Vitamin D metabolism, cartilage and bone fracture repair

Molecular and Cellular Endocrinology 347 (2011) 48–54

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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Review

Vitamin D metabolism, cartilage and bone fracture repair René St-Arnaud a,b,⇑, Roy Pascal Naja a,b a b

Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada H3G 1A6 Department of Human Genetics, McGill University, Montreal, Quebec, Canada H3A 2T5

a r t i c l e

i n f o

Article history: Received 16 February 2011 Received in revised form 26 April 2011 Accepted 2 May 2011 Available online 1 June 2011 Keywords: Chondrocytes Cyp27b1 1,25-Dihydroxyvitamin D Vitamin D receptor Cyp24a1 24,25-Dihydroxyvitamin D

a b s t r a c t The 1,25-(OH)2D metabolite mediates the endocrine actions of vitamin D by regulating in the small intestine the expression of target genes that play a critical role in intestinal calcium absorption. The major role of the vitamin D hormone on bone is indirect and mediated through its endocrine function on mineral homeostasis. However, genetic manipulation of the expression of Cyp27b1 or the VDR in chondrocytes strongly support a direct role for locally synthesized 1,25(OH)2D, acting through the VDR, in vascular invasion and osteoclastogenesis during endochondral bone development. Cells from the growth plate respond to the 24,25-(OH)2D and 1,25-(OH)2D metabolites in a cell maturation-dependent manner and the effects of 1,25-(OH)2D are thought to be mediated through binding to the membrane-associated receptor PDIA3 (protein disulfide isomerase associated 3). The physiological relevance of membranemediated 1,25-(OH)2D signaling is emerging and is discussed. Finally, preliminary results suggest that mice deficient for Cyp24a1 exhibit a delay in bone fracture healing and support a role for 24,25(OH)2D in mammalian fracture repair. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

Vitamin D metabolism and endocrine function . . . . . . . . 1,25-(OH)2D actions in bone . . . . . . . . . . . . . . . . . . . . . . . Direct effects of vitamin D metabolites in chondrocytes. 24,25(OH)2D and fracture repair . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Vitamin D metabolism and endocrine function Vitamin D is produced upon sunlight irradiation of skin or is ingested from the diet. The D compound is inactive and must be hydroxylated twice before it can exert biological activity (DeLuca, 2004). Vitamin D is first hydroxylated on carbon 25 in the liver. This produces 25-hydroxyvitamin D [25-(OH)D] (Cheng et al., 2003; Ohyama and Yamasaki, 2004). This inactive metabolite circulates in the bloodstream to reach the proximal kidney tubule where it is hydroxylated on carbon 1 by CYP27B1 to yield 1a,25-dihydroxyvitamin D [1,25-(OH)2D], the active, hormonal form of vitamin D (DeLuca, 2004; Ohyama and Yamasaki, 2004). Upon reaching a ⇑ Corresponding author. Address: Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montreal, Quebec, Canada H3G 1A6. Tel.: +1 514 282 7155; fax: +1 514 842 5581. E-mail address: [email protected] (R. St-Arnaud). 0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.05.018

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target tissue, 1,25-(OH)2D binds its receptor (vitamin D receptor or VDR) that acts to regulate the transcription of vitamin D target genes responsible for carrying out the physiological actions of 1,25-(OH)2D (Haussler et al., 1998; Norman, 2006). The VDR is a member of the superfamily of nuclear hormone receptors. This ligand-activated transcription factor belongs to a subfamily that also includes the triiodothyronine, retinoid-X and retinoic acid receptors. The VDR has been shown to mediate the effects of the active form of vitamin D by binding 1,25-(OH)2D, heterodimerizing with the retinoid-X receptor (RXR) and interacting with DNA sequences on target genes, thereby regulating their transcription. The nuclear VDR exists as a single gene, unlike other members of its nuclear hormone receptor subfamily, which each have more than one characterized isoform (a, b and c). The principal domains of the VDR protein are those involved in DNA binding, hormone binding, dimerization and transcriptional activation. Unlike most nuclear hormone receptors that contain a substantial amino termi-

