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Mechanism of Bone Mineralization

Monzur Murshed1,2,3

1Faculty of Dentistry, McGill University, Montreal, Quebec H3A 1G1, Canada 2Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, Quebec H4A 3J1, Canada 3Shriners Hospital for Children, Montreal, Quebec H4A 0A9, Canada Correspondence: [email protected]

Mineralized “hard” tissues of the skeleton possess unique biomechanical properties to support the body weight and movement and act as a source of essential minerals required for critical body functions. For a long time, extracellular matrix (ECM) mineralization in the vertebrate skeleton was considered as a passive process. However, the explosion of genetic studies during the past decades has established that this process is essentially controlled by multiple genetic pathways. These pathways regulate the homeostasis of ionic calcium and inorganic phosphate—two mineral components required for bone mineral formation, the synthesis of mineral scaffolding ECM, and the maintainence of the levels of the inhibitory organic and inorganic molecules controlling the process of mineral crystal formation and its growth. More recently, intracellular enzyme regulators of skeletal tissue mineralization have been identified. The current review will discuss the key determinants of ECM mineralization in bone and propose a unified model explaining this process.

he mineralized extracellular matrix (ECM) is The origin of biomineralization has been Ta unique feature of the vertebrate skeletoden- traced back to the late Precambrian period after tal system. The massive load-bearing capacity of tectonic activities caused a marked increase of the mineralized skeleton permitted the evolu- soluble minerals in the seawater (Wagner and tionary emergence of large vertebrates, such as Aspenberg 2011). It is commonly believed that

www.perspectivesinmedicine.org blue whales, which can weigh up to 180 tons. marine organisms first developed primitive exo- However, the functions of the mineralized tis- skeletons made up of calcium carbonate and/or sues are not limited to support the body mass, calcium phosphate minerals (Knoll 2003; Wag- protect the internal soft organs, or to facilitate ner and Aspenberg 2011). As part of the process locomotion and mastication only; they also of evolutionary adaptations, the skeletal tissues serve as a readily accessible reservoir for essen- were internalized, which paved the way for the tial minerals that are indispensable for many emergence of organisms with larger body sizes. physiologic activities. The current review will Whereas it is not clear what prompted some focus on the mechanism of calcium phosphate primitive organisms to deposit calcium phos- biomineralization in the vertebrate skeleton. phate instead of calcium carbonate minerals in

