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 DOI: 10.1177/10454411000110030101

2000 11: 279CROBMJ. Sodek, B. Ganss and M.D. McKee

Osteopontin  

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OSTEOPONTIN

J. Sodek1'2*B. Ganss1M.D. McKeel3

1MRC Group in Periodontal Physiology, Faculty of Dentistry, Room 239 Fitzgerald Building, and the 2Department of Biochemistry, University of Toronto, 150 (ollege Street, Toronto, ON,(anada M5S 3E2; and the 3Faculty of Dentistry and the Department of Anatomy and (ell Biology, McGill University, Montr6al, Canada; *corresponding author, joro.sodek@utoronto.ca

ABSTRACT: Osteopontin (OPN) is a highly phosphorylated sialoprotein that is a prominent component of the mineralizedextracellular matrices of bones and teeth. OPN is characterized by the presence of a polyaspartic acid sequence and sites ofSerlThr phosphorylation that mediate hydroxyapatite binding, and a highly conserved RGD motif that mediates cell attach-ment/signaling. Expression of OPN in a variety of tissues indicates a multiplicity of functions that involve one or more of theseconserved motifs. While the lack of a clear phenotype in OPN "knockout" mice has not established a definitive role for OPN inanv tissue, recent studies have provided some novel and intriguing insights into the versatility of this enigmatic protein indiverse biological events, including developmental processes, wound healing, immunological responses, tumorigenesis, boneresorption, and calcification. The ability of OPN to stimulate cell activity through multiple receptors linked to several interac-tive signaling pathways can account for much of the functional diversity. In this review, we discuss the structural features ofQPN that relate to its function in the formation, remodeling, and maintenance of bones and teeth.

Key words. Sialoprotein, cell signaling, cell attachment, bone remodeling, tooth.

(1) IntroductionOsteopontin (OPN) was first described as a secreted,

60-kDa transformation-specific phosphoprotein(Senger et al.! 1979) and subsequently was rediscovered bymolecular cloning of the transformation-associated gene2ar (Craig et a).l 1989). Meanwhile, OPN was identifiedindependently, together with bone sialoprotein (BSP), as amajor sialoprotein in the extracellular matrix of bone(Franzen and Hei negard, 1985; Fisher et al., 1987; Prince etal) 1987; Zhang et £l., 1990) and the 2 proteins initiallycalled bone sialoprotein (BSP I) and bone sialoprotein 11(BSP 11), respectively (Franz6n and Heinegard, 1985). Thename osteopontin" was introduced to reflect the poten-tial of the bone protein to serve as a bridge between cellsand hydroxyapatite through RGD and polyaspartic acidmotifs discovered in the primary sequence of the protein(Oldberg et Li) 1986). However, the same gene product wasicentified as a putative lymphokine produced by activatedlymphocytes and macrophages and called Eta-1 (early T-lymphocyte activation gene I; Palarca et al., 1989), andthus a more general pattern of expression for OPN waseinerging. Accordingly, secreted phosphoprotein (SPP 1)was introduced as an alternate narne, to reflect a broaderfuinctional role of this protein. Nevertheless, the nameosteopontin has largely been retained, in keeping withthe nomenclat-ure used for the human gene (Denhardt etail 195Q0.

In the past five years, several excellent reviews ofOPN and its potential significance in mineralized tissues(Butler et al., 1996; McKee and Nanci, 1996a, Denhardtand Noda, 1998; Gerstenfeld, 1999), the vascular system(Giachelli et al., 1995), the immune system (Weber andCantor, 1996), kidney (Rittling and Denhardt, 1999), andin cellular transformation and cancer (Oates et £4 1 997)have been published. Consequently, this review providesonly a brief overview of the general properties of OPNand focuses on the structure-function relationshipstogether with the implications of recent findings on therole of OPN in the formation and remodeling of mineral-ized connective tissues.

(i) GENERAL CHARACTERISTICS

OPN is expressed by a single-copy gene as a 34-kDanascent protein that is extensively modified by post-translational events. The human gene contains 7 exons,spans - 11.1 kb, and maps to the long arm of chromo-some 4 (4q13) (Young et al., 1990; Hijiya et £4l. 1994). Incomparison, the - 4.8-kb mouse gene is at the locus ofthe Rickettsia resistance gene Ricr on chromosome 5 (Fetet 4l., 1989; Miyazaki et al., 1989), and the pig gene is onchromosome 8 (Denhardt and Guo, 1993). Whereas themammalian and avian OPN proteins have a similar num-ber of amino acids, the reported size of the secreted pro-tein varies from 44 kDa to 75 kDa, due to differences in

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post-translational modifications and anomalous beha-vior on SDS-PAGE. The protein is rich in aspartic and glu-tamic acid and serine, and contains a polyaspartic acidmotif, through which the protein can bind to hydroxy-apatite and calcium ions, and an RGD sequence whichcan mediate cell attachment. In addition, multiple sites ofSer and Thr phosphorylation and sites of both N- and 0-linked glycosylation exist, together with a thrombin clea-vage site. Variations in phosphorylation, glycosylation,and sulphation generate different functional forms ofOPN which may be found in the same or different tissues.

(ii) TISSUE DISTRIBUTION

OPN is expressed by cells in a variety of tissues, inclu-ding bone, dentin, cementum, hypertrophic cartilage,kidney, brain, bone-marrow-derived metrial gland cells,vascular tissues and cytotrophoblasts of the chorionicvillus in the uterus and decidua, ganglia of the inner ear,brain cells and specialized epithelia found in mammary,salivary, and sweat glands, in bile and pancreatic ducts,and in distal renal tubules and in the gut, as well as inactivated macrophages and lymphocytes (Patarca et al.,1989, 1993; Giachelli et al., 1995; Butler et al., 1996; Weberand Cantor, 1996; Denhardt and Noda, 1998; Rittling andDenhardt, 1999). In bone, OPN is produced by osteoblas-tic cells at various stages of differentiation (Zohar et al.,1997a), by differentiated osteoblasts and osteocytes(McKee and Nanci, 1995a; Sodek et al., 1995), and also byosteoclasts (Dodds et al., 1995). OPN is also expressed byfibroblastic cells in embryonic stroma and in wound-healing sites (Giachelli et al., 1995). Although the secret-ed protein is incorporated into the matrix in mineralizedconnective tissues, in many situations it appears in bio-logical fluids, including blood (Bautista et al., 1996), milk(lactopontin; Senger et al., 1989a), urine (uropontin;Hoyer et al., 1995), and seminal fluid (Cancel et al., 1999).OPN is also expressed in the pre-implantation mouseembryo and during differentiation of embryonal stemcells (Botquin et al., 1998) and in the rostral hind brainand notochord during neuraxis formation (Thayer andSchoenwolf, 1998). The amount of OPN in normal plas-ma of women ranges from 22 to 122 pg/L, with a medianlevel of 47 pg/L (Singhal et al., 1997), whereas in urine,OPN ranges from 1.9 to 4.3 pg/mL, with - 4 mg excretedper day (Min et al., 1998). The concentration of serum OPNprotein is markedly elevated with increased tumor bur-den in cancer patients (Senger et al., 1989b; Singhal et al.,1997) and in patients with auto-immune diseases suchas systemic lupus erythematosus (lizuka et al., 1998).Recent studies have shown that OPN and bone sialopro-tein in serum bind tightly to Factor H and may help pro-tect normal and cancerous cells from complement-medi-ated attack (Fedarko et al., 2000)

Expression of OPN is characteristically observed in

pathological conditions and pathophysiological responses.Thus, OPN synthesis is induced in smooth-muscle cells andcardio-myocytes in cardiovascular diseases, including ath-erosclerosis (Giachelli et al., 1993), restenosis (Panda et al.,1997), and ventricular hypertrophy (Graf et al., 1997).Similarly, OPN is induced in kidney diseases, such as inter-stitial nephritis (Sibalic et al., 1997) and puromycinaminonucleoside nephrosis (Magil et al., 1997), and inpathological mineralization such as kidney stone formation(Kohri et al., 1993; McKee et al., 1995). The induction of OPNin various carcinomas and adenocarcinomas has beenrelated to the activity of various oncogenes such as Src(Tezuka et al., 1996) and Ras (Chambers et al., 1992), and itsexpression is directly correlated with the development andprogression of transformation processes (Chambers et al.,1996) and metastasis (Oates et al., 1997), although the stageat which it functions has not been established. Synthesis ofOPN by activated lymphocytes and macrophages (Patarca etal., 1989, 1993) can account for the presence of OPN in dis-eased and traumatized tissues, OPN being one of the mostabundant molecules synthesized by activated T-lympho-cytes (Weber and Cantor, 1996). Studies on granulomatousinflammation have indicated that, as well as acting as achemoattractant for T-cells, OPN can promote adhesion ofT-cells and possibly amplify a CD3-mediated proliferativeresponse (O'Regan et al., 1999). The increased OPN activityfollowing thrombin digestion observed in these studiessuggests a mechanism whereby OPN and thrombin canmodulate T-cell recruitment and activation. A critical rolefor OPN in type 1 cell-mediated immunity has been demon-strated recently in which OPN, acting through the a433 andCD44 receptors, respectively, increases IL- 12 and suppress-es IL-10 expression by macrophages (Ashkar et al., 2000).Together with its activation of B cells by OPN (lizuka et al.,1998), OPN could, therefore, contribute to the developmentof autoimmune diseases.

(iii) REGULATIONThe expression of OPN is affected by a large number of hor-mones, cytokines, and growth factors which can influencethe rate of gene transcription, mRNA processing, stability,and translation, as well as post-translational modifications.Many of these effects have been catalogued and referencedin a recent review (Denhardt and Noda 1998) and will beonly summarized here, with an emphasis on OPN expres-sion in mineralized tissues. Although regulation is general-ly similar in different tissues, there are some notable differ-ences, and, even within the same tissue, differential regula-tion has been observed which can be attributed, at least inpart, to the target cell and its stage of maturation.

