Matricellular proteins: Extracellular modulators of bone development, remodeling, and regeneration

Bone 38 (2006) 749 – 757

www.elsevier.com/locate/bone

Review

Matricellular proteins: Extracellular modulators of bone development, remodeling, and regeneration Andrea I. Alford a , Kurt D. Hankenson a,b,c,d,⁎ a

Department of Orthopaedic Surgery, Room G161, 400 North Ingalls Building, University of Michigan, Ann Arbor, MI 48109, USA b Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA c Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA d The Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI 48109, USA Received 25 July 2005; revised 7 October 2005; accepted 4 November 2005 Available online 18 January 2006

Abstract Matricellular proteins are components of the extracellular matrix which are highly expressed in the developing and mature skeleton. Members of this protein class serve as biological mediators of cell function by interacting directly with cells or by modulating the activity of growth factors, proteases, and other extracellular matrix proteins. Although skeletons of matricellular protein-null mice are grossly normal, they each display unique deficiencies that are often magnified under pathological conditions. In addition, bone cells from wild-type and matricellular protein-null mice behave differently in various in vitro models of bone matrix synthesis and turnover. In this review, osteopontin, bone sialoprotein, tenascin C, SPARC, and thrombospondins 1 and 2 will each be discussed in the context of bone cell biology. Because the biological effects of matricellular proteins are largely context dependent, in vivo and in vitro results must be considered together in order to fully appreciate the specific contributions that matricellular proteins make to bone physiology and pathophysiology. In particular, it is clear that although matricellular proteins are not required for bone development and function, the proteins act to modulate post-natal bone structure in response to aging, ovariectomy, mechanical loading, and bone regeneration. © 2005 Elsevier Inc. All rights reserved. Keywords: Extracellular matrix; Marrow stromal cell; MSC; Osteoblast; Osteoclast; Knockout mouse

Contents The matricellular concept . . . . . . . . . . . . . . . . . . . . . . . . Expression of MP in skeletal tissue. . . . . . . . . . . . . . . . . . . Expression of tenascin C in skeletal tissues . . . . . . . . . . . . Expression of osteopontin and bone sialoprotein in skeletal tissues Expression of SPARC in skeletal tissues . . . . . . . . . . . . . . Expression of thrombospondins in skeletal tissues . . . . . . . . . Effects of MP-deficiency on the skeleton and on bone cells . . . . . . Effects of tenascin C-deficiency on the skeleton and on bone cells Effects of OPN deficiency on the skeleton and on bone cells . . . Effects of SPARC deficiency on the skeleton and on bone cells . . Effects of thrombospondin deficiency on the skeleton and on bone Roles of MP in fracture healing . . . . . . . . . . . . . . . . . . . . Role of tenascin C in fracture healing . . . . . . . . . . . . . . . Role of SPARC in fracture healing . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. Department of Orthopaedic Surgery, Room G161, 400 North Ingalls Building, University of Michigan, Ann Arbor, MI 48109, USA. Fax: +1 734 647 0003. E-mail address: [email protected] (K.D. Hankenson). 8756-3282/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2005.11.017

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Role of OPN in fracture healing. . . . . . . . . . . . . Role of thrombospondins in fracture healing . . . . . . Role of MP in regulation of bone turnover . . . . . . . . . Role of tenascin C in regulation of bone turnover . . . Role of SPARC in regulation of bone turnover . . . . . Role of OPN and BSP in regulation of bone turnover . Role of thrombospondins in regulation of bone turnover Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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The matricellular concept Skeletal tissues are composed largely of extracellular matrix (ECM). Fibrillar ECM proteins, predominately type I collagen in bone and type II collagen in cartilage, provide structural integrity and account for mechanical strength. The ECM of bone also contains matricellular proteins (MP), which rather than playing structural roles, primarily serve as biological modulators [1]. MP interact with cell surface receptors, such as integrins, the structural matrix, and soluble extracellular factors such as growth factors and proteases. Through these multiple mechanisms, MP can influence cell function, as well as regulate the availability or activity of proteins sequestered in the matrix. Structural ECM proteins like fibronectin and collagen also bind to integrins, thus, MP have the potential to modulate their

