mechanism of bone mineralization - cshl p

Mechanism of Bone Mineralization

Monzur Murshed1,2,3

1Faculty of Dentistry, McGill University, Montreal, Quebec H3A 1G1, Canada2Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QuebecH4A 3J1, Canada

3Shriners Hospital for Children, Montreal, Quebec H4A 0A9, Canada

Correspondence: [email protected]

Mineralized “hard” tissues of the skeleton possess unique biomechanical properties tosupport the body weight and movement and act as a source of essential minerals requiredfor critical body functions. For a long time, extracellular matrix (ECM) mineralization in thevertebrate skeleton was considered as a passive process. However, the explosion of geneticstudies during the past decades has established that this process is essentially controlled bymultiple genetic pathways. These pathways regulate the homeostasis of ionic calcium andinorganic phosphate—two mineral components required for bone mineral formation, thesynthesis of mineral scaffolding ECM, and the maintainence of the levels of the inhibitoryorganic and inorganic molecules controlling the process of mineral crystal formation and itsgrowth. More recently, intracellular enzyme regulators of skeletal tissue mineralization havebeen identified. The current review will discuss the key determinants of ECMmineralizationin bone and propose a unified model explaining this process.

Themineralized extracellular matrix (ECM) isa unique feature of the vertebrate skeletoden-

tal system. The massive load-bearing capacity ofthe mineralized skeleton permitted the evolu-tionary emergence of large vertebrates, such asblue whales, which can weigh up to 180 tons.However, the functions of the mineralized tis-sues are not limited to support the body mass,protect the internal soft organs, or to facilitatelocomotion and mastication only; they alsoserve as a readily accessible reservoir for essen-tial minerals that are indispensable for manyphysiologic activities. The current review willfocus on the mechanism of calcium phosphatebiomineralization in the vertebrate skeleton.

The origin of biomineralization has beentraced back to the late Precambrian period aftertectonic activities caused a marked increase ofsoluble minerals in the seawater (Wagner andAspenberg 2011). It is commonly believed thatmarine organisms first developed primitive exo-skeletons made up of calcium carbonate and/orcalcium phosphate minerals (Knoll 2003; Wag-ner and Aspenberg 2011). As part of the processof evolutionary adaptations, the skeletal tissueswere internalized, which paved the way for theemergence of organisms with larger body sizes.Whereas it is not clear what prompted someprimitive organisms to deposit calcium phos-phate instead of calcium carbonate minerals in

Editors: Gerard Karsenty and David T. ScaddenAdditional Perspectives on Bone: A Regulator of Physiology available at www.perspectivesinmedicine.org

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their skeletal tissues, it is conceivable that thedeposited calcium phosphate minerals providedcertain physiological advantages. Indeed it hasbeen suggested that the presence of calciumphosphate minerals makes the skeleton morestable under acidic conditions (Wagner and As-penberg 2011). Vertebrate organisms rely onATP generation via anaerobic glycolysis for sud-den rapid movements during “fight-or-flight”situations. The activation of this pathway resultsin an acidic tissue environment, which wouldhave destabilized a skeleton made up of calciumcarbonate more readily.

MINERAL COMPOSITION OF BONE

In 1771, Scheele first reported the presence ofcalcium phosphate minerals in bone (Scheele1931). Later, bone mineral was described as aform of hydroxyapatite [(Ca)10 (PO4)6(OH)2]comparable to geological apatite (a group ofphosphate minerals) by both chemical compo-sition and X-ray diffraction pattern analyses (seeEliaz and Metoki 2017 for a comprehensive re-view on the historical perspective). However,subsequent studies demonstrated that mineralsin bone do not have a uniform composition (Reyet al. 1995; Pasteris et al. 2004). Although thereare still some disagreements about the initialphase of the deposited mineral and its time-de-pendent transition to apatite, it is now acceptedthat the matured bone mineral is a substitutedcrystalline phase of calcium phosphate, referredto as carbonated hydroxyapatite (Mahamid et al.2008). High-resolution transmission electronmicroscopy (TEM), 3D stereoscopic TEM, andatomic force microscopy on organic matrix-freebone samples provided accurate measurementsof bone crystals. These studies have firmly estab-lished that bone crystals are nanosized, long, andvery thin platelets (Rey et al. 2009).

