, this involvement has now been confirmed at hundreds of sites across both mouse and human genomes, as will be described in more detail in several sections to follow. These studies, however, have also revealed additional complexity in that while VDR DNA binding is largely dependent upon activation by 1,25(OH) 2 D 3 , DNA sites to which the VDR binds may already contain an RXR molecule, suggesting that the latter is capable of preoccupying VDR-binding sites for future activation. The molecular form of RXR (monomer, dimer, or heterodimer) bound to these sites is unknown. Finally, evidence also suggests that RXR may participate directly in the transcriptional activation process as well, although recent structural evidence suggests that only a single coregulatory factor is able to bind to the heterodimer when bound to DNA.
51.3.4
The Process of Transcriptional Modulation
The association of the VDR/RXR heterodimer with specific DNA sequences, as with other nuclear receptor family members, initiates a series of processes essential to the modulation of transcriptional output at target genes. Although early studies suggested that the interaction of gene regulators such as the VDR with basal transcriptional machinery was sufficient to modulate gene output, it is now known that the process of transcriptional modulation is exceedingly complex and involves the receptor-dependent recruitment of multiple comodulatory complexes, each with unique function ( Fig. 51.3 ). Overcoming and/or restoring the inherent repressive state of chromatin requires the presence of regulatory machinery able to shift and/or displace nucleosomes (chromatin remodeling), alter the condensation state and thus the architecture of chromatin (histone modifications), and/or facilitate the entry of RNA polymerase II (RNA pol II) at appropriate sites. At least three complexes are believed to accomplish these specific actions. The first are the vertebrate ATPase-containing homologues of the yeast SWI/SNF complex that utilize the energy of ATP to remodel and reposition nucleosomes. This action increases the availability of binding sites for additional transactivators and facilitates the changes necessary for transcription to proceed. The second are complexes that contain either acetyl- or methyltransferases, deacetyltransferases (HDAC), or demethylases each of which function to modify the lysine or arginine containing tails of histone three and/or histone four. Certain methyltransferases place unique covalent methylation marks on specific histones highlighting TSSs (promoters) at potentially regulated genes across the genome; others are known to install marks that represent the covalent signature function of key regulatory regions of genes. Interestingly, these enzymatic activities link the genomic processes to cellular energy metabolism. Demethylases remove these marks, instilling a dynamic nature to these regulatory marks. These processes comprise part of complex, location-dependent epigenetic layers that are present at specific histones that functions to coordinate and mediate the expression of gene networks across the genome that are essential to the differentiation of cells and to the development and maintenance of their unique functional phenotypes. Alterations in the presence of these marks are able either to provoke or to emerge as a consequence of disease. Acetyltransferases and their corresponding HDACs covalently modify histones through acetylation marks as well. The marking of histones H3 and H4 through acetylation promotes chromatin decondensation, thus facilitating transcription factor access and the modulation of genes. HDACs, on the other hand, remove these marks thereby limiting activation potential. These activities are coordinated by coregulatory factors such as the p160 family SRC-1, SRC-2, and SRC-3 as well as CBP/p300, the corepressors SMRT and NCoR as well as one of several HDACs. A third complex is Mediator, believed to facilitate the entry of RNA pol II and perhaps to play a role in transcriptional reinitiation and other processes. Many additional complexes are known to interact with nuclear receptors as well; their precise roles in transcriptional modulation are currently being defined. Importantly, activation of the VDR by 1,25(OH) 2 D 3 enables the VDR/RXR heterodimer to interact with both the above coregulators and others.
51.3.5
The Role of 1,25(OH) 2 D 3 in Activation of Gene Expression Through VDR
The previous sections have defined a role for 1,25(OH) 2 D 3 in promoting both RXR heterodimer formation and the recruitment of cofactors at target genes. Indeed, it is now clear from molecular as well as structural studies that the potentiation of these interactions by 1,25(OH) 2 D 3 is due to the ability of the ligand to induce changes in VDR conformation. Conformational changes in the VDR are likely induced upon entry of 1,25(OH) 2 D 3 into the carboxy-terminal ligand-binding pocket, where it is stabilized by direct contacts with specific VDR amino acids. These conformational changes induced by 1,25(OH) 2 D 3 result in a rearrangement of several of the 12 alpha helices, which create both a contact heterodimer surface for RXR and a binding site for the coregulators, the latter arising from an interaction between helices 3, 4, and 12. A molecular view of this relationship has been acquired recently through cocrystallization of the rat VDR ligand-binding domain (LBD) with a peptide sequence that mimics the coactivator-interacting domain of MED1. This sequence conforms to the leucine-charged receptor interacting region of most coregulators and is comprised of a motif whose central core contains the amino acid sequence LXXLL. The motif LXXXIXXX I/L found in corepressors also binds to the same region of the VDR. Most importantly, however, recent studies using cryo-EM have now been successful in providing a three-dimensional view of the VDR bound to its cognate DNA sequence element while paired to its heterodimer partner RXR. This successful 3D image and its comparison to similar structures of RAR and PPAR provide new insight into the structural specificities of these three mechanistically related nuclear receptors.