(a) Hormonal and cytokine control of OPN expressionGenerally, the steroids, retinoic acid and glucocortico-steroids, and particularly the seco-steroid hormone

280 Grit Rev Oral Biot Med11(3)279-303 (2000)

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1,25-dihydroxyvitamin D3, increase OPN expression inbone cells (Prince and Butler, 1987; Kasugai et al.,199!. The synthetic glucocortic.osteroid, dexametha-sone increases OPN expression in rat periosteal andmarrcow bone cell cultures, and in cultures of embryon-i( rat bones (Chen et al., 1996). Vitamin D3 is a particu-larly potent stimulator of OPN synthesis by bone cellsand epidermal cell lines, and a marked reduction ofCUPN mRNA expression is observed in vitamin-D3-defi-c'ent rachitic mice (Chen et al., 1999). Thus, while theimcrei se in OPN induced by glucocorticoids is linked tothe stimulation of bone formation, the vitamin D3effect is associated with massive bone resorption,reflecting the multifunctionality of OPN in boneremodleling iSodek et al., 1992, 1995). Retinoic acid alsoincreases OPN expression in bone (Manji et al., 1998),vN here..s the osteotropic parathyroid hormone, PTH,has marginal effects that appear to be bone cell type-dependent (Kasugai etal 1991)

Express lon of OPN is also up-regulated by variousgrowth andc differentiation factors, including epidermalgrowt(h facto)r (EGF), platelet-derived growth factorRl'DGA), transforming growth factor-3s (TGF-s), bonemorphogenetic proteins (BMPs), and inflammatorycvtokines (reviewed in Denhardt and Noda, 1998). Non-physiological agents such as phorbol esters and con-canavalin A also stimulate OPN expression. Increasedexpression of OPN induced by ELF, c-Src, and 12-0-tetradecano Iphorbol-l 3-acetate (TPA) appears to bemediated through protein kinase C (PKC), which acti-vates the earlv response genes, Jun and Fos, which inturn can increase OPN transcription through AP-s tes. The increase in OPN expression induced bymechanical stimuli iii vitro (Kubota et al., 1993; Klein-NulenId '1 ai. 1997; Carvalho et l, 1998) and in vivo(Miles. it (X.. 1998) has revealed a mechano-transduc-tion pathway involving stress on cell adhesion sites, inwxhich signals are mediated by protein tyrosine kinases.Taese signals require an intact cytoskeleton andir crease OPN transcription as a primary response(Koma et Li) 1997).

WVhereas OPN is generally up-regulated by hormonesa nd cvtokines it is suppressed by bisphosphonates inbone Sodek ct al., 1995) and kidney (Yasui et al., 1998).Tae (l-own-regulation of OPN by bisphosphonates inboane 1is c,nsis)tent with the suppiession of osteoclasticartivit, as ,vell as the reduced OPN expressionir hakalaparampil et al., 1996) ancl osteoclast activity inSi-1- nice (Soriano et cl.a 1991) and the suppression ofCPN synthesis and activity in osteoclasts by calcitoninKa)ji a 1)_I94). OPN expression is also suppressed inv,ascular moothamuscle cells by cGMP-dependent pro-te in ki tase, amediator of nitric oxide (NO), and by cGMPs.gnaling, vh.ich inhibits cell migration (Dey et al., 1998).

(b) OPN promoter and transcriptional regulationIncreased expression of OPN is frequently associated withan increase in transcription of the OPN gene, which is reg-ulated by transactivation of cis-acting elements in the genepromoter. The promoters for the human, pig, rat, mouse,and chicken have been sequenced and show some conser-vation in - 250 bp of the proximal promoter. Within thisregion is a conserved (TTTAAA) TATA box (nucleotides Ints(-27 to -19), an inverted CCAAT box (nts -52 to -46), and aCC box (nts -100 to -93). One (human, mouse, and chicken:Noda et al., 1990; Hijiya et al., 1994; Rafidi et al., 1994) or 2(rat and pig: Ridall et al., 1995; Li et al., 1998) 1,25-dihydroxy-vitamin D3 response elements (VDREs) that bind the vita-min D3 receptor (VDR) are present upstream of the inme-diate promoter region. In addition, activator protein (AP)motifs have been identified of which AP-R, a highly con-served element controlled by the proto-oncogene productsFos and Jun, mediates OPN transcription by the tumor-pro-moting phorbol ester, TPA, acting through PKC (Denlaardtand Guo, 1993). AP-2 is also a target of PKC-mediated sig-naling as well as signaling through cAMP-dependent pro-tein kinase A (PKA). There are 5 recognition sites for thepolyoma enhancer activator PEA-3. These sites are themajor targets of the Ets family of proto-oncogene tran-scription factors that are involved in T-cell activation andcould, therefore, account for the up-regulation of OPNexpression in activated lymphocytes and macrophages.Notably, OPN promoter activity is markedly eiahanced bypolyoma enhancer-binding protein/core binding factor(PEBP2xA/CBFAI), which functions as a master gene forosteogenesis. The CBFA1 acts synergistically with Ets I in amechanism that requires a direct interaction of the tran-scription factors with cognate enhancers that are criticallyspaced for transactivation (Sato et al., 1998) OPN expres-sion is also up-regulated during pre-implantation develop-ment by the POU transcription factor Oct-4 acting througha PORE element in the first intron and is repressed by Sox-2 acting on a closely located element (Botquin et a). 1998).

Regulation of OPN by Src was shown to increase OPNexpression through the inverted CCAAT box (Tezuka et al.,1996), which binds nuclear factor NF-Y (Kim and Sodek,1999). Since v-src is a transforming viral oncogene productoriginally identified in the Rous sarcoma virus (RSV), theseobservations provide a firm link between cellular transfor-mation and induction of OPN expression. Similarly, cellstransformed with v-Ras and v-Myc also increase transcrip-tion of OPN. A Ras-activated enhancer which interacts witha putative Ets-related transcription factor, the activity ofwhich correlates with the metastatic potential of the cell,has also been identified (Guo et a). 1995), AnotherCCAAT/enhancer binding protein family member, NuclearFactor-IL-6 (NF-IL6), which binds to the regulatory regionsof many genes induced in activated macrophages, inclu-ding macrophage inflammatory protein (MIPR- I alpha also

Crit Rev Oral Biol Med 281I I ) 7.979-3 3 I'000) at PENNSYLVANIA STATE UNIV on February 21, 2013 For personal use only. No other uses without permission.cro.sagepub.comDownloaded from

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Figure 1. Comparison of osteopontin protein sequences. The amino acid sequences from six mammalian (rat, mouse, cow, pig, human,and rabbit) and one avian (chicken) species hove been aligned by means of the multiple alignment program of Thompson et al. (1 994) tomaximize identical (dark shading) and similar (light shading) amino acids identified by JavaShade (Southern and Lewis, 1998;http://industry.ebi.ac.uk/JavaShade/) to reveal conserved structures. The 1 6-amino-acid leader sequence beginning with a methionine(position 1) is included with the first amino acid of the processed protein starting at position 1 7. The total number of amino acids in thesequence for each species is: chicken, 264; mouse, 294; rat, 317; rabbit, 311; cow, 278; pig, 303; and human, 300.

increases endogenous OPN gene expression (Matsumotoet a) 1998), thus providing a regulatory pathway for OPNexpression in inflammatory cells.

(2) Structural Characteristics

(i) AMINO ACID SEQUENCE COMPARISONS

Insights into the potential function of proteins can bederived from the conservation of structural motifs in theamino acid sequence. The amino acid sequences of OPNsderived from human (Kiefer et al., 1989), cow (Kerr et al.,1991), pig (Wrana et al 1989), rabbit (Tezuka et at, 1992), rat(Oldberg et al, 1986), mouse (Craig et al 1989), and chicken(Moore et a)l, 1991 ) cDNAs are currently available.Comparison of the mammalian OPN sequences reveals ahigh conservation in the amino- and carboxy-terminalregions and in the polyaspartate segment, as well as in theGRGDS and thrombin cleavage sites and in several poten-tial phosphorylation sites (Fig. I ). Of the 333 amino acids inthe consensus sequence for mammalian OPNs (CLUSTALW program, Thompson et al., 1994), 107 amino acids areidentical, with 59 amino acids retaining high similarity anda further 26 with lower similarity, for a conservation of 58%of the amino acids. Notably, there is also high identity(10/21 residues identical) in a sequence (220-240) that is

missing in bovine OPN and in a short sequence (312-316)that is missing in both the cow and the pig, When the aviansequence is included in the comparison, the number of per-fect matches is reduced considerably, to 52, with 48 aminoacids retaining high similarity and 37 with lower similarity,for a conservation of 39%. Despite the reduced conserva-tion, the GRGDS motif, the polyaspartic acid sequence, andthe thrombin cleavage sites are retained, together with sev-eral phosphorylation sites. Nevertheless, distinct differ-ences in the developmental expression have been reportedwhich could reflect functional differences between chickenand mammalian OPNs (Thayer and Schoenwolf, 1998)

(ii) CONSERVED MOTIFS

The conserved GRGDS sequence is flanked by several high-ly conserved sequences, including the thrombin cleavagesite, which is separated from the attachment sequence by aconserved "vaYgL" sequence On the amino terminal side,an "fTpvvPTv" sequence is conserved in which the prolineswould be anticipated to affect the structure of the cellattachmenUsignaling site. The thrombin cleavage site isfaithfully retained in all species, although the Arg-Ser recog-nition site is modified to an Arg-Ala in the chicken and aLys-Ser in the cow. Since thrombin is highly selective forArg-Gly sites in fibrinogen, it is likely that the transition

Cr1i Rev Oral Biol Med

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from a f-sheet to an (x-helix inthis region of OPN is conduciveto thrombin attack. The exis-tence of the thrombin site isindicative of an ancestral rela-tionship to proteins involved inhemostasis and the immunedefense system, while its conser-vation is consistent with animportant biological activity.Although the polyaspartateregion is not contiguous in allspecies, within a stretch of 8-10amino acids at least 7 are aspar-tates in all species, Four of eight(chicken) to 10 aspartates areconserved in all species, while aglutamic acid is an occasionalreplacement in a further threepositions, producing a stretch ofacidic amino acids in a region ofno definitive secondary struc-ture. However, the relative posi-tions of the aspartates in thelocal coil structure are more like-ly to be important functionally.

