Keywords

Introduction

Calcium , an essential ion for numerous physiological processes, is a major constituent of bone [1]. The intestine is the only source of new calcium. Absorption of dietary calcium, a process dependent on vitamin D, is essential for calcium homeostasis. The importance of vitamin D in this process is emphasized by the consequences of vitamin D deficiency which includes rickets in children and osteomalacia in adults [2,3,4]. Nearly a century ago, McCollum et al. identified vitamin D as the factor that cured rickets [5]. Solar or UVB irradiation is needed to convert 7-dehydrocholesterol in the skin to pre-vitamin D3 that ultimately is converted to vitamin D3 (cholecalciferol) by thermo-isomerization [3, 6]. Since the synthesis of vitamin D in the skin depends on the intensity of ultraviolet irradiation, geographical location and season play an important role in contributing to vitamin D sufficiency in man [3, 7]. Although cutaneous production of vitamin D remains an important source, the fortification of foods (including fortification of dairy products) largely contributed to the marked decrease in the incidence of vitamin D-dependent rickets in the Western world by the mid-twentieth century [5]. However, vitamin D deficiency in children and adults is still prevalent worldwide due, in part, to lack of exposure to sunlight and low vitamin D intake [3]. In this chapter the vitamin D endocrine system as well as the mechanisms by which 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) acts to maintain calcium homeostasis will be briefly reviewed followed by an emphasis on the intestinal actions of 1,25(OH)2D3 and effects of bone.

The Vitamin D Endocrine System : Bioactivation and Mechanism of Action

Vitamin D, which is taken in the diet or which is synthesized in the skin by UV irradiation, must be metabolized to its active form in order to regulate calcium homeostasis . Vitamin D is transported by vitamin D binding protein (DBP, which binds and transports vitamin D and its metabolites in the serum) to the liver where 25-hydroxyvitamin D3 (25(OH)D3), the major circulating form of vitamin D and an important biomarker for vitamin D status, is generated [6, 8, 9]. It has been suggested that the cytochrome P450 (CYP) enzyme CYP2R1 is the key enzyme involved in the conversion of vitamin D to 25(OH)D3 [10]. Patients with mutations in CYP2R1 are deficient in 25(OH)D3 and develop vitamin D-dependent rickets [11]. Studies in Cyp2r1 null mice which show that levels of 25(OH)D3 are diminished but not eliminated suggest the presence of additional vitamin D 25-hydroxylases is yet to be identified [12]. In the proximal renal tubule, CYP27B1 (25(OH)D3 1α hydroxylase) converts 25(OH)D3 to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D which is responsible for the biological actions of vitamin D [6, 8, 9]. Mutations in CYP27B1 cause vitamin D-dependent rickets type 1 (VDDR1), characterized by hypocalcemia, hypophosphatemia, decreased mineralization, and low circulating 1,25(OH)2D3 levels [13]. With regard to the regulation of vitamin D metabolism, parathyroid hormone (PTH) , whose synthesis is increased in response to hypocalcemia, induces CYP27B1 and is the major stimulator of 1,25(OH)2D3 production [8, 9]. 1,25(OH)2D3 and FGF23 (which promotes phosphate excretion) and its co-receptor α klotho negatively regulate CYP27B1. As an autoregulatory mechanism, 1,25(OH)2D3 induces CYP24A1 (25-hdyroxyvitamin D3 24-hydroxylase), the enzyme that accelerates the catabolism of 1,25(OH)2D3 preventing hypercalcemia resulting from high circulating 1,25(OH)2D3 (see ref. [9] for review of the regulation of vitamin D metabolism). Thus, vitamin D, FGF23/α klotho, and serum calcium and phosphate act together to regulate calcium homeostasis.

The actions of 1,25(OH)2D3, similar to other steroid hormones, are mediated by the vitamin D receptor (VDR) . 1,25(OH)2D3-occupied VDR heterodimerizes with the retinoid X receptor and together with chromatin active co-regulatory proteins interacts with vitamin D response elements in and around target genes resulting in the induction or suppression of gene expression [14].