51.3.6
Conclusive Evidence That VDR Mediates 1,25(OH) 2 D 3 Action
Despite delineation of the mechanism of action described above, unequivocal evidence for the central role of the VDR as the comprehensive mediator of the actions of 1,25(OH) 2 D 3 was derived from numerous experiments of nature. Early clinical studies defined a rare human syndrome termed hereditary 1,25(OH) 2 D 3 -resistant rickets or HVDRR that is characterized by hypocalcemia, hypophosphatemia, severe skeletal and dentitial deformities and, in a portion of these cases, alopecia as well. Additional features include reduced circulating levels of 24,25(OH) 2 D 3 and perhaps most strikingly, extremely high blood levels of 1,25(OH) 2 D 3 . Patients are generally unresponsive to treatment with 1,25(OH) 2 D 3 although the infusion of calcium and phosphorus can lead to normalization of calcium and phosphorus levels in the blood and improve the skeletal physiology. Fibroblastic cells derived from these affected individuals appear to recapitulate the transcriptional regulating capabilities of 1,25(OH) 2 D 3 (or lack thereof). It was subsequently shown that this syndrome is derived from genetic mutations in the VDR gene, first in the exons encoding the DNA-binding domain that prevent association with VDREs at target genes and later in exons that encode domains affecting protein stability, RXR heterodimerization, coregulator complex formation, or that lead to specific alterations in the VDR protein itself. Interestingly, only mutations that lead to the deletion of specific regions of the receptor or of the VDR protein in its entirety result in the alopecic phenotype. This observation, coupled with additional research in mice, revealed that an intact VDR molecule was essential for maintenance of the hair cycle but that this activity did not require activation through 1,25(OH) 2 D 3 . Many of the features of HVDRR, including those at the skeleton, have now been recapitulated through the successful preparation of mice nullizygous for the VDR alleles. Not surprisingly, these mutant mice all retain alopecia. To address this issue, recent studies have explored the expression of wildtype and mutant forms of the human VDR from large, comprehensive transgenes that contain all of the regulatory regions of the human gene as well. Importantly, when placed in a VDR-null mouse background, the wildtype gene is able to fully rescue the diverse VDR-null phenotype. In contrast, however, a 1,25(OH) 2 D 3 -resistant mutant VDR unable to bind the hormone is capable of rescuing the alopecia but not the skeletal deformities that arise due to the receptor’s inability to mediate the pervasive and essential transcriptional activities that are dependent upon 1,25(OH) 2 D 3 . These genetic strains of mice have proven useful in clarifying both the skeletal actions of 1,25(OH) 2 D 3 as well as new physiologic possibilities not previously recognized.
51.4
New Insights Into the Regulation of Gene Expression by 1,25(OH) 2 D 3
51.4.1
New Methodological Approaches
In this section, we describe new approaches to the study of transcription that have occurred over the past decade. When applied to the vitamin D system, they have extended our understanding of how the VDR modulates the expression of genes across entire cellular genomes and at important single gene loci as well. We also comment on how genomic sites of VDR and RXR-binding intersect with those of additional transcription factors that are activated via vitamin D-independent signaling pathways.
Although a number of techniques have emerged during the past decade perhaps the most significant is chromatin immunoprecipitation analysis (ChIP), linked early on to direct PCR methods (termed ChIP-qPCR) but more recently to tiled DNA microarray hybridization analysis (ChIP-chip) and now almost exclusively to massively parallel DNA sequencing methodologies (ChIP-seq). For ChIP analysis, proteins are bound directly or indirectly to DNA using cross-linking reagents such as formaldehyde, and the chromatin then fragmented to form short DNA segments of predetermined size (500–2000 bp). The protein/DNA complexes are subsequently precipitated using an antibody to a specific protein(s) or chromatin modification of interest. In ChIP-qPCR analysis, the harvested DNA is then quantitated by direct qPCR using primers to a small selected region(s) of the genome, and the abundance of the fragment used to assess the concentration of the protein through which the DNA fragment was initially precipitated. ChIP-chip analysis, on the other hand, involves the application of the entire precipitated DNA sample to a tiled oligonucleotide DNA microarray, thereby enabling a determination of the abundance of all DNA fragments within the sample. In the most current rendition of these analyses, ChIP-derived DNA is simply amplified, directly sequenced via “next generation” parallel sequencing techniques, and the individually sequenced segments of DNA (termed reads) are mapped to the genome based upon their homology to the DNA sequence of the genome itself. Assuming that the genome of the organism in question has been determined, these latter two applications of ChIP analysis are able to illuminate indirectly the relative levels of endogenous proteins and/or modifications to these proteins bound to chromatin across the entire genome in the sample in question. This approach, coupled to additional methodologies that measure the cellular transcriptome (the abundance of expressed RNAs within a cell) through similar “next-generation” sequencing techniques such as RNA-seq and GRO-seq, is transforming transcription research.
