We focus first on the regulation of 1,25(OH)₂D₃ production by the kidney. This hormone plays a central role in the regulation of mineral metabolism by virtue of its unique function to induce Ca and to a lesser extent P absorption from the diet via the intestinal enterocyte (15). Although this hormone also acts in bone and kidney as well, 1,25(OH)₂D₃ action in the intestine is novel. The primary regulator of 1,25(OH)₂D₃ production in the kidney is PTH which induces the expression of the Cyp27b1 gene whose enzymatic product 25(OH)-1α-vitamin D₃ hydroxylase (CYP27B1) is responsible for the synthesis of 1,25(OH)₂D₃ (16). As important, however, is the ability of PTH to reciprocally suppress the expression of Cyp24a1, whose product 1,25(OH)₂-24-vitamin D₃ hydroxylase (CYP24A1) is responsible for the initial degradation of 1,25(OH)₂D₃ (17). Accordingly, Cyp24a1 is suppressed by PTH thereby reducing the degradation rate of 1,25(OH)₂D₃ and raising the levels of 1,25(OH)₂D₃. That the regulation of Cyp24a1 is transcriptional in nature has only recently been defined in molecular terms, as will be discussed herein. Together, these two enzymes regulate the level of 1,25(OH)₂D₃, which is secreted into the blood where it functions as an endocrine hormone. Thus, the coordinated regulation of both Cyp27b1 and Cyp24a1 expression in the kidney by PTH is essential to both the production as well as the maintenance of appropriate levels of circulating hormonal 1,25(OH)₂D₃. Since the elaboration of PTH from the parathyroid glands is controlled by decreasing blood levels of Ca, an inverse relationship emerges between blood Ca and PTH, and thus control of circulating 1,25(OH)₂D₃ (18). As will be discussed in the next section, Cyp27b1 is suppressed by the reciprocal actions of FGF23 and 1,25(OH)₂D₃, while Cyp24a1 is coordinately induced by each of these hormones.

While studies several decades ago revealed the essential role for PTH in the control of renal production of 1,25(OH)₂D₃, it has been only recently that the mechanisms associated with the induction of Cyp27b1 and suppression of Cyp24a1 by this hormone have emerged. These findings were facilitated by the technics discussed above, including both ChIP-seq analysis and CRISPR-Cas9 mediated gene editing. To further the relevance of their application as well as our results, we applied these techniques to mice and conducted each of these analyses in vivo. As PTH is known to stimulate cAMP, we explored using ChIP-seq analysis the possibility that PTH might induce CREB at specific sites across the Cyp27b1 locus in tissue derived from the kidney cortex of normal wildtype mice (19). Following in vivo treatment with exogenous PTH, we used antibodies to phosphorylated S133 CREB (pCREB) and discovered that pCREB could be detected at several sites upstream of the Cyp27b1 gene, one located within an intron of the adjacent Mettl1 gene and three located further upstream in an intron of the Mettl21b (Eef1akmt3) gene ( Figure 2 ). None were found near the Cyp27b1 gene promoter region nor immediately upstream of the promoter in an intergenic region that had been the focus of efforts by many others. While pCREB levels were not induced initially by PTH at these sites, later time course experiments revealed that the actions of PTH were more rapid than anticipated, resulting in a striking and transient upregulation of pCREB followed by a return to baseline as tested in the initial experiment (20). PTH also exerted a profound increase in histone acetylation (H3K9ac and H3K27ac) across each of these pCREB-containing sites even at earliest times, suggesting that PTH likely exerted an increase in DNA accessibility around these factor-containing sites. Each of these activities with PTH, however, preceded an increase in the production of PTH-induced Cyp27b1 RNA transcripts. Most importantly, deletion of the pCREB containing site in the Mettl1 intron (M1) resulted in a profound mouse phenotype insensitive to exogenous PTH and consistent with a previously prepared mouse containing a mutation in Cyp27b1 that prevented the global expression of the active CYP27B1 enzyme (19, 21). The latter resulted in a total absence of circulating 