J Bone Miner Metab (2014) 32:1–9 DOI 10.1007/s00774-013-0508-z


Role of local vitamin D signaling and cellular calcium transport system in bone homeostasis Ritsuko Masuyama

Received: 28 June 2013 / Accepted: 7 August 2013 / Published online: 9 November 2013 Ó The Japanese Society for Bone and Mineral Research and Springer Japan 2013

Abstract Mouse genetic studies have demonstrated that the 1,25-dihydroxyvitamin D [1,25(OH)2D] endocrine system is required for calcium (Ca2?) and bone homeostasis. These studies reported severe hypocalcemia and impaired bone mineralization associated with rickets in mutant mice. Specific phenotypes of these mice with an engineered deletion of 1,25(OH)2D cell signaling resemble the features observed in humans with the same congenital disease or severe 1,25(OH)2D deficiency. Decreased active intestinal Ca2? absorption because of reduced expression of epithelial Ca2? channels is a crucial mechanism that contributes to the major phenotypes observed in the mutant mice. The importance of intestinal Ca2? absorption supported by 1,25(OH)2D-mediated transport was further emphasized by the observation that Ca2? supplementation rescues hypocalcemia and restores bone mineralization in both patients and mice lacking 1,25(OH)2D signaling. This observation questions the direct role of 1,25(OH)2D signaling in bone tissue. Studies regarding tissue-specific manipulation of 1,25(OH)2D function have provided a consensus on this issue by demonstrating a direct action of 1,25(OH)2D on cells in bone tissue through bone metabolism and mineral homeostasis. In addition, movement of Ca2? from the bone as a result of osteoclastic bone resorption also provides a large Ca2? supply in Ca2? homeostasis; however, the system controlling Ca2? homeostasis in osteoclasts has not been fully identified. Transient receptor potential vanilloid (TRPV) 4 mediates Ca2? influx during the late stage of osteoclast differentiation, thereby regulating the Ca2?

R. Masuyama (&) Department of Molecular Bone Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan e-mail: [email protected]

signaling essential for cellular events during osteoclast differentiation; however, the system-modifying effect of TRPV4 activity should be determined. Furthermore, it remains unknown how local Ca2? metabolism participates in systemic Ca2? homeostasis through bone remodeling. New insights are therefore required to understand this issue. Keywords Vitamin D receptor  Calcium absorption  Osteoclasts  Transient receptor potential vanilloid  TRPV

Introduction Although rickets was discovered in the 17th century, the critical factor needed to prevent this disease was not determined until the beginning of the 20th century when the action of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active form of vitamin D, was identified. In consequence of this discovery, 1,25(OH)2D deficiency-dependent calcium (Ca2?) malabsorption was identified as the physiological basis of rickets. Genetic manipulations of the vitamin D receptor (VDR) [1–4] or the 25-hydroxyvitamin D3 1alphahydroxylase cyp27b1 [5, 6] in mice provided concrete information on the molecular basis of the pathophysiology of 1,25(OH)2D deficiency; this led to recognition of the important biological function of 1,25(OH)2D in Ca2? homeostasis. Regulation of serum Ca2? levels is critical for physical function and depends on the coordinated handling of Ca2? in the target tissues of 1,25(OH)2D [7, 8]. In addition, the system which maintains intracellular Ca2? levels is essential for extensive biological reactions in every cell, that supported by inducing Ca2? entry from the extracellular fluid. In the past decade, the role of Ca2? signaling in bone cells and its contribution to normal bone remodeling has