R. St-Arnaud, R.P. Naja / Molecular and Cellular Endocrinology 347 (2011) 48–54

nal transactivation domain (Activation Function 1 or AF1), the VDR only sports a very short region N-terminal to the two zinc fingers that comprise the DNA binding domain. Amino acids motifs within the DNA binding domain (residues 24–90) are responsible for DNA binding as well as for nuclear localization and also have been shown to contribute to heterodimerization. The region of the VDR carboxy terminal to the DNA binding domain contains residues involved in hormone binding, heterodimerization, transactivation and interactions with nuclear receptor coactivators (reviewed in Haussler et al. (1998)). 1,25-(OH)2D plays a critical role in intestinal calcium absorption. Vitamin D deficient animals develop hypocalcemia, hypophosphatemia, hyperparathyroidism, rickets and osteomalacia (Halloran and DeLuca, 1981). To maintain serum calcium at physiological levels, the 1,25-(OH)2D-liganded VDR induces in the small intestine the expression of the TRPV6 epithelial calcium channel (Song et al., 2003; Van Cromphaut et al., 2001). In parallel, 1,25-(OH)2D induces transcription of genes whose products facilitate the movement of calcium through the enterocyte cytoplasm and transfer the calcium across the basolateral membrane into the circulation, such as calbindin D9k and the basolateral membrane pump, PMCA1b (Christakos et al., 2003; Raval-Pandya et al., 1998; Wasserman et al., 1992). Reduction in circulating phosphate levels stimulate 1,25-(OH)2D synthesis which in turn enhances the absorption of dietary phosphorus (Portale et al., 1989). For a more in-depth review of the molecular mechanisms involved in the role of 1,25-(OH)2D for the control of mineral ion homeostasis, the reader is referred to recent reviews specifically addressing this topic (Christakos et al., 2003; Canadillas et al., 2007; Liu et al., 2007). In target cells, the 1,25-(OH)2D hormone also induces the expression of the gene encoding the key effector of its catabolic breakdown: 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) Makin et al., 1989; Omdahl et al., 2001. This insures attenuation of the 1,25-(OH)2D biological signal and helps regulate vitamin D homeostasis.

2. 1,25-(OH)2D actions in bone Numerous studies have examined the skeletal actions of 1,25-(OH)2D. Vitamin D deficiency results in rickets with disorganized growth plates, similar to what is observed in hypophosphatemia. Rickets is characterized by the inadequate calcification of the growth plate and adjacent metaphysis. The impaired mineralization of the growth plate cartilage in the zone of provisional calcification prevents this zone from being resorbed. As the cartilage continues to be formed, but not resorbed, the growth plate begins to widen. Simultaneously, the trabecular bone directly underneath the cartilage fails to mineralize properly and vascularization of this tissue becomes aberrant. These defects are accompanied by similar abnormalities in cortical bone leading to the full spectrum of skeletal symptoms associated with the condition. The ricketic growth plates are characterized by an expansion of the hypertrophic chondrocyte layer (Halloran and DeLuca, 1981). Healing of the growth plate is seen with normalization of calcium and phosphorus levels in vitamin D deficient animals and children, suggesting that impaired mineral ion homeostasis contributes to the rachitic changes (Al-Aqeel et al., 1993; Amling et al., 1999; Balsan et al., 1986; Weinstein et al., 1984). Murine studies directed at clarifying the cellular and molecular basis for the rachitic changes demonstrated that expansion of the growth plate is due to impaired hypertrophic chondrocyte apoptosis (Donohue and Demay, 2002). This impaired apoptosis is a result of hypophosphatemia and not perturbations of the circulating PTH or calcium levels (Sabbagh et al., 2005). Osteoblasts express the VDR and 1,25-(OH)2D plays a critical role in the regulation of several genes encoding matrix proteins