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function through competitive binding. Further, the presence of cryptic cell-binding sites in some MP make protein conformation and biologically active proteolytic products two additional factors that contribute to MP function [2–4]. Presumably, the complement of structural ECM, MP, cell surface receptors, and soluble factors determines specific downstream effects on gene expression and cell phenotype. Table 1 summarizes MP–protein interactions deduced from work with a variety of cell types (reviewed in [1,2,5–8]). While these data contribute to our overall understanding of MP function, given that MP interact in a context-dependent manner with a wide-variety of proteins, the interpretation of in vitro data is often difficult. Therefore, examples of documented physiologically relevant outcomes of MP-mediated interactions are also included in Table 1. Finally, Table 1 summarizes available examples of MP-induced changes

Table 1 Matricellular protein interactions and function MP

ECM interactions

Tenascin C Fibronectin

SPARC

Collagens, Ca2+

OPN

Fibronectin, collagens, Ca2+ hydroxyapatite

BSP

Collagen, hydroxyapatite Ca2+

TSP1

Fibrinogen, collagens, heparan sulfated proteoglycans, fibronectin, laminin, Ca2+ Collagens, proteoglycans

TSP2

Cell surface interactions Integrins, contactin/F11, annexin II, heparan sulfated proteoglycans A cell surface receptor for SPARC has not been identified Integrins, CD44

Soluble factor binding

Signaling mechanisms

Biological contributions that may influence bone

Established effects on bone cell phenotype

Tenascin C effects on fibronectin deposition and turnover contribute to wound healing [40]

Promotes osteoblast differentiation [25]

Promotes osteoblast differentiation and survival, inhibits adipogenesis [29] Promotes osteoblast and osteoclast adhesion, differentiation and function [2,44,55] Promotes osteoclast adhesion, differentiation and function [2,7] Promotes angiogenesis [17] Promotes osteoclast function [50]

PDGF, TGFβ1, VEGF, MMP2, bFGF, IGF

Cytoskeleton-dependent

SPARC affects synthesis and assembly of collagen I containing ECM [40]

MMP3, complement factor H, EGF, PTH

Cytoskeleton-dependent, PI3K, Ca2+, calmodulin

Integrins

MMP2, complement factor H

Ca2+, Calmodulin

OPN-αvβ3-cytoskeleton pathway is required for osteoclastic bone resorption Regulation of mineralization

Integrins, HSPG, CD47/IAP, CD36, calreticulin, chondroitin sulfate, LRP, syndecan 1, Thy-1

Clotting cascade factors, TGF-β1, cathepsin, elastase, PDGF, bFGF, MMP2, IGF-1, IGF-BP

Focal adhesion kinase, G protein, PKC, PI3K, p38-MAP kinase, ERK1/2, JNK, Caspase, src

Consistent with a role in regulating inflammation, TSP1 inhibits the release of IL-12, TNFα and IL10 via CD36 and CD47 on dendritic cells

Integrins, HSPG, CD47, CD36, chondroitin sulfate, LRP

MMP2

Caspase

A role for TSP2 in collagen fibrilogenesis is suggested by altered collagen fibril morphology in TSP2-null mice

Inhibits MSC proliferation, promotes osteoblast differentiation, inhibits adipogenesis [32,33]

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in bone cell function. Together with careful analysis of MP knockout mice, in vitro studies suggest that MP contribute to skeletal development, homeostasis, and fracture healing, and in this review, these molecules will be discussed in the context of bone cell function and skeletal biology. Expression of MP in skeletal tissue MP are expressed at high levels during embryonic development where they contribute to tissue growth and morphogenesis. In healthy adult tissues, MP are expressed at lower levels, but during inflammation, wound healing [2,9,10], and aging [8], MP levels increase. Inappropriate MP expression is also associated with cancer. The specific effects of MP on disease progression are variable (reviewed in [6,11,12]). For example, increased OPN and BSP levels are seen with inflammation and tissue remodeling, as well as ectopic mineralization, that is associated with multiple pathologies [2,7]. Recent data suggest that BSP and OPN specifically bind to and promote activation of matrix metalloproteinases (MMP)2 and -3, respectively, even in the presence of MMP-specific tissue inhibitors of metalloproteinases (TIMP) [13]. Taken together, these observations suggest that increased OPN or BSP levels might contribute to increased MMP activity associated with tumor metastasis to bone. While MP gene expression does decline in adult musculoskeletal tissues, they contain higher levels under normal conditions than do most other organ systems [9]. Since MP are involved in tissue development, their constitutive expression in bone might reflect this tissue's constant adaptive remodeling. Fig. 2 summarizes MP expression in cells that contribute to bone formation.

