Regulation of 1 and 24 hydroxylation of vitamin D metabolites in the proximal tubule

Abstract

Calcium and phosphate are critical for numerous physiological processes. Consequently, the plasma concentration of these ions are tightly regulated. Calcitriol, the active form of vitamin D, is a positive modulator of mineralization as well as calcium and phosphate metabolism. The molecular and physiological effects of calcitriol are well documented. Calcitriol increases blood calcium and phosphate levels by increasing absorption from the intestine, and resorption of bone. Calcitriol synthesis is a multistep process. A precursor is first made via skin exposure to UV, it is then 25-hydroxylated in the liver to form 25-hydroxyitamin D. The next hydroxylation step occurs in the renal proximal tubule via the 1-αhydroxylase enzyme (encoded by CYP27B1) thereby generating 1,25-dihydroxyvitamin D, that is, calcitriol. At the same site, the 25-hydroxyvitamin D 24-hydroxlase enzyme encoded by CYP24A1 can hydroxylate 25-hydroxyvitamin D or calcitriol to deactivate the hormone. Plasma calcitriol levels are primarily determined by the regulated expression of CYP27B1 and CYP24A1. This occurs in response to parathyroid hormone (increases CYP27B1), calcitriol itself (decreases CYP27B1 and increases CYP24A1), calcitonin (increases or decreases CYP24A1 and increases CYP27B1), FGF23 (decreases CYP27B1 and increases CYP24A1) and potentially plasma calcium and phosphate levels themselves (mixed effects). Herein, we review the regulation of CYP27B1 and CYP24A1 transcription in response to the action of classic phophocalciotropic hormones and explore the possibility of direct regulation by plasma calcium.

Impact Statement

Fundamental to the maintenance of calcium and phosphate homeostasis is the regulation of the phosphocalciotropic hormone 1,25-dihydroxyvitamin D or calcitriol. This occurs in the renal proximal tubule via the regulation of the expression of two enzymes. 1-alpha-hyrdoxylase encoded by CYP27B1 results in the 1-hydroxylation of calcitriol and thus more active vitamin D. In contrast, 24-hydroxylase encoded by CYP24A1, results in the 24- hydroxylation of calcitriol and its precursor 25-hydroxyvitamin D, decreasing the levels of the circulating active hormone. This review summarizes what is known about the regulation of these enzymes.

Introduction

Calcium and phosphate homeostasis

Calcium is a vital mineral found predominantly in the structural matrix of bone. However, calcium is also essential for a diversity of physiological functions including muscle contraction, neurotransmitter release, intracellular signal transduction, and blood clotting.^1,2 Phosphate is also a vital mineral and a significant structural element in bone. It is also a component of ATP, nucleic acids and phospholipids. ¹ Due to their critical importance in physiological and cellular processes, calcium and phosphate concentrations are tightly regulated within a narrow range in the circulation. Moreover, when the level of one ion becomes sufficiently elevated they can precipitate forming extraosseous calcifications as seen in patients with renal insufficiency. ³ As such, the plasma levels of both minerals are regulated via the coordinated action of a group of hormones, referred to as phosphocalciotropic hormones that includes calcitriol (active vitamin D or 1,25-dihydroxyvitamin D), parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23) (N.B. sex hormones also affect calcium and mineral homeostasis but are not considered phophocalciotropic hormones). Importantly, plasma concentrations of calcium and phosphate are interdependent. Many of these hormones regulate calcium and phosphate balance by increasing the production or inactivation of calcitriol (Figure 1). This is thought to occur primarily by regulating transcription of the 1- or 24-hydroxylating enzymes that activate and deactivate 25-hydoxyvitamin D and calcitriol respectively. Although there is evidence that 1-alpha hydroxylase can be phosphorylated, the effect of this on protein abundance and function is unclear due to a lack of adequate antibodies to study the phosphoenzyme.^4,5 1-alpha hydroxylase is a mixed function oxidase encoded by the gene CYP27B1 within the nuclear genome. ⁶ This enzyme localizes to the inner membrane of mitochondria where it hydroxylates 25-hydroxyvitamin D at the 1-alpha position to produce 1,25-dihydroxyvitamin₂D₃ (calcitriol), the biologically active form of the hormone. ⁷ Calcitriol is deactivated by a 25-hydroxyvitamin D-24-hydroxylase enzyme which is encoded by the gene CYP24A1. It is also a mitochondrial enzyme that catalyzes the hydroxylation of both calcitriol and its precursor 25-hydroxyvitamin D₃ to 1,24,25-trihydroxyvitamin D₃ or 24,25-dihydroxyvitamin D₃. 24 hydroxylation inactivates these hormones as these forms of vitamin D are unable to bind to the vitamin D receptor (VDR). ⁸

Figure 1.