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that are expressed by the bone-forming cells. 1,25-(OH)2D has been shown to downregulate the transcription of the alpha1(I) collagen gene in osteoblasts (Harrison et al., 1989) and is a potent transcriptional activator of the genes encoding osteocalcin and osteopontin (Noda et al., 1990). Thus vitamin D deficiency might be expected to impact the amount of matrix protein in bone. However, experiments performed in vitamin D deficient rats have failed to show significant differences in the bone content of numerous matrix proteins, including osteocalcin (Wientroub et al., 1987). Studies performed in vitamin D deficient rats (Underwood and DeLuca, 1984) and in humans (Balsan et al., 1986) with vitamin D receptor mutations have demonstrated that normalization of circulating calcium and phosphorus levels can correct the hypomineralized bone matrix in the absence of 1,25-(OH)2D action, suggesting that the hormone is not required for mineralization. Similarly, preventing hypocalcemia can prevent the mineralization defects observed in mice with Cyp27b1 or VDR mutations (Amling et al., 1999; Dardenne et al., 2003). Several laboratories have generated mice strains with targeted ablation of the vitamin D receptor (Van Cromphaut et al., 2001; Li et al., 1997; Yoshizawa et al., 1997). These strains have proven to be valid animal models for hereditary vitamin D resistance rickets (HVDRR), also termed vitamin D-dependent rickets type II, a human disease associated with vitamin D receptor mutations. The mice develop hypocalcemia, hypophosphatemia and secondary hyperparathyroidism (Van Cromphaut et al., 2001; Li et al., 1997; Yoshizawa et al., 1997). VDR null mice develop severe rickets and osteomalacia (accumulation of unmineralized osteoid at sites other than the growth plate). As observed in some patients with HVDRR, VDR-deficient animals have normal hair neonatally, but gradually develop alopecia (Li et al., 1997, 1998; Cianferotti et al., 2007; Sakai et al., 2001; Skorija et al., 2005). The onset of phenotypic manifestations has been closely studied in VDR targeted mice. The pups are phenotypically indistinguishable from their wild type and heterozygous littermates at birth, and thus skeletal development does not depend on genomic VDR signaling. At the end of the third week of life, the mice develop hyperparathyroidism, secondary to impaired intestinal calcium absorption, which leads to hypocalcemia and hypophosphatemia (Van Cromphaut et al., 2001). Expansion of the hypertrophic chondrocyte layer of the growth plate is evident as early as 21 days, two days after the development of secondary hyperparathyroidism. Chondrocyte proliferation or differentiation are not altered in the VDR null mice, but changes in hypertrophic chondrocyte apoptosis were observed (Donohue and Demay, 2002). The absence of a growth plate phenotype prior to the development of impaired mineral ion homeostasis supports the hypothesis that these rachitic changes are a direct consequence of hypocalcemia, hypophosphatemia or secondary hyperparathyroidism, rather than impaired genomic actions of 1,25-(OH)2D. Indeed, normalizing mineral ion homeostasis by feeding the mice with a rescue diet consisting of high calcium, high phosphate and lactose prevents the growth plate abnormalities in the VDR null mice (Li et al., 1998). These data strongly suggest that the role of the vitamin D hormone on bone is indirect and mediated through its endocrine function on mineral homeostasis. However, chondrocytes and osteoblasts express the VDR (Boivin et al., 1987; Johnson et al., 1996; Narbaitz et al., 1983), and thus a specific, albeit non-essential, role for 1,25-(OH)2D-mediated signaling in endochondral bone formation remains a formal possibility. A role for 1,25-(OH)2D in growth plate function is further supported by the observation that rescuing mineral homeostasis of Cyp27b1-deficient mice with a high calcium, high lactose diet corrected all aspects of the phenotype, except long bone growth (Dardenne et al., 2003). The paracrine/intracrine role of 1,25-(OH)2D in the growth plate has been tested using recently engineered mutant mice models.