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Expression of tenascin C in skeletal tissues Tenascin C is a large hexameric MP, primarily associated with sites of new tissue formation [14]. The tenascin C monomer is a modular glycoprotein with epidermal growth factor-like repeats, fibronectin type III repeats, and a globular fibrinogen-like C-terminus (Fig. 1). Alternative splicing of tenascin C transcripts generates multiple isoforms which display tissue and cell type-specific patterns of expression [14]. At the onset of skeletal development, tenascin C is expressed by condensing mesenchyme (Fig. 2). Osteoblasts continue to synthesize tenascin C during the phase of bone growth and morphogenesis [14], but it does not appear to be present in mineralized matrix. In mature bone, most tenascin C expression is confined to the pericellular space surrounding some osteocytes and to articular cartilage [14]. Expression of osteopontin and bone sialoprotein in skeletal tissues Osteopontin (OPN) and bone sialoprotein (BSP) are both acidic, phosphorylated glycoproteins which are highly expressed in bone matrix [2,7] (Fig. 2). Analysis of OPN-null mice has revealed important contributions that these MP make to bone physiology and pathophysiology. However, since BSP and OPN share structural and functional similarities (Fig. 1), the generation of BSP-null and BSP/OPN-double null animals would help further clarify the biological roles of these MP in the skeleton. Both OPN and BSP have also been shown to modulate the structure of mineralized matrix in vitro. Specifically, while BSP appears to be required for nucleation of hydroxyapatite crystal formation, OPN inhibits mineralization by physically

Fig. 1. MP structure. Each MP is represented as series of functional domains arranged from the amino terminus on the left to the carboxyl terminus on the right. Six tenascin C monomers form hexamers through covalent interactions near the amino terminus. BSP and OPN are structurally very similar, except that BSP is larger and the predominant acidic amino acids differ, as indicated. The SPARC homolog, hevin, has a longer acidic domain at the amino terminus, as indicated. The thrombospondins each form homotrimers. See text for additional details.

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blocking crystal growth [2,7]. During skeletal development, immature and mature osteoblasts secrete OPN into the matrix [15,16]. BSP mRNA levels peak just prior to the onset of mineralization, and the protein is secreted by osteoblasts which are actively synthesizing matrix [15]. At this time, the proangiogenic effects of BSP [17] potentially modulate vascularization of new bone. In healthy mature bone, BSP and OPN are relatively restricted to osteoblasts and osteoclasts participating in active remodeling [18]. Expression of SPARC in skeletal tissues SPARC (secreted protein, acidic and rich in cysteine)/ osteonectin is a calcium-binding MP (Fig. 1) found in many tissues undergoing morphogenesis or remodeling. SPARC modulates ECM synthesis and turnover through its effects on collagen and extracellular proteases [6]. Moreover, peptides generated by protease digestion of SPARC differentially affect angiogenesis [6]. SPARC expression parallels that of OPN during skeletal development. In mature bone, SPARC is found in the pericellular matrix surrounding osteoblasts and osteocytes (Fig. 2).