1,25(OH)₂D₃ in these mice. Additional phenotypic features in both this unique Cyp27b1 mutant mouse and the M1 deleted mouse included hypocalcemia, hypophosphatemia, and striking hypocalcemic bone disease. Deletion of the three collective sites in the Mettl21b intron (M21) in the genome of mice resulted in a much more modest phenotype comprised of only slightly reduced sensitivity to PTH, and normal levels of Ca, P, and 1,25(OH)₂D₃ (19). These and additional studies, together with the profound epigenetic observations induced by PTH, suggest that each of these pCREB sites mediate PTH action, but that the pCREB site in M1 is likely a dominant requirement for physiologically impaired PTH action (20). Importantly, a double mutant containing deletions of the pCREB site in M1 and the multiple sites in M21 resulted in a decreased basal level, a profound resistance to PTH, strikingly reduced circulating levels of 1,25(OH)₂D₃ and an extreme phenotype similar to that of the Cyp27b1 null mouse; it was also slightly more severe than that seen in the M1 enhancer deleted mouse (21). Perhaps as interesting, a genomic analysis using ENCODE-produced DNase hypersensitivity (DHS) experiment revealed that all sites containing PTH-regulated pCREB exhibited an open chromatin profile present only in normal mouse kidney and absent in all other tissues examined (19, 22). More recent studies in isolated cells using ATAC-seq analysis revealed that this Cyp27b1 enhancer profile could be seen only in renal proximal tubules and not in other renal cell types nor in a variety of other tissue cell types as well (2). Finally, that these enhancers were active was confirmed most recently through single cell RNA expression studies that revealed that Cyp27b1 expression was limited to the renal proximal tubule segments S1 as well as S2 and S3 (23). While Cyp27b1 was expressed at very low levels in many non-renal tissues (NRTCs), it was not responsive to PTH in any of these tissues (19). Thus, we conclude that the kidney module responsible for PTH mediated upregulation of Cyp27b1 is unique to the kidney and responsible for the selective upregulation of Cyp27b1 by PTH.

Our most recent studies using ChIP-seq analysis have revealed that while the levels of pCREB are not strongly induced by PTH, the mechanism through which the transcriptional activity of PKA induced pCREB is manifested is via the promoted recruitment of CBP, a CREB coactivator that exhibits histone H3K27 acetylation activity and is perhaps responsible for the ability of PTH to induce H3K27ac across the M1 and M21 sites (20, 23). Interestingly, additional chemical and genetic studies revealed that PTH via PKA also inhibits the salt-induced kinases (SIK) that result in the release of the cytoplasmic CREB regulated transcriptional coactivator (CRTC) class of CREB coactivators such as CRTC2 that also translocate to the nucleus where they bind to the CREB modules in M1 and M21 to facilitate Cyp27b1 activation. Thus, activation of Cyp27b1 in this case is mediated largely via the PTH mediated upregulation of two known coactivators of DNA-bound pCREB. The distinct role of each of these coactivators remains to be determined, however. Although widely expressed CBP retains multiple functions, one of which may be in the acetylation of histone H3K27; a second hypothetical function in the case of Cyp27b1 may be to participate in the 3D repositioning of the four distal CREB enhancer sites at or near the Cyp27b1 promoter (24). Perhaps as important in linking PTH to the upregulation of Cyp27b1 is our observation that treatment with PTH results in the recruitment of BRD4 at each of the M1 and M21 pCREB sites (20). This histone H3 lysine acetylase reader participates in the initial phases of the recruitment of RNA polymerase II to Cyp271b. Accordingly, we observed that PTH also induced a striking increase in RNA polymerase II and an increase in enrichment of the epigenetic histone methylation mark H3K36me3 across the Cyp27b1 gene body. The appearance of both factors reflects an increase in Cyp27b1 transcription and provides direct links to the PTH-induced Cyp27b1 RNA transcripts that are elevated in response to PTH treatment.