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VDR-mediated signaling was elegantly shown to transiently regulate chondrocyte differentiation or function in the context of normal vitamin D endocrine actions. Masuyama et al. (2006) targeted the VDR gene with loxP sites and crossed mice with the floxed allele to transgenic mice expressing the Cre recombinase under the control of the type II collagen promoter and enhancer. This strategy led to inactivation of the VDR gene in proliferating chondrocytes. Growth plate morphology was not affected in 15-day-old mutant mice (chondrocyte-specific VDR-KO). A surprising finding was the measure of an increased bone volume in mutant newborn and 15-dayold mice when compared to littermate controls. This phenotype was transient as adult chondrocyte-specific VDR-KO mice did not manifest it (Masuyama et al., 2006). After ruling out that the augmented bone volume was caused by increases in osteoblast-mediated bone formation, the authors examined the interesting possibility that defects in vascular invasion and/or osteoclastogenesis could be responsible for the observed phenotype. Immunodetection of endothelial cells in developing tibiae of chondrocyte-specific VDR-KO mutant mice revealed a reduction in the number of invading blood vessels in the growth plate, and VEGF mRNA levels were significantly reduced in mutant newborn growth plates (Masuyama et al., 2006). This was accompanied by a reduction in the numbers of osteoclasts and a decrease in RANKL expression. Elegant cell culture studies determined that 1,25-(OH)2D induced RANKL expression in chondrocytes in a VDR-dependent manner, confirming previous results (Takamoto et al., 2003). Blood biochemistry was analyzed before weaning in chondrocyte-specific VDR-KO mutant mice. Augmented circulating phosphate and 1,25(OH)2D levels were measured. These changes were also transient as levels normalized in older mice (Masuyama et al., 2006). The measured biochemical changes were somewhat surprising considering that the mutation targeted the VDR gene in chondrocytes and should not have affected endocrine vitamin D function. Mechanistic investigation of this phenotypic manifestation revealed that chondrocyte-specific VDR-KO mice had increased renal expression of the phosphate co-transporter Npt2a and increased Cyp27b1 mRNA levels (Masuyama et al., 2006). The impact of a localized mutation such as chondrocyte-specific inactivation of the VDR on the regulation of gene expression in a distant tissue such as the kidney suggested the involvement of an endocrine regulator and the obvious candidate was FGF23, as this phosphaturic factor inhibits renal expression of Npt2a and Cyp27b1 (Liu et al., 2007; Saito et al., 2003). The expression of FGF23 was reduced in the metaphysis of chondrocyte-specific VDR mutant mice. Chondrocytes do not express FGF23 (Masuyama et al., 2006). Rather, it was shown that FGF23 expression in osteoblasts is regulated by a chondrocyte-derived secreted factor whose expression requires VDR activity (Masuyama et al., 2006). These results established a direct role for 1,25-(OH)2D genomic actions in bone, showing that VDR signaling in chondrocytes is necessary for timely vascular invasion and osteoclastogenesis during development, and for proper endocrine action of bone in phosphate homeostasis. While these studies demonstrated the role of VDR-mediated signaling in chondrocytes, the source of the 1,25-(OH)2D ligand remained undetermined. Since the CYP27B1 enzyme that synthesizes 1,25-(OH)2D is expressed in chondrocytes, it was suggested that local production of 1,25-(OH)2D could play an autocrine or paracrine role in the differentiation of these cells and explain the partial rescue of the phenotype when mineral homeostasis is corrected (Dardenne et al., 2003). To test this hypothesis, we generated mutant mice that do not express the Cyp27b1 gene in chondrocytes (Naja et al., 2009). We observed an increase in the width of the growth plate’s hypertrophic zone at E15.5. Concomitantly, we detected a reduction in the number of activated osteoclasts migrating towards the primary ossification center. The reduction in activated osteo-

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Fig. 1. Chondrocyte-specific inactivation of Cyp27b1 leads to transient changes at the chondro-osseous junction. (A) Hematoxylin/eosin stained sections of femurs from 2 day-old control and Cyp27b1/CH4 mice. R, resting zone; P, proliferative zone; H, hypertrophic zone. There is no statistical difference between the size of each zone when all samples are compared. Bar, 200 lm. (B–D) Microcomputed tomography (lCT) showing increased bone volume in 2 day-old long bones of Cyp27b1/CH4 mice as compared to controls. The difference in bone volume were not observed at day 14 and 42 after birth (n = 7 for all genotypes). (E–F) RT-qPCR on RNA extracted from 2 day-old (E) or 9 day-old (F) rib cartilage was used to quantify mRNA levels of vascular endothelial growth factor a (VEGFa) in Cyp27b1/CH4 mice (n = 9) and controls (n = 14). ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001. BV/TV, bone volume/tissue volume; P2, postnatal day 2; P9, postnatal day 9; P14, postnatal day 14; P42, postnatal day 42.