Regulation of CYP27B1 and CYP24A1 transcription by phosphocalciotropic hormones, calcium and phosphate. Calcitonin and PTH are secreted from the thyroid and parathyroid glands, respectively. Both act to upregulate the transcription of CYP27B1. In addition, calcitonin affects CYP24A1 by either upregulating or attenuating its transcription depending on the situation. High extracellular calcium has been associated with a decrease in CYP27B1 transcription, while low extracellular calcium and hypophosphatemia are associated with increased CYP27B1 transcription. FGF23 production in bone suppresses CYP27B1 expression while enhancing CYP24A1 production. Finally, calcitriol itself feedback inhibits its own production by inhibiting CYP27B1 transcription and promoting its own inactivation by increasing CYP24A1 transcription. (Created with BioRender.com, supported by previous studies^9–15). (A color version of this figure is available in the online journal.)

Calcitriol synthesis

The synthesis of calcitriol occurs in multiple steps. First, pre-vitamin D₃ is formed from 7-dehydrocholesterol in the skin following UV irradiation or absorption from the diet. Following thermal isomerization to produce vitamin D₃, the compound then travels, bound to vitamin D binding protein, to the liver where it is hydroxylated to form 25-hydroxyvitamin D by 25-hydroxylase.^16–18 This initial hydroxylation step is not tightly regulated, and is only limited by the amount of pre-vitamin D₃ in the circulation. ¹⁹ The secondary hydroxylation step, which produces 1,25-dihydroxyvitamin D₃ or calcitriol, is the most regulated and occurs most substantially in the mitochondria of the renal proximal tubule by 1-alpha hydroxylase encoded by CYP27B1.^7,20,21 For this to occur 25-hydroxyvitamin D bound to the vitamin D-binding protein (DBP) must be filtered through the glomerulus and subsequently endocytosed into the proximal tubule after binding to megalin and cubilin. Cubilin sequesters the 25-hydroxyvitamin D-DBP and megalin stimulates endocytosis of the complex. After trafficking to lysosomes 25-hydroxytamin D is liberated and can be shuttled to the mitochondria. ²² Calcitriol is the active form of vitamin D, which can bind to the vitamin D receptor (VDR) and exert its downstream effects on calcium and phosphate metabolism.^23–26 CYP27B1 is primarily expressed in the proximal tubule, where the majority of calcitriol production occurs. ²¹ Interestingly the proximal tubule is also the site of the majority (60–70%) of calcium reabsorption from the glomerular filtrate, via paracellular pathways.^27–29 The majority of tubular phosphate reabsorption also occurs in this segment but by a transcellular pathway. ³⁰ There is also extra-renal expression of 1-alpha hydroxylase, encoded by CYP27B1, though the effect on plasma calcitriol levels is negligible and thus these sites of calcitriol synthesis are mostly relevant for local, paracrine vitamin D action.^9,31–33 Consistent with this, a kidney-specific Cyp27b1 pseudo-null mouse model displays a phenotype similar to the global null animal. ³² Extra-renal synthesis of 1-alpha hydroxylase appears to be regulated separately to the proximal tubule, as they do not contain the same regulatory module as the kidney gene, and is not considered herein. ³² Importantly, an increase in expression of Cyp27b1 is closely tied to increased calcitriol production, consistent with transcription being the major mode of regulation in calcitriol production. ³⁴