Table 2 MP knockout mice MP gene disrupted

Tenascin C Abnormal behavior [9], altered excisional wound healing [40] OPN Altered excisional wound healing and T-cell-mediated immunity, context-specific changes in macrophage-induced debridement [2]

SPARC

Expression of thrombospondins in skeletal tissues Thbs1

Thrombospondins (TSP) are a small family of secreted, modular glycoproteins. TSP1 and TSP2 each form 450-kDa homotrimers, and they display considerable sequence homology. TSP1 and TSP2 are both expressed by mesenchymal cells and chondrocytes in developing cartilage [14,19]. As invading osteoblasts replace the mineralized cartilage scaffold, TSP2 immunoreactivity diminishes in chondrocytes and increases, to variable degrees, in the matrix of areas undergoing ossification [19] (Fig. 2). TSP1 and 2 are each potent anti-angiogenic factors [20], suggesting that they might regulate blood vessel formation in new bone [21]. Effects of MP-deficiency on the skeleton and on bone cells Physiologically relevant functions of MP have largely been deduced using knockout mice (Table 2). Mice deficient in any single MP are viable, and they reproduce normally, but each displays a variety of connective tissue abnormalities under normal conditions and markedly defective tissue regeneration following injury. The bone marrow of adult mammals contains mesenchymal cells which are thought to provide a source of osteoblast lineage cells that contribute to routine renewal of bone, as well as to fracture healing (Fig. 2). These marrow stromal cells (MSC) can be isolated and, depending on culture conditions, will differentiate into osteoblast-, chondrocyte-, or adipocyte-like cells [22]. Assembly of a unique ECM is associated with each cell type, and MP potentially modulate MSC differentiation by affecting matrix composition and structure. Primary MSC grown with ascorbate and betaglycerophosphate produce nodules of mineralized matrix. In a manner qualitatively similar to that seen during bone development, alkaline phosphatase, type I collagen, SPARC, and OPN levels increase prior to the onset of mineralization, while BSP

General phenotype

Thbs2

Decreased collagen and altered fibril structure, altered distribution of collagen IV and laminin in epithelial basement membranes, early onset cataractogenesis, accelerated wound healing, increased angiogenesis, greater age-associated increases in adipose tissue and serum leptin levels [6] Reduced viability and fecundity, pulmonary inflammatory disorders, prolonged wound healing Disordered collagen fibrillogenesis, increased vascular density, bleeding disorder, accelerated wound healing

Skeletal phenotype Altered fracture healing [38]

Brittle bones, increased trabecular volume, dysfunctional osteoclasts, abnormal bone resorption, resistance to unloading-induced bone loss, increased ectopic mineralization [2], increased marrow cellularity, altered hematopoesis [56] Age-associated osteopenia due to decreased osteoclast and osteoblast numbers

Curvature of the spine, craniofacial dysmorphism

Increased cortical bone density, increased MSC numbers, accelerated fracture healing, resistance to OVX-induced bone loss, Altered pattern of loadinduced bone formation

and osteocalcin levels peak as nodules appear and mineralization continues [23]. Analysis of the skeletal phenotypes of MPnull mice, as well as comparison of wild-type and MP-null MSC and osteoblasts in vitro, has helped elucidate specific contributions that MP make to bone physiology and pathophysiology. Effects of tenascin C-deficiency on the skeleton and on bone cells Despite early expression during embryonic bone development and reappearance at sites of new bone formation [24], tenascin C-null mice do not display any developmental skeletal deficiencies [9]. However, in vitro data support the premise that tenascin C might regulate differentiation of osteoblast lineage cells. For example, tenascin C promotes alkaline phosphatase activity and collagen synthesis in osteosarcoma-derived osteoblast-like cells [25]. The observation that osteoblasts cultured on tenascin C remain rounded suggests that these MP might regulate cell phenotype by modulating cell shape. Interestingly, induction of cell rounding alone does not mimic the effects of tenascin C, implicating a specific osteoblast cell surface receptor in the downstream effects of this MP on bone cell phenotype [25].

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Fig. 2. Expression of MP by bone lineage cells. Expression of MP genes are indicated in boxes next to the various bone lineage cells. The MP compositions of newly formed osteoid and mineralized bone matrix are also indicated. Osteoblasts synthesize osteoid during development, as well as during normal metabolism of adult bone. See text for details. Abbreviations: MC (mesenchymal condensates); MSC (marrow stromal cell); OB (osteoblast); OC (osteocyte), OCL (osteoclast), TNC (tenascin C); OPN (osteopontin), TSP (thrombospondin).