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their skeletal tissues, it is conceivable that the X-ray diffraction analysis. This pattern suggest- deposited calcium phosphate minerals provided ed that the precipitated mineral is amorphous certain physiological advantages. Indeed it has calcium phosphate (ACP), not apatite. Interest- been suggested that the presence of calcium ingly, when X-ray diffraction analyses were per- phosphate minerals makes the skeleton more formed on the same samples 2 days later, the stable under acidic conditions (Wagner and As- precipitates were found to be a poorly formed penberg 2011). Vertebrate organisms rely on crystalline apatite (Boskey 1997). This initial ATP generation via anaerobic glycolysis for sud- finding of mineral phase transition in vitro den rapid movements during “fight-or-flight” prompted follow-up experiments using miner- situations. The activation of this pathway results alized tissue samples and, subsequently, the in an acidic tissue environment, which would presence of ACP in the embryonic chick bones have destabilized a skeleton made up of calcium was reported by the same group (Eanes et al. carbonate more readily. 1965). However, the controversy on whether bone contains ACP continued as later studies concluded that the embryonic bones do not con- MINERAL COMPOSITION OF BONE tain ACP (Bonar et al. 1983; Grynpas et al. In 1771, Scheele first reported the presence of 1984). Since then, more sophisticated analyses calcium phosphate minerals in bone (Scheele of bone minerals using Raman spectra, Fourier- 1931). Later, bone mineral was described as a transform infrared spectroscopy (FTIR), and form of hydroxyapatite [(Ca)10 (PO4)6(OH)2] synchrotron-generated X-ray diffraction tech- comparable to geological apatite (a group of niques have been performed; however, a consen- phosphate minerals) by both chemical compo- sus on the issue is still elusive (Rey et al. 2009). sition and X-ray diffraction pattern analyses (see The presence of amorphous mineral phase Eliaz and Metoki 2017 for a comprehensive re- has been reported in some invertebrates (Becker view on the historical perspective). However, et al. 1976, 2005). Additionally, ACP has been subsequent studies demonstrated that minerals detected at the sites of ectopic calcification in the in bone do not have a uniform composition (Rey vertebrates (Marulanda et al. 2017). In two stud- et al. 1995; Pasteris et al. 2004). Although there ies published by Mahamid et al. (2008, 2010), are still some disagreements about the initial ACP has been identified as a major mineral phase of the deposited mineral and its time-de- phase in zebrafish fin bones, suggesting that pendent transition to apatite, it is now accepted amorphous to crystalline phase transition oc- that the matured bone mineral is a substituted curs in vivo. On the other hand, concerns have crystalline phase of calcium phosphate, referred been raised about this latter finding on the www.perspectivesinmedicine.org to as carbonated hydroxyapatite (Mahamid et al. ground of technical limitations, including pos- 2008). High-resolution transmission electron sible mineral phase transition during sample microscopy (TEM), 3D stereoscopic TEM, and collection and processing (Rey et al. 2009). atomic force microscopy on organic matrix-free bone samples provided accurate measurements DETERMINANTS OF SKELETAL TISSUE of bone crystals. These studies have firmly estab- MINERALIZATION lished that bone crystals are nanosized, long, and very thin platelets (Rey et al. 2009). In human embryos, although primary ossifica- The source of the dispute regarding the early tion centers of endochondral bones (e.g., verte- mineral phase in bone appears to be a simple in brae and long bones) appear between 8 and 12 vitro experiment performed in the mid-sixties in weeks of gestation, the bulk mineralization of the laboratory of Dr. Posner (Boskey 1997). The skeletal tissues does not occur until the third mixing of concentrated solutions of calcium trimester (Kovacs 2003). The type I collagen- chloride and sodium acid phosphate resulted rich ECM in the intramembranous bones min- in the precipitation of calcium phosphate salts, eralizes directly, whereas in the endochondral which showed a broad and diffused pattern by bones, mineralization starts concomitantly at

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Mechanism of Bone Mineralization

two sites: within the core region of the cartilage or XLH patients leads to an increase of circulat- anlagen and along the bone collar surrounding ing fibroblast growth factor 23 (FGF23) pro- it (Karsenty and Wagner 2002). In the cartilage, duced by osteoblasts and osteocytes (Xia et al. the type X collagen matrix synthesized by the 2007). Increased FGF23, in turn, down-regulates hypertrophic chondrocytes serves as the mineral the expression of sodium phosphate transport- scaffold, whereas in the bone collar (and later ers in the kidney tubules preventing Pi reabsorp- in the trabecular bones), the primary mineral tion and causing hypophosphatemia. As expect- scaffolding protein is the osteoblast-derived ed, in ApoE-Fgf23 transgenic mice in which type I collagen (Karsenty and Wagner 2002). systemic FGF23 level increases in the absence A brief discussion on the key determinants reg- of any PHEX mutations, a reduction of systemic ulating skeletal ECM mineralization is pre- Pi level and osteomalacia comparable to that of sented below. Hyp mice have been reported (Bai et al. 2004). The similarity of the osteomalaciatraits in ApoE- Fgf23 and Hyp mice suggests that the accompa- Systemic Levels of Calcium nied hypophosphatemia is the main cause of the and Phosphate Ions phenotype in these two models. The chemical structure of bone mineral implies More recently, osteopontin, a protein be- that the extracellular levels of ionic calcium longing to the family of small integrin-binding 2+ (Ca ) and inorganic phosphate (Pi) will be ligand, N-linked glycoproteins (SIBLINGs), has two critical determinants for bone mineraliza- been shown as a substrate for PHEX (Barros tion. Indeed, data from patients and animal et al. 2013). It has been proposed that in the models of human diseases clearly demonstrates absence of PHEX, accumulation of osteopontin “ ” that the reduction of systemic Pi levels with or in the ECM may contribute to the hard tissue without any alteration of the Ca2+ levels lead to mineralization defects seen in the XLH patients osteomalacia with the characteristic increase of and in Hyp mice. Another SIBLING protein, unmineralized osteoid volume. The importance dentin matrix protein 1 (DMP1), has been of systemic levels of these mineral ions in bone shown to be an important regulator of circulat- mineralization was demonstrated by impair- ing FGF23 levels (Martin et al. 2011). As is the ment of the 1,25 dihydroxy vitamin D3 signaling case with the inactivating mutations in PHEX, pathway. For example, mutations in 25, hydroxy DMP1 deficiency leads to severe osteomalacia. α D3-1 -hydroxylase (required for functional vi- A severe osteomalacia phenotype is also seen tamin D synthesis) or inactivating mutations in in the calcium sensing receptor (CaSR) knock- vitamin D receptor, impair the absorption of out mice. The ablation of the Casr gene leads to a