Calcitriol actions

Calcitriol is a steroid hormone that acts by crossing the plasma membrane and binding the VDR, which heterodimerizes with the retinoid X receptor (RXR) (Figure 1). This complex then translocates to the nucleus where it transcriptionally regulates target genes via binding to vitamin D response elements (VDREs).^24,25,35 Through this process, calcitriol has a significant impact on calcium and phosphate homeostasis by indirectly influencing the handling of these minerals in the intestine, kidney, and bone.^{16,19,25,35–39} The effects of calcitriol are primarily observed in the duodenum and colon, where transcellular calcium absorption is increased via increasing TRPV6 expression. ³⁷ Calcitriol likely also increases paracellular calcium absorption from the jejunum and ileum by increasing claudin 2 expression. ⁴⁰ In the distal renal tubule calcitriol increases TRPV5 expression thereby increasing the predominant transcellular reabsorption pathway for calcium. ⁴¹ Calcitriol also increases the reabsorption of phosphate from the proximal tubule and absorption of phosphate from the intestine, although the mechanism behind the former processes requires further elucidation. ⁴² In the intestine, calcitriol increases the expression and/or posttranscriptional modulation of the sodium-dependent phosphate transport protein 2b (NaPi-IIb), resulting in increased absorption of phosphate and increased serum phosphate concentrations. ⁴³ In bone, high levels of calcitriol can trigger resorption to mobilize calcium and phosphate stores into the circulation when required, but calcitriol can also support bone mineralization by raising calcium and phosphate levels through the stimulation of intestinal absorption.^{25,26,36,44,45}

Clinical importance of calcitriol

Abnormalities in calcitriol metabolism highlight the importance of this hormone in the maintenance of calcium and phosphate homeostasis. Calcitriol deficiency results in hypocalcemia, hypophosphatemia, and vitamin D-dependent rickets. Rickets is associated with abnormalities of bone weakness, fractures, pain, as well as abnormal bone bending and deformity of the tibiae and femora. Consistent with this, mutations in the CYP27B1 gene cause vitamin D hydroxylation-deficient rickets type IA, an autosomal recessive disorder characterized by an inability to synthesize 1,25-dihydroxyvitamin D, and consequently hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and rickets. ⁴⁶ 1-alpha hydroxylase deficiency is rare, but there is an unusually high frequency in the French-Canadian population due to a founder effect. Fortunately, this condition responds to treatment with exogenous calcitriol.^47,48 However, calcitriol supplementation is more commonly prescribed to patients with chronic kidney disease to treat secondary hyperparathyroidism and hypocalcemia which is the result of decreased calcitriol production secondary to lower levels of 1-alpha hydroxylase occurring as a result of decreasing renal mass. ⁴⁹

In contrast, loss of function mutations in the CYP24A1 gene cause hypercalcemia due to the inability to inactivate calcitriol and can present as idiopathic infantile hypercalcemia or as a rare genetic cause of nephrolithiasis.^8,50–53 Granulomatous diseases such as subcutaneous fat necrosis, sarcoidosis, tuberculosis, and lymphoma can contribute excess 1-alpha hydroxylase production due to macrophage activation. This results in increased calcitriol levels and hypercalcemia.^54–56 These diseases highlight the importance of CYP27B1 and CYP24A1 in maintaining appropriate concentrations of calcitriol in the circulation and the impact of dysregulated activity on calcium and phosphate homeostasis. The remainder of this review therefore focuses on the signaling mechanisms regulating the expression of the CYP27B1 and CYP24A1 genes in the renal proximal tubule, and thus calcitriol levels in plasma.

Classic signaling pathways

Parathyroid hormone-mediated regulation of CYP27B1 and CYP24A1

PTH is a peptide hormone secreted by the chief and oxyphil cells of the parathyroid gland. PTH acts to increase plasma calcium by increasing renal reabsorption and bone resorption while promoting excretion of phosphate. The secretion of PTH is predominantly regulated by the extracellular calcium concentration, which is detected by the calcium-sensing receptor (CaSR) on the surface of chief and oxyphil cells of the parathyroid.^57,58 Calcium ions bind to the extracellular domain of the CaSR, which at high levels activates the receptor, thereby suppressing PTH secretion when the blood calcium concentration is sufficiently high.^59,60

Phosphate also affects CaSR signaling as a noncompetitive inhibitor, whereas calcium is an agonist to the receptor. ⁶⁰ Elevated blood phosphate levels inhibit CaSR signaling, thus stimulating PTH secretion. ⁶¹ In the kidney, PTH inhibits phosphate reabsorption from the proximal tubule and increases renal calcium reabsorption from the distal nephron. These actions in the proximal tubule are the result of PTH binding to the G-protein-coupled type 1 PTH receptor (PTHR1) on both the apical and basolateral surfaces of proximal tubule epithelial cells. The major effects of PTH are mediated by the coupling of PTHR1 to G_s- and G_q/11-proteins, stimulating the protein kinase A (PKA) and protein kinase C (PKC) pathways, respectively. These kinases phosphorylate the sodium hydrogen exchanger regulatory factor 1 (NHERF1) a PDZ domain-containing scaffold protein, which triggers degradation of sodium-dependent phosphate transport protein 2A (NaPi-IIa), thereby attenuating phosphate reabsorption from the proximal tubule. ⁶²