51.4.2
Genome-Wide Analysis Reveals New Concepts of Vitamin D Action
ChIP-seq analyses in particular are now capable of providing important genome-wide insight into both genomic features and overarching principles of gene regulation by a broad variety of tissue states and signaling pathways. Embedded within the data sets that emerge from these genome-wide analyses are exquisite details into the structural and functional features of virtually any gene of interest. On a genome-wide scale, ChIP-seq analyses can identify and permit quantitation of the total number of detectable binding sites for a given transcription factor in a cell (a property termed a cellular cistrome), provide precise information as to the location of these sites across the genome, determine the frequency with which given transcription factors form dimers with additional protein species, identify candidate response elements located within the individual peaks of activity, and annotate many other features of the genome. Perhaps as important, when paired to genome-wide transcriptome analysis, these techniques can facilitate the establishment of linkage between identified regulatory elements and the genes that they may regulate, although unequivocal establishment of this relationship requires much additional work. Accordingly, these analyses provide crucial information regarding the regulation of gene networks and the underlying mechanisms that are responsible.
ChIP-seq analyses of the VDR and its partner RXR have been performed on the genomes of murine MSCs and their differentiated adipocytic, bone osteoblastic, and osteocytic derivatives, primary murine intestinal epithelial cells, colorectal cancer cell lines, an activated stellate cell line, and a human monocytic cell line. As general features of these analyses for the VDR are similar, we will discuss the results obtained in bone cells in this section and discuss specific details of intestinal and colorectal cell analyses in a subsequent section. While VDR is present at over 1000 sites in MSCs and bone osteoblasts in the absence of 1,25(OH) 2 D 3 , over 7000 sites can be identified in the presence of ligand, suggesting that VDR binding to specific DNA sequences is highly 1,25(OH) 2 D 3 -dependent. RXR, on the other hand, binds extensively to the genome in the absence of 1,25(OH) 2 D 3 , but is likewise induced at many additional sites in the presence of the hormone. Colocalization of VDR with RXR is extensive, however, comprising over 60% of VDR-binding sites, supporting the concept that VDR DNA binding requires RXR. Additional factors such as the bone differentiation master regulators RUNX2 and C/EBPβ and others are also bound near many of these sites of heterodimer occupancy. Importantly, de novo motif finding analysis has revealed that the most frequent DNA sequence found within these sites is the classic VDRE comprised of two hexameric half-sites separate by three base pairs. Perhaps most interesting, nearest neighbor analyses as well as inspection indicates that while these VDR/RXR bound regions can be found near gene promoters, they are most frequently found at sites located intergenically and within introns 10s if not 100s of kilobases distant from potential target genes. Of considerable importance, the locations of regulatory regions identified previously for a number of known vitamin D target genes are fully confirmed by these analyses, although many new sites of regulatory action for these genes have been identified as well. Thus, this genome-wide study and the others enumerated above support previously developed hypotheses but also demonstrate that some of these early principles of vitamin D action were factually incorrect. A summary of the overarching principles derived from these genome-wide studies, which will be highlighted in the studies discussed in the next section, can be found in Table 51.1 .
| VDR binding sites (the cistrome): 2000–8000 1,25(OH) 2 D 3 -sensitive binding sites/genome whose number and location are determined as a function of cell-type |
| Active transcription unit for induction: The VDR/RXR heterodimer |
| Distal binding site locations: Dispersed in cis -regulatory modules (CRMs or enhancers) across the genome; located in a cell-type specific manner near promoters, but predominantly within introns and distal intergenic regions; frequently located in clusters of elements |
| VDR/RXR binding site sequence (VDRE): Induction mediated by classic hexameric half-sites (AGGTCA) separated by three base pairs; repression mediated by divergent sites |
| Mode of DNA binding: Predominantly, but not exclusively, 1,25(OH) 2 D 3 -dependent Modular features: CRMs contain binding sites for multiple transcription factors that facilitate either independent or synergistic interaction |
| Epigenetic CRM signatures: Defined by the dynamically regulated posttranslational histone H3 and H4 modifications |
| VDR cistromes are highly dynamic: Cistromes change during cell differentiation, maturation, and disease activation and thus have consequential effects on gene expression |
51.4.3
ChIP-seq Analysis Provides New Insights Into Specific Targets of 1,25(OH) 2 D 3 Action
51.4.3.1
The CYP24A1 Gene