While PTH induces Cyp27b1 expression in the kidney, this hormone also imposes a reciprocal negative action on renal Cyp24a1 levels (19). This reduction in Cyp24a1 expression leads to reduced degradation of 1,25(OH)₂D₃ thereby facilitating the overall upregulation of renal 1,25(OH)₂D₃ that raises the hormones levels in the blood. While it has long been known that PTH suppresses Cyp24a1 production, the mechanism of this decrease has evaded discovery; indeed, studies during the past decade suggested a post-transcriptional mechanism involving Cyp24a1 transcript stabilization (25, 26). Interestingly, our recent ChIP-seq analyses of the renal Cyp24a1 gene revealed that PTH suppressed Cyp24a1 expression via a transcriptional mechanism, and that this downregulation was mediated via a large DNA segment that contains at least three widely spaced pCREB sites that are located immediately downstream of the Cyp24a1 gene body ( Figure 3 ) (27). Like Cyp27b1, an open chromatin feature also characterized the DNA at each of these pCREB sites along this downstream segment, but only in the kidney proximal tubules (2). Importantly, however, deletion of this large DNA segment (DS1) containing these pCREB sites in the mouse using CRISPR/CAS9 gene editing revealed a loss of PTH mediated Cyp24a1 suppression, indicating that the pCREB features of this segment likely participated in renal Cyp24a1 downregulation. Mechanistically, however, recent ChIP-seq studies revealed only modest suppression of small amounts of residual pCREB together with an apparent blockade of CBP and CRTC2 binding at these pCREB sites as well as a site at the Cyp24a1 promoter in response to PTH (20). These limited changes in coregulator levels likely lead to the accompanying suppression of residual histone H3K9ac and H3K27ac and the absence of BRD4 observed at these sites, and therefore an inability of this gene to recruit RNA polymerase II. The corresponding absence of the transcriptional mark H3K36me3 indicated a lack of transcription. These features support the idea that PTH reduces Cyp24a1 expression in the kidney at the genetic level, consistent with our observed suppression of Cyp24a1 RNA transcripts, and does so by opposing the ability of the pCREB modules to interact with CREB coregulators essential for Cyp24a1 expression. Further studies will be necessary to identify the specific sites of action of PTH at one or more of these pCREB modules and to delineate the potential involvement of additional factors that may play a role in this suppression. Interestingly, as with pCREB components in the Cyp27b1 gene, the pCREB modules in the Cyp24a1 gene are uniquely absent in non-renal cells (NRTCs), supporting the concept that PTH is only capable of downregulating Cyp24a1 in the kidney (27). Thus, as with Cyp27b1, the Cyp24a1 gene retains regulatory features unique to the kidney. This reciprocal regulatory mechanism, together with the identified presence of both Cyp27b1 and Cyp24a1 in the same renal proximal tubules, support the coordinated involvement of both genes in the control of circulating 1,25(OH)₂D₃.