PTH also acts to increase transcription of the rate-limiting enzyme in calcitriol production, 1-alpha hydroxylase, in the proximal tubule (Figure 2).^{10,11,63–71} This enables PTH to further increase the calcium concentration in the blood through the actions of calcitriol on the intestine and bone. This is the classical pathway through which PTH indirectly increases intestinal calcium absorption. Simultaneously, the half-life of CYP24A1 mRNA is reduced approximately fourfold in the presence of PTH, thereby slowing the inactivation of calcitriol. ⁷²

Figure 2.

Transcriptional regulation of CYP27B1 and CYP24A1 by PTH and calcitriol in proximal tubular epithelial cells. PTH-induced transcriptional regulation occurs primarily by a PKA-mediated pathway. Upon binding to the G-protein-coupled receptor in the apical or basolateral membrane, adenylate cyclase is activated producing cAMP, which in turn activates protein kinase A (PKA). PKA phosphorylates CREB, activating it and permitting binding to CRE sites in the promoter of CYP27B1. Calcitriol signaling occurs via binding to the intracellular vitamin D receptor (VDR), which heterodimerizes with the retinoid X receptor (RXR) prior to DNA binding. Together, liganded VDR-RXR enter the nucleus where it can bind to vitamin D response elements of target genes. This increases CYP24A1 transcription, resulting in decreased calcitriol levels, and decreases CYP27B1 transcription, reducing calcitriol production. (Created with BioRender.com, supported by previous studies^{11,24,68,71,73}). (A color version of this figure is available in the online journal.)

Evidence of the important role PTH plays in regulating calcitriol production comes from animal models. When parathyroidectomized animals are fed a low calcium diet, they fail to increase circulating levels of calcitriol, yet have increased levels of the inactive hormone produced by 24-hydroxylase. ¹⁰ However, calcitriol production was induced by administering either a parathyroid extract or PTH, consistent with PTH stimulating the production of calcitriol via increasing Cyp27b1 and reducing Cyp24a1 expression. ¹⁰ These experiments highlight the important role the parathyroid glands contribute through PTH which positively modulates the levels of calcitriol.

Further studies on parathyroidectomized and calcitriol-deficient rats have implicated cAMP as an important intracellular signal involved in the stimulation of Cyp27b1 transcription in response to PTH. ⁷¹ Notably, infusion of cAMP into parathyroidectomized rats mimicked the effect of PTH infusion by stimulating calcitriol production in a dose-dependent manner. ⁷¹ Moreover, renal adenylate cyclase activity was enhanced by PTH in rats made calcitriol-deficient by feeding them a vitamin D-deficient diet. In this study, the administration of PTH caused an immediate increase in renal cAMP levels. ⁷¹ Brenza and DeLuca ¹¹ also provide evidence that cAMP is a second messenger involved in the induction of CYP27B1 transcription in a pig cell line, AOK-B50 cells, as well as the human HCK-8 proximal tubule cell line. In this cell culture work, forskolin, an adenylate cyclase activator that increases intracellular cAMP, increased the activity of a CYP27B1 reporter. ¹¹ However, direct application of PTH to these cell models resulted in a significantly greater response, suggesting that PTH is potentially activating more than one second-messenger pathway to increase transcription of CYP27B1.^11,68 Work done by Korkor et al. in mouse cortical kidney cell cultures (a model system composed predominantly of proximal tubule epithelial cells) further implicates cAMP as an important second messenger for PTH signaling. They found that calcitriol synthesis was dependent on de novo production of 1-alpha hydroxylase, and that calcitriol synthesis was proportional to the amount of cellular cAMP. ⁷⁴ Furthermore, addition of protein or mRNA synthesis inhibitors prevented cAMP-mediated stimulation of calcitriol production, leading to the conclusion that increased calcitriol production is dependent on an increase in novel protein synthesis. ⁷⁴ Brenza et al. ⁶⁸ also found that the promoter for CYP27B1 contains cyclic AMP response element (CRE) sites. These are DNA binding sites for the CREB transcription factor, which is activated in response to phosphorylation by the PKA pathway. ⁷⁵ These findings further implicate cAMP as the predominant second messenger mediating PTH-induced stimulation of CYP27B1 transcription. ⁶⁸