Figure 2. Osteopontin structure. A hypothetical structure for osteopontin, based on the amino acidsequence and secondary structure prediction (SOPM, Self-Optimized Prediction Method, GOR IV,and PREDATOR in Thompson et al., 1 994) of the mammalian proteins, is shown together with thepositions of demonstrated and potential post-translational modifications. The actual amino acidsequences of the conserved regions for the GRGDS cell attachment motif, the polyaspartatesequence, sites of phosphorylation and gclycosylation and thrombin susceptibility, and the transglu-taminase cross-linking site are given with identical amino acids capitalized and conserved aminoacids in lower case. The additional sequence present in rat OPN is shown with a dashed line.

Seven sites of casein kinase 11 phosphorylation are con-served in most species, with 6 of the sites involving serinephosphorylation and the other a threonine. In addition, inthe mammalian sequences, 9 serines are in conserved sites(Ser-X-Glu/PSer) for Golgi-casein kinase or mammary glandcasein kinase (Ser-X-Glu/PSer/Asp), with several additionalsites that are conserved in some species. Potential phos-phorylation sites for protein kinase C and tyrosine kinaseare present in different species with no strong conservation.Although sites of N-linked glycosylation are present in allspecies, the conservation is generally poor, with only oneAsn-X-Ser site conserved in five of the mammalian species.Two glutamines (positions 60 and 62 in the alignedsequences) separated by a lysine serve as substrates fortransglutaminase cross-linking (Sorensen et al., 1994) andare present in a conserved region of OPN extending fromresidue 56 in the mammalian sequence. Interestingly, theGly in position 12 of the highly conserved leader sequencein the mammalian OPNs is a potential site for N-myristoy-lation, which can attach proteins to membrane structures.

(iii) SECONDARY STRUCTURE PREDICTIONS

Analysis of secondary structure based on the nascentprotein sequences, with three different algorithms(SOPM, Self-Optimized Prediction Method, GOR IV, andPREDATOR), available in the CLUSTAL W program(Thompson et al., 1994), reveals few areas with consis-tent secondary structure prediction through the

species. Most conserved sequences are in regions pre-dicted to form coil structures with some sheet structurebetween the GRGDS and thrombin cleavage site. Thepossibility of helical structure is increased toward theC-terminus, with a high probability of helix within theterminal 30 amino acids. Short sections of helix forma-tion are also seen in association with sites of caseinkinase 11 phosphorylation.

(iV) STRUCTURAL MODEL

Based on both the consensus sequence, shown in Fig. 1,and the secondary structure prediction, a model for theOPN molecule is depicted in Fig. 2. Due to the paucity ofhydrophobic amino acids together with the high numberof charged amino acids, including the post-translationalmodifications on Thr, Ser, and Asn residues, an openextended and flexible structure is anticipated, as foundfor the bone sialoprotein molecule (Ganss et al., 1999).

(V) POST-TRANSLATIONAL MODIFICATIONS

Extensive alteration through post-translational modifi-cations can have significant effects on the structure ofthe OPN molecule. Phosphorylation and sulphation willincrease the anionic surface characteristics, while exten-sive glycosylation can limit flexibility. These modifica-tions can also affect the biological properties of OPN, asdiscussed later.

Crit Rev Oral Biol Med

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(a) PhosphorylationPhosphorylation of OPN, catalyzed by several kinases, cantake place on tyrosine, or on serine and threonine residues(Saavedra et al., 1995; Lasa et al., 1997). Potential serine andthreonine phosphorylating enzymes include casein kinasesI and 11, Golgi kinases, cAMP- and GMP-dependent kinases,protein kinase C, and ectokinases. Thus, there is consider-able potential for phosphorylation reactions to modify thestructure and properties of OPN, including its signalingactivities. The initial characterization of rat bone OPNrevealed 12 phosphoserines and a phosphothreonine(Prince et al., 1987), while subsequent analyses haverevealed considerable heterogeneity (Neame and Butler,1996). Approximately 8 phosphates have been estimated inbovine bone OPN (Salih et al., 1996), whereas 27 phospho-rylated serines have been identified in bovine milk OPN(Sorensen et al., 1995), with 19 of the phosphorylation sitesbeing fully conserved in rat OPN (Lasa et al., 1997) andretaining the consensus sequence (S-x-E/Sp) for Golgiapparatus casein kinase (G-CK). The location of G-CK andits high activity on OPN relative to the casein kinases indi-cate that it is the major enzyme involved in the phospho-rylation of secreted OPN (Lasa et al., 1997). Ectokinases onthe cell surface can also phosphorylate secreted OPN(Mikuni-Takagaki et al., 1995; Zhu et al., 1997), and recombi-nant nascent OPN has been reported to catalyze autophos-phorylation of tyrosines (Ashkar et al., 1993). However, thelack of a kinase domain in OPN necessitates verification ofthe autophosphorylation activity.

(b) GlycosylationSignificant glycosylation of OPN, estimated at > 33% of theweight of the protein (Oldberg et al., 1986), occurs in mosttissues. Thus, treatment of OPN with glycosidases increas-es the relative mobility of the protein markedly on SDS-PAGE (Shanmugam et al., 1997). In addition to the singleAsn-X-Ser site for N-linked glycosylation, there are - 26Ser-X-Glu sites for 0-linked glycosylation. In bovine milkOPN, 3 0-linked oligosaccharides have been identified(Sorensen et al., 1995), whereas 5 or 6 0-linked and a singleN-linked oligosaccharide are present in rat bone OPN(Prince et al., 1987). Glycosylation of OPN synthesized bytransformed cells may prevent phosphorylation and gener-ate non-phosphorylated OPN (Singh et al., 1990). In Roussarcoma virus-transformed Rat-1 cells, sialic acid in theOPN is removed, reducing the relative mobility from 69kDa to 62 kDa and abrogating the cell-binding propertieswhich may be important in the invasive behavior of OPN-expressing cancer cells (Shanmugan et al., 1997).

(c) SulfationStudies of bone formation in vitro have indicated that sul-phation of OPN occurs predominantly in the highly phos-phorylated form of OPN, and this has been suggested as

a potential marker for differentiated osteoblasts (Nagataet al., 1989). Sulphate is likely to occur in the sialylatedoligosaccharide side-chains. Although tyrosine can besulphated, the tyrosines in OPN do not exist withinsequences recognized by sulphotransferase enzymes.

(d) Transglutaminase cross-linkingOPN is a substrate for tissue transglutaminase, as demon-strated by Prince et al. (1991), indicating that polymeriza-tion of OPN occurs in connective tissues. Subsequently, 2glutamines near the amino terminus have been identifiedas the transglutaminase targets (Sorensen et al., 1994,1995). Both intra- and intermolecular cross-linking tofibronectin can occur (Beninati et al., 1994), and cross-link-ing of OPN has been reported to alter its conformationand to increase its binding to collagen (Kaartinen et al.,1999). Although a pair of glutamines is also present in theavian sequence, with a second pair close by, the sequenceis not as well-conserved, and it is not known whether thechicken OPN is a transglutaminase substrate.

(Vi) MULTIPLE FORMS OF OPNMultiple forms of OPN have been identified as productsof normal (Kubota et al., 1989; Kasugai et al., 1991) andtransformed cells derived from rodent tissues (Craig etal., 1989; Nemir et al., 1989; Singh et al., 1990). Rat bonecells produce high- and low-phosphorylated forms ofOPN, the high-phosphorylated form being associatedwith differentiated osteoblasts (Kubota et al., 1989; Sodeket al., 1995), while a low-phosphorylated form is producedin response to a vitamin-D3-induced Ca2+ influx (Safranet al., 1998). In transformed cells, two forms of phospho-rylated OPN, termed pp62 and pp69, and a non-phos-phorylated form, np69, have been characterized (Nemir etal., 1989). Also, a non-phosphorylated form of OPN hasbeen reported in bovine seminal fluid (Cancel et al.,1999). Although the physiological significance of theseforms is not clear, it is likely that post-translational dif-ferences reflect different functional roles. Thus, sialyla-ted forms of OPN, such as pp69, have been shown toassociate with cell-surface fibronectin (Shanmugam et al.,1997), whereas the non-phosphorylated, np69, but notpp62, binds to soluble fibronectin (Singh et al., 1990).Moreover, a switch from the synthesis of non-phosphory-lated to phosphorylated OPN in phorbol-ester-treatedmouse epidermal cells (Chang et al., 1995), and from anon-sialylated to a sialylated form in oncogene-trans-fected cells (Shanmugam et al., 1997), suggests that post-translational modifications of OPN may be significant incellular transformation.