The source of the dispute regarding the earlymineral phase in bone appears to be a simple invitro experiment performed in themid-sixties inthe laboratory of Dr. Posner (Boskey 1997). Themixing of concentrated solutions of calciumchloride and sodium acid phosphate resultedin the precipitation of calcium phosphate salts,which showed a broad and diffused pattern by

X-ray diffraction analysis. This pattern suggest-ed that the precipitated mineral is amorphouscalcium phosphate (ACP), not apatite. Interest-ingly, when X-ray diffraction analyses were per-formed on the same samples 2 days later, theprecipitates were found to be a poorly formedcrystalline apatite (Boskey 1997). This initialfinding of mineral phase transition in vitroprompted follow-up experiments using miner-alized tissue samples and, subsequently, thepresence of ACP in the embryonic chick boneswas reported by the same group (Eanes et al.1965). However, the controversy on whetherbone contains ACP continued as later studiesconcluded that the embryonic bones do not con-tain ACP (Bonar et al. 1983; Grynpas et al.1984). Since then, more sophisticated analysesof bone minerals using Raman spectra, Fourier-transform infrared spectroscopy (FTIR), andsynchrotron-generated X-ray diffraction tech-niques have been performed; however, a consen-sus on the issue is still elusive (Rey et al. 2009).

The presence of amorphous mineral phasehas been reported in some invertebrates (Beckeret al. 1976, 2005). Additionally, ACP has beendetected at the sites of ectopic calcification in thevertebrates (Marulanda et al. 2017). In two stud-ies published by Mahamid et al. (2008, 2010),ACP has been identified as a major mineralphase in zebrafish fin bones, suggesting thatamorphous to crystalline phase transition oc-curs in vivo. On the other hand, concerns havebeen raised about this latter finding on theground of technical limitations, including pos-sible mineral phase transition during samplecollection and processing (Rey et al. 2009).

DETERMINANTS OF SKELETAL TISSUEMINERALIZATION

In human embryos, although primary ossifica-tion centers of endochondral bones (e.g., verte-brae and long bones) appear between 8 and 12weeks of gestation, the bulk mineralization ofskeletal tissues does not occur until the thirdtrimester (Kovacs 2003). The type I collagen-rich ECM in the intramembranous bones min-eralizes directly, whereas in the endochondralbones, mineralization starts concomitantly at

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two sites: within the core region of the cartilageanlagen and along the bone collar surroundingit (Karsenty andWagner 2002). In the cartilage,the type X collagen matrix synthesized by thehypertrophic chondrocytes serves as themineralscaffold, whereas in the bone collar (and laterin the trabecular bones), the primary mineralscaffolding protein is the osteoblast-derivedtype I collagen (Karsenty and Wagner 2002).A brief discussion on the key determinants reg-ulating skeletal ECM mineralization is pre-sented below.

Systemic Levels of Calciumand Phosphate Ions

The chemical structure of bone mineral impliesthat the extracellular levels of ionic calcium(Ca2+) and inorganic phosphate (Pi) will betwo critical determinants for bone mineraliza-tion. Indeed, data from patients and animalmodels of human diseases clearly demonstratesthat the reduction of systemic Pi levels with orwithout any alteration of the Ca2+ levels lead toosteomalacia with the characteristic increase ofunmineralized osteoid volume. The importanceof systemic levels of these mineral ions in bonemineralization was demonstrated by impair-ment of the 1,25 dihydroxy vitaminD3 signalingpathway. For example, mutations in 25, hydroxyD3-1 α-hydroxylase (required for functional vi-tamin D synthesis) or inactivating mutations invitamin D receptor, impair the absorption ofCa2+ and Pi in the gut (Dardenne et al. 2001;Masuyama et al. 2001; Panda et al. 2001). Addi-tionally, these mutations also reduce the mobi-lization of these ions from the bone by restrictingbone resorption (Suda et al. 1992).