www.perspectivesinmedicine.org 2+ Ca and Pi in the gut (Dardenne et al. 2001; marked increase of systemic parathyroid hor- Masuyama et al. 2001; Panda et al. 2001). Addi- mone (PTH) levels leading to hypercalcemia ac- tionally, these mutations also reduce the mobi- companied by hypophosphatemia as a result of lization of these ions from the bone by restricting increased urinary excretion of Pi (Tu et al. 2003). bone resorption (Suda et al. 1992). Interestingly, the observed increase of Ca2+ in 2+ Although Ca and Pi are both integral parts the circulation failed to prevent osteomalacia of bone mineral, genetic experiments in mice in these mice. On the other hand, an opposite suggest that circulating Pi may have a more phenotype has been seen in mice lacking glial prominent role in the regulation of bone miner- cell missing 2 (GCM2), a transcription factor alization. For example, in Hyp mice with a mu- required for the development of the parathyroid tation in the Phex gene (a model of human X- gland (Gunther et al. 2000). The ablation of linked hypophosphatemia [XLH]), the calcium Gcm2 impairs the development of the parathy- level is normal, but the systemic Pi level is re- roid gland causing a marked reduction of PTH duced to almost half, causing a severe osteoma- level in the circulation. A basal PTH level is lacia phenotype (Eicher et al. 1976; Costa et al. maintained as the thymus acts as a secondary 1981). The inactivation of PHEX in the Hyp mice source for the hormone. As expected, the sur-