An alternative spliced form of OPN mRNA, in whicha 42-nucleotide sequence coding for 14 amino acidsbeginning at residue 58 is removed, has been reported inhuman bone and decidual cells (Kiefer et al., 1989; Young

284CitRvOlBilMd1()7933(0)284 Crit Rev Oral Biol Med 11(3):279-303 (2000) at PENNSYLVANIA STATE UNIV on February 21, 2013 For personal use only. No other uses without permission.cro.sagepub.comDownloaded from

et a)l.1990), aid three splice variants are reported to beexpressed in human glioma tumors (Saitoh et al., 1995).Analysis of cDNAs prepared from Kirsten sarcoma virus-transformed normal rat kidney cells (KNRK) and ratosteosarcoma cells (ROS 17/2.8) revealed a 52-nucleotide insert in the 5'-non-coding region of theKNRK OPN cDNA (Singh et al 1992). While the differen-tially spliced torms expressed by t-he human cells couldgive rise to different proteins, the alternative splicing inthe non-coding region of the rat transcripts would notalter the translated protein,

(3) Structure-Function AnalysisAlthough the precise functions of the OPN are unknown,the uniquje conserved regions of the OPN molecule (i.e.,the RGD site, th-irombin cleavage site, polyaspartic acidsequence, adu serine/threonine phosphorylation sites)cc n providle insights into the diverse functions predictedfcr th is pt-ein

(i) REGULATION OF THE FORMATION AND GROWTHOF CALCIUM PHOSPHATE AND OXALATE CRYSTALS

The associatlon between the expression of OPN and theformantion of mnineral crystals is highly suggestive of afunctic)n in the regulation of miueralization in normalanid pathological situations (Shiraga ct al, 1992; Kohri etal, 1993; Bellahcdne et al., 1994; Goldberg and Hunter,1095; M1cKee Ct al., 1995; McKee anc3 Nanci, 1995b; Hunteretall 19960 Mohleretal_ 1997 Shenet al., 1997).ThatOPNis often enri hed in biological fluids (milk, urine, bile,seminal fluich having elevated levels of calcium salts isalso indhaicaie of a role in preventing spontaneous pre-cio itation if caicium salts. Also, .n the kidney, OPN ispredomiinantly localized to the thin limb of the loop ofHenle IKin<1emran et al i1995, McKee et al., 1995),upstream ot region with a high propensity for sponta-neour precipitation of calcium salts. Consistent withthese (bserv,ticns, osteopontin from bone (Boskey et al.,1(9:93, Hunrater it all- 1994, 1996) and uropontin (Shiraga etcal 190)2: \Vorcester ot al- 1995; Arsenault et al 1996;Aspiir N &l.1a)998) inhibit crystal nucleation and growthof HA and cc-liiJm oxalate (CO), respectively. This inhi-bition whici, believed to relate to the calcium-bindingproperties (of the polyaspartic acic sequence and serinepl-osphorTlat io0r sites (Singh et al_, 1993) appears toin.olve selective binding to a specific crystal face-acidicpioteirote i (lind preferentially to the {100l face of HA,w iereas phoSphorylated proteins bind to the (010) face(Fuj iscawa a-mI1n Kaboki 1991, Furedi-Milhofer et al., 1994)Thfat the pih (p ate groups aretie predominant moi-eties Mvolved ii the inhibition of crystal formation andgrowth isrin cated by the low inhibitory activity ofrecombinant huj-nan OPN. which lacks post-translationalm)dlifdtionB IGoldberg personal communication), and

the loss of inhibitory activity when OPN is dephosphory-lated (Boskey et al., 1993; Hunter ft al., 1994) In contrast,modification of OPN carboxyl groups has a more modesteffect (Hunter et al., 1994). Although polyaspartate caninhibit HA and COM nucleation and growth (Hunter t al.,1994, 1996), the minimal number of Asp residuesrequired is not known

(i;) CELL ATTACHMENT AND SIGNALINGTHROUGH INTEGRINS

Interaction of the GRGDS sequence in OPN with aA 3 5(Ross et a.l, 1993; Liaw et al., 1995), cX,5j (Smith ot al!1996), and oX8X1 integrins (Denda et al, 19981 can medi-ate cell attachment, cell migration, chemotaxis, andintracellular signaling in various cells, includingendothelial smooth-muscle cells (Liaw et al. 1995), pla-cental trophoblasts (Daiter et al, 1996), kidney cells(Rabb et al 1996; Denda ot al., 1998), platelets andmacrophages (Giachelli et al, 1995 as wel asosteoblasts (Oldberg ot al. 1986) and osteoclasts(Miyauchi et al., 1991; Ross et al., 1993) The different sig-nals that OPN can elicit in the same and different cellscan be explained by the existence of multiple het-erodimeric combinations of integrin chains that car lig-ate OPN and by variant forms of OPN Thus, an aimino-terminal fragment of OPN, generated by thrombin diges-tion, that retains the RGD motif within 6 amino acidsfrom the cleavage site, has been reported to increase theattachment and spreading properties of OPN possiblyby facilitating accessibility of the RGD (Senger andPerruzzi, 1996) However, the thrombin effects are likelyto be receptor-specific, since other studies (Xuan tI al.,1994) have reported that thrombin digestion reducesthe ability of OPN to promote cell attachment, while themigration and adhesion activities of OPN mediated byinteraction of the RGD sequence with cx(3, are observedonly with the thrombin-cleaved amino-terminal frag-ment (Smith and Giachelli, 1998) That the enhancedadhesion of cells mediated by the N-terminal fragmentis suppressed by the presence of the C-terminal frag-ment also suggests that the C-terminal domain mayregulate OPN functions by suppressing RGD-dependentcell adhesion (Takahashi et al., 1998) In addition to theROD interactions, OPN has also been shown to mediatecell attachment through non-GRGDS sites that arelocated in the amino- and carboxy-terminal parts of themolecule (Katagiri et al., 1996) and in a 28-kDa fragmentof OPN isolated following proteolytic digestion (van Dilket al., 1993). Attachment of activated leukocytic cell linessuch as HL-60 and Ramos, by OPN has also been local-ized to the amino-terminal half of OPN This interactioninvolves the a4i integrin which recognizes a sequencethat is in competition with a leu asp-val iLDV1 peptide(Bayless et al., 1 998)

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P13-kinase (triton-insol)

/ Ptdlns gelsolinPI3-kinase actinc-Src ERM

/ / >Rho ElI Rac

IY'AK Ra -------------

5 p-Src *NRaf

N.

Ca2+]

.55.

MAPK

NFkB

Phagocytosis

Figure 3. Osteopontin signaling through the ct,[3 integrin. A summary of potential signalingpathways for OPN that are mediated through the c0v33 integrin, which can influence cell prolif-eration, cytoskeletal organization, motility, apoptosis, and phagocytosis in different cells. Bothsoluble and immobilized forms of OPN can interact with the (x03 integrin. The evidence forthese pathways is discussed in the text. Pathways that may be cell-type-specific or are not well-established are shown with dashed lines.

The 0J33 integrin, which can be co-ordinately regu- Ras and Src (Scatelated with OPN (Liaw et a{., 1995, Srivatsa et al., 1997; can regulate theEllison et a)., 1998), is believed to be primarily responsi- (Zhang et al, 1995)ble for the adhesion and migratory properties of OPN tivity for °UA3 has Iand can also generate intracellular signals through while anti- a 3 arautocrine and paracrine mechanisms. Notably, activation inhibit angiogenesof afi3. through inside-out signaling may be needed to effect (Scatena et cstimulate adhesion and migration induced by OPN ment correlates wil(Faccio et al., 1998). OPN is more effective than other kinase which actsECM ligands in promoting haptotaxis through the fi_33 Viciana et al., 1997integrin, the thrombin fragment being twice as effective associated Ptdlnsas the intact OPN molecule (Senger and Peruzzi, 1996, Hruska, 1996). MoiSenger et al., 1996). OPN ligation to c0j33 can also modu- required for stimulate intracellular Ca2l by stimulating release of Call from kinase in osteoclaslintracellular compartments and by regulating extracellu-lar Ca2l influx via calmodulin-dependent, Ca2+-ATPase (iii) CELL ATTAC(Miyauchi et al., 1991; Zimolo et al., 1994). Possible path- THROUGH CD44ways for OPN signaling through integrins such as cn,j3 OPN is also an extrare depicted in Fig. 3. Ligation of integrins such as (x 33 1996), which is tby OPN leads to phosphorylation of focal adhesion hyaluronate. CD44 arkinase (FAK) (Frisch et al., 1996: Craig and Johnson, 1996), sively studied as a capaxillin, tensin, and Src (Lopez et al., 1995), which initi- cluding myeloid ceates signals for proliferation, cytoskeletal organization, However, CD44 is alsmotility, and blockage of apoptosis By utilizing distinct osteoclasts, fibrobla&

FocalIContactI

Crit Rev Orcil Biol Med

OPN

|cytoskeletal|I organizationI

motilitytembrane ruffling

A Iprolifferation|

I Apotoisblock

-I

effectors of Ras activation, discretesignaling pathways have beenrevealed for activation of MAPkinase and cell ruffling, both path-ways being required for mitogenicactivity of Ras (loneson et al.1996). The proliferative effects ofOPN have been demonstrated in asubpopulation of prostate epithe-lial cells which would proliferateonly under growth-restricted con-ditions when grown on an OPNsubstratum (Elgavish et al., 1998).Ras, P13-kinase, and Akt pathwaysactivated by V03-mediated cellattachment have also been impli-cated in cell survival (Khwaja andDownward, 1997), as have MEKK-I/INK (Cardone ct al., 1997) andBcl-2 (Stromblad et al., 1996). ThatOPN can prevent cell death wasfirst indicated in studies of kidneyproximal tubule epithelial cellsexposed to hypoxia and re-oxy-genation (Denhardt et al., 1995). Apotential mechanism wherebyOPN can prevent apoptosis hasbeen demonstrated in endothelialcells in which OPN binding to avf3increases NFKB activity through

na et al, 1998). Integrin engagementanti-apoptotic gene Bcl-2 in COSand endothelial cells in which selec-been shown (Stromblad et cil., 1996),itibodies, specifically anti-f3, may;is by blocking the anti-apoptoticil., 1998). Notably, actin re-arrange-ih the ability of Ras to activate P13-through Rac and Rho (Rodriguez-

), and OPN can stimulate gelsolin-through P13-kinase (Chellaiah andreover, c-Src has been shown to beilating the gelsolin-associated P13-fs (Chellaiah et al, 1998).