Although Ca2+ and Pi are both integral partsof bone mineral, genetic experiments in micesuggest that circulating Pi may have a moreprominent role in the regulation of bone miner-alization. For example, in Hyp mice with a mu-tation in the Phex gene (a model of human X-linked hypophosphatemia [XLH]), the calciumlevel is normal, but the systemic Pi level is re-duced to almost half, causing a severe osteoma-lacia phenotype (Eicher et al. 1976; Costa et al.1981). The inactivationofPHEX in theHypmice

or XLH patients leads to an increase of circulat-ing fibroblast growth factor 23 (FGF23) pro-duced by osteoblasts and osteocytes (Xia et al.2007). Increased FGF23, in turn, down-regulatesthe expression of sodium phosphate transport-ers in the kidney tubules preventing Pi reabsorp-tion and causing hypophosphatemia. As expect-ed, in ApoE-Fgf23 transgenic mice in whichsystemic FGF23 level increases in the absenceof any PHEXmutations, a reduction of systemicPi level and osteomalacia comparable to that ofHyp mice have been reported (Bai et al. 2004).The similarity of the osteomalacia traits inApoE-Fgf23 and Hyp mice suggests that the accompa-nied hypophosphatemia is the main cause of thephenotype in these two models.

More recently, osteopontin, a protein be-longing to the family of small integrin-bindingligand, N-linked glycoproteins (SIBLINGs), hasbeen shown as a substrate for PHEX (Barroset al. 2013). It has been proposed that in theabsence of PHEX, accumulation of osteopontinin the ECM may contribute to the “hard” tissuemineralization defects seen in the XLH patientsand in Hyp mice. Another SIBLING protein,dentin matrix protein 1 (DMP1), has beenshown to be an important regulator of circulat-ing FGF23 levels (Martin et al. 2011). As is thecase with the inactivating mutations in PHEX,DMP1 deficiency leads to severe osteomalacia.

A severe osteomalacia phenotype is also seenin the calcium sensing receptor (CaSR) knock-outmice. The ablation of theCasr gene leads to amarked increase of systemic parathyroid hor-mone (PTH) levels leading to hypercalcemia ac-companied by hypophosphatemia as a result ofincreased urinary excretion of Pi (Tu et al. 2003).Interestingly, the observed increase of Ca2+ inthe circulation failed to prevent osteomalaciain these mice. On the other hand, an oppositephenotype has been seen in mice lacking glialcell missing 2 (GCM2), a transcription factorrequired for the development of the parathyroidgland (Gunther et al. 2000). The ablation ofGcm2 impairs the development of the parathy-roid gland causing a marked reduction of PTHlevel in the circulation. A basal PTH level ismaintained as the thymus acts as a secondarysource for the hormone. As expected, the sur-


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vivingGcm2−/−mice show a reduction of serumCa2+ levels and an increase of serum Pi levels.However, despite the significant reduction ofsystemic Ca2+ levels, no osteomalacia was ob-served in these mice (Gunther et al. 2000).

Currently, a mouse model showing isolatedhypocalcemia with a normal serum Pi level isunavailable. Considering that Ca2+ is an integralcomponent of bone mineral, it is possible thatsuch a model of isolated hypocalcemia, if exist-ed, would have shown impaired bone minerali-zation. However, phenotypic comparisons ofthe mouse models with abnormal Ca2+ and/orPi homeostasis discussed above suggest that theosteomalacia phenotype in a model of isolatedhypocalcemia may not be as severe as seen inisolated hypophosphatemia. Because of the crit-ical role of Ca2+ in many physiologic activities,its serum level ismore tightly regulated than thatof Pi. Moreover, ubiquitously present inorganicpyrophosphate (PPi), a potent inhibitor of ECMmineralization, is a Pi derivative (see below)(Terkeltaub 2001). These observations suggestthat early organisms depositing calcium phos-phate minerals in the skeletal tissues evolved toregulate ECMmineralization by modulating thePi levels and its incorporation into the growingmineral crystals.