Returning to our investigation of renal Cyp27b1 regulation, we also explored the role of FGF23 and 1,25(OH)₂D₃ itself in renal Cyp27b1 expression. Interestingly, while PTH speaks to the unique and direct upregulatory control of renal 1,25(OH)₂D₃ production in response to an increased need to elevate blood Ca levels, both FGF23 and 1,25(OH)₂D₃ itself are negative regulators that seek to downregulate the production of 1,25(OH)₂D₃ in response to unique physiologic/metabolic determinants (28–30). Despite this, while both suppress Cyp27b1 expression, they may do so for different reasons, and as will be seen, by different mechanisms. Importantly, this suppression of Cyp27b1is also potentiated by the ability of FGF23 and 1,25(OH)₂D₃ to reciprocally induce Cyp24a1, thus collectively reducing blood 1,25(OH)₂D₃, as discussed in the next section (31). It is also worth noting that there is likely a distinction in the nature of the two signals in that while FGF23 represent a distal feedback mechanism prompted by circulating levels of P, 1,25(OH)₂D₃ and perhaps other regulatory components in the blood that control FGF23 expression from bone, identify a distal bone-kidney feedback axis. 1,25(OH)₂D₃ likely exerts its effects exclusively in the kidney via a short negative feedback mechanism. Nevertheless, despite an early understanding of the actions of 1,25(OH)₂D₃ and more recently of FGF23 to reduce 1,25(OH)₂D₃ production in the kidney via a downregulation of Cyp27b1, a delineation of the mechanisms involved have generally resisted research efforts. Importantly, while both FGF23 and 1,25(OH)₂D₃ suppress Cyp27b1 expression in wildtype mice, deletion of either the M1 or M21enhancer regions using CRISPR/Cas9 gene editing in vivo reduced but did not eliminate the ability of either to suppress Cyp27b1 expression (19). Deletion of both the M1 and M21 enhancers, on the other hand, fully abrogated the ability of both hormones to suppress renal Cyp27b1 expression, suggesting that both hormones may act at similar sites ( Figure 2 ) (21). Unfortunately, while we confirmed the presence of 1,25(OH)₂D₃-inducible VDR and RXR at each of these sites in M1 and M21 using ChIP-seq analysis, the lack of identification of a factor responsible for FGF23 suppression prevented unequivocal confirmation that these sites were the precise targets of FGF23. Moreover, since there were no striking differences between FGF23 and 1,25(OH)₂D₃ as measured by changes in residual pCREB abundance, or through striking suppression in the levels of residual CBP or CRTC2 at the pCREB sites within these M1 or M21enhancers, or in the ability of FGF23 to confer a differential profile of histone acetylation, these studies did not advance our understanding (20). Finally, as suppressors neither hormone was able to alter BRD4 binding at each of the pCREB sites, to influence residual levels of RNA polymerase II or to affect H3K36me3 levels across the gene body (32). We also observed that both FGF23 and 1,25(OH)₂D₃ oppose the upregulation of Cyp27b1 in response to simultaneous treatment with PTH, although 1,25(OH)₂D₃ is more effective (20). These results suggest that renal Cyp27b1 is upregulated by PTH and that both FGF23 and 1,25(OH)₂D₃ appear to prevent both residual actions as well as the upregulation of Cyp27b1 by PTH. These results provide novel mechanistic evidence for how these three hormones dynamically regulate the expression of renal Cyp27b1 and therefore the production of circulating 1,25(OH)₂D₃.

Cyp24a1 expression has long been known to be positively regulated by 1,25(OH)₂D₃ in tissues that include not only the kidney but in most VDR containing NRTCs as well. In contrast, while the more recent discovery that FGF23 regulates Cyp24a1 has suggested that this may occur in several tissue types, the bulk of evidence suggests that FGF23 regulation of Cyp24a1 is restricted to the kidney where FGF23 is biologically active (27). While FGF23 is known to mediate its extracellular actions via a coupling with Klotho that facilitates direct high affinity interaction with FGFR1C and FGFR3, the identity of the intracellular transcription factor and the mechanism whereby FGF23 induces Cyp24a1 and certain other genes as stated earlier remain to be clarified. This may contrast with the suggestion that FGF23 activates the ERK1/ERK2 pathway that induces the transcription factor EGR1 (33–36). Importantly, however, the deletion of EGR1 in the mouse led to only a minor suppression in renal Cyp27b1 expression while effects on Cyp24a1 were not reported (35). And not surprisingly, many other immediate early genes are induced by numerous cellular regulators such as FGF23. Regardless, our efforts to identify the general genomic site(s) of