clasts was also observed at the chondro-osseous junction of twoday-old tibiae. The mRNA levels of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor were increased at postnatal day 2 along with a decrease in VEGF levels (Fig. 1E). This decrease in the angiogenic VEGF was accompanied by a decrease in blood vessel formation at the chondro-osseous junction as observed by a decrease in PECAM-1 immunostaining (Naja et al., 2009). The major phenotype observed at postnatal day 2 was a significant increase in femoral trabecular bone mass (Fig. 1A and B) Naja et al., 2009. These phenotypic manifestations were transient (Fig. 1C, D and F). The decrease in molecular angiogenic markers (VEGF and its receptor Kdr), endothelial cell/blood vessel marker (PECAM-1), osteoclast recruitment, and increase of metaphyseal bone mass in long bones from chondrocyte-specific Cyp27b1-deficient mice (Cyp27b1/CH4) suggests the following model: in Cyp27b1/CH4 mice, the reduction in VEGF expression and signaling leads to a decrease in blood vessel formation. This

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vasculature delay in turn reduces osteoclast invasion at the chondro-osseous junction. Since osteoclasts resorb the calcified cartilaginous matrix that serves as the scaffold for woven bone formation, a reduction in osteoclast invasion would result in an increased scaffold size that would lead to a larger bone volume at the primary spongiosa. This phenotype is transient as blood vessels eventually invade the chondro-osseous junction and normalize osteoclast recruitment and bone turnover. These results agree with the phenotype observed in chondrocyte-specific VDR-ablated mice (Masuyama et al., 2006) and support an autocrine/paracrine role of 1,25-(OH)2D in endochondral ossification and chondrocyte development in vivo. 3. Direct effects of vitamin D metabolites in chondrocytes The gene encoding the vitamin D 24-hydroxylase, Cyp24a1, is expressed in growth plate chondrocytes and cells from the growth plate respond to 24,25-(OH)2D in a cell maturation-dependent manner (reviewed in Boyan et al. (2002, 2004)). Studies examining the direct effect of vitamin D metabolites on chondrocytes were mostly performed using the in vitro rat costochondral primary culture system. Dissection of the tissue allows isolation of cells from different regions of the growth plate. Each region represents a different maturation stage along the chondrocytic differentiation pathway. In this model system, the less differentiated cells of the resting zone, also called the reserve zone, respond to 24,25-(OH)2D. The more mature cells of the growth zone, including the prehypertrophic and hypertrophic compartments, respond primarily to 1,25-(OH)2D. The effects of 24,25-(OH)2D were also recently observed in the well characterized prechondrocytic cell line ATDC5 (Atsumi et al., 1990; Denison et al., 2009). In resting zone cells, 24,25-(OH)2D decreases cell proliferation but stimulates differentiation and maturation (Schwartz et al., 1989, 1995), with increased alkaline phosphatase activity and increased synthesis of sulfated glycosaminoglycans (Schwartz et al., 2001). The mechanisms have been studied in detail and involve activation of protein kinase C that is dependent upon conversion of phosphatidylcholine to phosphatidic acid by phospholipase D (Schwartz et al., 2001; Helm et al., 1996; Sylvia et al., 2001). Phospholipase D activity also leads to lysophosphatidic acid (LPA) production that acts as a second messenger through LPA receptors 1 and 3 (Hurst-Kennedy et al., 2009). LPA protects resting zone cells from apoptotic cell death by decreasing p53 abundance and increasing the Bcl-2/Bax ratio (Boyan et al., 2010; Hurst-Kennedy et al., 2010). Inhibition of LPA receptors 1 and 3 attenuates responsiveness of resting zone chondrocytes to 24,25-(OH)2D Hurst-Kennedy et al., 2009. Interestingly, treatment of resting zone chondrocytes with 24,25-(OH)2D induces a change in maturation state, resulting in down-regulation of responsiveness to 24,25-(OH)2D and up-regulation of responsiveness to 1,25-(OH)2D Schwartz et al., 1995. These observations support the hypothesis that 24,25-(OH)2D plays a role in cartilage development. This may be salient to the actions of the metabolite during bone fracture healing (see below). It is worth mentioning that growth plates from Cyp24a1/ mice do not show major defects (St-Arnaud et al., 2000; St-Arnaud, 2005). These observations suggest that the absence of CYP24A1 activity does not affect growth plate development and that 24,25-(OH)2D is not required for chondrocyte maturation in vivo (St-Arnaud et al., 2000). It remains possible, however, that a redundant endocrine system is able to compensate for the function of 24,25(OH)2D in animals. Studies using primary cultures of costochondral chondrocytes from Cyp24a1/ animals would be useful in elucidating the role of 24,25-(OH)2D in chondrocyte maturation. As previously mentioned, in vitro studies show that growth plate chondrocytes exhibit differential responsiveness to 1,25-