Effects of OPN deficiency on the skeleton and on bone cells The most well-characterized effects of OPN in bone are on osteoclasts and bone turnover. Nevertheless, some evidence suggests that OPN also modulates osteoblast phenotype. In vitro, OPN stimulates MSC attachment and migration [26], and it induces osteocalcin expression and alkaline phosphatase activity via αvβ3 integrin and focal adhesion kinase (FAK) in these cells [27]. Similarly, overexpression of OPN in MSC cultures leads to greater culture time-dependent increases in alkaline phosphatase activity and mineral deposition [28]. Conversely, normal suppression of osteoblast synthetic activity under pathological conditions such as unloading is not observed in OPN-null mice [2]. These apparently contradictory observations suggest that the specific downstream affects of OPN on osteoblasts might depend on the differentiation state of the cell. Alternatively, or in addition, the downstream effects of OPN on osteoblastic cells may be differentially affected by the extracellular environments of normal and pathologic bone. Effects of SPARC deficiency on the skeleton and on bone cells The observation that SPARC-deficiency has a variety of tissue-specific effects in mice (Table 2) illustrates the concept that the effects of MP on cells are largely context-dependent [1]. In the context of mesenchymal cell differentiation, SPARC-null mice display an increased volume of adipose tissue [6] and a marked age-associated osteopenia due to decreased osteoblast

and osteoclast numbers [1]. Similarly, primary MSC isolated from SPARC-null mice display an increased likelihood to differentiate into adipocytes rather than osteoblasts [29]. While MSC isolated from wild-type and SPARC-null mice have similar rates of proliferation, a larger fraction of wild-type MSC survive under conditions of serum starvation [29]. Together, these data implicate SPARC as an important modulator of MSC proliferation, differentiation, and survival. Effects of thrombospondin deficiency on the skeleton and on bone cells In vivo, TSP1 and 2 have different temporal and spatial expression patterns [14,19,30], as a result of distinct proximal promoter sequences [31]. Thus, TSP1 and 2 knockout animals have largely distinct phenotypes (Table 2), and there is no apparent compensatory increase in expression of the other thrombospondin [1]. With regards to skeletal development, mice lacking TSP1 are born with curvature of the spine, and they display minor abnormalities in trabecular bone [1]. The skeletal phenotype of TSP2-null mice further supports the premise that MP functions are largely dependent upon the physiological context in which they are expressed. TSP2-null mice display increased endocortical, but not periosteal, bone formation rates compared to wild-type mice, as a result of a larger pool of marrow osteoprogenitor cells [32]. Interestingly, TSP2 deficiency results in an altered loading-induced pattern of bone formation compared to wild-type mice. In normal mice,

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loading (0.225 N and 0.5 N) results in increased periosteal, but not endosteal bone formation. In TSP2-null mice, this pattern of loading-induced bone formation is reversed: the endosteal surface displays significant increases in bone formation, while the periosteal surface is not affected [57]. Differential effects of TSP2 deficiency on periosteal and endocortical bone surfaces suggest that the function of TSP2 might depend on whether it is acting as a soluble marrow-derived factor or as part of the bone matrix. In vitro mineralization experiments using primary MSC isolated from wild-type and TSP2-null mice suggest that the increased cortical bone density of TSP2-null mice is due to increased numbers of osteoblast precursors, rather than to an altered synthetic capacity of the cells. As such, MSC from normal and TSP2-null mice express identical cell surface markers, and they achieve similar levels of mineralization over extended time in culture. However, despite greater numbers of osteoblast precursor cells, the onset of mineralization is actually delayed in cultures of TSP2-null MSC [32]. Because TSP2 inhibits DNA synthesis in vitro, increased MSC numbers and delayed mineralization in the absence of TSP2 may reflect a mutually exclusive relationship between proliferation and mineralization in vivo. Thus, in the context of bone formation, the anti-proliferative effects of TSP2 might precipitate temporal patterns of gene expression associated with maturation of osteoblast lineage cells [32,33]. In support of this hypothesis, osteoinduction of the MSC line, ST2 with BMP and dexamethasone induces TSP2 expression. Temporally, the appearance of TSP2 in the medium occurs just prior to and overlaps the onset of mineralization (Hailu Shitaye and KDH, unpublished observations). In addition, the possibility that TSP2 affects MSC cell phenotype through mechanisms other than proliferation cannot be excluded. For example, when cultured under a mild adipogenic stimulus, MSC isolated from TSP2-null mice produce higher levels of lipid compared to wild-type MSC [34]. Together with the observed skeletal effects of TSP2 deficiency discussed above, these in vitro data suggest that the role of TSP2 in bone is largely context-dependent.