HMENT AND SIGNALING

acellular ligand for CD44 (Weber et al.,he main cell-surface receptor fornd its various isoforms have been exten-ell-surface marker for immune cells, in-ells, lymphocytes, and macrophagesso expressed in osteoblasts, osteocytes,;ts, smooth-muscle cells, and epithelial

4

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and endothelial cells. TheCD44 family of receptorsmediates cellular respons- ies similar to those of OPN ( -

integrins, including adhe-sion, migration, and stim-ulation of both normaland transformed cells.Indeed, recent studies EGFindicate that CD44 vari- kiants co-operate with [31-containing integrins tobind multiple domains in CD44OPN to stimulate motilityand chemotaxis (Katagiri HAet al., 1999). Expression ofCD44 and its interactionwith hyaluronate occur in OPNearly development of tis-sues as well as in repairand regenerative process-es, while its expression inmigrating immune cells islinked to tumor growthand progression, althoughthe exact role of CD44 inthis process is unknownWhile OPN and hyaluro-nan can both mediate cellattachment through CD44,the aggregation of homoty,hyaluronan, whereas OPN sactivity. Thus, the up-regulimmune and tumor cells mation of these cells induced bL

(jV) INTRACELLULAR OFRecently, immunocytochenmicroscopy have revealed aperi-membranous distributwith several OPN antibodieCD44 and ezrin-radixin-nmigrating embryonic fibmacrophages, and metastatet a), 1998a; Zohar, 2000) Bethe specificity of OPN ant1998), the identity of the imimust be confirmed. Neverlevidence to support the prement complex. For exampleCD44 is up-regulated inextravasating immune celland metastatic cells (Oateswhile synthesis of OPN, (

Figure 4. Association of intracellular osteopontin with the CD44-ERM complex. Although a cytosolicform of OPN, distinct from the secreted protein, is yet to be confirmed, a hypothetical interaction ofintracellular OPN with components of the CD44-ERM and potential signaling pathways, involving crosstalk with focal adhesions and the epidermal growth factor receptor (EGF-R), that are involved in cellmigration is shown. Evidence for this model is discussed in the text.

Dic cells is observed only for increased by EGF (Zhang et al, 1997), the over-activity of;electively exerts chemotactic which is frequently linked with cell transformation.ation of CD44 and OPN in Moreover, co-expression of OPN and CD44 is seen in dif-y relate to the directed migra- ferentiating monocytic HL-60 cells (Atkins et a)., 1998),/ OPN ligation. while co-immunoreaction has been reported in gastric

cancer cells (Ue et al), 1998). Although a possible intra-'N cellular function for OPN may appear surprising, there-ical studies with confocal are similarities with calreticulin, a calcium-binding ERn intracellular protein with a protein that acts as a molecular chaperone, but whichion that is immunoreactive also has a multiplicity of functions, including the regula-s and which co-localizes with tion of cellular signaling through integrins (Dedhar et al,noesin (ERM) proteins in 1994; Coppolino et a), 1997).)roblastic cells, activated A possible association of OPN with the CD44-ERMic breast cancer cells (Zohar complex is depicted in Fig. 4, and the inter-relationshipecause of concerns regarding with OPN:av[33 interactions in cell signaling and motilityJibodies (Rittling and Feng, is indicated. The formation of the hyaluronan:CD44:ERMnunoreactive protein as OPN complex is regulated by various phosphorylations medi-theless, there is substantial ated by serine/threonine kinases (Nakamura et al), 1995),sence of OPN in this attach- associated with Rac and Rho (Mackay et a)., 1997), whichexpression of both OPN and are downstream mediators of Ras, and by the tyrosinemigratory cells, including kinase activity of the EGF receptor. Moreover, hyaluronan(Weber and Cantor, 1996) binding to CD44 is dependent on the phosphorylation of

et al., 1997; Naot et al, 1997), the cytoplasmic tail of CD44 (Pure et al, 1995). ERM andD44, and ERM proteins is CD44 can associate in the presence of phosphoinositides

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(Ptlnds) generated by phosphotidylinositol 3-hydroxylkinase (P13-kinase), which is stimulated by OPN in celllysates (Chellaiah and Hruska, 1996). In this complex,OPN can also be phosphorylated at Ser/Thr sites and,through its Ca2+-binding abilities, can modulate localintracellular Ca2+ levels, thereby regulating the bindingof gelsolin to actin, which modulates actin-severingactivity (lamney et al., 1990).

In addition to a role in linking CD44 complexes withactin filaments, ERM proteins have also been implicatedin the formation of stress fibers associated with focaladhesions, both systems being regulated through Racand Rho (Mackay et al., 1997). Thus, the CD44-ERM-OPNcomplex may represent a novel adhesion complex thatforms close contacts, as described by Abercrombie andDunn (1975), that are transiently formed in rapidlymigrating cells, consistent with the co-localization ofCD44, ERM, and OPN in the leading edges of migratingcells (Zohar et at., 1998a). The extracellular OPN can pro-vide temporary (CD44) or more substantial (ox3A) attach-ment complexes required for motility as well as functionin the chemotaxis of the migrant cells. Moreover, theextracellular OPN can regulate the migratory processesby activating intracellular signaling pathways throughthese receptors. In this manner, the involvement of OPNas an attachment protein, chemoattractant, and signal-ing molecule in diverse biological activities involvingdevelopmental, regenerative/repair processes, immunedefense, and tumor cell survival and metastasis, and itsrelationship to integrin and CD44 receptors, can be morereadily appreciated.

(V) POTENTIAL FUNCTIONSA broad range of activities involving the conservedmotifs has been reported for OPN. The functionality ofthe polyaspartic acid sequence and the presence ofphosphate groups are clearly related to the binding ofOPN to mineral crystals in normal and pathologicalstates. Additionally, the phosphate groups can alsomodulate the attachment activity of OPN in osteoclasts,as will be discussed. OPN appears to prevent bothnucleation of mineral crystal formation and the growthof pre-existing crystals. The RGD motif, together withother sites on the OPN molecule, can mediate cellattachment, migration, and signaling. In combinationwith its hydroxyapatite and calcium oxalate bindingproperties, OPN can also mediate attachment of cells tomineral crystal structures, as originally suggested forthis protein (Oldberg et al., 1986). OPN expressed byactivated macrophages (Patarca et al., 1989, 1993) andother phagocytic cells, such as absorptive epithelialcells (Qu-Hong et al., 1997), has opsonizing activity(McKee and Nanci, 1996b), which likely involves attach-ment to cell-surface receptors in these cells. OPN is also

a chemoattractant for macrophages (Singh et al., 1990;Giachelli et al., 1998), as well as for smooth-muscle (Liawet al., 1995) and tumor (Senger and Peruzzi, 1996; Weberet al., 1996) cells. Moreover, OPN produced by immunecells can stimulate B-cells and antibody production(Weber and Cantor, 1996; lizuka et al., 1998). Expressionof OPN can suppress the cytotoxic effects ofmacrophages by blocking production of reactive oxygenspecies (Weber and Cantor, 1996). Thus, OPN can inhib-it the induction of inducible nitrogen oxide synthase(iNOS) mRNA synthesis by lipopolysaccharide andinterferon-gamma in macrophages (Rollo et al., 1996)and kidney proximal tubule epithelial cells (Hwang et al.,1994), which is also blocked by OPN in vascular tissues(Scott et al., 1998). OPN has been shown to preventapoptosis by activating NFKB in endothelial cells(Scatena et al., 1998), preventing the cellular redistribu-tion of Bcl-x, a member of the Bcl-2 family that isbelieved to inhibit activation of procaspases, and main-taining membrane integrity (Denhardt and Noda, 1998).

Conservation of the thrombin cleavage site and theability of OPN to serve as a substrate for plasma trans-glutaminase (Factor XllIa) also indicate functional signifi-cance and a relationship to the vascular system.Thrombin can regulate potential adhesive and migratoryfunctions of OPN by exposing cryptic cell-binding sitesand facilitating access to others, including the RGD site(Senger and Peruzzi, 1996; Senger et al., 1996; Smith andGiachelli, 1998). Also, osteocalcin, which is related tocoagulation factors through the vitamin-K-dependent for-mation of y-carboxylic acid groups and is reported tobind to OPN (Butler et al., 1996), is a competitive inhibitorof tissue transglutaminase-mediated cross-linking ofOPN in vitro (Kaartinen et al., 1997).

Despite the potential for a broad range of functionsfor OPN in development, bone remodeling, calcification,homeostasis, and immunoprotection, ablation of theOPN gene in two transgenic mouse lines has notrevealed an obvious phenotype that can be clearly con-nected to any specific function (Liaw et al., 1998; Rittlinget al., 1998). While compensatory mechanisms may maskOPN functions, embryological development is notaffected in double knockouts of the OPN and vitronectingenes, indicating that vitronectin, which shares thesame cell-surface receptors (most notably CoVf3), is notcompensating for OPN in early development (Liaw et al.,1998). However, defects in wound healing have beenobserved in both mouse lines. In addition, effects onosteoclast formation and bone resorption (Rittling et al.,1998), as well as significant effects on tumorigenesis(Crawford et al., 1998), have been observed.