Collagen Scaffold

The organic part of the bone ECM is primarilycomposed of type I collagen. Two genes, Col1a1andCol1a2, encode the α1 and α2 chains of typeI collagen, respectively. Two α1 and one α2chains assemble together to form the collagentriple helix in the extracellular spaces of the bonemicroenvironment. In a hierarchical fashion,these helices are first arranged axially in a stag-gered manner as collagen fibrils, which are thenbundled together to form the collagen fibers(Shoulders and Raines 2009). In a healthy indi-vidual, the mineralization of the unmineralizedcollagen (osteoid) occurs seamlessly in contin-uation of the existing mineralized matrix onwhich the newly synthesized osteoid is depos-ited by the osteoblasts.

The involvement of collagen in bone miner-alization came from the initial electron micros-

copy of the mineralizing bones from newbornmice by Sheldon and Robinson (1957). Al-though earlier studies provided circumstantialevidence that mineral crystals are depositedwithin the collagen fibrils, this study first dem-onstrated that there are two sites on the collagenmatrix in bone where mineral deposition starts(1) at the intrafibrillar gap spaces, where the car-boxy- and amino-terminal ends of two seriallyarranged collagen triple helices meet, and (2)interfibrillar spaces between the fibrils (Sheldonand Robinson 1957). Since then, numerousstudies have confirmed the critical role of colla-gen matrix as a scaffold for bone mineraldeposition. These include the initial studiesdemonstrating that collagen sponges can bemineralized in vitro, implanted collagen canmineralize in vivo, and demineralized bone col-lagen can be mineralized under a cell-free con-dition (Mergenhagen et al. 1960; Bachra andFischer 1968a,b). More recently, genetic experi-ments have demonstrated that the reduction ofcollagen synthesis in bone results in a reductionof mineralized bone mass (Yang et al. 2004).

The exact mechanism by which the collagenlattice facilitates mineral deposition is still un-known. The amino acid side chains exposed atthe intrafibrillar gap space and at the interfibril-lar space may regulate this process. However, itis possible that the dense packaging of collagenmolecules and their hierarchical organization,rather than the primary structure of the proteinis the driving force underlying the nucleation ofhydroxyapatite. Indeed, one theory attempts toexplain the mineralization of bone ECM by thesize-exclusion properties of the collagen scaffold(Toroian et al. 2007; Price et al. 2009). Accord-ing to this theory, the nanoscale gaps presentwithin a collagen fibril and in between the fibrilsarranged in a fiber allow the access of Ca2+ andPi ions, but not the large proteins, which caninhibit the formation and growth of the nascenthydroxyapatite crystals inside the scaffold.

Mineralization Inhibitors

Considering that the concentrations of variousions in all the tissues of the body are at equilib-rium to that of blood, it is likely that Ca2+ and Pi

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levels do not differ significantly between the“soft” and skeletal “hard” tissues. This may raisethe question—why do some “soft” tissues, suchas blood vessels and some cartilaginous tissues,never mineralize despite their high collagencontent? The most straightforward answer tothis question can be obtained from several hu-man diseases of pathologic “soft” tissue miner-alization and their animal models. For example,generalized arterial calcification in infancy(GACI) and progressive ankylosis in humansare both caused by the reduced level of PPi inthe bone joints (Ho et al. 2000; Albright et al.2015). PPi is a mineralization inhibitor that isknown for its potent antimineralization proper-ties for over 50 years (Fleisch and Bisaz 1962).A wide distribution of the PPi synthesis and/or extracellular transport machinery in verte-brate organisms prevents pathologic “soft” tis-sue mineralization.