Work by Zierold et al. ⁷⁰ in a porcine kidney cell line (AOK-B50) identified other specific transcription factors that upregulate CYP27B1 gene production following exposure to PTH. NR4A2 (Nurr1), a nuclear receptor that binds to DNA sequences in the promoter region of the genes it regulates, has been implicated in the upregulation of CYP27B1 in response to PTH. NR4A2 over-expression increased CYP27B1 mRNA by binding to the promoter and NR4A2 mRNA levels were increased in response to PTH. ⁷⁰ C/EBP $\beta$ was also implicated in the regulation of CYP27B1 transcription in these studies, acting directly on the promoter to decrease transcription and indirectly by reducing the amount of NR4A2 produced. ⁷⁰ C/EBP $\beta$ is also known to upregulate CYP24A1 transcription. The overall effect of C/EBP $\beta$ in this work was thus a decrease in calcitriol production. However, these results are in contrast to work interrogating the role of calcitonin in regulating calcitriol expression, which is discussed below.

Feedback inhibition by calcitriol

Calcitriol exerts negative feedback on CYP27B1 transcription thereby inhibiting its own production. This negative effect on CYP27B1 expression occurs downstream of the binding of calcitriol to VDR. Calcitriol-bound VDR dimerizes with the retinoid X receptor (RXR), which in turn can bind to vitamin D response elements (VDREs) within DNA. Consistent with this concept of calcitriol feedback inhibition via nuclear signaling, inhibition of Cyp27b1 expression is absent in VDR knockout mice, who have significantly higher calcitriol levels compared with wild-type animals.^24–26,36 Further, animals made calcitriol deficient by being fed a low vitamin D containing diet and limiting exposure to UV radiation were supplemented with calcitriol, they had reduced Cyp27b1 expression. ²⁴

This is supported by work in rats. The administration of calcitriol to calcitriol-deficient rats reduced the transcription of Cyp27b1 in both the absence and presence of PTH. ¹¹ Calcitriol also inhibits PTH gene transcription in the parathyroid to further suppress its own production.^11,76 This suggests that calcitriol can override PTH-mediated stimulation of CYP27B1 expression when the concentration of calcitriol is sufficiently high, although the exact level is currently unclear. ¹¹ It is unclear whether the ultimate effect of these two competing inputs depends on their relative concentrations or another input. Clearly, more studies are needed to determine their roles in different conditions. Finally, calcitriol also upregulates C/EBP $\beta$ in rat kidney, which acts directly on the Cyp27b1 promoter to inhibit its activity and reduce CYP27B1 transcription. ⁷⁰ C/EBP $\beta$ also inhibits the stimulatory action of NR4A2 on the promoter, thereby reducing the impact of NR4A2 in the presence of PTH and reducing CYP27B1 transcription. ⁷⁰

Calcitriol also acts to limit its own production by increasing the expression of CYP24A1, which encodes the 24-hydroxylase that inactivates calcitriol.^6,11,77 When porcine AOK-B50 kidney cells were treated with calcitriol, CYP24A1 gene expression was stimulated in concert with suppressed CYP27B1 gene expression. ¹¹ Transcription of CYP24A1 was upregulated by calcitriol via VDR-dependent control of VDREs in the CYP24A1 promoter.^11,73 Calcitriol-induced upregulation of C/EBP $\beta$ in the kidney not only serves to repress CYP27B1 transcription, but also to upregulate CYP24A1 expression via C/EBP $\beta$ sites in the 24-hydroxylase promoter. ⁷⁸ This is further supported by the observation that there is marked attenuation of CYP24A1 transcription when this site is mutated. ⁷⁸ Thus, not only is there a feedback loop that inhibits further production of calcitriol, there is also feedback to promote the inactivation of calcitriol and its precursor, 25-hydroxyitamin D.