been extensively investigated [9–12]. This review highlights the direct action of 1,25(OH)2D on local tissue in the regulation of bone and mineral metabolism, and the role of Ca2? transport in sustaining two distinct regulatory systems— systemic mineral homeostasis and cellular function. Role of systemic and cellular Ca21 homeostasis in bone metabolism Locomotive function is a significant factor limiting the quality of life in an aging society. Hence, there is considerable urgency to understand the molecular pathology of locomotive disease and develop therapeutic strategies for these patients. Bone homeostasis is maintained by the balance between the activities of osteoblasts and osteoclasts that perform bone formation and resorption, respectively. Disharmony in bone homeostasis leads to bone loss and is often caused by increased osteoclast activity. On the other hand, since bone has an important role as a large biological store of Ca2? in the body, increased osteoclast activity transfers Ca2? from bone to blood when blood Ca2? levels are decreased [13]. Bone resorption by osteoclasts is a major process for destroying bone tissue, although alternatively, this system is indispensable for maintaining calcium homeostasis. Furthermore, Ca2? channels and transporters located in the intestinal epithelium and renal tubular cells constantly regulate extracellular Ca2? concentrations. Accordingly, the functions of the Ca2? transport systems involving 1,25(OH)2D signaling should be elucidated for both local tissues and cells. Signaling mediated by intracellular Ca2? is a crucial system for initiating physiological reactions in every cell and also supports major cellular events during the life of osteoclasts. Differentiation of osteoclasts is closely associated with Ca2? signaling activities. In the first step of osteoclast differentiation, Ca2? oscillations originating from alternative Ca2? release and reloading into intracellular stores, induces cellular Ca2? signaling [12]. This oscillatory effect is gradually lost during differentiation, and at the later stages in mature osteoclasts Ca2? influx from the extracellular space through Ca2? channels becomes necessary to sustain Ca2? signaling. As expected, many of these Ca2? channels are localized in the membrane of osteoclasts, with increased extracellular Ca2? often inducing osteoclast retraction and even apoptosis [14]. For these reasons, the Ca2? permeable channels involved in the control of Ca2? signaling during osteoclast differentiation remain elusive. Calcium homeostasis maintained by 1,25(OH)2D Studies regarding Ca2? homeostasis and bone phenotype in mice with genomic ablation of VDR have identified the


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Ca2? transporters that respond to 1,25(OH)2D signaling as well as the role of intestinal Ca2? absorption on bone metabolism [3]. 1,25(OH)2D-induced genomic actions are crucial for normal bone metabolism, primarily by regulating active Ca2? transport in the intestinal epithelium [15]. Disorders of the 1,25(OH)2D endocrine system due to inactivation of VDR [1–3] or cyp27b1 [5, 6] result in profound disturbances in mineral homeostasis and bone mineralization. Decreased active intestinal Ca2? absorption due to reduced expression of epithelial Ca2? channels is a crucial mechanism that is involved in the phenotype [3, 16]. The importance of Ca2? absorption was further supported by the fact that Ca2? supplementation rescued hypocalcemia and hyperparathyroidism and restored bone mineralization in both patients [17, 18] and mice [19–21] with a loss of VDR function. These findings raised questions regarding the role and necessity of VDR in bone metabolism. However, these data do not exclude the possibility that VDR might have a specific although not an essential role in bone metabolism. To elucidate the genomic action of 1,25(OH)2D in intestinal epithelial cells, mice lacking VDR specifically in the intestine, were generated and compared with particular phenotypes of global VDR null mice [22]. Loss of intestinal 1,25(OH)2D signaling reduced both Ca2? absorption and expression of the intestinal Ca2? transport proteins that was strongly induced in epithelial cells by a 1,25(OH)2DVDR-dependent action (Fig. 1a). Although intestinal Ca2? absorption was severely decreased (Fig. 1b), serum Ca2? levels remained normal in mice lacking VDR in the intestine. This result was in contrast to the hypocalcemia observed in global VDR null mice [3], while Ca2? absorption was similarly reduced in both mice. Remarkably, osteoclast bone resorption was increased in mice lacking intestinal VDR (Fig. 2), to redistribute Ca2? into the blood to preserve normocalcemia as a compensatory mechanism. The induction of osteoclast bone resorption in these mice was due to the increased expression of receptor activator of nuclear factor kappa-B ligand (RANKL) caused by high levels of parathyroid hormone (PTH) and 1,25(OH)2D after intestinal Ca2? absorption was reduced. The decrease in 1,25(OH)2D-dependent intestinal Ca2? absorption led to subsequent stimulation of PTH secretion.