PPi is composed of two inorganic Pi groupsjoined by an ester linkage (Terkeltaub 2001).This inorganic mineralization inhibitor is pro-duced both intracellularly and extracellularlyvia enzymatic reactions. Intracellularly, PPican be generated as a byproduct of enzymaticactivities in numerous metabolic pathways. In-tracellular PPi is transported to the extracellularspace through a transmembrane transportercalled ANK (Ho et al. 2000). On the otherhand, extracellularly, a direct cleavage of thephosphodiester bond in purine and pyrimidinenucleoside triphosphates (e.g., ATP) by nucle-otide pyrophosphatase/phosphodiesterase en-zymes can also generate PPi (Hessle et al.2002).

At a threshold concentration, PPi preventsthe incorporation of Ca2+ and Pi into nascentapatitic crystals and inhibits their growth (Ter-keltaub 2001). Studies on the genetic modelshave identified the key proteins involved inPPi-mediated inhibition of pathologic calcifica-tion. In a mouse model of human progressiveankylosis with homozygous mutations in theAnk gene (ank/ank), massive mineral deposi-tion is seen in the joints and other soft tissues(Ho et al. 2000). A similar phenotype has beenseen in ttw/ttw mice, which carry homozygousmutations in the ectonucleotide phosphodies-

terase nucleotide pyrophosphatase I (Enpp1)gene (Koshizuka et al. 2001).

Interestingly, both Ank and Enpp1 genes arehighly expressed in bone,more specifically in theosteoblasts (Murshed et al. 2005). Although thepresence of a mineralization inhibitor in bonemay appear counterintuitive, this quandary canbe explained by the presence of a strong alkalinephosphatase (ALPL) activity in bone. The car-dinal role of ALPL during skeletal mineraliza-tion in humans was demonstrated by many dif-ferent mutations identified in the ALPL gene inhypophosphatasia patients, causing awide rangeof phenotypic severity. Some of these patientsshow very severe osteomalacia and fetal/perina-tal lethality, while others show milder, progres-sive osteomalacia later in life (Mornet 2000; Tail-landier et al. 2000, 2001). The genetic modelslacking ALPL activity further confirmed the re-quirement of this enzyme for bone mineraliza-tion (Fedde et al. 1999; Anderson et al. 2004).

ALPL-mediated hydrolysis of PPi has twoimplications—first, it reduces the amount ofPPi in the bone microenvironment and, second,it increases the amount of mineralization-pro-moting Pi ions by liberating them from PPi.These coupled activities alter the Pi/PPi ratio inthe bone ECM in such a way that the formationand growth of the apatite crystal is promoted(Hessle et al. 2002; Murshed et al. 2005). Thecritical effect of this ratio in regulating ECMmineralization has been demonstrated in ank/ank;Hyp compound-mutant mice, which showa marked reduction of joint mineralization(Murshed et al. 2005). The low extracellular Pi/PPi ratio in these mice in comparison to theoriginal ank/ank mice prevented the ectopicmineral deposition.

The ALPL “knockout” mice were used tovalidate the therapeutic effectiveness of a genet-ically engineered ALPLmolecule with the abilityto bind to the bone matrix in vivo. These miceshowed a remarkable improvement of the bonephenotype, as the hypophosphatasia-associatedosteomalaciawas prevented (Yadav et al. 2011a).This approach of enzyme-replacement therapyhas been successfully used to treat the hypo-phosphatasia-associated skeletal abnormalitiesin human clinical trials (Kitaoka et al. 2017).



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Matrix Gla protein (MGP) and fetuin, twoproteins involved in the prevention of “soft” tis-suemineralization are also present in the skeletalhard tissues (Jahnen-Dechent et al. 1997). How-ever, considering that fetuin “knockout”mice donot show any overt bone mineralization defectsor skeletal anomalies, the current discussion willfocus onMGPonly. Inhumans, thehomozygousloss-of-function mutations in MGP leads to arare genetic disorder known as Keutel syndrome(Fryns et al. 1984; Munroe et al. 1999). The pa-tients show abnormal mineralization of theircartilaginous tissues leading tomidface hypopla-sia, shortening of terminal phalanges, tracheo-bronchial calcification, and vascular abnormali-ties, including calcification of the arterial media.MGP-deficient mice recapitulated all of theseanomalies albeit with amore severe vascular cal-cification phenotype (Luo et al. 1997).