action of FGF23 at the Cyp24a1 gene suggest that FGF23 may act at open chromatin sites within the large DNA segment DS1 located downstream of the Cyp24a1 gene that contains multiple pCREB binding sites. We speculate that the localization of FGF23 action in this region may facilitate in future studies the identification of the FGF23 transcription factor itself. On the other hand, 1,25(OH)₂D₃ has long been known to induce Cyp24a1 expression via a specific couplet of VDREs located immediately upstream of the Cyp24a1 promoter (37–40). These sites bind the VDR and its heterodimer partner RXR and appear to function in all tissues that express Cyp24a1. Our more recent studies using ChIP-seq analysis and novel cell-based and in vivo assays, however, have now revealed an additional large intergenic region located even further downstream of the Cyp24a1 gene that contains numerous additional sites that bind the VDR/RXR heterodimer ( Figure 3 ) (41). This discovery led to additional research that showed that many of these specific VDREs were functionally active in transfection assays. To functionally corroborate these earlier studies in vivo, we separately deleted the pCREB containing DS1 region downstream of Cyp24a1 as well as the downstream region containing VDR binding sites (DS2) using CRISPR/Cas9 gene editing in the mouse genome and assessed the ability of FGF23 and 1,25(OH)₂D₃ to induce Cyp24a1 following hormonal injection (27). Deletion of the DS1 segment resulted in the loss of FGF23 induction of Cyp24a1 in the kidney, as seen similarly for PTH suppression, but resulted in only a modest effect on the ability of 1,25(OH)₂D₃ to induce Cyp24a1. Loss of the DS2 region also resulted in a modest decrease in 1,25(OH)₂D₃ induction in the kidney and other tissues as well. While these results may appear to conflict, it is worth noting that retention of the apparently dominant VDRE located near the promoter remained active in vivo and thus likely underlined the limited loss of 1,25(OH)₂D₃ response seen in the DS2-deleted mouse. This complexity suggests that additional experiments are needed to dissect the contributions of the promoter proximal VDREs in the Cyp24a1 gene. However, we conclude that the sites of action of FGF23 and 1,25(OH)₂D₃ across this gene locus are likely distinct, with FGF23 sites located within the DS1 region and the 1,25(OH)₂D₃ inducible sites located within the DS2 region as well as at the promoter. Importantly, however, further analysis using ChIP-seq analysis revealed additional relevant information. Although both FGF23 and 1,25(OH)₂D₃ are strong agonists at the Cyp24a1 gene, 1,25(OH)₂D₃ is a much more robust inducer of Cyp24a1 than FGF23 (20). Thus, while each displayed common as well as distinct mechanistic effects on the gene, certain features appear to correlate directly with the more robust nature of 1,25(OH)₂D₃. For example, while neither affected the levels of pCREB present at the gene, both appeared to modestly induce CBP both across the downstream region as well as at the gene promoter, although 1,25(OH)₂D₃ exhibited more activity at DS2 and at the promoter proximal region. Interestingly, we noted that while both hormones exerted only nominal action on CRTC2, 1,25(OH)₂D₃ was much more effective at the Cyp24a1 promoter. This surprising finding suggests that in addition to PTH, 1,25(OH)₂D₃ may also be able to influence CRTC2 migration to the nucleus. While the actions of FGF23 and 1,25(OH)₂D₃ are not entirely clear at the levels of pCREB downstream factors, the two hormones’ actions are largely similar at the level of epigenetic modification and the consequences that lead to RNA upregulation. In short, both hormones induce H3K27ac across similar regions of the gene, although 1,25(OH)₂D₃ is unique in that it also strongly induces H3K9ac. Finally, both hormones induce the recruitment of BRD4, although FGF23 is most active at DS1 whereas 1,25(OH)₂D₃ is most active at DS2, yet both are active at the promoter. Finally, RNA polymerase II is strongly recruited to the Cyp24a1 gene under both hormone treatments, and this is reflected in the appearance of H3K36me3 across the gene body itself. Thus, there are both common as well as unique properties of both hormones, although it is clear 1,25(OH)₂D₃ is much more active at the Cyp24a1 gene than FGF23, a feature that can be seen in the relative ability of the two hormones to induce Cyp24a1 RNA transcripts. These data provide significant insight into the mechanisms through which FGF23 and 1,25(OH)₂D₃ regulate Cyp24a1, although additional work will be required to place the two hormones in perspective, not the least of which will be the identification of the transcription factor that mediates FGF23 action.