Impaired osteoblast and osteoclast function in global VDR null mice In mice with a systemic lack of 1,25(OH)2D signaling, osteoblasts are abundantly observed in bone sections [1–6]. The bone area in these mice was reported to have increased compared with that in wild-type (WT) mice and was associated with an expanded osteoid caused by impaired

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Fig. 1 Lack of VDR signaling in the intestine decreases calcium absorption. a Duodenum TRPV6 immunostaining (green) of wildtype (WT) and intestinal VDR null (knockout; KO) mice. Nuclei were

stained with TOPRO-3 (red). b Apparent calcium absorption was measured for 4 weeks (n = 8, means ? SEMs). *P \ 0.01 vs. WT

bone mineralization. A possible explanation for these changes is that elevated PTH levels because of severe hyperparathyroidism in the mutant mice might have caused anabolic changes in osteogenesis. These results are supported by the finding that expression of runt-related transcription factor 2 (Runx2) [23] was also upregulated. This factor is essential for both osteoblast differentiation and the anabolic effects of PTH on bone [24]. To date, although the interaction between Runx2, PTH, and 1,25(OH)2D has been studied extensively, a complete understanding of the system has not been achieved, particularly regarding transcriptional regulation [25]. The results of osteoblast pathology in mutant mice suggest that hypocalcemia is the primary cause of hyperparathyroidism-induced abnormal osteoblastogenesis, except for direct regulation by 1,25(OH)2D. More significantly, both PTH and 1,25(OH)2D signaling have profound effects on osteoclastogenesis by promoting RANKL expression. Formation of intact osteoclasts was confirmed in co-cultures of osteoblasts and osteoclast progenitors derived from WT and global VDR null mice with different genetic combinations [26]. Treatment with 1,25(OH)2D induces osteoclast formation as a result of VDR-mediated actions in osteoblasts, whereas bone resorption in osteoclasts occurs without 1,25(OH)2D when PTH is present; therefore, mutant mice with hyperparathyroidism would be expected to have an increased number of osteoclasts in the bone. In fact, PTH levels were reported to be increased in mice without 1,25(OH)2D signaling, whereas the number of osteoclasts was not manifestly increased compared with those in WT mice with normal calcium homeostasis [27–29]. In global VDR null mice, the absence of precise induction of osteoclastogenesis with severe hyperparathyroidism might be explained by a blunted skeletal response to PTH, a phenomenon frequently observed following prolonged hyperparathyroidism [30]. Although the pathogenesis of this desensitization remains unknown, it is most likely attributable to down-regulation of the PTH receptor [30]. Another possibility for the reduced response of

osteoclastogenesis to PTH might be the absence of 1,25(OH)2D signaling. The mechanisms for transcriptional regulation of RANKL have been well demonstrated, with several distinct regions that respond strongly to identification of gene expression. A distal enhancer region located beyond the 70-kb upstream of the murine RANKL transcription start site contains a functional cAMP-binding domain that mediates the PTH [31] and 1,25(OH)2D [32] responsive elements, respectively. Deletion of this region diminishes the response of PTH and 1,25(OH)2D to induce RANKL mRNA expression and osteoclastogenesis in bone. This indicates that the responsiveness of the region involves cross-talk between PTH and 1,25(OH)2D [33]. As a consequence, 1,25(OH)2D signaling appears necessary for maximal PTH-induced osteoclast production. These findings provide an explanation for the decreased response to PTH observed in the absence of 1,25(OH)2D signaling in bone. Although osteoblasts are abundantly present in bone, impaired mineralization coupled with hyperosteoidosis are evident in mice lacking 1,25(OH)2D signaling. In addition, osteoclast numbers in these mice were inappropriately low, resulting in uncoupling of bone turnover. Therefore, an imbalance between osteoblasts and osteoclasts cannot preserve bone remodeling systems without the local functions of 1,25(OH)2D.