By assimilating the properties of OPN outlined aboveand considering these in the context of the informationgleaned so far from the manipulation of OPN expression

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in vito, one call formulate multi-functional roles for OPNin bone remodeling, wound healing, and tumorigenesis.Here, paradigms for the role of OPN in wound healingand tumorigenesis will be discussed, while the roles ofOPN in mineralization and mineralized tissues will bediscussed in more detail in Section 4 below.

(a) OPN and wound healing

Although there is no obvious impairment of wound healingin OPN-null mice, the formation of srnaller, more irregular-ly organized collagen fibrils and the slower removal of tissue debris have been reported (Liaw et al., 1998). It is con-ceivable that expression of OPN, which is elevated at dis-eased and traumatized sites (Carlson et al, 1997; Nau et )l.,1997), attracts macrophages, the N-formyl-met-leu-phe(FMLP)-induced accumulation of wh.ch can be blocked byanti-OPN antibodies (Giachelli et al, 1998). Expression ofOPN by activated macrophages and lymphocytes occurs aspart of the host defense mechanism, in which OPN acts asa cytokine that stimulates the cellular and humoral branch-es of the immu-ne system (Weber and Cantor, 1996). ThatOPN has a roie in resistance to bacterial infection is indi-cated by its relationship to Ric resistance in mice (Weberand Cantor, 1996). Moreover, secretion of OPN by phago-cytic cells such as macrophages (McKee and Nanci, 1996b),osteoclasts (Dodds et al., 1995), and absorptive epithelialcells IOu-H-Ion tof ., 1997) indicates an opsonizing func-tion in which OPN coats foreign particles and particulatedebiis for subsequent receptor-mediated phagocytosis.OPN sec reted by macrophages and/or derived from bloodalso coats exposed fracture surfaces of mineralized tissuesof bones arnci teeth and biomaterials (McKee and Nanci,1996a,hL Lekic t7t a)l_ 1996) following wounding or surgicalimplantatio.n In these circumstances, OPN appears tomediate the attachment of cells as a prerequisite to tissuerepair anc implant integration. Intracellular OPN inrnacrophages -wndc in fibroblasts populating a wound sitemav also be in olved in the migratory activity of these cells.It is also notable that OPN and 03 integrin expression inendothelial cells are up-regulated by vascular permeabilityfactor/'ascular endothelial cell growth factor (VPFNEGF).VECF dran also influence microvascular permeabilitythroughl the extrinsic coagulation pathway, which stimu-lates thrombin digestion of the OPN. This, in turn, can pro-mote olatelet aggregation (Bennett et a)., 1997) andincrease the OPN-stimulated migration and survival of theendothelial ceils involved in vascular repair and neo-vas-cularization enger et al.! 1996).

(b) OPN and tumorigenesis

Early in the development of cancerous cells, local stromalcells express OPN that acts as a signal to attractmacrophages -nC perhaps also lymphoctyes. The expres-sion of OPN bKV local cells also provicdes protection against

the cytotoxic products of macrophages, while abnormal cellswill be killed and removed. However, OPN expression is fre-quently induced early during the initiation and progressionof cancerous growth by transforming agents. High expres-sion of OPN is observed in high-grade metastatic malignanthuman gliomas (Tuckeretal., 1998) and in the highly invasiveand metastatic human breast cancer cell lines LCC15-MBand MDA-MB-435 (Sung et a)., 1998). Also, transfection ofOPN-negative breast cancer cells with an OPN expressionvector has been shown to increase the tumorigenicity of thecells and their metastasis to bone, through a mechanismthat appears to involve a promotion of angiogenesis(Yoneda et al., 1998). The production of OPN can also con-tribute to the metastatic phenotype through autocrine andparacrine effects. Thus, autocrine effects can influence cellproliferation and survival, converting benign tumor cells intohighly metastatic cells (Oates et al., 1996)1 whereas paracrineeffects can provide protection from cytotoxic macrophages,possibly by inhibiting the production of nitric oxide(Denhardt and Chambers, 1994). Collectively, these mecha-nisms could explain how tumor cells not expressing OPNare eliminated, while providing a mechanism of tumor cellescape and selection of OPN-producing clones in secondarymetastases (Crawford et al., 1998). In this regard, highermedian levels of plasma OPN in metastatic breast cancerhave been associated with increased tumor burden anddecreased survival (Singhal et al., 1997).

(4) Function of OPN in Bones and Teeth

(i) DISTRIBUTION OF OPNIN THE CELLULAR MATRIX OF BONES AND TEETH

(a) The interfacial distribution of OPN in bone

Since its original identification in bone by biochemicalmeans (Franzen and Heinegard, 1985; Fisher et al., 1987,Prince et al., 1987), OPN in hard tissues has been consis-tently localized to specific regions of the extracellularmatrix (ECM) in bones and teeth by immunohistochemi-cal and immunocytochemical methods (reviewed inMcKee and Nanci, 1993). Importantly, whereas most non-collagenous proteins are more or less homogeneouslydispersed throughout bone, ultrastructural immunocvto-chemical studies have consistently highlighted theunique, interfacial distribution of OPN relative to otherECM proteins found in bone-the most prominent accu-mulation being at cement lines in remodeling bone, andat laminae limitantes at bone surfaces (McKee and Nanci,1996b,c). These two planar accumulations of non-collage-nous protein define matrix-matrix and cell-matrix bound-aries, respectively, and therefore represent important loca-tions in the formative history of the bone.

Cement lines (or layers/planes) indicate sites wherebone formation has been quiescent (resting line) or wherea major reversal event in cell activity has occurred

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between the formative and resorptive phases of this tissue(reversal line). Initially, at reversal sites, osteoclasticactivity results in the enzymatic degradation of the organ-ic bone matrix, together with the dissolution of the inor-ganic phase by cell-mediated acidification of the mineral-containing extracellular space (Baron et al., 1993). In thismanner, bone is asynchronously lost at different anatomi-cal sites by an excavation process that results in resorp-tion cavities that are continuously refilled with new bonesuch that overall bone mass is maintained (Parfitt, 1994).Cement layers demarcate the boundary between the olderbone and newer bone, and characteristically contain ahigh content of OPN. The cellular origin of the OPN in thecement layer has been controversial (Dodds et al., 1996;McKee and Nanci, 1996d), yet it is likely that both osteo-clasts and osteoblasts contribute (albeit unequally) to theformation of this interfacial structure. Whereas OPN pro-duction by osteoclasts is well-documented (Tezuka et al.,1992; Merry et al., 1993; Dodds et al., 1995), the majority ofwhich likely binds in an autocrine/paracrine fashion tointegrin receptors a-3A and/or CD44 on the basolateralaspect of the osteoclast (Horton et al., 1995; Helfrich et al.,1996; Nakamura and Ozawa, 1996; Duong and Rodan,1999), vectorial secretion of OPN and its assembly into thecement layer have been convincingly demonstrated forcells of the osteoblastic lineage (McKee and Nanci,1996a). In the latter case, relatively undifferentiated, early-stage osteoblastic cells migrate to the resorbed bone sur-face and commence matrix secretion and assembly as acement layer on the exposed bone surface. The cementlayer is deficient in collagen fibrils but rich in OPN, andsometimes contains bone sialoprotein. Initially, OPN inthe cement layer may be utilized for osteoblast cell adhe-sion, or to guide early calcification events at this junction.Here, OPN immobilized onto pre-existing mineral and/ororganic moieties of the underlying bone may act to initi-ate a new cycle of mineralization at this site. Studies areongoing to determine the macromolecular interactionsspanning this critical interfacial region that allow for calci-fication, cell attachment, and adhesion of the nascentbone matrix to its underlying, pre-existing substratum.Following cement layer deposition, bone matrix properaccumulates in the resorption lacunae from the net secre-tory activity of cohorts of fully differentiated osteoblasts.Notably, reversal lines (cement layers) are prominentmatrix structures in regions of high bone remodeling, suchas observed in the alveolar bone of the periodontium.

(b) OPN as an interfibrillar proteinin the collagenous matrix of bone

OPN is particularly prevalent within the interfibrillar(with respect to type I collagen fibrils) compartment ofthe extracellular matrix of woven (non-lamellar) bone.OPN first appears at small calcification foci within the

generally unmineralized osteoid seam, with high levelsbeing observed at the mineralization front and extendinginto the mineralized bone matrix proper (McKee andNanci, 1995b). By way of analogy, it has been proposedthat OPN incorporated into bone beyond the mineraliza-tion front, together with other non-collagenous proteins,acts as the "mortar between the collagen bricks", impart-ing molecular cohesion to the matrix as a whole, whilesimultaneously guiding mineralization events within thismatrix compartment. With regard to its cohesive/adhe-sive properties, OPN serves as a substrate for transglu-taminase (Prince et al., 1991; Beninati et al., 1994;Sorensen et al., 1994), whose covalent cross-linkingactivity provides for homotypic OPN-OPN binding andheterotypic binding to collagen (Kaartinen et al., 1999).Additionally, the mineral-binding domains of OPN maybe functional in linking matrix to mineral, and in pre-cisely guiding and regulating hydroxyapatite crystalgrowth within the interfibrillar spaces of the bone matrix.Importantly, the physical properties of the bone matrixare in large measure defined by the orientation of miner-al both within and between the collagen fibrils. Wherethe collagen fibrils of lamellar bone are more highlyordered and closely packed, non-collagenous proteincontent is diminished, and OPN likely plays a more sub-tle role in defining the biomechanical properties of thebone. In addition to mineral regulation and cohesivefunctions, the more diffuse distribution of OPN through-out the bone matrix may be of some importance in influ-encing osteoclast activity during resorption into wovenand lamellar bone.