As discussed earlier in this chapter, CYP24A1 encodes the 25-dihydroxyvitamin D 3 -24 hydroxylase, a wide-spread p450 enzyme which catalyzes the initial steps that result in the inactivation of 1,25(OH) 2 D 3 . Accordingly, this enzyme represents a dynamic determinant of the circulating levels of 1,25(OH) 2 D 3 through activity in the kidney and establishes the set point for the level of response to 1,25(OH) 2 D 3 in all vitamin D target cells both in vitro and in vivo. Indeed, global genetic deletion of CYP24A1 expression in mice or the production of mutant hydroxylases as observed in young children with idiopathic familial hypercalcemic result in exaggerated levels of 1,25(OH) 2 D 3 that potentiate homeostatic activity and can cause hypercalcemia. Importantly, CYP24A 1 expression is strongly regulated by 1,25(OH) 2 D 3 . Traditional studies of this gene in 1993–95 initially revealed the presence of two highly conserved VDREs located within the first 260 bp of both the mouse and human homologues that were capable of binding both VDR and RXR and mediating strong induction by 1,25(OH) 2 D 3 . Importantly, the presence of these two sites of 1,25(OH) 2 D 3 action was confirmed in 2010 following extensive ChIP-chip and ChIP-seq analyses of VDR and RXR binding in both mouse and human bone and other cell types as well. These studies, however, also revealed the presence of an intergenic cluster of VDR/RXR-binding sites downstream of the gene spanning + 35 to + 37 kb in the mouse and + 50 to + 66 kb in the human genomes. Detailed studies identified several of the multiple VDREs that were contained within these complex regulatory regions. In addition, they also linked the functional activity of these regions directly to the enhancement of the Cyp24a1 expression by 1,25(OH) 2 D 3 . Accordingly, osteoblastic cells were stably transfected with large mouse and human BAC clone minigenes containing the CYP24A1 genes and adjacent contiguous intergenic segments. Stable transfection of additional mouse and human BAC clones containing either mutations within both promoter proximal VDREs, within the downstream segments containing the cluster of VDR-binding sites or both revealed that both regulatory regions contributed to the regulation of CYP24A1 by 1,25(OH) 2 D 3 . We have now created transgenic mice containing each of these mouse or human CYP24A1 BACs in the genetic background of a Cyp24a1 -null mouse and assessed their capacity to rescue the Cyp24a1 -null phenotype. The Cyp24a1 -null phenotype is characterized by the absence of circulating 24,25(OH) 2 D 3 , the inability to metabolize and degrade an injected bolus of 1,25(OH) 2 D 3 , and the absence of the potential to reduce 1,25(OH) 2 D 3 levels and toxicity in the mouse. Both transgenes were fully regulated by exogenous treatment with 1,25(OH) 2 D 3 and fully restored normal control of 1,25(OH) 2 D 3 metabolism (Carlson and Pike, unpublished). Interestingly, the mouse but not the human transgene was also able to upregulate the levels of the 25(OH)D 3 -26,23-lactone, a synthetic capability found in the mouse enzyme. These studies both in vitro and in vivo provide additional evidence that the Cyp24a1 gene is controlled by not only a promoter proximal set of VDREs but also by a downstream intergenic cluster. From a general point of view, these results also highlight the ability of the human CYP24A1 gene to replace the endogenous mouse gene and confirm both similarities and differences that are evidence between the mouse and human genes themselves. The humanized mouse BAC clone approach can now be used to explore mutations within the human CYP24A1 gene that cause familial idiopathic hypercalcemia under conditions of normal regulatory and tissue expression.
51.4.3.2
The VDR Gene
The organizational features of the VDR gene emerged during the decade of the 1990s using traditional molecular biologic techniques. Despite considerable effort, the mechanisms whereby the VDR gene was regulated by the many hormones that were shown previously to induce the gene’s expression, including 1,25(OH) 2 D 3 , failed to emerge. Given the size and complexity of this gene, it was easy to speculate that many of the key regulatory components for VDR expression might be located far from the proximal promoter, although at the time there were no unbiased methods that would permit a rational yet unbiased search for these regulatory elements across many kilobases of DNA. Following the emergence of ChIP-chip analysis in early 2005, however, we used this method to explore the mouse VDR gene, focusing first on the identification of enhancers within the VDR gene locus that might be capable of mediating the autoregulatory effects of 1,25(OH) 2 D 3 and its cognate receptor. Surprisingly, strong VDR and RXR-binding activity was discovered within two large introns of the VDR gene; these regions were termed the S1/S2 and the S3 enhancers, respectively. These regions and two additional sites, including one located approximately 6 kb upstream of the TSS (U1), were all confirmed using a subsequent ChIP-seq analysis. Importantly, it was also discovered in subsequent analyses that subsets of these enhancers also mediated upregulation of the VDR gene by retinoic acid, glucocorticoids, and PTH. Additional analyses revealed that 1,25(OH) 2 D 3 -induced VDR/RXR binding at these sites also resulted in the recruitment of coregulators such as SRC-1 and MED1 and increased levels of RNA pol II as well. Importantly, further direct study of the S1/S2 and the S3 enhancers revealed the presence of both functional VDREs in the former capable of mediating the autoregulatory actions of 1,25(OH) 2 D 3 in bone cells and retinoic acid response elements (RAREs) in the latter capable of mediating retinoid response. Regulation mediated by PTH was also confined to the enhancer located at − 6 kb. The unique role of each of these enhancers has now been fully confirmed through the use of recombineered normal and mutant mouse VDR BAC clones stably integrated into bone cells in vitro and as transgenes into the mouse genome in mice. These observations highlight the utility of these unbiased genomic approaches in the discovery of regions that regulate the expression of complex genes such as the VDR .