qualitatively similar to that seen during embryonic bone development [35,36]. Interestingly, application of tension to the fracture site has been shown to increase and prolong the rise in MP gene expression associated with fracture healing and to accelerate repair [10,37].

Roles of MP in fracture healing

During both normal and distraction-enhanced fracture healing, OPN gene expression is restricted to terminally differentiated chondrocytes, osteoblasts, and osteoclasts participating in endochondral bone formation [10,35]. As mineralization begins, OPN mRNA levels increase [35,36], and the protein is deposited into the matrix as it is mineralized [37]. As with SPARC, distraction prolongs the increased OPN expression associated with new bone formation. Although the volume of the fracture callus is decreased in OPN-null mice, they display normal levels of mineralization during fracture repair. One possible explanation for a smaller callus is that OPN-null fractures display a reduced average vessel diameter within sites of neovascularization. In addition, mechanical testing reveals that regenerated bones of OPN-null mice are brittle compared to those of wild-type mice [39]. Together with the observed effects of OPN on osteoblast differentiation in vitro outlined above, these data suggest that OPN potentially modulates formation of new bone through multiple mechanisms.

Despite distinct temporal expression patterns during osteogenesis, and clear effects on osteoblasts in vitro, MP-null mice do not display overt skeletal defects. Instead, the effects of MP deficiency are manifested to a greater degree under pathological conditions, such as fracture healing and estrogen depletion. As with embryonic development, new bone formation following injury occurs by both intramembraneous and endochondral pathways. During repair of a fractured long bone, a cartilaginous provisional matrix, called a fracture callus, is deposited by chondrocytes at the wound site. With time, the site becomes calcified and is replaced by bone as osteoblasts and osteoclasts remodel the provisional matrix. In other parts of the callus and in mechanically stable defects, mesenchymal precursor cells differentiate into bone forming osteoblasts. During both intramembraneous and endochondral repair, matrix protein expression patterns change in a defined manner, which is

Role of tenascin C in fracture healing In contrast to the normal skeletal development of tenascin Cnull mice, bone regeneration in these mice is accelerated and uncontrolled. Osteoblastic cells invade the defect and lay down new bone sooner in tenascin C-null mice compared to wild-type mice, and the new trabecular bone eventually occludes the marrow cavity. In addition, osteoclasts begin resorbing the new bone before osteocytes have been incorporated into the mineralized matrix [38]. These observations implicate tenascin C as an important attenuator of osteogenesis, as well as of osteoclast maturation. Role of SPARC in fracture healing SPARC mRNA is detected immediately after injury as periostial cells begin proliferating and depositing ECM [35]. Similarly, in a rat model of distraction osteogenesis, SPARC is expressed by mesenchymal cells and osteoblasts as they begin the process of endochondral ossification [10]. Unlike nondistracted controls, gene expression remains elevated in cells participating in intramembraneous bone formation at later stages of distraction [10]. In contrast, hypertrophic chondrocytes participating in endochondral ossification express relatively low levels of SPARC [35,36]. Fracture healing in SPARC-null mice has not been investigated, but its upregulation throughout the fracture healing process, sensitivity to mechanical tension, and effects on MSC in vitro suggest that SPARC might be an important modulator of new bone formation following injury. Role of OPN in fracture healing