The above discussion defines the reciprocal co-regulation of Cyp27b1 and Cyp24a1 expression in the kidney that is central to the production and maintenance of endocrine 1,25(OH)₂D₃ by PTH, FGF23 and 1,25(OH)₂D₃ itself. These events are summarized in Figure 4 . This importance is stressed at the in vivo level in mice when the M1 and M21 enhancers are deleted in the mouse, preventing the production of 1,25(OH)₂D₃ from being synthesized by CYP27B1. Under these conditions, the circulating levels of 1,25(OH)₂D₃ are strikingly reduced, but not eliminated. This level is insufficient to maintain the skeleton due to high PTH levels and reveals a striking Cyp27b1 null-like phenotype (21). The residual blood levels of 1,25(OH)₂D₃ are due, however, to the substantial co-reduction in Cyp24a1 expression that permits the small amounts of 1,25(OH)₂D₃ synthesized in the kidney to be sustained. We ultimately provide evidence of this hypothesis by rescuing this M1/M21-deleted mouse with a diet rich in Ca and P, which brings down PTH and raises FGF23 to normal levels thereby increasing renal Cyp24a1 expression. Under these conditions, the levels of endocrine 1,25(OH)₂D₃ in the blood are reduced to undetectable concentrations, indicating the importance of the regulation of both renal Cyp27b1 and Cyp24a1 levels in the maintenance of circulating 1,25(OH)₂D₃.

The literature is replete with studies suggesting that FGF23 regulates PTH. These studies support both positive as well as negative effects and are almost always conditional (54). Given that lack of insight into the impact of environment on FGF23 action on PTH, a definitive impact remains to be established mechanistically.

This short summary outlines what is largely known historically of PTH gene regulation. It is not surprising that key details remain to be explored, as useful parathyroid cells are generally not available thereby restricting the use of current techniques described earlier in this manuscript.

Early exploration of the Fgf23 gene using ChIP-seq analysis revealed four regions across the Fgf23 gene locus in osteocytes that were enriched for H3K27ac, an epigenetic mark that has been associated with an open chromatin state critical for enhancer function ( Figure 6 ) (65). Two sites were located upstream of Fgf23 gene at -16 kb and -38 kb, one near the promoter region, and one in a downstream intron at +7kb. This discovery prompted the CRISPR mediated deletion of each region and a search for loss of functional activation by PTH. While PTH induction of Fgf23 mRNA in bone was lost in the distal -16kb (DE16K) deleted mouse, no effect was seen in any of the other mouse mutants. Further ChIP-seq analysis of osteocytes revealed the presence of pCREB exclusively at this distal enhancer. Interestingly, recent studies suggest that several SIK inhibitors that promote the recruitment of the CREB receptor transcriptional coactivator 2, (CRTC2) to the Cyp27b1 gene in mouse kidney also induces Fgf23 in the bones of those same mice (20). These results suggest that the mediator of PTH action may indeed be pCREB seen bound at DE16K. While further work will be necessary to establish the precise role for pCREB at the Fgf23 gene, its location 16kb upstream does not contain a NURR1 site. Additional ChIP-seq analyses also fail to identify the presence of NURR1 across the entire Fgf23 binding motif while revealing binding at other additional known sites for NURR1 including the osteocalcin gene in response to PTH. Further studies of NURR1 activation by PTH and 1,25(OH)₂D₃ in rat UMR bone cells revealed that the pattern of temporal induction of NURR1, as well as NURR2 and NURR3 and other immediate early genes such as EGR1, 2 and 3 are also upregulated (Pike, unpublished)). These studies allow us to conclude that it is unlikely that the mediator of PTH action at Fgf23 in bone is NURR1. In mice, the rapid actions of exogenous PTH resulted in PTH induction of Fgf23 bone mRNA, and prevented the delayed but coordinated detection of upregulated iFGF23 in the blood, as will be seen for 1,25(OH)₂D₃ and P.