51.4.3.3
Characterizing the Regulatory Elements of the Tnfsf11 (RANKL) Gene
Early studies by Suda and colleagues suggested that a factor derived from osteoblast-lineage cells was capable of regulating osteoclast differentiation. Importantly, the expression of this factor was controlled by 1,25(OH) 2 D 3 as well as other calciotropic agents including PTH and several of the inflammatory cytokines. As indicated earlier, this factor termed RANKL is a membrane-bound and sometimes soluble TNFα-like factor derived from the TNFSF11 gene that strongly induces osteoclast differentiation from hematopoietic precursors. Indeed, the signaling pathways that mediate activation of this complex differentiation pathway are now well described. Importantly, the actions of RANKL over the intervening years have been dramatically expanded to include immune regulation, mammary gland maturation, thermogenesis, and cardiovascular calcification, to name a few. Despite considerable effort, however, early attempts using traditional methods to identify regions mediating the regulation of the TNFSF11 gene by 1,25(OH) 2 D 3 as well as PTH and cytokines such as IL-6 and OSM were largely unsuccessful. Indeed, virtually all of these studies using traditional Tnfsf11 promoter-based approaches yielded the identification of elements that could not be reproduced. As the mouse and human TNFSF11 genes are located in gene deserts and bounded on each side by nearly 200 kb of intergenic DNA, we explored this gene in osteoblasts for regulation by the vitamin D hormone using ChIP-chip analysis of both VDR and RXR, extending our query to over 500 kb of DNA surrounding the mouse Tnfsf11 transcription unit. This initial study, which has been fully confirmed through subsequent studies using VDR ChIP-seq analysis was highly revealing. Accordingly, while neither VDR nor RXR were present near the promoter region following administration of 1,25(OH) 2 D 3 , both were strongly detected − 16 (termed D1), − 22 (D2), − 60 (D3), − 69 (D4) and − 75/76 (D5) kb upstream of the Tnfsf11 gene promoter. Each of these regions, as well as a more recently defined site at − 40 kb (D7), was extensively characterized and the VDR-binding sites that mediated vitamin D hormone action in a subset of these sites were identified. Follow up ChIP-chip studies of PTH action at the Tnfsf11 gene focused upon CREB were also performed. As with the VDR, these studies revealed that PTH induced CREB binding at several but not all of these regulatory regions. We also demonstrated that cytokine induction in osteoblasts was mediated through STAT3 induction at both the D5 region and at a novel region located − 88 kb (D6) upstream. Numerous additional hormones were similarly shown to regulate Tnfsf11 expression as well. Interestingly, O’Brien and colleague while studying PTH action identified the upstream regulatory region located at D5 simultaneously using an entirely independent approach, further demonstrating the presence of not only CREB but also RUNX2 occupancy. Importantly, this enhancer region was deleted from the mouse genome in vivo and shown to recapitulate the functions ascribed to its actions in cell culture. These studies provided conclusive evidence that the regulation of Tnfsf11 expression by 1,25(OH) 2 D 3 , PTH and other hormonal factors was mediated by multiple independent enhancers located at significant distances from the gene’s TSS. Importantly, deletion of 2 kb of the promoter proximal region of the Tnfsf11 gene has no effect on the ability of 1,25(OH) 2 D 3 , PTH, or the cytokines to modulate the gene’s expression in cells in vitro or in mice in vivo (Meyer, Onal, O’Brien, and Pike, unpublished). Interestingly, additional ChIP-chip studies in T cells revealed a second set of three regulatory enhancers located intergenically even further upstream of the Tnfsf11 TSS between − 123 and − 155 kb (termed T1–T3). These enhancers bind several transcription factors including c-Fos and NFATc, and together with the enhancer at D5 mediate the expression of Tnfsf11 exclusively in hematopoietic B and T cells. Thus, a set of at least 10 independent enhancers have now been shown to regulate Tnfsf11 expression in osteoblast- and hematopoietic-lineage cells when assessed in cultured cells. Interestingly, it is these regions that contain single nucleotide variants (SNPs) of genome-wide significance that appear to influence TNFSF11 expression and bone mineral density in human populations.