(c) OPN as a bone-surface protein:cell-matrix relationships in vivo

In addition to its accumulation in the cement layer, OPNis a prominent component of the laminae limitantesthat line both internal and external bone surfaces(McKee and Nanci, 1996c). These surfaces are critical inthat they define the interface between bone cells andmatrix/mineral, and thus are strategically positioned toinfluence cell attachment and signaling. The OPN-richlamina limitans is a thin organic structure that is readi-ly apparent only by electron microscopy after decalcifi-cation of the bone, and which exists at the very marginsof calcified bone interfacing with osteocytes, with bone-lining cells, and, where remodeling occurs, with osteo-clasts. Such an important location implies a role in ter-minating mineralization at the bone surface, and wheremineral encroaches upon osteocytes in their lacunae, orcell processes in their canaliculi. Concomitant with itsbinding mineral, OPN at these sites would be availablefor ligation with the cell-surface receptors of bone cellssuch as integrins and/or CD44 (Nakamura and Ozawa,1996; Weber et al., 1996). For osteocytes and their cell

290 Crit Rev Oral Biol Med 1 l(3):279-303290 Crit Rev Oral Biol Med 11(3):279-303 (2000) at PENNSYLVANIA STATE UNIV on February 21, 2013 For personal use only. No other uses without permission.cro.sagepub.comDownloaded from

processes within the lacunar and canalicular systemenmanating throughout bone, such receptor occupancymight provide a means by which these strategicallylocated and most numerous cells detect and respond tobone tissue strain, thereby acting as mechano-sensorscapable of initiating signaling cascades to the bone sur-face and activating the bone remodeling cycle (McKeeand Nanci, 1995a, 1996a). Perhaps most importantly,surface-located OPN subjacent to bone-lining cellswould be exposed upon retraction or death of thesecelIs, thereby being available for the initial events ofosteoclast attachment, migration, and resorption at thebone surface.

(d) OPN in teeth

OPN present in tooth extracellular matrices residesprincipally within the cementum that lines root sur-faces and which serves as the tooth-related attachmentpoint for periodontal ligament fibers (McKee et al., 1996;D'Errico et il., 1997; Bosshardt et al., 1998). Both acellu-lar and cellular cementum types contain abundant OPNat levels which are substantially higher than that inbone, particularly for the acellular cementum variety.OPN accumulation in cementum typically occursbetween collagen fibrils and is thought to guide andstabilize the interfibrillar mineralization pattern withinthiy tissue. An additional accumulation of OPN is fre-quently observed at the dentino-cementum junction, asite where non-collagenous cementum proteins accu-mulate in a variable pattern according to the surfacetopography and packing of the underlying mantleden-tin collagen fibrils (McKee et al., 1996; Bosshardt etal., 1998). At the dentino-cementum junction, cemento-blasts secrete OPN and coordinate the integration ofcementum collagen fibrils with those of dentin in amanner that may be likened to the extracellular matrixassembly events occurring at cement lines in bone.While a role ftr OPN in cell adhesion is less obvious atthe tooth root surface, its ability to act in this mannerhacK been suggested (Somerman et fl., 1990).

In dentin, the large majority of OPN appears to beassociated with the initial sites of calcification thatoccur within the so-called mantle dentin of the devel-oping root (McKee et al., 1996). Additional, moderateamrounts of OPN appear diffusely (listributed through-out the thin layer of mantle dentin, with very little beingpresent in the peritubular dentin itself. This OPN mayoriginate cellularly from both the odontoblasts and thecementoblasts. The implication of this distribution isthat while OPN may regulate early calcification eventsin -oot dentin, its role in the ongoing mineralizationprocess in the bulk of this tissue is less obvious, andother matrix constituents in dentin may function in asimilar marner

(ii) TEMPORAL EXPRESSION OF OPNIN BONE FORMATION

Studies on the temporal expression of OPN during the for-mation of bone in vitro and during the formation ofintramembranous and endochondral bone in vivo haverevealed a biphasic pattern in which OPN is producedearly in the differentiation of bone cells, with higher levelsexpressed after mineralization has been initiated (McKeeand Nanci, 1995b; Sodek et al., 1995). The high levels ofexpression continue in association with the remodeling ofbone Studies of OPN expression in populations of fetalrat calvarial cells have revealed a high proportion of cellsexpressing OPN through the various stages of differentia-tion (Zohar et al., 1997a,b, 1998a,b), with different, post-translationally-modified forms of OPN being expressed byundifferentiated and differentiated osteogenic cells(Kubota et al., 1989; Nagata et al., 1991). In addition toOPN-secreting cells, proliferating osteogenic cells expressintracellular OPN associated with cell migration (Zohar etal., 2000). Although the nature of the intracellular OPN hasnot been characterized, OPN secreted at this stage ofosteogenic differentiation is less phosphorylated than theprotein secreted by osteoblasts; it also migrates moreslowly on SDS-PAGE and may have a lower affinity forhydroxyapatite. Compared with embryonic calvariae-derived cells, marrow-derived stromal cells stimulated todifferentiate along the osteogenic pathway produce muchhigher levels of OPN during the early proliferative stage.The high expression of OPN by the marrow cells is associ-ated with the formation of a collagen-free cement layer invitro (Davies, 1996) upon which bone is subsequentlydeposited, following the differentiation of osteoblasts(Sodek et al., 1995). The cement layer formed in vitro isthought to correspond to the cement (reversal) line struc-ture formed during bone growth and remodeling (Zhou etal., 1994; McKee and Nanci, 1995a, 1996a), and on implantsurfaces prior to the formation of bone (Nanci et al., 1994).Since this OPN co-migrates with highly phosphorylatedOPN produced by osteoblasts and is associated with min-eral deposits, it is clearly different from the OPN producedby undifferentiated calvarial cells. Following the formationof bone and the initiation of mineral deposition produc-tion of the highly phosphorylated OPN is increased withosteoblast maturation, When bone formation is complet-ed, OPN forms a surface coating, the lamina limitans,residing between the mineral at the bone surface and theqUiescent bone-lining cells. The temporo-spatial expres-sion of OPN and its different forms during the formationof bone is depicted in Fig. 5.

(;ii) ROLE OF OPN IN THE FORMATIONOF MINERALIZED TISSUES

Expression of intracellular OPN, together with CD44, inproliferating osteogenic cells appears to be related to the

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migratory properties of these cells. In comparison, the low-phosphorylated form of extracellular OPN could conceiv-ably have regulatory functions involving integrin ligation,as depicted in Fig. 3. Also, OPN expressed during cementlayer formation is likely to regulate hydroxyapatite crystalgrowth, which would be consistent with the demonstratedability of phosphorylated OPN to be a potent regulator ofhydroxyapatite crystal growth (Hunter et al., 1994, 1996;Wada et al., 1999). However, in the absence of collagen andBSP in the cemnent layer, OPN could conceivably act as anucleator, especially if it were immobilized on the substra-tum or the cell surface. Similarly, the highly phosphorylat-ed OPN expressed following the initiation of mineraliza-tion is thought to control the sizes and shapes of mineralcrystals. Whether it is the same OPN that forms the laminalimitans layer is unknown. However, the ability of OPN,which is prominent in the lamina limitans, to mediate cellattachment is well-established. Although the expression ofOPN is clearly not essential for cement layer formation andtissue integrity, as evident in Opn-/- mice, its absence in thelamina limitans structure may contribute to impairedosteoclast act,vitv

(iv) OPN IN BONE RESORPTION

Remodeling of bone, which occurs throughout life,requires the maintenance of a careful balance betweenformation and resorption. Althougfi the coupling of osteo-clast and osteoblast activities in the homeostasis of bonemass has been questioned by recent studies in whichselective ablation of osteoblasts failed to affect bone masssignificantl, Corral et al., 1998), an influence of osteogeniccells on the fcrmation and function of osteoclasts is, nev-ertheless a likely scenario. Moreooer, OPN appears to bean important component of the communication betweenthese cells, and there is strong evidence for the involve-n ent of OPN n the formation, migration, and attachmento' osteoclasts and for their resorotive activity. The per-ceived role of OPN in osteoclast foimation and function issu-pported by the increased capacity of spleen cells fromOpn-/- mice( generate osteoclasi-s (Rittling et al., 1998).

This may reflect compensation for a deficiency in osteo-clastic resorption (Noda et al., 1998) associated withincreased osteoprotegerin ligand (TRANCE/RANKL/ODF)and its receptor in the bones of Opn-/- mice (Yoshitake etal., 1998). Impaired bone resorption has also beenobserved in Opn-/- mice treated with IL-1, an inflammato-ry cytokine which, like TNF- c, strongly stimulates OPNexpression and bone resorption in normal animals(Takazawa et al., 1998). Similarly, the enhanced boneresorption due to the effects of estrogen deficiency onosteoclastic activity in ovariectomized mice, which is alsomediated by inflammatory cytokines, is not evident in theOpn-/- mice (Yoshitake et al., 1999). Thus, while it is clearthat OPN is not essential for normal bone resorption, itappears, nevertheless, to be required for stimulatingosteoclast generation, as illustrated in Fig. 6.