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− − viving Gcm2 / mice show a reduction of serum copy of the mineralizing bones from newborn 2+ Ca levels and an increase of serum Pi levels. mice by Sheldon and Robinson (1957). Al- However, despite the significant reduction of though earlier studies provided circumstantial systemic Ca2+ levels, no osteomalacia was ob- evidence that mineral crystals are deposited served in these mice (Gunther et al. 2000). within the collagen fibrils, this study first dem- Currently, a mouse model showing isolated onstrated that there are two sites on the collagen hypocalcemia with a normal serum Pi level is matrix in bone where mineral deposition starts unavailable. Considering that Ca2+ is an integral (1) at the intrafibrillar gap spaces, where the car- component of bone mineral, it is possible that boxy- and amino-terminal ends of two serially such a model of isolated hypocalcemia, if exist- arranged collagen triple helices meet, and (2) ed, would have shown impaired bone minerali- interfibrillar spaces between the fibrils (Sheldon zation. However, phenotypic comparisons of and Robinson 1957). Since then, numerous the mouse models with abnormal Ca2+ and/or studies have confirmed the critical role of colla- Pi homeostasis discussed above suggest that the gen matrix as a scaffold for bone mineral osteomalacia phenotype in a model of isolated deposition. These include the initial studies hypocalcemia may not be as severe as seen in demonstrating that collagen sponges can be isolated hypophosphatemia. Because of the crit- mineralized in vitro, implanted collagen can ical role of Ca2+ in many physiologic activities, mineralize in vivo, and demineralized bone col- its serum level is more tightly regulated than that lagen can be mineralized under a cell-free con- of Pi. Moreover, ubiquitously present inorganic dition (Mergenhagen et al. 1960; Bachra and pyrophosphate (PPi), a potent inhibitor of ECM Fischer 1968a,b). More recently, genetic experi- mineralization, is a Pi derivative (see below) ments have demonstrated that the reduction of (Terkeltaub 2001). These observations suggest collagen synthesis in bone results in a reduction that early organisms depositing calcium phos- of mineralized bone mass (Yang et al. 2004). phate minerals in the skeletal tissues evolved to The exact mechanism by which the collagen regulate ECM mineralization by modulating the lattice facilitates mineral deposition is still un- Pi levels and its incorporation into the growing known. The amino acid side chains exposed at mineral crystals. the intrafibrillar gap space and at the interfibril- lar space may regulate this process. However, it is possible that the dense packaging of collagen Collagen Scaffold molecules and their hierarchical organization, The organic part of the bone ECM is primarily rather than the primary structure of the protein composed of type I collagen. Two genes, Col1a1 is the driving force underlying the nucleation of www.perspectivesinmedicine.org and Col2a1, encode the α1 and α2 chains of type hydroxyapatite. Indeed, one theory attempts to I collagen, respectively. Two α1 and one α2 explain the mineralization of bone ECM by the chains assemble together to form the collagen size-exclusion properties of the collagen scaffold triple helix in the extracellular spaces of the bone (Toroian et al. 2007; Price et al. 2009). Accord- microenvironment. In a hierarchical fashion, ing to this theory, the nanoscale gaps present these helices are first arranged axially in a stag- within a collagen fibril and in between the fibrils gered manner as collagen fibrils, which are then arranged in a fiber allow the access of Ca2+ and fi bundled together to form the collagen bers Pi ions, but not the large proteins, which can (Shoulders and Raines 2009). In a healthy indi- inhibit the formation and growth of the nascent vidual, the mineralization of the unmineralized hydroxyapatite crystals inside the scaffold. collagen (osteoid) occurs seamlessly in contin- uation of the existing mineralized matrix on Mineralization Inhibitors which the newly synthesized osteoid is depos- ited by the osteoblasts. Considering that the concentrations of various The involvement of collagen in bone miner- ions in all the tissues of the body are at equilib- 2+ alization came from the initial electron micros- rium to that of blood, it is likely that Ca and Pi

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Mechanism of Bone Mineralization