To confirm that the Tnfsf11 gene and the multiple regulatory components located upstream carry all the necessary genetic information to mediate tissue-wide expression of RANKL in vivo, we constructed a large segment of DNA by merging two overlapping mouse BAC clones and prepared transgenic mice using this construct. We then crossed this genetically modified mouse expressing normal levels of RANKL into a RANKL-null mouse background and determined whether the construct could rescue the extensive RANKL regulatory and biological phenotype of the null strain. Our results suggest that this segment of DNA fully recapitulated the regulatory response of the endogenous Tnfsf11 gene to PTH, 1,25(OH) 2 D 3 , and the cytokines, and that it produced sufficient endogenous-like tissue-specific levels of RANKL to rescue the phenotypic effects imposed by endogenous RANKL deletion. These results suggest that the transgene contains all the information necessary for endogenous-like expression of the Tnfsf11 gene, however, they do not prove definitively that the regulatory regions within the construct mediate the activities defined in cell culture. To confirm these functional activities, we individually deleted from the mouse genome two of the key osteoblast-specific enhancers D2 and D6 as well as a single hematopoietic enhancer at − 123 kb (T1). Mice bearing these deletions as well as the enhancer deletion at D5 explored earlier in vivo were then subjected to extensive regulatory and biological phenotyping. The results of these analyses indicate that (1) the D2 enhancer mediates PTH action in osteoblasts, (2) the D5 enhancer reduces basal expression of RANKL in both osteoblasts and hematopoietic cells and mediates both PTH and 1,25(OH) 2 D 3 action in the former, (3) the enhancer at D6 mediates the regulation of RANKL by inflammatory cytokines, and (4) the enhancer at T1 reduces the basal expression of RANKL in hematopoietic cells. Biologically, the osteoblast-active enhancers exerted profound effects of the skeleton, whereas the hematopoietic cell-active enhancer T1 had no effect. Numerous additional phenotypic responses were identified as well. We conclude from these studies that the Tnfsf11 gene is regulated both in vitro and in vivo by multiple distal enhancers that retain features that mediate the regulation of Tnfsf11 expression in a unique temporal, hormonal, and tissue-specific fashion. Ongoing studies are aimed at understanding the regulation of the Tnfsf11 gene and its mechanism of action in diseased tissues such as in atherosclerosis where aortic plaques develop in genetically modified mice maintained on high-fat diets (Shamsuzzaman, Onal, and Pike, unpublished).
The details of the studies just described provide new mechanistic insight into the actions of 1,25(OH) 2 D 3 in bone and other cell types. We will discuss the actions of vitamin D in the intestinal epithelium and colon in Section 51.7 .
51.5
The Transport of Calcium Across the Intestinal Epithelium
51.5.1
The Calcium Transport Process
As indicated earlier, the maintenance of extracellular calcium and phosphorus is vital to the proper functioning of virtually all cell types in vertebrates and other organisms. Although both the kidney and bone participate in mineral homeostasis, it is the intestine that is ultimately charged with making both minerals available from external sources. Thus, this section will focus on the events that occur in the intestine, although the process of conserving minerals at the kidney is also highly related. Not surprisingly, the essentiality of calcium absorption, coupled to a requirement for this process to respond dynamically to the changing needs of the organism, places this process under the regulatory control of a wide variety of developmental, physiological, homeostatic, and endocrine regulators, including 1,25(OH) 2 D 3 . Due to the complexity of the subject, we will focus almost exclusively on the regulation of calcium. The reader is referred to extensive reviews of the regulation of phosphorus metabolism by 1,25(OH) 2 D 3 in the intestine, kidney and bone regarding phosphate handling. It is worth highlighting, however, that there has been significant progress in this latter area during the past decade.
Calcium absorption occurs across the intestinal enterocyte. While both paracellular and transcellular routes are operable, it is the transcellular route, facilitating the net movement of calcium from the gut lumen into the circulation, that is the most highly regulated. Transepithelial calcium transport is a complex multistep process that entails the movement of cations through the glycocalyx region and across the brush-border membrane into the cell interior, the translocation of the cation through the cytoplasm to the basolateral membrane, and finally extrusion of the cation into the extracellular fluid of the lamina propria where it is absorbed into the circulation ( Fig. 51.4 ). A number of physicochemical and thermodynamic parameters pertain to this uptake process, such that energy is required despite the presence of a steep downhill lumen to blood gradient.
51.5.2
The Calcium Transporters
51.5.2.1
Calcium Entry
Although a number of transport mechanisms have been postulated to account for the uptake of calcium into the intestinal epithelial cell, the identity of the key components and the mechanisms of their action have only recently come together. It is now believed that two ion channels termed transient receptor potential, vanilloid, type 5 (TRPV5), and type 6 (TRPV6), the latter perhaps most important, play paramount roles in the entry of calcium into the intestinal cell ( Fig. 51.4 ). TRPV5 and TRPV6 were identified via functional expression cloning techniques by Hoenderop et al. and Peng et al. respectively, and shown to retain an appropriate level of calcium-selective gating activity. TRPV5 and TRPV6 are members of a large superfamily of genes whose products are nonvoltage-gated ion channels that function as receptors for a diverse set of external stimuli and facilitate a wide range of biological processes. Perhaps most importantly, the tissue expression profiles as well as calcium-transferring capabilities of TRPV5 and TRPV6 support the idea that these ion channels represent the anticipated proteins responsible for the transcellular transport of calcium across both kidney and intestinal epithelia. Extensive work since the cloning of these genes has revealed important biophysical properties of the channels, their pore-forming properties and the mechanisms by which they manifest calcium selectivity and inward rectifying and gating capabilities. The mechanisms through which these protein channel activities are modulated and the components that are essential to their modulation are rapidly emerging. The discovery of these channels and the subsequent characterization of their mode of action in the intestine as well as in kidney begin to fill a crucial gap that has existed for several decades in our understanding of transepithelial calcium transport. Whether these proteins represent the key transporters that mediate vitamin D dependent actions in the intestine, however, remains unclear.