Nonclassic signaling pathways

Calcitonin

Calcitonin is a hormone released from the C-cells of the thyroid gland in response to high blood calcium levels. It binds to the G-protein-coupled calcitonin receptor, which then signals through both the PKC and PKA pathways. ¹² Calcitonin protects against hypercalcemia largely by exerting a negative effect on osteoclasts in bone to prevent bone resorption and thus increases bone mineralization an effect potentially augmented by decreasing urinary calcium excretion.^79–81 In this way, calcitonin may redirect calcium from the blood and urine into bone mineralization. There is evidence, however, that calcitonin also plays a role in regulating calcitriol production by increasing CYP27B1 transcription. Under hypercalcemic conditions, calcitonin may be a significant regulator of CYP27B1 expression, rather than PTH which is likely more relevant in the hypocalcemic state. ³⁴ While PTH signals through both the PKA and PKC pathways to induce CYP27B1 transcription, calcitonin preferentially increases CYP27B1 expression via the PKC pathway as demonstrated in renal porcine LLCPK cells. ¹² Although administration of the PKA activator 8-bromo-cAMP had a stimulatory effect on CYP27B1 mRNA expression, the PKA inhibitors Rp-cAMPS and H-89 had no effect on calcitonin-induced CYP27B1 expression. In contrast, the PKC activator phorbol 12-myristate 13-acetate (PMA) increased CYP27B1 mRNA levels to a similar extent as seen with calcitonin treatment, while administration of the PKC inhibitor staurosporine attenuated CYP27B1 transcription induced by calcitonin in a dose-dependent manner. ¹² This strongly implicates PKC in mediating the stimulatory effect of calcitonin on calcitriol production.

The transcription factor C/EBP $\beta$ is also a downstream effector of calcitonin (Figure 3). Expression of this transcription factor increased following exposure to calcitonin. ¹³ C/EBP $\beta$ can bind to the promoter of CYP27B1 thereby increasing 1-alpha hydroxylase production. Further, the transfection with a dominant negative modulator of C/EBPs binding sites inhibited calcitonin induced Cyp27b1 transcription in a dose-dependent manner. ¹³ This is a potential mechanism by which calcium retention is increased despite a normal blood calcium concentration, thereby increasing calcium availability in the circulation in times of increased demand, such as pregnancy and lactation. ¹³ There is evidence therefore, supporting a role for C/EBP $\beta$ in both positive and negative regulatory pathways, as it is also associated with decreased CYP27B1 expression after calcitriol exposure. These seemingly contradictory effects might be explained by other yet to be identified signaling events affecting C/EBPβ gene modulation. This issue requires further investigation to elucidate the differing effects of C/EBP $\beta$ on Cyp27b1 expression.

Figure 3.

Transcriptional regulation of CYP27B1 and CYP24A1 by calcium, phosphate, FGF23, and calcitonin. Extracellular calcium activates the calcium sensing receptor, which in turn activates mitogen-activated protein kinase (MAPK) and protein kinase C pathways. These pathways are proposed to interact with the CYP27B1 promoter to increase its activity at low extracellular calcium concentrations and to decrease promoter activity at high extracellular calcium concentrations. Hypophosphatemia is linked to increased expression of CYP27B1. However, the mechanism behind this is unknown. Fibroblast growth factor 23 (FGF23) binds to the FGF receptor (FGFR) and its cofactor klotho to activate the MAPK pathway, phosphorylating ERK1/2, which inhibits CYP27B1 transcription. FGF23 signaling in the proximal tubule is also associated with increased CYP24A1 levels, although the mechanism behind this is unknown. Calcitonin binds to its G-protein-coupled receptor, activating both the PKA and PKC pathways. PKC increases the expression of the C/EBP $\beta$ transcription factor thereby promoting CYP27B1 transcription. Calcitonin can also signal through the Sp1 and NF-Y transcription factors to upregulate CYP24A1 transcription, although there may be an additional mechanism whereby CYP24A1 transcription is attenuated. (Created with BioRender.com, supported by references^{9,12–15,82,83}.) (A color version of this figure is available in the online journal.)