levels do not differ significantly between the terase nucleotide pyrophosphatase I (Enpp1) “soft” and skeletal “hard” tissues. This may raise gene (Koshizuka et al. 2001). the question—why do some “soft” tissues, such Interestingly, both Ank and Enpp1 genes are as blood vessels and some cartilaginous tissues, highly expressed in bone, more specifically in the never mineralize despite their high collagen osteoblasts (Murshed et al. 2005). Although the content? The most straightforward answer to presence of a mineralization inhibitor in bone this question can be obtained from several hu- may appear counterintuitive, this quandary can man diseases of pathologic “soft” tissue miner- be explained by the presence of a strong alkaline alization and their animal models. For example, phosphatase (ALPL) activity in bone. The car- generalized arterial calcification in infancy dinal role of ALPL during skeletal mineraliza- (GACI) and progressive ankylosis in humans tion in humans was demonstrated by many dif- fi are both caused by the reduced level of PPi in ferent mutations identi ed in the ALPL gene in the bone joints (Ho et al. 2000; Albright et al. hypophosphatasia patients, causing awide range 2015). PPi is a mineralization inhibitor that is of phenotypic severity. Some of these patients known for its potent antimineralization proper- show very severe osteomalacia and fetal/perina- ties for over 50 years (Fleisch and Bisaz 1962). tal lethality, while others show milder, progres- A wide distribution of the PPi synthesis and/ sive osteomalacia later in life (Mornet 2000; Tail- or extracellular transport machinery in verte- landier et al. 2000, 2001). The genetic models brate organisms prevents pathologic “soft” tis- lacking ALPL activity further confirmed the re- sue mineralization. quirement of this enzyme for bone mineraliza- PPi is composed of two inorganic Pi groups tion (Fedde et al. 1999; Anderson et al. 2004). joined by an ester linkage (Terkeltaub 2001). ALPL-mediated hydrolysis of PPi has two This inorganic mineralization inhibitor is pro- implications—first, it reduces the amount of duced both intracellularly and extracellularly PPi in the bone microenvironment and, second, via enzymatic reactions. Intracellularly, PPi it increases the amount of mineralization-pro- can be generated as a byproduct of enzymatic moting Pi ions by liberating them from PPi. activities in numerous metabolic pathways. In- These coupled activities alter the Pi/PPi ratio in tracellular PPi is transported to the extracellular the bone ECM in such a way that the formation space through a transmembrane transporter and growth of the apatite crystal is promoted called ANK (Ho et al. 2000). On the other (Hessle et al. 2002; Murshed et al. 2005). The hand, extracellularly, a direct cleavage of the critical effect of this ratio in regulating ECM phosphodiester bond in purine and pyrimidine mineralization has been demonstrated in ank/ nucleoside triphosphates (e.g., ATP) by nucle- ank;Hyp compound-mutant mice, which show www.perspectivesinmedicine.org otide pyrophosphatase/phosphodiesterase en- a marked reduction of joint mineralization zymes can also generate PPi (Hessle et al. (Murshed et al. 2005). The low extracellular Pi/ 2002). PPi ratio in these mice in comparison to the At a threshold concentration, PPi prevents original ank/ank mice prevented the ectopic 2+ the incorporation of Ca and Pi into nascent mineral deposition. apatitic crystals and inhibits their growth (Ter- The ALPL “knockout” mice were used to keltaub 2001). Studies on the genetic models validate the therapeutic effectiveness of a genet- have identified the key proteins involved in ically engineered ALPL molecule with the ability fi PPi-mediated inhibition of pathologic calci ca- to bind to the bone matrix in vivo. These mice tion. In a mouse model of human progressive showed a remarkable improvement of the bone ankylosis with homozygous mutations in the phenotype, as the hypophosphatasia-associated Ank gene (ank/ank), massive mineral deposi- osteomalacia was prevented (Yadav et al. 2011a). tion is seen in the joints and other soft tissues This approach of enzyme-replacement therapy (Ho et al. 2000). A similar phenotype has been has been successfully used to treat the hypo- seen in ttw/ttw mice, which carry homozygous phosphatasia-associated skeletal abnormalities mutations in the ectonucleotide phosphodies- in human clinical trials (Kitaoka et al. 2017).