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Role of thrombospondins in fracture healing Excisional wounding and fracture both lead to immediate activation of the coagulation cascade and formation of a provisional extracellular matrix. At the same time, inflammatory cells and stem cells are recruited to the injury or fracture site [40,41]. Because early cellular events following both forms of injury are similar, noted accelerated dermal wound healing in both TSP1 and TSP2-null mice suggests that these MP might also modulate fracture healing. Indeed, TSP2-null mice display accelerated fracture healing. Specifically, TSP2-null mice have a reduction in cartilage and enhanced bone at an earlier time point (day 10 post-fracture) than wild-type mice. Significantly, the TSP2-null fractures show an associated increase in torsional strength. Altered fracture healing might reflect increased angiogenesis at the callus site, as well as increased numbers of mesenchymal progenitor cells in TSP2-null bones [21,32]. Alternatively, altered dermal wound healing is associated with increased MMP2 levels in both TSP1 and 2-null mice, and this protease could also contribute to accelerated fracture healing observed in TSP2-null animals. Role of MP in regulation of bone turnover Adult bone undergoes continual adaptive remodeling via simultaneous osteoclast-mediated resorption and osteoblastinduced synthesis. Osteoclasts and osteoblasts each produce factors which regulate bone turnover through autocrine and paracrine pathways. Following bone resorption, osteoblast lineage cells deposit a matrix, called the reversal line, that is rich in BSP and OPN [42], but also contains tenascin C [24]. Contrary to normal turnover, estrogen loss associated with menopause, or with ovariectomy (OVX) in animal models, leads to increased bone synthesis and resorption. Under these conditions, resorption outpaces synthesis and leads to osteoporosis. Using MP knockout mice, several groups have shown that MP contribute to OVX-induced bone loss. In the following sections, the influences of MP on osteoclast function are discussed. Role of tenascin C in regulation of bone turnover The possible contribution of tenascin C to OVX-induced bone loss has not been reported. However, the observations that tenascin C-null mice display inappropriate bone resorption during fracture repair [38] and that osteoclasts do not adhere to tenascin C in vitro [43] suggest that this MP might affect osteoclast function by modulating these cells' attachment to bone. Role of SPARC in regulation of bone turnover The osteopenia of SPARC-null mice reflects decreased bone synthesis and turnover as the result of reductions in both osteoblast and osteoclast numbers [29]. Because osteoblasts promote osteoclast maturation, reduced osteoblast numbers associated with SPARC deficiency may, by itself, lead to

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reduced osteoclast numbers. On the other hand, unlike those derived from wild-type mice, SPARC-deficient osteoclasts do not express cyclin A or proliferate in response to PDGF [29], suggesting direct effects of SPARC on osteoclast phenotype. Together, these observations suggest that SPARC might affect bone turnover through its effects on both osteoblasts and osteoclasts. Role of OPN and BSP in regulation of bone turnover OPN-null mice display various abnormalities in bone turnover under both normal and pathological conditions. For example, unlike wild-type mice, OPN-null mice subjected to hind-limb suspension or to ovariectomy do not lose bone mass [2,44]. Osteoclasts synthesize OPN, as well as BSP [7,18], and they bind RGD-containing peptides derived from both MP within the mineralized matrix [2,7]. Although BSP has been shown to inhibit the differentiation of immature precursor cells to osteoclasts in vitro [7], OPN and BSP both stimulate osteoclast-induced bone resorption. Antibody blocking studies have shown that αvβ3 integrin-OPN binding is required for osteoclasts to attach to and resorb bone. Subsequent studies illustrated that this MP–integrin interaction induces the cytoskeletal rearrangements required for podosome formation by activating gelsolin via pp60src and PI3K [2]. Interestingly, an intracellular pool of OPN appears to associate with CD44 and contribute to cell process formation and migration in osteoclasts [45,46]. Together these data suggest that the phenotype of OPNnull mice (Table 2) is due in part to the absence of OPN-integrin– cytoskeleton interactions (Table 1) necessary for osteoclast activation. As indicated in Table 1, OPN also modulates maturation of osteoclast lineage cells directly by stimulating transcription of genes encoding resorptive enzymes [47] and by promoting acidification of the extracellular environment [48]. Role of thrombospondins in regulation of bone turnover As a result of decreased bone resorption and increased bone synthesis, TSP2-null mice are protected from OVX-induced bone loss and in fact display no significant changes in bone geometry or density in response to estrogen depletion [49]. Increased bone formation in OVX-TSP2-null mice is associated with expansion of marrow-derived osteoblast lineage cells following estrogen depletion. TSP2-null mice also display lower levels of OVX-induced bone resorption in comparison to wild-type mice, thus suggesting a role for TSP2 in the regulation of osteoclast function under estrogen deprivation. While osteoclasts do not adhere to intact TSP [43], a peptide containing the CD36 binding sequence present in both TSP1 and 2 promotes resorption in vitro [50]. CD36 receptor-binding recruits components of c-src signaling in platelets [51], and this pathway is essential for normal bone resorption in mice [52,53]. Whether TSP2 modulates osteoclast-induced bone resporption via CD36 and c-src remains to be determined. Nevertheless, these data suggest that TSP2, present in MSC, osteoblasts and/ or osteoclasts, might modulate bone resorption by affecting osteoclast–osteoblast or osteoclast–ECM interactions.