MGP, a small mineral-binding protein ishighly expressed by chondrocytes and vascularsmooth muscle cells (Luo et al. 1995, 1997). Al-though MGP was initially purified from the bo-vine bones, its expression is markedly lower inthe trabecular bones in comparison to the pre-hypertrophic zone of the developing growthplate (Price and Williamson 1985; Luo et al.1995). This expression pattern suggests thatMGP present in bone might have originatedelsewhere (e.g., in the vascular tissues) andtransported to bone via blood. Transgenic over-expression ofMgp in the osteoblasts resulted in amoderate level of osteomalacia affecting bothintramembranous and endochondral bones(Murshed et al. 2004). This observation high-lights the importance of themechanisms to con-trol the expression of mineralization inhibitorsin the mineralizing skeletal tissues.

In agreement with the observation thatMgpis weakly expressed in bone in comparison to thecartilaginous tissues, the whole-body ablation ofMgp in mice does not overtly affect bone min-eralization, but shows skeletal anomalies associ-ated with abnormal cartilage mineralization(Marulanda et al. 2017). The most prominentof these anomalies is midface hypoplasia, whichis also seen in the patients with Keutel syn-drome. Cephalometric analyses of the micro-computed tomography (CT) images of the

head revealed that the phenotype is caused bythe impaired growth of the maxillary and pala-tine bones in comparison to the mandibularbones. Marulanda et al. (2017) reported thatthe hypoplastic midface was not a result of cra-niosynostosis as seen in several other mousemodels of midface hypoplasia, but a result ofectopic mineralization of the cartilaginous partof the nasal septum, which normally does notmineralize. This abnormal mineralization in-duced apoptosis of the chondrocytes presentin the nasal septum (Marulanda et al. 2017).The loss of the matrix synthesizing active chon-drocytes during the early growth phase and theincreased rigidity of the ECM upon mineraliza-tionmight have impaired the growth of the nasalseptum and that of the maxillary complex. In-terestingly, the deposited minerals in the septalcartilage of the mutant mice show the presenceof ACP, suggesting that MGP-mediated inhibi-tion of mineralization occurs during the earlyphases of mineral precipitation.

THE ROLE OF INTRACELLULAR ENZYMES INSKELETAL MINERALIZATION

Whereas most of the published work on themechanism of cartilage and bonemineralizationfocuses primarily on the extracellular determi-nants, newer studies have identified several in-tracellular enzymes as important regulators ofthis process. The main strength of these studiesis the use of in vivo gene ablation models exhib-iting obvious “hard” tissue mineralization de-fects, which are not caused by the alterationsof the known determinants discussed above.The intracellular enzymes, sphingomyelin phos-phodiesterase 3 (SMPD3) and phosphatase or-phan 1 (PHOSPHO1) reported by these studiesare both involved in the metabolism of phos-pholipids and/or associated metabolites (Aubinet al. 2005; Macrae et al. 2010).

SMPD3 (also known as neutral sphingo-myelinase 2) is a cell membrane–bound lipid-metabolizing enzyme expressed in high amountsin the brain, cartilage, and bone. Among variousacid, basic, and neutral sphingomyelinases, onlySMPD3 deficiency leads to impaired minerali-zation of the skeletal tissues (Stoffel et al. 2005,

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2007; Macrae et al. 2010; Khavandgar andMurshed 2015). SMPD3 cleaves sphingomyelinpresent in the cell membrane to generate cer-amide, a bioactive lipid molecule, and phospho-choline, an essential nutrient. Emerging datafrom the analysis of genetically altered mousemodels suggest that SMPD3 activity during em-bryonic development plays a critical role innormal skeletogenesis. This insight originallycame from two different mouse models lackingSMPD3. One of these models, fro/fro, carries achemically induced deletion encompassingpart of intron 8 and most of exon 9 of theSmpd3 gene, whereas the other mouse model(Smpd3−/−) was generated by the conventionalgene-targeting approach (Aubin et al. 2005;Stoffel et al. 2005, 2007).