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Matrix Gla protein (MGP) and fetuin, two head revealed that the phenotype is caused by proteins involved in the prevention of “soft” tis- the impaired growth of the maxillary and pala- sue mineralization are also present in the skeletal tine bones in comparison to the mandibular hard tissues (Jahnen-Dechent et al. 1997). How- bones. Marulanda et al. (2017) reported that ever, considering that fetuin “knockout” mice do the hypoplastic midface was not a result of cra- not show any overt bone mineralization defects niosynostosis as seen in several other mouse or skeletal anomalies, the current discussion will models of midface hypoplasia, but a result of focus on MGPonly. In humans, the homozygous ectopic mineralization of the cartilaginous part loss-of-function mutations in MGP leads to a of the nasal septum, which normally does not rare genetic disorder known as Keutel syndrome mineralize. This abnormal mineralization in- (Fryns et al. 1984; Munroe et al. 1999). The pa- duced apoptosis of the chondrocytes present tients show abnormal mineralization of their in the nasal septum (Marulanda et al. 2017). cartilaginoustissues leading to midface hypopla- The loss of the matrix synthesizing active chon- sia, shortening of terminal phalanges, tracheo- drocytes during the early growth phase and the bronchial calcification, and vascular abnormali- increased rigidity of the ECM upon mineraliza- ties, including calcification of the arterial media. tion might have impaired the growth of the nasal MGP-deficient mice recapitulated all of these septum and that of the maxillary complex. In- anomalies albeit with a more severe vascular cal- terestingly, the deposited minerals in the septal cification phenotype (Luo et al. 1997). cartilage of the mutant mice show the presence MGP, a small mineral-binding protein is of ACP, suggesting that MGP-mediated inhibi- highly expressed by chondrocytes and vascular tion of mineralization occurs during the early smooth muscle cells (Luo et al. 1995, 1997). Al- phases of mineral precipitation. though MGP was initially purified from the bo- vine bones, its expression is markedly lower in THE ROLE OF INTRACELLULAR ENZYMES IN the trabecular bones in comparison to the pre- SKELETAL MINERALIZATION hypertrophic zone of the developing growth plate (Price and Williamson 1985; Luo et al. Whereas most of the published work on the 1995). This expression pattern suggests that mechanism of cartilage and bone mineralization MGP present in bone might have originated focuses primarily on the extracellular determi- elsewhere (e.g., in the vascular tissues) and nants, newer studies have identified several in- transported to bone via blood. Transgenic over- tracellular enzymes as important regulators of expression of Mgp in the osteoblasts resulted in a this process. The main strength of these studies moderate level of osteomalacia affecting both is the use of in vivo gene ablation models exhib- www.perspectivesinmedicine.org intramembranous and endochondral bones iting obvious “hard” tissue mineralization de- (Murshed et al. 2004). This observation high- fects, which are not caused by the alterations lights the importance of the mechanisms to con- of the known determinants discussed above. trol the expression of mineralization inhibitors The intracellular enzymes, sphingomyelin phos- in the mineralizing skeletal tissues. phodiesterase 3 (SMPD3) and phosphatase or- In agreement with the observation that Mgp phan 1 (PHOSPHO1) reported by these studies is weakly expressed in bone in comparison to the are both involved in the metabolism of phos- cartilaginous tissues, the whole-body ablation of pholipids and/or associated metabolites (Aubin Mgp in mice does not overtly affect bone min- et al. 2005; Macrae et al. 2010). eralization, but shows skeletal anomalies associ- SMPD3 (also known as neutral sphingo- ated with abnormal cartilage mineralization myelinase 2) is a cell membrane–bound lipid- (Marulanda et al. 2017). The most prominent metabolizing enzyme expressed in high amounts of these anomalies is midface hypoplasia, which in the brain, cartilage, and bone. Among various is also seen in the patients with Keutel syn- acid, basic, and neutral sphingomyelinases, only drome. Cephalometric analyses of the micro- SMPD3 deficiency leads to impaired minerali- computed tomography (CT) images of the zation of the skeletal tissues (Stoffel et al. 2005,

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Mechanism of Bone Mineralization