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Conclusions Taken together, published data indicate that MP modulate bone cell function in multiple contexts. While MP are expressed at high levels during development, where they appear to affect ECM remodeling, survival of knockout animals suggests that MP are not required for structural integrity. Multiple growth factors, proteases, structural ECM, and MP play an orchestrated role in tissue repair and remodeling. While MP are not required for bone development or function, they clearly modulate normal physiology, healing, and remodeling. Understanding the modulatory nature of MP is essential for fully understanding the mechanisms of other, more highly studied anabolic and catabolic agents. Knowledge of the contributions that MP make to tissue homeostasis should be helpful in designing pharmaceutical therapies and tissue engineered scaffolds to improve bone healing, reduce bone resorption, and restore bone mass. Indeed, localized delivery of antisense-TSP2 DNA has been shown to alter the foreign body response to implanted biomaterials by promoting angiogenesis and the formation of a loose network of collagen fibers [8]. Conversely, in an animal model of rheumatoid arthritis, the presence of TSP2 in the synovium correlates with decreased vascularization and inflammation [54]. The biological effects of MP are clearly cell- and tissue-specific, and careful consideration of the contexts in which they are expressed should be made in the design of therapeutic interventions. Acknowledgments K.D.H. is supported by NIH grants AR8562 and RR00161. A.I.A is supported by the University of Michigan Regenerative Science Training Program (T90DK070071). We would like to thank Drs. Joshua D. Miller and Steven A. Goldstein for the insightful discussions and for the review of the manuscript. References [1] Bornstein P, Sage EH. Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol 2002;14:608–16. [2] Giachelli CM, Steitz S. Osteopontin: a versatile regulator of inflammation and biomineralization. Matrix Biol 2000;19:615–22. [3] Yamamoto N, Sakai F, Kon S, Morimoto J, Kimura C, Yamazaki H, et al. Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis. J Clin Invest 2003;112:181–8. [4] Hotchkiss KA, Matthias LJ, Hogg PJ. Exposure of the cryptic Arg-GlyAsp sequence in thrombospondin-1 by protein disulfide isomerase. Biochim Biophys Acta 1998;1388:478–88. [5] Mackie EJ. Molecules in focus: tenascin-C. Int J Biochem Cell Biol 1997;29:1133–7. [6] Framson PE, Sage EH. SPARC and tumor growth: where the seed meets the soil? J Cell Biochem 2004;92:679–90. [7] Ganss B, Kim RH, Sodek J. Bone sialoprotein. Crit Rev Oral Biol Med 1999;10:79–98. [8] Bornstein P, Agah A, Kyriakides TR. The role of thrombospondins 1 and 2 in the regulation of cell-matrix interactions, collagen fibril formation, and the response to injury. Int J Biochem Cell Biol 2004;36:1115–25. [9] Mackie EJ, Tucker RP. The tenascin-C knockout revisited. J Cell Sci 1999;112:3847–53. [10] Sato M, Yasui N, Nakase T, Kawahata H, Sugimoto M, Hirota S, et al. Expression of bone matrix proteins mRNA during distraction osteogenesis. J Bone Miner Res 1998;13:1221–31.

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