The fro mutation completely abolishes theenzymatic activity of SMPD3, but does not affectits membrane localization (Khavandgar et al.2011). A severe mineralization defect affectingboth intramembranous and endochondralbones, and an abnormal delay of apoptosis ofthe hypertrophic chondrocytes during the earlystages of skeletal development are the hallmarksof the skeletal phenotypes in fro/fro mice. Re-cently, Li et al. have demonstrated that SMPD3activity in both chondrocytes and osteoblasts arerequired for a normal bone development (Liet al. 2016). The poor mineralization of the skel-etal tissues in fro/fro mice is seen without anyalterations of the homeostasis of Ca2+, Pi, andPPi (Khavandgar et al. 2011). This observationsuggests that the loss of SMPD3 function affectsECM mineralization through a novel, yet un-known, mechanism.

As mentioned above, SMPD3 cleaves sphin-gomyelin to generate many different species ofceramides that regulate a myriad of cell func-tions. SMPD3 activity also liberates an essentialnutrient, phosphocholine. Whereas the role ofceramide is well documented in the regulation ofapoptosis (Obeid et al. 1993), it is not yet clearwhether both ceramide and phosphocholine oronly one of these metabolites is involved in skel-etal tissue mineralization. Interestingly, geneknockout experiments reducing ceramide bio-synthesis via an alternative pathway without in-volving SMPD3 activity did not report any ECM

mineralization defects (Holland et al. 2007).These findings lead to two conclusions: it is pos-sible that the ceramides generated from thesetwo pathways contribute to different poolswith distinct cellular functions; alternatively, itis possible that ceramide does not play a role inthe process of bone mineralization. While atpresent, no experimental data directly suggeststhe involvement of ceramide in ECM minerali-zation, the importance of phosphocholine inthis process has been convincingly shown byanimal experiments (Macrae et al. 2010; Yadavet al. 2011b). Phosphocholine generated fromsphingomyelin by SMPD3, or from dietary cho-line by two isoforms of choline kinases, can becleaved by PHOSPHO1, an intracellular enzymewith phosphatase activity. The deficiency ofPHOSPHO1 in mice has been shown to causesimilar bone mineralization defects as seen infro/fro mice (Yadav et al. 2011b).

INTRACELLULAR REGULATORS OF ECMMINERALIZATION AND THE “MATRIXVESICLE THEORY”

Whereas the regulatory roles of two intracellularenzymes, SMPD3 and PHOSPHO1, in ECMmineralization are now well established, thequestion remains how these intracellular en-zymesmay regulate a process that occurs outsidethe cells. A possible mechanism may involvematrix vesicles (MVs), which are nanosized(20–200 nm) vesicles released by the cells in amineralizing tissue (Anderson et al. 2004, 2005;Golub 2009; Wu et al. 2002). Since their initialdiscoveries as chondrocyte-derived vesicularbodies promoting the initiation of mineral nu-cleation, there have been considerable efforts toexplain various types of ECM mineralization,including pathologic mineralization of “soft tis-sues” using the “matrix vesicle theory” (Ander-son 1984). Although there is no general consen-sus, it has been suggested that MVs provide anisolated microenvironment to facilitate the ini-tial nucleation of apatite mineral. Mineral crys-tals formed inside the MVs grow progressivelyin size by the addition of Ca2+ and Pi ions andeventually rupture the MV membrane to be de-posited on the collagen scaffold.



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Wu et al. (2002) showed extensive phospho-lipid degradation in the mineralizing MVs witha concomitant increase of free fatty acids. Thisobservation suggested the presence of phospho-lipase activity inside theMVs and demonstrateda link between phospholipid metabolism andthe initiation of ECM mineralization. Phospho-lipase activity generates phosphocholine andphosphoethanolamine, which can be cleavedby PHOSPHO1 releasing free Pi inside theMVs. The increase of Pi inside the MVs mayalter the Pi/PPi ratio to favor the seeding of earlymineral crystals.