2007; Macrae et al. 2010; Khavandgar and mineralization defects (Holland et al. 2007). Murshed 2015). SMPD3 cleaves sphingomyelin These findings lead to two conclusions: it is pos- present in the cell membrane to generate cer- sible that the ceramides generated from these amide, a bioactive lipid molecule, and phospho- two pathways contribute to different pools choline, an essential nutrient. Emerging data with distinct cellular functions; alternatively, it from the analysis of genetically altered mouse is possible that ceramide does not play a role in models suggest that SMPD3 activity during em- the process of bone mineralization. While at bryonic development plays a critical role in present, no experimental data directly suggests normal skeletogenesis. This insight originally the involvement of ceramide in ECM minerali- came from two different mouse models lacking zation, the importance of phosphocholine in SMPD3. One of these models, fro/fro, carries a this process has been convincingly shown by chemically induced deletion encompassing animal experiments (Macrae et al. 2010; Yadav part of intron 8 and most of exon 9 of the et al. 2011b). Phosphocholine generated from Smpd3 gene, whereas the other mouse model sphingomyelin by SMPD3, or from dietary cho- − − (Smpd3 / ) was generated by the conventional line by two isoforms of choline kinases, can be gene-targeting approach (Aubin et al. 2005; cleaved by PHOSPHO1, an intracellular enzyme Stoffel et al. 2005, 2007). with phosphatase activity. The deficiency of The fro mutation completely abolishes the PHOSPHO1 in mice has been shown to cause enzymatic activity of SMPD3, but does not affect similar bone mineralization defects as seen in its membrane localization (Khavandgar et al. fro/fro mice (Yadav et al. 2011b). 2011). A severe mineralization defect affecting both intramembranous and endochondral bones, and an abnormal delay of apoptosis of INTRACELLULAR REGULATORS OF ECM MINERALIZATION AND THE “MATRIX the hypertrophic chondrocytes during the early VESICLE THEORY” stages of skeletal development are the hallmarks of the skeletal phenotypes in fro/fro mice. Re- Whereas the regulatory roles of two intracellular cently, Li et al. have demonstrated that SMPD3 enzymes, SMPD3 and PHOSPHO1, in ECM activity in both chondrocytes and osteoblasts are mineralization are now well established, the required for a normal bone development (Li question remains how these intracellular en- et al. 2016). The poor mineralization of the skel- zymes may regulate a process that occurs outside etal tissues in fro/fro mice is seen without any the cells. A possible mechanism may involve 2+ alterations of the homeostasis of Ca ,Pi, and matrix vesicles (MVs), which are nanosized – PPi (Khavandgar et al. 2011). This observation (20 200 nm) vesicles released by the cells in a www.perspectivesinmedicine.org suggests that the loss of SMPD3 function affects mineralizing tissue (Anderson et al. 2004, 2005; ECM mineralization through a novel, yet un- Golub 2009; Wu et al. 2002). Since their initial known, mechanism. discoveries as chondrocyte-derived vesicular As mentioned above, SMPD3 cleaves sphin- bodies promoting the initiation of mineral nu- gomyelin to generate many different species of cleation, there have been considerable efforts to ceramides that regulate a myriad of cell func- explain various types of ECM mineralization, tions. SMPD3 activity also liberates an essential including pathologic mineralization of “soft tis- nutrient, phosphocholine. Whereas the role of sues” using the “matrix vesicle theory” (Ander- ceramide is well documented in the regulation of son 1984). Although there is no general consen- apoptosis (Obeid et al. 1993), it is not yet clear sus, it has been suggested that MVs provide an whether both ceramide and phosphocholine or isolated microenvironment to facilitate the ini- only one of these metabolites is involved in skel- tial nucleation of apatite mineral. Mineral crys- etal tissue mineralization. Interestingly, gene tals formed inside the MVs grow progressively 2+ knockout experiments reducing ceramide bio- in size by the addition of Ca and Pi ions and synthesis via an alternative pathway without in- eventually rupture the MV membrane to be de- volving SMPD3 activity did not report any ECM posited on the collagen scaffold.

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Wu et al. (2002) showed extensive phospho- SMPD3 may generate additional phosphocho- lipid degradation in the mineralizing MVs with line to be cleaved by PHOSPHO1. Although a concomitant increase of free fatty acids. This further studies will be needed to establish that observation suggested the presence of phospho- SMPD3 and PHOSPHO1 works in a relay in the lipase activity inside the MVs and demonstrated MVs, such a possibility is supported by the ob- a link between phospholipid metabolism and servation that both PHOSPHO1 and SMPD3 the initiation of ECM mineralization. Phospho- are present in the MV preparations (Mebarek lipase activity generates phosphocholine and et al. 2013). Apart from its role described above, phosphoethanolamine, which can be cleaved SMPD3 might be involved in the biogenesis of by PHOSPHO1 releasing free Pi inside the the MVs. MVs. The increase of Pi inside the MVs may alter the P /PP ratio to favor the seeding of early i i SUMMARY mineral crystals. Being a membrane-bound enzyme with an Based on the discussion above, a unified model intracellular catalytic domain, SMPD3 is expect- for bone mineralization can be proposed. When ed to be present inside the MVs. The cleavage of present at physiologic concentrations, two min- — 2+ — sphingomyelin present in the MV membrane by eral ions Ca and Pi will promote hydroxy-