Vitamin D and Intestinal Calcium Absorption

The principal function of 1,25(OH)2D3 in the maintenance of calcium homeostasis is to increase calcium absorption from the intestine. This conclusion was made from studies in VDR null mice which showed that rickets, osteomalacia, hypocalcemia, and hyperparathyroidism were prevented when VDR null mice were fed a rescue diet which included high calcium (2%), indicating that the skeletal abnormalities of VDR ablation are primarily the result of impaired intestinal calcium absorption (and resultant hyperparathyroidism and hypophosphatemia) [15, 16]. The abnormalities in the VDR null mice were reported to develop only after weaning [17] consistent with previous studies showing that intestinal VDR and the calcium binding protein calbindin-D9k are induced at weaning, the time of onset of active intestinal calcium absorption [18, 19]. Although there is an increase in PTH and in the number of osteoblasts, osteoclast number is not increased in VDR null mice, suggesting that 1,25(OH)2D3/VDR is needed for PTH-induced osteoclastogenesis [15]. Similar to VDR null mice, serum calcium and PTH were normalized in CYP27B1 null mice and in CYP27B1/VDR double null mice fed the high calcium rescue diet, confirming the importance of both 1,25(OH)2D3 and VDR in intestinal calcium absorption [20, 21]. In addition, in CYP27B1 null mice, in spite of markedly elevated PTH levels, osteoclast numbers were also not increased above levels in normal wild-type (WT) mice, indicating that both VDR and 1,25(OH)2D3 are necessary for PTH-mediated production of osteoclasts [21].

Direct evidence for the critical role of 1,25(OH)2D3-mediated intestinal calcium absorption in bone homeostasis was noted in studies in transgenic mice with VDR expression limited to the intestine [22]. Transgenic expression of VDR in the intestine of VDR null mice restored calcium homeostasis and prevented the rachitic phenotype of the VDR null mice [22]. Thus, intestinal VDR is essential for controlling bone formation. In addition, when VDR is deleted specifically from the intestine (Vdrint-), there is a decrease in intestinal calcium absorption, an inhibition of bone mineralization , and an increase in bone fractures in the Vdrint-mice [23]. In these mice serum calcium is normal indicating that in the absence of VDR-mediated intestinal calcium absorption normal serum calcium will be maintained at the expense of skeletal integrity.

When there is an increased need for calcium (under low dietary calcium conditions, during growth, pregnancy, or lactation), the synthesis of 1,25(OH)2D3 is increased and 1,25(OH)2D3 acts at the intestine to increase active calcium absorption [6, 8]. The major defect from the loss of VDR is decreased intestinal calcium absorption resulting in decreased bone mineralization [15, 16]. If normal serum calcium cannot be maintained by intestinal calcium absorption, then 1,25(OH)2D3 acts together with PTH to stimulate osteoclastogenesis resulting in the removal of calcium from the bone and to increase calcium reabsorption from the distal tubules of the kidney [6, 8].

Mechanisms Involved in 1,25(OH)2D3 Regulation of Intestinal Calcium Absorption

Intestinal calcium absorption occurs by an active, saturable, transcellular mechanism or by a nonsaturable passive process which occurs through tight junctions and structures within intercellular spaces and requires a high luminal calcium concentration (>2–6 mM) [24]. 1,25(OH)2D3 regulates the transcellular process by inducing TRPV6 (an apical membrane calcium channel), the calcium binding protein calbindin-D9k , and the basolateral membrane calcium ATPase (PMCA1b) [8, 25, 26] (Fig. 1). Although it has been suggested that calbindin-D9k mediates intracellular calcium diffusion, other studies suggest that a principal function of calbindin-D9k is to buffer calcium preventing toxic levels from accumulating in the cell [27, 28]. When calcium is low, the 1,25(OH)2D3-mediated transcellular calcium transport process is the predominant mechanism of calcium absorption [24]. Although TRPV6 and calbindin-D9k are induced by 1,25(OH)2D3, TRPV6 or calbindin-D9k null mice have normal serum calcium and show no change in active intestinal calcium absorption compared to WT mice [29,30,31]. However, studies in TRPV6/calbindin-D9k double null mice under conditions of low dietary calcium have shown that intestinal calcium absorption is least efficient in the absence of both proteins (compared to single null mice and WT mice), suggesting that TRPV6 and calbindin can act together in certain aspects of the absorptive process [29]. Findings in the single null mice suggest that in the absence of calbindin or TRPV6, there is compensation by other channels or proteins yet to be identified. Although other apical membrane calcium transporters may compensate for the loss of TRPV6, intestine-specific transgenic expression of TRPV6 has been shown to result in a marked increase in intestinal calcium absorption and bone density in VDR null mice, indicating a direct role for TRPV6 in the calcium absorptive process and that a primary defect in the VDR null mouse is low apical membrane calcium uptake [28].