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(OH)2D and 24,25-(OH)2D Boyan and Schwartz, 2005. The response of rat costochondral chondrocytes to vitamin D metabolites depends on the zone of maturation from which the cells were originally derived (Boyan et al., 1988). The prehypertrophic and upper hypertrophic zones (defined as the ‘growth zone’ in this culture system) respond primarily to 1,25-(OH)2D. Treatment of growth zone chondrocytes with 1,25-(OH)2D inhibits proliferation, but increases membrane fluidity (Swain et al., 1993), and stimulates alkaline phosphatase activity, collagen synthesis, proteoglycan synthesis and arachidonic acid turnover (Schwartz and Boyan, 1988; Schwartz et al., 1990). These effects are thought to be mediated through binding of 1,25-(OH)2D to the membrane-associated receptor PDIA3 (protein disulfide isomerase associated 3, also known as ERp60, Erp57, Grp58, or membrane associated rapid response steroid binding protein, MARRS) (Nemere et al., 2004). Binding of 1,25-(OH)2D to the membrane receptor leads to protein kinase Ca signaling mediated by phospholipase A2 (PLA2) activation and PLA2-activating protein (Schwartz et al., 2005, 2003). The physiological relevance of membrane-mediated 1,25(OH)2D signaling is emerging. VDR-deficient growth zone chondrocytes respond to 1,25-(OH)2D, but their response is blocked by specific antibodies against ERp60 (Boyan et al., 2003). Rapid 1,25(OH)2D membrane signaling has also been reported in osteoblasts, where the metabolite regulates voltage gated ion channels (Zanello and Norman, 2004) and activates phospholipase C, protein kinase C, phospholipase A2 activity and prostaglandin production (Baran, 1994; Wali et al., 2003; Schwartz et al., 1992). Interestingly, inhibition of PDIA3 expression using RNA knockdown technology abolishes 1,25-(OH)2D membrane signaling in osteoblasts (Chen et al., 2010). Preliminary results suggest that Pdia3 gene dosage reduction as achieved in mice heterozygous for targeted inactivation of the Pdia3 gene results in bone abnormalities (Wang et al., 2010). Complete inactivation of Pdia3 appears to be embryonic lethal (Wang et al., 2010). Finally, many of the signaling molecules involved in rapid 1,25-(OH)2D membrane signaling are found in specialized regions of the plasma membrane called lipid rafts or caveolae. A major scaffolding component of these structures is caveolin-1 (Shaul and Anderson, 1998; van Deurs et al., 2003). Interestingly, caveolin-1-deficient growth zone chondrocytes do not respond to 1,25-(OH)2D, demonstrating that the membrane-mediated effects of 1,25-(OH)2D require caveolae and caveolin-1 (Boyan et al., 2006). 4. 24,25(OH)2D and fracture repair It has been proposed that 24,25-(OH)2D, the enzymatic product of the CYP24A1 activity on the 25-(OH)D substrate, might also play a role in fracture repair, but this putative function of the metabolite has not been extensively studied. The healing of fractures is a unique postnatal biological repair process resulting in the restoration of injured skeletal tissue to a state of normal structure and function. Fracture repair involves a complex multistep process that involves response to injury, intramembranous bone formation, chondrogenesis, endochondral bone formation, and bone remodelling. Several studies have described a complex pattern of gene expression that occurs during the course of these events (Hadjiargyrou et al., 2002; Hatano et al., 2004; Nakazawa et al., 2004; Rundle et al., 2006). Taken together, results from gene expression monitoring during bone repair suggest that the molecular regulation of fracture healing is complex but recapitulates some aspects of embryonic skeletal formation (Colnot et al., 2003; Ferguson et al., 1999). A role for 24,25-(OH)2D in fracture repair is supported by the observation that the circulating levels of 24,25-(OH)2D increase during fracture repair in chickens due to an increase in CYP24A1 activity (Seo and Norman, 1997). When the effect of various vitamin D metabolites on the mechanical properties of healed bones was tested, treatment with 1,25-(OH)2D alone resulted in poor healing