(a) Attraction, migration, and maturationof osteoclast precursors

Although OPN is not essential for osteoclast activity,impaired recruitment of osteoclasts to ectopicallyimplanted bone from Opn-/- mice (Noda et al., 1998) isindicative of the requirement of OPN expression attargeted sites of bone resorption to attract osteoclastprecursors. This can occur through a combination of achemotactic response involving the CD44 receptor(Weber et al., 1996) and by haptotaxis through theintegrin (xv3 (Senger et al., 1996, both receptorsbeing expressed at high levels in osteoclasts. In con-trast to the cxvA3 receptor, the CD44 receptor does notappear to be involved in cell attachment Katayama etal., 1998) but may be more important in the migratoryresponse, in which OPN can function as an extracellu-lar ligand, and also possibly inside migrating cells, asdiscussed earlier. High levels of OPN are expressed bydifferentiated osteoblasts, including bone-lining cellsand osteocytes, as well as by osteoclasts themselves,and, in both osteogenic and osteoclastic cell lin-eages, OPN expression is strongly stimulated by vita-min D3, which promotes osteoclast differentiation

Figure 5. Post-embedding, colloidal-gold immunocytochemistry for OPN in bone and teeth. Electron micrographs illustrate the most promi-nent sites of OPN accumulation in the calcified extracellular matrices of mineralized tissues where OPN predominantly resides at variouscell- and matrix-matrix interfaces, and in the bulk of the matrix. (a-d) Within the bone, OPN accumulates primarily in cement (reversal)lines (CL) and in patches (arrowheads) of non-collagenous proteins found among the collagen fibrils and in the osteoid at small calcifica-tion foci (inset in b). In the alveolar bone of the periodontium, a frequent observation is the presence of a seam of bone rich in OPN (brack-et in c) at sites of periodontal ligament (PDL) insertion. Osteocytes and their cell processes (cp) are found within lacunae and canaliculi,respectively, both of which are lned by a thin, OPN-containing coating termed the lamina limitans (LL). (e-f) At external bone surfaces,bone-lining cells (BLC) and osteoclasts (OCL) are typically observed in direct apposition to the OPN-rich lamina limitans (LL). More specif-ically, in the case of osteoclasts, well-developed lamellipodia (OCL-Lam) extend beneath periosteal cells (PC) to follow the contour of thelamina limitans at the bone surface. (g) In the tooth, immunolabeling for OPN is particularly abundant in cementum (CEM, bracket) thatlines the root surface and serves as the tooth attachment site for the periodontal ligament (PDL). At certain locations along the root, anOPN-containing cement line (CL), but not a reversal line in this case, is present at the dentino-cementum junction. The mantle dentin (mDEN)of the root, immediately adjacent to the cementum, also exhibits small patches (arrows) of non-collagenous matrix containing OPN thatrepresent the initial sites of calcification that occur along the advancing root edge during tooth development. All electron micrographs aretoken from thin sections of aldehyde-fixed, decalcified rat bone embedded in LR White acrylic resin, immunolabeled with OPN antibodiesand colloidal-gold conjugates by means of the post-embedding approach, and conventionally stained with uranyl acetate and lead citrate.

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Figure 6. Temporal expression of osteopontin during bone remodeling. The resorptionof bone by osteoclasts, and the subsequent replacement by new bone, is shown in thisFig. Osteoclasts are formed from pluripotential mononuclear precursors (CFU-GM)stimulated by monocyte-macrophage colony-stimulating factor (M-CSF). Expression ofOPN, which is associated with the early development of osteoclasts, is up-regulatedwith CD44 in differentiating osteoclast/macrophages and can regulate osteoclast for-mation, activity, and survival (see Fig. 7). Osteogenic precursors express intracellularOPN, associated with cell motility, and secrete a low-phosphorylated OPN which mayact as a cytokine in osteodifferentiation. Osteogenic cells express OPN that associateswith the non-collagenous cement layer, while osteoblasts produce a highly phospho-rylated OPN which regulates hydroxyapatite crystal growth and which is enriched atthe mineralization front and becomes incorporated into the interfibrillar matrix. OPNis also deposited as a lamina limitans layer between the mineralized bone surface andboth osteocytes and bone-lining cells. The major sites of OPN deposition in bone,including the mineralization front, the cement layer, the interfibrillar matrix, and thelamina limitans, are shown in the enlarged section.

and bone resorption, The expression of OPN is asso-ciated with the early development of osteoclasts Ca2+-ATPase (M(Yamate et al., 1997) and macrophages (Krause et al., subunit of cUA31996), and together with CD44, OPN synthesis is pl 30cas) througincreased in promyelocytic leukemia HL-60 cells stim- domains. Uponulated by PMA to differentiate along the X-100 insolublemonocyte/macrophage pathway (Atkins et a)l, 1998). with pl30cas, iNotably, macrophages and osteoclast precursors osteoclastic bordemonstrate strong similarities in their chemoattrac- Rodan, 1999) Iftive responses to OPN and the expression of OPN. other signalingMoreover, the expression of OPN can regulate the vation of NFKBactivity of the macrophages/osteoclasts at different effects of OPN irdevelopmental stages as well as provide a substratum also contributefor osteoclast attachment and opsonin activity in link with the osphagocytic mechanisms. In the subsequent fusion of mation (Kong etpre-osteoclasts to form multi-nucleated osteoclasts, tory cytokines (Ithe non-ligated CD44 receptor may have an important recently beenrole (Sterling et al 1998). (Burgessetal, 11

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(b) Regulation of osteoclastic activity

Since an OPN-deficient bone matrix ispoorly resorbed and has a diminishedcapacity to support cell attachment tobone (Noda et al., 1998), OPN in the lami-na limitans appears to be important forattachment of osteoclasts through the(v3 receptor in vivo, Moreover, phospho-rylation of OPN has been shown to beimportant in mediating osteoclast, butnot osteoblast, cell attachment throughafi, (Ek-Rylander el al., 1994; Katayama ctat., 1998), and it has been suggested(Dodds et al, 1995) that the tartrate-resis-tant acid phosphatase (TRAP) character-istically expressed by osteoclasts maycontrol osteoclast attachment and motil-ity by dephosphorylating OPNRegulation of osteoclast activity by OPNappears to occur primarily through thehighly expressed afi3, which mediatesintracellular signaling pathways (Yamateet al., 1997, Duong and Rodan, 1999),shown in Fig. 7. Moreover, an autocrinepathway is implicated by the inhibition ofosteoclast activity by OPN anti-sensedeoxyoligonucleotides (Tani-Ishii ct al.,1997) The activation of the axfi receptoris Ca2"-dependent and is thought to pro-vide a regulation point for osteoclastsduring bone resorption (Faccio et al.,1998) Thus, OPN can modulate intracel-lular Ca2l in osteoclasts through ligationto x.)fi which stimulates release of Ca2+from intracellular compartments (Zimoloet al., 1994) via a calmodulin-dependent,

iyauchi et al., 19911 Ligation through the Dalso activates the FAK-related PYK2 (and

gh c-Src, which binds PYK2 through the SH2activation, PYK2 translocates to the Tritoncytoskeletal compartment and, together

s found in the sealing zone required forie resorption (Duong et al., 1998. Duong andInaddition, OPN can potentially stimulatepathways in osteoclasts. For example, acti-by OPN, which mediates anti-apoptotic

i endothelial cells (Scatena et al., 1998), mayLo osteoclast survival as well as providing ateoprotegerin regulation of osteoclast for-al., 1999) and the effects on pro-inflamma-Franzoso et al., 1997). Moreover, OPGL hasshown to activate osteoclasts directly999). In addition, OPN can stimulate matrix

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metalloproteinase (MMP) pro-duction through the ERK path-way, which stimulates Fos expres-sion through an SRE, as well asthe activation of MMP-2, whichhas been shown to be mediatedby the cxfi33 integrin in GTC23giant tumor cells (Teti et al, 1998).These pathways are also relevantto phagocytosis by macrophages

(5) SummaryThe functional diversity of OPNin bone formation and remod-eling appears to relate to fun-damental roles of this proteinin host defenses and tissuerepair, the bone remodelingsequence having many featuresof repair processes involvinginflammatory responses. Thus,a primary role of OPN appearsto be that of facilitating recov-ery of the organism after injuryor infection, which generallycauses an increase in itsexpression. OPN stimulatescellular signaling pathways viavarious receptors found onmost cell types, including thecells of mineralized tissues. Itcan regulate cell proliferationand phagocytic activity, andcan both promote and partici-pate in cell migration.Moreover, its apparent abilityto enhance cell survival byinhibiting apoptosis mayexplain why the metastatic pro-ficiency of tumor cells increas-es with increased OPN expres-

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Figure 7. Osteopontin regulation of osteoclost formation and activity. Possible pathways for theregulation of osteoclast formation and activity by OPN, as a matrix protein or as a cytokine, areshown in this diagram. Together with CD44, the at,l3 integrin is highly expressed in osteoclasts andosteoclast precursors, both receptors being a primary target for OPN in signaling, cell attachment,and also possibly for osteoclast chemotaxis (haptotaxis) and migration. Ligation of OPN to pre-osteoclasts could modulate osteoclast development through the OPG/OPGL/RANKL regulatorypathway, which is also influenced by inflammatory cytokines, including TNF-a and IL-1. Ligation ofOPN to osteoclasts through the 1 subunit of the a l33 integrin can activate the FAK-related PYK2(protein tyrosine kinase 2) through c-Src, which binds PYK2 through the SH2 domains. Upon acti-vation, PYK2 translocates to the Triton X- 100 insoluble cytoskeletal compartment and, together withpl 30as, is found in the sealing zone required for osteoclastic bone resorption. Abbreviations:OPG, Osteoprotegerin (decoy receptor for RANK); also known as OCIF, Osteoclast InhibitoryFactor: RANK, Receptor Activator of NFKB, also known as ODAR, Osteoclast Differentiation andActivation Receptor, and as TNFR, Tumor Necrosis Factor Receptor: RANKL, Receptor Activator ofNFKB Ligand, which is identical to TRANCE, Tumor Necrosis Factor (TNF(x)-Related Activation-induced Cytokine; and ODF, Osteoclast Differentiation Factor; and TRAIL, TNF-Related Apoptosis-Inducing Ligand: TRAF, TNFcx-Receptor-Associated Factor.

sion. Modulation of immune and inflammatoryresponses by OPN is reflected in the formation andactivity of macrophages and also osteoclasts. As anextracellular matrix component, it can regulate mineralcrystal formation and growth and can formsupramolecular structures by covalent interactions withother matrix macromolecules. However, more detailedstudies are needed to determine precisely how OPN isable to perform these diverse functions.

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