There is conflicting evidence surrounding the role of calcitonin on CYP24A1 expression. Research in thyroparathyroidectomized rats, in which both PTH and calcitonin production are lost, fed a low calcium diet found twofold increased Cyp24a1 expression, however, calcitonin administration reduced Cyp24a1 mRNA expression. ⁸² In contrast to this, in vitro work in human embryonic kidney (HEK-293) cells that were transfected with the calcitonin receptor found that calcitonin stimulates CYP24A1 expression. In addition, H89 and calphostin C, inhibitors of the PKA and PKC pathways respectively, reduced calcitonin-induced CYP24A1 expression by 60%. ⁸³ It was proposed that calcitonin induced the PKA or PKC pathway which phosphorylates and activates the transcription factors Sp1 and NF-Y, which were both shown to increase expression of CYP24A1. ⁸³ Although these studies are seemingly contradictory, there is a possibility that calcitonin can exert a different effect depending on calcium and calcitriol levels, promoting calcitriol degradation when blood calcium levels are high and suppressing degradation of calcitriol when calcium levels are low. Further work is required to determine the role of calcitonin in calcitriol metabolism.

FGF23/klotho

Fibroblast growth factor-23 (FGF23) is a peptide hormone produced in bone by osteoblasts and osteocytes that inhibits renal tubular phosphate reabsorption and calcitriol production to reduce intestinal calcium and phosphate absorption.^64,84 FGF23 additionally indirectly lowers serum calcium levels by decreasing PTH levels. FGF23 binds to fibroblast growth factor receptor isoforms 3 and 4, thereby activating the receptors and inducing tyrosine autophosphorylation and the stimulation of its intrinsic tyrosine kinase activity.^85,86 This leads to the activation of the MAP kinase pathway and downstream phosphorylation of extracellular signal-regulated kinase-1 and -2 (ERK1/2). ⁸⁷ The coreceptor, klotho, is required for binding of FGF23 to the receptor enabling its subsequent activation in the kidney.^87,88 FGF23 and klotho have a suppressive role in renal CYP27B1 transcription through ERK1/2.^84,87,88 HEK-293 cells transfected with the CYP27B1 promoter had suppressed CYP27B1 promoter activity when exposed to FGF23. The suppressive effect of FGF23 was blocked by a ERK1/2 inhibitor. Moreover, FGF23-null mice display threefold increased CYP27B1 promoter activity in the kidney compared with WT mice with normal FGF23 levels, consistent with FGF23 suppressing CYP27B1 expression. ⁹ This supports the role of FGF23 in suppressing CYP27B1 transcription.

FGF23 also lowers blood calcitriol levels by promoting renal CYP24A1 transcription, resulting in calcitriol inactivation.^9,89 Consistent with this, FGF23-null mice display 63% lower Cyp24a1 mRNA expression in the kidney compared with wild-type mice. ⁹ Further work is needed to elucidate the mechanism driving the FGF23-mediated increase in CYP24A1 expression. Research into this area is of great interest because of potential implications in chronic kidney disease, where elevated FGF23 presents in the early phases and is accompanied by severe calcitriol deficiency. ⁹⁰ Increased CYP24A1 transcription, in response to FGF23, may exacerbate calcitriol deficiency and contribute to the progression of chronic kidney disease.^91,92

Hypophosphatemia

Regulation of CYP27B1 expression also occurs in response to alterations in serum phosphate concentration. Hypophosphatemia, as caused by a low phosphate containing diet, results in increased Cyp27b1 expression independently of PTH and markedly elevated serum calcitriol levels.^93,14 This is supported by observations that phosphate-depleted mice exhibit enhanced calcitriol production threefold compared with control-fed mice. ⁹⁴ However, this effect is abolished in these mice post-hypophysectomy suggesting that the pituitary gland has a role in sensing circulating phosphate levels and altering serum calcitriol production in response. ¹⁴ Consistent with this is evidence that pituitary hormones may exert an effect on calcitriol production through transcriptional regulation of Cyp27b1. ¹⁴ Due to the previous association of growth hormone deficiency with hypovitaminosis D, growth hormone represents a potential candidate for this role. ⁹⁵ However, the exact mechanism whereby extracellular phosphate sensing occurs, or how pituitary hormones affect CYP27B1 expression remains to be elucidated.

Calcium and the calcium-sensing receptor

In addition to plasma phosphate, plasma calcium concentration also modulates calcitriol production, independent of PTH or other calciotropic hormones. Thyroparathyroidectomized rats are unable to secrete PTH or calcitonin in response to altered blood calcium levels. However, the direct infusion of CaCl₂ into these animals resulted in suppression of renal Cyp27b1 expression. ⁶⁵ Further, infusion of these rats with calcium and PTH simultaneously resulted in increased blood calcium levels, however, Cyp27b1 activity was suppressed compared with rats given PTH alone. Conversely, when PTH was administered with EGTA, a calcium chelator that prevents a rise in blood calcium levels, Cyp27b1 expression was stimulated. ⁶⁵ These results demonstrate that higher blood calcium levels counteract the effects of PTH to suppress production of calcitriol when blood calcium levels are high. Further work is needed to fully delineate the effect of increased blood calcium on CYP24A1 expression.