Fig. 1
figure 1

Model of vitamin D-mediated intestinal calcium absorption . The transcellular pathway consists of influx through the apical calcium channel TRPV6, diffusion through the cytosol, and active extrusion at the basolateral membrane by the plasma membrane calcium ATPase (PMCA1b). Studies using TRPV6 and calbindin-D9k (CaBP9k) null mice indicate that our understanding of the vitamin D-mediated calcium transport process remains incomplete. In the absence of TRPV6 or calbindin-D9k 1,25(OH)2D3-mediated active transport still occurs, suggesting compensation by another channel or protein. Intracellular calcium transfer may involve calcium bound to calbindin as well as other calcium binding proteins. Calbindin as well as other calcium binding proteins may also act to prevent toxic levels of calcium from accumulating in the cell. It has been suggested that 1,25(OH)2D3 can regulate the paracellular pathway by regulating tight junction proteins

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The duodenum has been a focus of research related to 1,25(OH)2D3 regulation of calcium absorption. However, it is the distal intestine where most of the ingested calcium is absorbed [24]. VDR, TRPV6 , and calbindin-D9k are expressed in all segments of the intestine and 1,25(OH)2D3-regulated active calcium absorption occurs in the ileum, cecum, and colon [22, 32,33,34,35,36]. Recent studies have shown that transgenic expression of VDR specifically in the ileum, cecum, and colon can prevent abnormal calcium homeostasis and rickets in VDR null mice [37]. In addition, when VDR is deleted specifically from the distal region of the intestine, altered calcium metabolism is observed [38]. These findings indicate that the distal as well as the proximal segments of the intestine are important in vitamin D-mediated calcium homoeostasis and bone mineralization . Future studies related to mechanisms involved in 1,25(OH)2D3-mediated regulation of calcium absorption in the distal intestine may suggest new strategies to increase the efficiency of calcium absorption in individuals at risk for bone loss including those with reduced calcium absorption due to small bowel resection or following menopause.

In addition to regulation by vitamin D of the active transcellular process, early studies suggested that the passive, nonsaturable process of intestinal calcium absorption can also be enhanced by vitamin D [39]. More recent studies have shown that 1,25(OH)2D3 can regulate intestinal proteins that are involved in tight junctions or cell adhesion. Claudin-2 and claudin-12 are induced and cadherin-17 is inhibited by 1,25(OH)2D3 [40, 41]. These findings suggest that 1,25(OH)2D3, by regulating these proteins , can facilitate calcium absorption through the paracellular path (Fig. 1). The identification of multiple mechanisms and novel vitamin D targets involved in 1,25(OH)2D3-mediated calcium absorption in different segments of the intestine is needed in order to identify new approaches to maximize calcium absorption and minimize bone loss.

Vitamin D : Direct Effects on Bone

Although the primary role of the vitamin D endocrine system on bone is indirect (providing calcium to bone by stimulating intestinal calcium absorption), direct effects of 1,25(OH)2D3 on bone cells have also been demonstrated (see [42] for review). As indicated above in studies in VDR and CYP27B1 null mice, VDR and 1,25(OH)2D3 are needed for PTH-mediated osteoclastogenesis [15, 21]. Osteoclastogenesis mediated by 1,25(OH)2D3 as well as by PTH involves upregulation of receptor activator of nuclear kB ligand (RANKL) in osteoblastic cells and requires cell to cell contact between osteoblasts and osteoclast precursors [43]. In addition 1,25(OH)2D3 can also stimulate the production in osteoblasts of the calcium binding proteins osteocalcin and osteopontin (OPN) [8]. OPN has been reported to inhibit bone matrix mineralization [44]. Thus, during a negative calcium balance, 1,25(OH)2D3 action can promote increased bone resorption and reduced bone matrix mineralization in order to maintain normal serum calcium levels [23]. 1,25(OH)2D3 has also been shown to induce LRP5 (low density lipoprotein receptor-related 5) which facilitates β catenin activation and exerts an anabolic effect on bone formation [45]. These findings indicate that the effects of 1,25(OH)2D3 on osteogenic cells are complex and can result in either bone resorption or formation.