51.5.2.2
Calcium Diffusion
The influx of calcium, representing the first step in transepithelial transport, must be buffered such that it does not perturb low intracellular calcium levels and transported through the cytoplasm to the basolateral membranes where it can be extruded into the circulation ( Fig. 51.4 ). Several hypotheses have been developed to account for these two events, and although the actual mechanisms are yet to be defined, it is clear that the calcium-binding proteins calbindin D9k and calbindin D28K play essential roles in these processes. The calbindins are highly conserved soluble calcium-binding proteins that are rather ubiquitously expressed. They were proposed early on to act as cytosolic buffers capable of maintaining low intracellular calcium levels during transcellular calcium transport and to serve as shuttles for the diffusion of calcium. Physical interactions between the calbindins and both TRPV5 and TRPV6 as well as the basolateral membrane calcium ATPase PMCA1b have been postulated, based upon the coexpression of calbindin D9K and TRPV channels in tissues and through the ability of calbindin D28K to enhance the calcium ATPase activity. Solid demonstration of these interactions would bolster the concept of a role for the calbindins in calcium transfer across the enterocyte. As will be discussed later, however, calcium transport does not correlate directly with the levels of calcium-binding proteins and can occur in their absence as well. It is unclear presently whether the calbindins are essential components of the calcium transfer process or whether they and many of their related family members simply play redundant roles.
51.5.2.3
Calcium Extrusion at the Basolateral Surface
Calcium is extruded from the enterocyte against a significant electrochemical gradient ( Fig. 51.4 ). Thus, this process requires the energy of ATP hydrolysis. Members of both the sodium/calcium exchangers (NCXs) and the plasma membrane calcium ATPases (PMCAs) are expressed in the intestine, although it appears that the former function primarily in the reabsorption of calcium by the kidney. These proteins likely mediate the extrusion of calcium from the enterocyte. PMCAs are expressed ubiquitously and are comprised of at least four isoforms, two of which are believed to function in the general maintenance of cellular calcium homeostasis. PMCA1b, the isoform that is expressed predominantly in the intestine, is likely the calcium-regulated pump that extrudes calcium from the enterocyte into the circulation.
51.5.3
Genetic Deletion of the Components of Transepithelial Calcium Transport in the Mouse
The previous sections describe known components of the calcium absorption process in the intestine, including apical TRPV6 (and TRPV5), cytosolic calbindin D9K (and 28K), and basolateral PMCA1b. Importantly, several of these genes have been deleted in mice through homologous recombination techniques. While the phenotypes of these gene-knockout mice are illuminating, they unfortunately do not provide unequivocal evidence that any one of the three components represents the direct and essential determinant of calcium uptake that is regulated by vitamin D. These mice do, however, provide evidence that each of these components is involved in calcium metabolism. Perhaps most illuminating is the phenotype of TRPV6-knockout mice. When placed on a calcium-deficient diet, TRPV6-null mice exhibit a significantly reduced capacity to absorb calcium, suggesting initially that this factor could be the long sought after “vitamin D-dependent” component of intestinal calcium absorption. However, while TRPV6 is highly regulated by 1,25(OH) 2 D 3 (see section below), deletion of TRPV6 in the small intestine reduced the amount of calcium uptake that occurred across the isolated intestinal epithelium but did not alter the vitamin D hormone’s ability to upregulate calcium absorption further. Likewise, while a phenotype in the kidney and other tissues was apparent upon deletion of the calbindin D28K gene, a relationship between this gene and vitamin D also did not emerge. One complication is the possibility that other calcium-binding proteins, of which there are many, may function redundantly in the absence of expression of the calbindin D9K and D28K genes. Finally, deletion of PMCA1b and NCX1 results in embryonic lethality; thus, we must await the preparation of conditional knockouts of these two potentially important genes in mice to determine if these genes retain the properties essential to vitamin D-dependent regulation of calcium absorption. Strikingly, simultaneous deletion of both TRPV6 and calbindin D9K expression in the intestine were similarly ineffective in preventing additional intestinal calcium response to 1,25(OH) 2 D 3 . Accordingly, the search continues for the elusive factor(s) essential to vitamin D-mediated calcium uptake in the intestine, and the identity of this component(s) has yet to emerge. However, it has also prompted a “gene network hypothesis” that will be discussed as an alternative explanation.