1,25(OH)₂D₃ upregulates Fgf23 expression at the transcriptional level and is known to require the presence of VDR in both mice and in cells in culture. In cells, this induction is rapid and reminiscent of most other genes that are induced by this hormone (57). Nevertheless, our earliest studies of Fgf23 in the osteocyte revealed that despite this classic temporal regulation of Fgf23 by 1,25(OH)₂D₃, we were unable to identify the presence of the VDR at any sites across the Fgf23 gene locus, despite its presence at numerous known vitamin D target genes in these bone cells (66). Our most recent attempts to identify VDR as well as RXR binding sites at Fgf23 using ChIP-seq analysis in other bone cell lines were also unable to identify the presence of these two receptors, again despite the presence of this heterodimer at all known genes we inspected. These data suggested that the induction of Fgf23 by 1,25(OH)₂D₃ might be indirect, and mediated by the ability of this hormone and its receptor to rapidly induce one or more secondary transcription factors that might interact directly with the Fgf23 gene. This result prompted further exploration of the Fgf23 gene and revealed that deletion of only the promoter proximal (PPE) but not the DE16K region of Fgf23 in mice using CRISPR/Cas9 gene editing was essential for loss of 1,25(OH)₂D₃ induction of Fgf23 (67). The unusual nature of this activity of 1,25(OH)₂D₃ dictated a further unbiased dissection of the PPE by creating a series of mouse mutants containing unique genomic deletions upstream of the Fgf23 start site. Numerous internal mutations and specific deletions were also created as well. These series of mutant mice were then explored for loss of function at the Fgf23 gene as above to map in an unbiased manner response to not only 1,25(OH)₂D₃ but to P (see below) and other activators as well. Accompanying the quantification of bone Fgf23 mRNA, we also determined whether any changes in the level of mRNA resulted in a change in the circulating levels of intact (i) FGF23 (iFGF23); a change in both was necessary for us to conclude that the deletion resulted in a loss of function. The loss of response to 1,25(OH)₂D₃ occurred in mutant mice in which deletions were created downstream of the VDRE-like sequence proposed by Liu et al. (58) and upstream of a site suggested to contain a proposed NURR1-like site termed the NURR1/VDRE like sequence as discussed above which was believed to be a potential target for PTH as well as 1,25(OH)₂D₃ (57, 59). Internal base mutations of this NURR1/VDRE sequence alone as well as small deletions both upstream and downstream of this site had no effect on the ability of 1,25(OH)₂D₃ to induce bone Fgf23 mRNA or to upregulate blood levels of iFGF23. These data suggest that neither the upstream VDRE-like sequences proposed earlier nor that of the NURR1/VDRE sequence proposed more recently, nor sites more proximal to this latter proposed site mediates the actions of 1,25(OH)₂D_3. A recent alternative evaluation of this region appears to concur, speculating in detail that this site and transcription factors other than NURR1 may be involved (15). Perhaps more importantly, the DNA segment located between these two hypothetical mediators of 1,25(OH)₂D₃ does not contain a potential VDRE-like DNA motif capable of directly binding the VDR, supporting our earlier suggestion that the VDR does not bind directly within the Fgf23 gene locus. Thus, we are currently exploring this mapped region of 1,25(OH)₂D₃ sensitive genomic space that appears to be functionally relevant in vivo in an unbiased search for potential transcription factor candidates that are amenable to technical confirmation as mediators of 1,25(OH)₂D₃ action. Importantly, these data suggest a novel, yet transcriptionally consistent, genomic mechanism whereby 1,25(OH)₂D₃ induces the expression of an important gene involved in mineral homeostasis.