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(Seo et al., 1997). However, the strength of healed bones in animals fed 24,25-(OH)2D in combination with 1,25-(OH)2D was equivalent to that measured in a control population fed 25-(OH)D (Seo et al., 1997). These results support a role for 24,25-(OH)2D as being an essential vitamin D metabolite important for fracture repair. It is likely that 24,25-(OH)2D would act through receptor-mediated signaling (as do other vitamin D metabolites and hormones), and circumstantial evidence suggests the presence of a nonnuclear membrane receptor for 24,25-(OH)2D in the chick tibial fracturehealing callus (Seo et al., 1996; Kato et al., 1998). Cell fractionation to isolate a membrane fraction followed by ligand binding studies using hydroxylapatite to separate bound and free ligands described a receptor/binding protein for 24,25-(OH)2D in the fracture-healing callus membrane fraction from vitamin D-depleted chicks (Kato et al., 1998). These observations were never followed through and to date, no molecular entity corresponding to this binding activity has ever been cloned or purified for complete characterization. The Cyp24a1-deficient mouse strain (St-Arnaud et al., 2000) represents an invaluable tool to examine the putative role of 24,25-(OH)2D in mammalian fracture repair. Cyp24a1 mutant animals that survive past weaning appear to use an alternative pathway of 1,25-(OH)2D catabolism to regulate circulating levels of the hormone (Masuda et al., 2005) and are normocalcemic and normophosphatemic when fed regular rodent chow. This allows to study bone healing in these animals. As a first step, the induction of the expression of the Cyp24a1 gene during fracture repair was confirmed in mice. Wild-type mice were subjected to a stabilized, transverse mid-diaphysial fracture of the tibia. To stabilize the fracture without disrupting the bone marrow microenvironment, we used a small-scale version of the Ilizarov distraction osteogenesis device used in orthopaedic patients (Aronson, 1994; Tay et al., 1998; Thompson et al., 2002). RNA was extracted from the fracture callus at 14 days post-osteotomy, reverse-transcribed, and analyzed by RT-qPCR. Cyp24a1 mRNA levels were significantly elevated in the fracture callus as compared to the undamaged contra-lateral bone (data not shown). Importantly, these results confirm the data reported previously for chicken (Seo and Norman, 1997) and support the hypothesis that the activity of the CYP24A1 enzyme is important for bone fracture repair. Fracture repair was then compared between Cyp24a1/ mice and wild-type controls. We have observed a delay in the mineralization of the cartilaginous matrix of the soft callus in Cyp24a1/ mutant animals, accompanied by altered expression of differentiation marker genes (data not shown). The repair delay and the aberrant pattern of gene expression could be rescued by treatment with 24,25-(OH)2D. We then used the Cyp24a1-deficient mice as a source of tissue to identify differentially expressed genes in the callus of Cyp24a1-deficient mice. This has led to the identification of a restricted set of genes which we are currently characterizing for their ability to bind 24,25-(OH)2D. Taken together, our preliminary results strongly support a role for 24,25-(OH)2D in mammalian fracture repair. 5. Perspectives The power of mouse molecular genetics has helped determine a specific, direct role for 1,25-(OH)2D genomic signaling in chondrocytes (Masuyama et al., 2006). Similar techniques are now being used to study the physiological role of non-genomic (membrane) 1,25-(OH)2D signaling. A floxed allele of Pdia3 has been generated (Nemere et al., 2010) and it will be interesting to selectively inactivate the gene in chondrocytes to examine the impact of such a mutation on growth plate maturation and function. Specific inactivation in osteoblasts should be performed in parallel to ascertain the biological relevance of rapid 1,25-(OH)2D membrane signaling in bone-forming cells. Since the differential membrane effects of