Vitamin D: The Kidney and Calcium Homeostasis

Vitamin D-mediated calcium homeostasis is regulated by an integrated system involving not only the intestine and bone but also the kidney. Although most of the filtered calcium is reabsorbed by a passive, paracellular path in the proximal renal tubule that is independent of 1,25(OH)2D3, 10–15% of the filtered calcium is reabsorbed in the distal convoluted tubule and connecting tubule and is regulated by PTH and 1,25(OH)2D3 [46]. Similar to studies in the intestine, 1,25(OH)2D3 regulates an active, transcellular process in the distal portion of the nephron by inducing the apical calcium channel TRPV5 (which shares 75% sequence homology with TRPV6) and by inducing the calbindins [both calbindin-D9k (9,000 Mr) and calbindin-D28k (28,000 Mr) are present in the mouse kidney and only calbindin-D28k is present in rat and human kidney] [8]. Calcium is extruded via PMCA1b and the Na+Ca++ exchanger [8]. It has been shown that calbindin-D28k binds to TRPV5 and modulates calcium influx [47]. PTH has been reported to activate TRPV5 via protein kinase A phosphorylation [48]. The kidney is also the major site of production of 1,25(OH)2D3 and its regulation [9]. Thus, the kidney, by regulating transport processes and as a major site of synthesis of 1,25(OH)2D3, plays an essential role in the maintenance of calcium homeostasis.

Vitamin D: The Intestine and Bone Health

In aging intestinal calcium absorption declines which results in secondary hyperparathyroidism and increased fracture risk [49]. Decreased intestinal calcium absorption with age has been shown to correlate with decreased expression of TRPV6 and calbindin-D9k , the two major targets of 1,25(OH)2D3 in the intestine [50, 51] (Fig. 2). Increasing evidence indicates that the reason for disturbed calcium balance with age is that vitamin D status is often inadequate in the elderly [52]. With age there is a decline in the ability of the kidney to synthesize 1,25(OH)2D3 and an increase in CYP24A1 which would result in enhanced catabolism of 1,25(OH)2D3 [53,54,55] (Fig. 2). Intestinal resistance to 1,25(OH)2D3 with age has also been reported [56,57,58]. It has been suggested that this resistance is due to a decrease in the content of intestinal VDR with age [57]. However, this has been a matter of debate [58]. It is possible that the resistance to 1,25(OH)2D3 may also be due to altered recruitment by 1,25(OH)2D3 of VDR and VDR coactivators to intestinal vitamin D target genes and/or to epigenetic changes. To reduce fracture risk, a combination of calcium and vitamin D supplementation has been recommended [59]. The current recommended daily doses for vitamin D-sufficient individuals are 800 IU calcium and 1,000 mg calcium [60].

Fig. 2
figure 2

Age-related effects on the vitamin D endocrine system

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In addition to effects of aging, gastric bypass surger y has also been reported to result in calcium malabsorption and decreased bone mineral density in patients [61,62,63]. Animal studies have also noted gastric bypass-associated bone resorption which is due to several factors including vitamin D and calcium malabsorption and acid/base dysregulation [64, 65]. Patients with inflammatory bowel disease are also at risk for bone disease due in part to impaired intestinal calcium absorption as well as to proinflammatory cytokines which are involved in the intestinal immune response but can also enhance bone resorption [66]. Future studies related to mechanisms involved in VDR-mediated activation of intestinal calcium absorption may suggest new mechanisms to compensate for calcium malabsorption in order to minimize bone loss due to aging, bariatric surgery, or inflammatory bowel disease.