51.6
Regulation of Calcium Transporter Expression by 1,25(OH) 2 D 3
51.6.1
Hormonal Regulation of the Expression of Calcium Transporting Genes
1,25(OH) 2 D 3 is known to regulate each of the components that mediates transepithelial calcium transport, including PMCA1b , the calbindins D9K and D28K, and both TRPV5 and TRPV6 , despite the fact that independently, none of these may represent the postulated central factor for vitamin D action in the intestine. The degree of regulation of PMCA1b by 1,25(OH) 2 D 3 is modest. The calbindins D9K and D28K, in contrast, are well-recognized targets of vitamin D action, having been discovered originally as a result of the physiologic activities of vitamin D itself. Importantly, the expression levels of these proteins are dramatically reduced as a result of both vitamin D deficiency as well as genetic deletion of Cyp27b1 . Moreover, a single dose of 1,25(OH) 2 D 3 is effective in upregulating calbindin D9K expression in the intestine and calbindin D28K in the kidney. Perhaps most interesting, however, is the capacity of 1,25(OH) 2 D 3 to regulate the expression of renal TRPV5 and intestinal TRPV6. Although numerous experiments have been conducted, the most compelling are those carried out in both Cyp27b1- and VDR -null mice. Both mouse strains show a dramatic depletion in the expression of TRPV5 and TRPV6. This reduced expression, however, can be rescued directly in the Cyp27b1 -null mouse following acute treatment with 1,25(OH) 2 D 3 . The genetic deletion of the VDR prevents 1,25(OH) 2 D 3 action and thus fully abrogates rescue by 1,25(OH) 2 D 3 . Additional support for the regulatory role of 1,25(OH) 2 D 3 in TRPV5 and TRPV6 expression derives from studies by Song et al. who demonstrated that 1,25(OH) 2 D 3 was able to upregulate the expression of the two genes as well as the calbindins as a function of both dose and time. These as well as other studies provide unequivocal support for the ability of 1,25(OH) 2 D 3 to regulate the expression of genes whose products are essential for the transport process in both intestine and kidney.
51.6.2
Mechanisms of 1,25(OH) 2 D 3 Regulation of Pmca1, Calbindins D9K, and TRPV5 and TRPV6
51.6.2.1
Historical and Contemporary Regulation of Pmca1 and S100g (Calbindin D 9K)
The mammalian calbindins are expressed in a wide variety of tissues including the calcium homeostatic organs intestine, kidney, and bone. Importantly, their expression is regulated by a number of hormones and factors including 1,25(OH) 2 D 3 , although early studies suggested that 1,25(OH) 2 D 3 might regulate their expression through both transcriptional and posttranslation mechanisms. Nevertheless, the transcriptional mechanism of regulation largely prevailed when it was shown that VDREs were present between − 445 and − 489 bp upstream in the rat S100g gene and between − 170 and − 200 bp upstream in the mouse Calb1 (calbindin D 28K) gene. However, although these elements exhibit some similarity to that of bone fide VDREs in many other genes, there remained considerable divergence both in the half-site sequence as well as in nucleotide spacing characterized. Moreover, none of these sequences were capable of driving significant transcriptional output in traditional transfections studies, suggesting the possibility that legitimate VDREs might be found located at sites distal to their promoters. Support for this concept appeared to be present in the observation that an S100g promoter gene construct containing the proximal regulatory VDRE described above failed to mediate response to 1,25(OH) 2 D 3 when introduced as a transgene into mice in vivo, whereas inclusion of sequences further upstream of the promoter appeared in part to regenerate this response. The mechanisms through which Pmca1 expression in the intestine is regulated by vitamin D, on the other hand, have never been explored. As a consequence, we recently conducted an in vivo study in which we treated mice with either vehicle or a single dose of 1,25(OH) 2 D 3 , isolated intestinal tract tissues, and subjected these tissues to a genome-wide ChIP-seq analysis using antibodies to the VDR. ChIP-seq data tracks for the S100g and Pmca1 genes revealed the locations of key enhancers that are residually occupied by the VDR in wildtype (vitamin D sufficient) mice and can be further occupied by ligand-induced VDR; it is these sites that most likely mediate the expression of S100g and Pmca1 in the intestine ( Fig. 51.5 ). These regulatory sequences, which are recapitulated in more recent studies of Cyp27b1 -null mice (data not shown), are also highlighted by epigenetic histone signatures that mark enhancers, are both multiple in nature, and located distal to the TSSs of both the S100g and Pmca1 genes. These data suggest that both of these genes, like so many others indicated earlier, are regulated by distal elements. Studies as conduced in Section 51.4 will be necessary to prove that these distal elements are linked functionally to their respective genes. Nevertheless, these data show convincingly that both the S100g and Pmca1 genes are not regulated through the proximal elements identified in early studies.