Local function of 1,25(OH)2D signaling on bone and mineral homeostasis Unlike global VDR null mice, 1,25(OH)2D signaling in bone of intestinal VDR null mice functions normally, although these mice also have high serum PTH levels [22]. As a result, renal 25-hydroxylation was stimulated by PTH leading to an increase in serum 1,25(OH)2D levels. The abundance of osteoclasts was caused by upregulation of RANKL levels in bone of intestinal VDR null mice. Since 1,25(OH)2D signaling in osteoblasts is active in these mice, the action of both PTH and 1,25(OH)2D strongly support osteoclast genesis. However, it remains to be clarified



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Fig. 2 Intestinal VDR inactivation decreases bone volume in mice because of increased osteoclastic bone resorption. a H&E staining images of WT and intestinal VDR null (knockout; KO) tibia. b TRAP staining images of osteoclasts located on the proximal tibia. c Trabecular image of proximal tibia reconstructed by microcomputed tomography (microCT) detection. d, e Quantification of trabecular bone mineral content (BMC) (d) and cortical bone mineral density (BMD) (e) analyzed by micro-CT (n = 8, means ? SEMs). *P \ 0.01 vs. WT

whether or not the reduction in bone volume associated with calcium malabsorption or intestinal 1,25(OH)2D inactivation is solely attributable to the increased bone resorption by osteoclasts. As growth plate phenotypes appear before the onset of hypocalcemia in VDR null mice [2], a direct action of 1,25(OH)2D signaling in bone tissue has been proposed. This possibility was investigated by tissue-specific ablation or by enhancing 1,25(OH)2D signaling. Genetic ablation of


VDR in osteoblasts using the collagen type 1 gene promoter, which is expressed during the early stage of osteoblast differentiation, was reported to increase bone mass and bone mineral density [34]. These results demonstrate that 1,25(OH)2D signaling inhibits osteoblast function. In contrast, VDR transgene expression in mature osteoblasts using the osteocalcin gene promoter also increases bone volume, suggesting an anabolic action of 1,25(OH)2D signaling in bone [35, 36].

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of 1,25(OH)2D signaling in renal tubular cells along with the functional interaction between the intestine, bone, and kidney.

Role of Ca21 channels and cellular homeostasis on bone metabolism

Fig. 3 Function of 1,25(OH)2D signaling in calcium homeostasis. While PTH and 1,25(OH)2D increase RANKL expression and release of Ca2? by promoting osteoclast bone resorption, inhibition of bone mineralization is controlled by 1,25(OH)2D. These are beneficial processes for maintaining calcium homeostasis

Analysis of intestinal and mature osteoblasts/osteocytes in VDR null mice established a novel regulatory mechanism for local calcium metabolism—1,25(OH)2D-dependent inhibition of bone mineralization [22]. Bone matrix mineralization was reported to be suppressed by a mineralization inhibitor induced by 1,25(OH)2D signaling [22, 37]. The increase in 1,25(OH)2D in intestinal VDR null mice resulted in extensive hypersteroidosis with hypomineralization. Furthermore, although injections of 1,25(OH)2D maintained high serum levels, ablation of VDR signaling in osteoblasts and osteocytes precluded this inhibitory system for local mineralization and preserved bone mass. Taken together, these findings suggest that 1,25(OH)2D signaling stimulates Ca2? release by promoting osteoclast bone resorption, and inhibition of bone matrix mineralization, which are beneficial processes for maintaining normocalcemia (Fig. 3). Although there are major differences in the Ca2? homeostasis phenotype between intestinal VDR null mice and global null mice, serum Ca2? levels are maintained within the normal range in intestinal VDR null mice, indicating that the physiological function of 1,25(OH)2D on Ca2? homeostasis contributes to increased serum Ca2? levels. In order to solve the other parts of the puzzle on ‘Ca2? homeostasis’, it is also necessary to consider the contribution of renal Ca2? handling. Future research using conditional VDR null mice should elucidate the direct role