Being a membrane-bound enzyme with anintracellular catalytic domain, SMPD3 is expect-ed to be present inside the MVs. The cleavage ofsphingomyelin present in theMVmembrane by

SMPD3 may generate additional phosphocho-line to be cleaved by PHOSPHO1. Althoughfurther studies will be needed to establish thatSMPD3 and PHOSPHO1 works in a relay in theMVs, such a possibility is supported by the ob-servation that both PHOSPHO1 and SMPD3are present in the MV preparations (Mebareket al. 2013). Apart from its role described above,SMPD3 might be involved in the biogenesis ofthe MVs.

SUMMARY

Based on the discussion above, a unified modelfor bone mineralization can be proposed. Whenpresent at physiologic concentrations, two min-eral ions—Ca2+ and Pi—will promote hydroxy-

PPi

ALPL

Collagen

SM SMPD3

Phosphocholine

PHOSPHO1

HA

Pi + Choline

2PiCa2+

Ca2+

Figure 1. Model of bone mineralization. Serum calcium (Ca2+), inorganic phosphate (Pi) levels, and a mineralscaffolding 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:first, it reduces the level of amineralization inhibitor, and second, in the process generates Pi, an activator of ECMmineralization. This coupled ALPL activity alters the Pi/PPi ratio in the bone microenvironment to favor bonemineralization. Compact hierarchical assembly of collagenmolecules in the fibrils and fibers results in both intra-and interfibrillar nanoscale gaps. These gaps are accessible by Ca2+ and Pi ions, but not by the large proteininhibitors of ECMmineralization. This may explain why there aremineral deposits both inside and in the gaps inbetween the collagen fibrils. Matrix vesicle (MV)–mediated mineralization may serve as an auxiliary mechanismfor bone mineralization. These nanoscale vesicles carrying the intracellular mineralization-promoting enzymesbud off from themineralizing cells. Enzymes like SMPD3 and phospholipases present inside theMVsmay cleavethe phospholipids (e.g., sphingomyelin [SM]) to generate phosphocholine, which in turn can be cleaved byanother cytosolic enzyme PHOSPHO1 liberating free Pi. An increase of intravesicular Pi leads to its precipitationwith Ca2+ to form the nascent hydroxyapatite (HA) crystals.

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apatite crystal growth within and between thenewly synthesized collagen fibrils in the skeletalECM. PPi, a chemical derivative of Pi, can inhibitthe mineralization process. The presence of ascaffolding matrix and a defined extracellularratio of Pi to PPi are two critical determinantsof ECM mineralization. ALPL, an ectoenzymebound to the osteoblast cell membrane, cleavesPPi to generate Pi and alters the local Pi to PPiratio to favor mineral precipitation (Fig. 1).

The abovemodel suggests that the specificityof skeletal mineralization can be explained, inpart, by the unique coexpression of tissue-non-specific genes encoding type I collagen andALPL. Indeed as in vivo proof for this hypoth-esis, it was demonstrated that when the PPicleaving enzyme, ALPL, was mis-expressed inthe dermis and arterial media, tissues rich infibrillar collagen, it caused rapid ECM mineral-ization (Murshed et al. 2005). However, itshould be noted that this finding does not ruleout the existence of auxiliarymechanisms work-ing together tomineralize the skeletal ECM. Thepossibility of additional mechanism(s) is sup-ported by the fact that both SMPD3- andPHOSPHO1-deficient mice show bone miner-alization defects. These enzymes may work inconcert to increase the Pi levels to facilitate thenucleation of minerals inside the MVs (Fig. 1).

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2, 20182018; doi: 10.1101/cshperspect.a031229 originally published online AprilCold Spring Harb Perspect Med

Monzur Murshed Mechanism of Bone Mineralization

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