ALPL SM PPi SMPD3

2Pi Ca2+ Phosphocholine

PHOSPHO1

2+ Ca Pi + Choline

HA

www.perspectivesinmedicine.org Collagen

2+ Figure 1. Model of bone mineralization. Serum calcium (Ca ), inorganic phosphate (Pi) levels, and a mineral scaffolding collagen-rich extracellular matrix (ECM) are important determinants of bone mineralization. Alka- line phosphatase (ALPL), an ectoenzyme tethered to the osteoblast cell membrane, cleaves inorganic pyrophos- phate (PPi), a small, but potent mineralization inhibitor. This facilitates bone ECM mineralization in two ways: fi rst, it reduces the level of a mineralization inhibitor, and second, in the process generates Pi, an activator of ECM mineralization. This coupled ALPL activity alters the Pi/PPi ratio in the bone microenvironment to favor bone mineralization. Compact hierarchical assembly of collagen molecules in the fibrils and fibers results in both intra- fi 2+ and inter brillar nanoscale gaps. These gaps are accessible by Ca and Pi ions, but not by the large protein inhibitors of ECM mineralization. This may explain why there are mineral deposits both inside and in the gaps in between the collagen fibrils. Matrix vesicle (MV)–mediated mineralization may serve as an auxiliary mechanism for bone mineralization. These nanoscale vesicles carrying the intracellular mineralization-promoting enzymes bud off from the mineralizing cells. Enzymes like SMPD3 and phospholipases present inside the MVs may cleave the phospholipids (e.g., sphingomyelin [SM]) to generate phosphocholine, which in turn can be cleaved by another cytosolic enzyme PHOSPHO1 liberating free Pi. An increase of intravesicular Pi leads to its precipitation with Ca2+ to form the nascent hydroxyapatite (HA) crystals.

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Mechanism of Bone Mineralization

apatite crystal growth within and between the Bachra BN, Fischer HR. 1968a. Mineral deposition in colla- 2: – newly synthesized collagen fibrils in the skeletal gen in vitro. Calcif Tissue Res 343 352. Bachra BN, Fischer HR. 1968b. Recalcification of decalcified ECM. PPi, a chemical derivative of Pi, can inhibit bone collagen in vitro as a model for biologic calcification. the mineralization process. The presence of a Calcif Tissue Res 2: 7. scaffolding matrix and a defined extracellular Bai X, Miao D, Li J, Goltzman D, Karaplis AC. 2004. Trans- fi ratio of Pi to PPi are two critical determinants genic mice overexpressing human broblast growth factor of ECM mineralization. ALPL, an ectoenzyme 23 (R176Q) delineate a putative role for parathyroid hor- mone in renal phosphate wasting disorders. Endocrinol- bound to the osteoblast cell membrane, cleaves ogy 145: 5269–5279. PPi to generate Pi and alters the local Pi to PPi Barros NM, Hoac B, Neves RL, Addison WN, Assis DM, ratio to favor mineral precipitation (Fig. 1). Murshed M, Carmona AK, McKee MD. 2013. Proteolytic The above model suggests that the specificity processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine of skeletal mineralization can be explained, in model of X-linked hypophosphatemia. J Bone Miner Res part, by the unique coexpression of tissue-non- 28: 688–699. specific genes encoding type I collagen and Becker GL, Termine JD, Eanes ED. 1976. Comparative stud- ALPL. Indeed as in vivo proof for this hypoth- ies of intra- and extramitochondrial calcium phosphates from the hepatopancreas of the blue crab (Callinectes esis, it was demonstrated that when the PPi sapidus). Calcif Tissue Res 21: 105–113. cleaving enzyme, ALPL, was mis-expressed in Becker A, Ziegler A, Epple M. 2005. 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Mechanism of Bone Mineralization

Monzur Murshed

Cold Spring Harb Perspect Med published online April 2, 2018

Subject Collection Bone: A Regulator of Physiology

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