Compared to systemic Ca2? homeostasis largely contributing to bone tissue development, the intracellular Ca2? concentrations ([Ca2?]i) in bone cells and the external Ca2? balance are crucial factors for normal bone homeostasis and are well regulated by Ca2? entry from the extracellular fluid into cells. Ca2? influx subsequently leads to Ca2? signaling that is necessary for a wide range of cellular processes [38], and the importance of [Ca2?]i regulation for bone cell function is most prominent in osteoclasts. RANKL signaling evokes Ca2? oscillations that result in Ca2?/calcineurin-dependent dephosphorylation and activation of nuclear factor activated T cells (NFAT)c1 [9]. Thereafter, NFATc1 translocate to the nucleus and induce osteoclast-specific gene transcription, which promotes osteoclast differentiation [9, 12]. Ca2? entry involves plasma membrane-localized Ca2?permeable channels such as the transient receptor potentials (TRPs) [38]. To explore the importance of Ca2? entry, systemic phenotypes were investigated in mice with genetic ablations of TRP vanilloid 4 (TRPV4), a widely expressed Ca2?-permeable cation channel that is activated by diverse skeletal tissue familiar physical and chemical stimuli, including cell swelling, heat, mechanical stress, and phorbol esters such as 4a-PDD [39–42]. These items of the TRPV4 activation process are closely involved in the working environment of the skeletal tissue. The pathologies of patients with genetic mutations in TRPV4 have recently been reported [43–48], and certain features of their skeletal muscles were identified [43, 44]. Thus, physiological targets of this channel are being sought. Mice lacking TRPV4 exhibited increased bone mass resulting from impaired terminal differentiation of osteoclasts but no changes in serum levels of Ca2? and calcitropic hormones such as PTH and 1,25(OH)2D [49]. These findings suggest a novel role of TRPV4 in bone remodeling. Impaired osteoclast differentiation has been reported to result in increased bone mass in TRPV4 null mice (Fig. 4); this raises a concern to understand how TRPV4 changes cell function during osteoclast differentiation. In early osteoclasts, Ca2? oscillation evoked by Ca2? efflux from the endoplasmic reticulum is a crucial event for supporting cell function. Mechanistically, the basolaterally localized TRPV4 channel mediates Ca2? influx during osteoclast maturation, at a stage when Ca2? oscillations disappear. Thus, TRPV4 maintains basal [Ca2?]i which regulates



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Fig. 4 TRPV4 inactivation increases bone mass in mice due to decreased osteoclast bone resorption. a Von Kossa stained tibia section of WT and TRPV4 knockout (KO) mice. b Quantification of trabecular bone mineral content (BMC) by micro-CT analysis (n = 8, means ? SEMs). c Serum CTX level in WT and TRPV4 KO mice (n = 8, means ? SEMs). *P \ 0.05 vs. WT

Fig. 5 Maintenance of intracellular Ca2? concentration and Ca2? signaling during osteoclast differentiation. At the start, Ca2? oscillations regulate cellular Ca level. When osteoclasts become larger Ca2? oscillations diminish and TRPV4-mediated Ca2? influx becomes operational. This process is required to sustain cellular Ca2? level and NFATc1 activation during terminal differentiation of osteoclasts

NFATc1 activity and induces differentiation and resorptive activity of osteoclasts (Fig. 5). Osteoclasts exhibit characteristic morphological changes throughout the differentiation process initiated by migration, including an enlarged size accompanied by multinucleation after fusion. Changes in the Ca2? supply system are associated with morphological changes in osteoclasts. Initially, Ca2? oscillations are present persistently and have a major effect on [Ca2?]i regulation [50].