P induces an upregulation of Fgf23, even in mice devoid of the VDR (31). The mechanism by which this mineral functions to induce Fgf23 as well as other genes is not known. This appears to be largely because the study of this mineral over many decades in vivo has involved dietary P loading experiments of a generally prolonged nature, wherein lower or elevated P levels in the diet were delivered to mice. This delivery system results in a lengthy delay (days) in the measurement of P response, including the upregulation of Fgf23 and prevented exploration of mechanisms whereby P might induce Fgf23. To overcome this issue, we developed a method whereby elevated dietary P could be rapidly delivered to mice and the upregulation of Fgf23 mRNA evaluated within several hours as described previously; importantly, both the upregulation of Fgf23 mRNA as well as blood iFGF23 could be detected within that time frame (60). Subsequent studies revealed an elevated response to high P as early as 1 h. The rapidity of this response suggested that the mechanisms might be transcriptional in nature. More definitively, however, we also discovered that deletion of the PPE but not the DE16K of the FGF23 gene resulted in a loss of this rapid induction by high P, providing strong support for the idea that this mechanism likely involved transcriptional regulation. Surprisingly, however, despite this loss of acute induction, P appeared to upregulate Fgf23 mRNA and iFGF23 through an additional chronic mechanism apparent in this PPE mouse mutant beginning within 24 h that resulted in normal levels of Fgf23 mRNA and blood iFGF23 by 48 h. It is worth noting here that these results did not appear to arise through an unusual underlying feeding mechanism inherent to the approach, as diet without P was used as feeding controls. Thus, high P appears to induce Fgf23 by two different processes that differ temporally and mechanistically.

To further explore the site of acute action of P at the Fgf23 gene, we mapped the loss of P induced response of Fgf23 mRNA and iFGF23 in each of the mutant mice developed for our studies described above with 1,25(OH)₂D₃. As with the steroid hormone, a loss of P response was identified both upstream of the NURR1/VDRE-like site. Deletion of the NURR1/VDR-like site also had no effect on loss of Fgf23 mRNA or Fgf23 induction. Thus, a single upstream DNA segment identical to that for 1,25(OH)₂D₃ induction was apparent (67). It is worth noting, however, that this segment was relatively lengthy. In addition, the time course for P was much more rapid for P than for 1,25(OH)₂D₃. Therefore, it cannot be concluded that both P and 1,25(OH)₂D₃ target the same DNA sequence(s) or utilize the same transcription factor(s). Studies are in progress with additional mutant mice to refine the sites of action of P at the Fgf23 gene and to identify functional motifs useful for further exploration of the mechanism of acute P action.

In order to gain additional support for the identity of possible targets of both 1,25)OH)₂D₃ and P, we also explored the general response in bone via RNA-seq analysis Our working hypothesis is that a genome wide response as determined by an RNA-seq analysis and subsequent analysis of regulated RNAs might provide information regarding not only a relationship between the two inducers, but might reveal additional useful pathways for P in particular, and possible reveal novel transcription factor candidates as well. Of course, the actions of each of these inducers may not involve the induction of regulators via transcription but rather the activation of other signaling pathways as well. One pathway that is common to both 1,25(OH)₂D₃ and P is an upregulation of Wnt/β-catenin signaling, observed via an increase in the expression of numerous Wnts and the downregulation of numerous Wnt inhibitors (68). However, this overlap is not broadly displayed and many of the genes that are regulated by 1,25(OH)₂D₃ do not overlap those regulated by P. Future studies will be necessary to determine the relevance of these data with those derived from our mechanistic studies of Fgf23 regulation in the mutant mice.