The calcium-sensing receptor (CaSR) is a seven transmembrane G-protein-coupled receptor that senses the extracellular calcium concentration and negatively regulates PTH production and secretion from the parathyroid gland in response. The CaSR also has effects outside of the parathyroid gland, with expression in organs such as the brain, kidney, and intestines where it has effects on neuropathological conditions and modulate renal calcium reabsorption and intestinal calcium absorption.^60,96–99 It acts to modulate the expression and activity of calcium and phosphate transporters, channels and pores, including tight junction proteins that regulate both paracellular and transcellular calcium (re)absorption across renal and intestinal epitehlia.^{59,97,100–102} CaSR activity has also recently been linked to the modulation of CYP27B1 expression in a HEK-293 cell model. CaSR expressing HEK-293 cells were transfected with a CYP27B1 promoter reporter construct and exposed to increasing levels of extracellular calcium. Up to and including 3 mM extracellular calcium resulted in increased reporter activity. ¹⁵ This suggests that activation of the CaSR at higher calcium concentrations signals to the CYP27B1 promoter to increase gene transcription. This is in contrast to the in vivo experiments described in the above section whereby increased blood calcium levels supressed CYP27B1 expression. Interestingly, when the extracellular concentration of calcium was raised above 3 mM there was suppression of promoter activity. ¹⁵ This group further suggests that the CaSR signals through either a PKC or ERK_1/2 pathway to regulate CYP27B1 expression, since administration of inhibitors of these pathways simultaneously blocked the effect of calcium on CYP27B1 transcription, though administration of each inhibitor individually had little effect. ¹⁵ Further, whether CaSR alters CYP24A1 expression is not known. Thus, further work is required to determine the exact effects of renal CaSR activation on CYP27B1 and CYP24A1 expression and the signaling pathways involved.

Future directions

Regulation of 1-alpha and 24-hydroxylases and therefore circulating calcitriol levels is relatively unexplored beyond the actions of PTH and calcitriol. Thus, several questions remain to be answered. The signaling pathways that regulate CYP27B1 and CYP24A1 transcription need to be further delineated, particularly with respect to the direct effects of calcium and phosphate and the potential role of the CaSR in this regulation. In addition, given that the majority of the reabsorption of these minerals occurs in the proximal tubule, the same site of the majority of 1-alpha hydroxylase production, there may be a potential connection between the renal reabsorption of calcium and phosphate and the production of the enzymes that modulate calcitriol production. Further studies should investigate a potential link.

The curious results that phosphate depleted mice are unable to increase calcitriol production post-hypophysectomy requires confirmation and follow up. Further studies aimed to determine the roles of pituitary hormones on calcitriol metabolism should be completed, in particular to determine the potential role of growth hormone. Moreover, the mechanisms behind phosphate sensing and pituitary hormone effects on CYP27B1 and CYP24A1 expression remains to be determined.

Given the important role of 24-hydroxylase in regulating circulating calcitriol levels, the effect of phosphocalciotropic hormones on the expression of CYP24A1 and the mechanisms behind these actions also demands further attention.

Conclusions

This review highlights our knowledge of the regulation of renal calcitriol production. Due to its importance in calcium and phosphate regulation, it is critical to tightly control the expression of CYP27B1, which encodes the rate limiting enzyme in calcitriol production. PTH signals increased CYP27B1 expression and thus calcitriol production through cAMP and the transcription factors CREB and NR4A2. Calcitriol inhibits its own production and promotes its inactivation by decreasing CYP27B1 and increasing CYP24A1 expression, respectively. There are a number of less well-studied effectors of CYP27B1 expression and thus calcitriol levels. This includes repression by FGF23, and calcium itself possibly through CaSR signaling and stimulation by calcitonin. These less-studied pathways are key to fully understanding the regulation of calcitriol metabolism. This knowledge will be crucial in understanding vitamin D associated pathologies and help uncover new drug targets for their treatment.