The Ca2? oscillations subsequently diminish, and TRPV4mediated Ca2? influx becomes operational after the differentiated osteoclasts become larger. These results indicate that TRPV4 activity is increased during osteoclast differentiation and suggest a system in which TRPV4 activation regulates cell differentiation. Furthermore, it has been suggested that calmodulin (CaM) might be a possible regulator, as this action has been demonstrated in combination with some TRP family members [51–53]. CaM is a ubiquitous intracellular Ca2? sensor that forms complexes with Ca2? and activates various protein kinase-mediated Ca2? signaling activities [54]. In osteoclasts, the dynamic upregulation of CaM protein levels in mature osteoclasts supports the Ca2?/CaMdependent signaling. In other words, the role of CaM becomes even more important during terminal differentiation of osteoclasts. Ca2?/CaM frequently exert negative feedback regulation that prevents excessive Ca2? influx into cells and defines the time course of channel activity. In fact, the functions of TRP cation channels are inhibited by the Ca2?/CaM complex, leading to [Ca2?]i normalization [51, 52]. CaM protein is abundantly present in osteoclasts and the levels are increasing by cells differentiation [55]. The study of osteoclasts lacking a CaM-binding domain in TRPV4 demonstrated that TRPV4 activation was abrogated by the loss of interaction between the Ca2?/CaM complex and TRPV4 [56]. This finding emphasized the necessity of this domain for channel activity. Furthermore, Ca2?/CaM initiate cellular phosphorylation networks,

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mice and global VDR null mice, this raises several unanswered questions; for example, how is the release of PTH stimulated despite normal serum calcium in intestine VDR null mice, and what are the in vivo regulators of TRPV (particularly TRPV5-6) localization and activation in intestinal epithelial cells? Thus, the system modifying Ca2? channel activation is a potential therapeutic target for improving Ca2? and bone homeostasis in individuals suffering from Ca2? malnutrition and metabolic disorder by insufficient Ca2? absorption. Conflict of interest

Author has no conflict of interest.

References Fig. 6 TRPV4-mediated Ca2?/CaM signaling and the system of TRPV4 activation by CaM signaling during osteoclast differentiation. TRPV4-mediated Ca2? influx supports Ca2?/CaM signaling in osteoclasts. Once CaM is activated, cell signaling altered by the Ca2?/CaM complex accesses the CaM-binding domain (CaMBD) of TRPV4 and supports its activation. MYH9, a heavy chain subunit of non-muscle myosin isoforms IIa (MYOII), is a potential mediator that is affected by myosin light chain kinase (MLCK) phosphorylation of myosin isoforms and binds to the CaMBD of TRPV4

resulting in a transduced signal that accesses the CaMbinding domain of TRPV4, thereby allowing further Ca2? entry into the cell (Fig. 6). These results suggest a reciprocal activation between regulation of Ca2? entry through TRPV4 and generation of CaM signaling in mature osteoclasts exhibiting sufficient bone resorption activity that is required for normal bone remodeling. These results only demonstrate a portion of the cellular signal activated by the CaM-binding domain of TRPV4. However, several calcium channels that regulate calcium entry are located in osteoclasts. Cell signaling mediated by Ca2?/CaM is therefore considered to be a therapeutic target for controlling the activation of calcium channels and maintaining local tissue function including bone. Because genetic TRPV4 activation in osteoclasts decreased mouse bone mass, elucidating the regulatory mechanisms of TRPV4 may provide an effective approach for maintaining bone mass concomitant with normal bone remodeling. Blocking the accessing signals to the CaM-binding domain of TRPV4 might be another option to modify TRPV4 activity. Ca2? homeostasis is well maintained in the normal range by local Ca2? metabolism, which in turn, is coordinated by the function of cellular Ca2? channels located in the membrane, intracellular transporters, and the Ca2? pump. Studies regarding vitamin D-dependent Ca2? transport have identified the factors involved in local calcium metabolism. However, as vitamin D-independent transcellular Ca2? transport exists in conditional VDR null

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Role of local vitamin D signaling and cellular calcium transport system in bone homeostasis.

Mouse genetic studies have demonstrated that the 1,25-dihydroxyvitamin D [1,25(OH)2D] endocrine system is required for calcium (Ca(2+)) and bone homeo...
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