vitamin D metabolites in cells from bone appear to be dependent on cell type maturation (Schwartz et al., 1999), it will be interesting to study the effect of selective inactivation of PDIA3 at various cell differentiation stages using the variety of Cre-expressing transgenic lines available for such studies. One critical piece of missing information is the identity of the 1,25-(OH)2D-induced secreted factor from chondrocytes that modulates FGF23 secretion in osteoblasts. Protein purification from the media of 1,25-(OH)2D-treated chondrocyte cultures coupled with mass spectrometry could help characterize this important regulatory factor. Alternatively, gene expression monitoring with cDNA microarrays between 1,25-(OH)2D-treated wild-type and VDR-deficient chondrocytes may serve to identify the gene encoding this factor. Further characterization of this molecule will help determine its potential as a therapeutic target to modulate phosphate homeostasis. Finally, the observation that mice deficient for Cyp24a1 exhibit a delay in bone fracture healing that can be corrected by exogenous administration of 24,25-(OH)2D suggests that treatment with vitamin D metabolites hydroxylated at position 24, such as 24,25(OH)2D, could be useful in the treatment of bone fractures subsequent to trauma or metabolic bone diseases. It can be argued that 24,25-(OH)2D is an abundant circulating vitamin D metabolite and that it is present in sufficient amounts to efficiently promote bone healing without the need for additional supplementation. However, it is now recognized that a sizeable proportion of the population suffers from vitamin D insufficiency (Holick et al., 2005; Rucker et al., 2002; Thomas et al., 1998), which may have deleterious effects for optimized fracture repair. Thus fracture healing could benefit from supplementation with 24,25-(OH)2D or a suitable analog. Acknowledgements Work from the laboratory of the authors was supported by the Shriners of North America through grant No. 8560 to R.StA. Guylaine Bédard prepared the figure. R.P. Naja was a Canada Bone Scholar supported in part by the CIHR Skeletal Health Training Grant. We used the SkyScan lCT instrument from the Centre for Bone and Periodontal Research of McGill University. References Al-Aqeel, A., Ozand, P., Sobki, S., Sewairi, W., Marx, S., 1993. The combined use of intravenous and oral calcium for the treatment of vitamin D dependent rickets type II (VDDRII). Clin. Endocrinol. 39, 229–237. Amling, M., Priemel, M., Holzmann, T., Chapin, K., Rueger, J.M., Baron, R., Demay, M.B., 1999. Rescue of the skeletal phenotype of vitamin D receptor-ablated mice in the setting of normal mineral ion homeostasis: formal histomorphometric and biomechanical analyses. Endocrinology 140, 4982–4987. Aronson, J., 1994. Experimental and clinical experience with distraction osteogenesis. Cleft Palate Craniofac. J. 31, 473–481 (discussion 481–482). Atsumi, T., Miwa, Y., Kimata, K., Ikawa, Y., 1990. A chondrogenic cell line derived from a differentiating culture of AT805 teratocarcinoma cells. Cell Differ. Dev. 30, 109–116. Balsan, S., Garabedian, M., Larchet, M., Gorski, A.M., Cournot, G., Tau, C., Bourdeau, A., Silve, C., Ricour, C., 1986. Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1, 25dihydroxyvitamin D. J. Clin. Invest. 77, 1661–1667. Baran, D.T., 1994. Nongenomic actions of the steroid hormone 1 alpha, 25dihydroxyvitamin D3. J. Cell Biochem. 56, 303–306. Boivin, G., Mesguich, P., Pike, J.W., Bouillon, R., Meunier, P.J., Haussler, M.R., Dubois, P.M., Morel, G., 1987. Ultrastructural immunocytochemical localization of endogenous 1, 25- dihydroxyvitamin D3 and its receptors in osteoblasts and osteocytes from neonatal mouse and rat calvaria. Bone Miner. 3, 125–136. Boyan, B.D., Schwartz, Z., 2005. Cartilage and vitamin D: genomic and nongenomic regulation by 1,25(OH)2 D3 and 24,25(OH)2 D3. In: Feldman, D. et al. (Eds.), Vitamin D, second ed. Elsevier, Academic Press, San Diego, pp. 575–597. Boyan, B.D., Schwartz, Z., Carnes Jr., D.L., Ramirez, V., 1988. The effects of vitamin D metabolites on the plasma and matrix vesicle membranes of growth and resting cartilage cells in vitro. Endocrinology 122, 2851–2860. Boyan, B.D., Sylvia, V.L., Dean, D.D., Del Toro, F., Schwartz, Z., 2002. Differential regulation of growth plate chondrocytes by 1alpha, 25-(OH)2D3 and 24R, 25-

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