Integrins and other cell surface attachment molecules of bone cells

Chapter 17

Integrins and other cell surface attachment molecules of bone cells Pierre J. Marie1 and Anna Teti2 1

UMR-1132 Inserm (Institut national de la Santé et de la Recherche Médicale) and University Paris Diderot, Sorbonne Paris Cité, Paris, France;

2

Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy

Chapter outline Introduction Role of integrins in bone cells Osteoblasts and osteocytes Osteoclasts Chondrocytes Role of cadherins in bone cells Osteoblasts and osteocytes Osteoclasts Chondrocytes Roles of other attachment molecules in bone cells

401 401 401 404 406 406 406 408 409 410

Syndecans Glypicans and perlecan CD44 Immunoglobulin superfamily members Osteoactivin Chondroadherin Conclusion Acknowledgments References

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Introduction Cell adhesion molecules play important roles in bone cell functions and fate. Integrins are a family of cell surface adhesion transmembrane molecules composed of a chains and b chains that assemble noncovalently as heterodimers (Campbell and Humphries, 2011), allowing the attachment of osteoclasts and osteoblasts to the extracellular matrix (ECM). Binding of integrins to ECM proteins is essential for the function of bone cells (Bennett et al., 2001; Horton, 2001), and contributes to activating a number of intracellular signals that govern bone cell fate and activity (Marie et al., 2014a). Cadherins are other attachment molecules that control the functions of bone cells through cellecell adhesion, interactions with other cell surface molecules, and modulation of intracellular pathways (Marie et al., 2014b). During recent years, progress in cell biology and mouse genetics has led to significant advances in our understanding of the roles of integrins, cadherins, and other cell attachment molecules in the control of bone cell activity in vitro and in vivo, and the mechanisms involved in these functions are now better understood. This chapter updates our knowledge of the role of these molecules in bone cell functions and discusses potential therapeutic strategies that emerged from these findings.

Role of integrins in bone cells Osteoblasts and osteocytes Osteoblasts and osteocytes express several integrins, although the pattern of expression varies with the stage of cell differentiation (Clover et al., 1992; Hughes et al., 1993; Hultenby et al., 1993; Grzesik and Robey, 1994). Integrin binds to the ArgeGlyeAsp (RGD) sequence present in bone matrix proteins such as fibronectin, type I collagen, bone sialoprotein, and osteopontin (Gronthos et al., 2001; Grzesik, 1997; Puleo and Bizios, 1991; Schaffner and Dard, 2003). Integrins control cell adherence to the ECM through the assembly of intracellular proteins linked to the cytoskeleton. In addition to allowing

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cellematrix adherence, integrins play a key role in osteoblast differentiation (Moursi et al., 1997; Jikko et al., 1999; Mizuno et al., 2000), an effect that is mediated by intracellular signals generated by ECMeosteoblast interactions. IntegrineECM interaction leads to the phosphorylation of focal adhesion kinase (FAK) and subsequent activation of mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K), or GTPases of the Rho family (Lai et al., 2001; Salasznyk et al., 2007; Khatiwala et al., 2009). These signals converge to promote specific gene expression and osteoblast differentiation. In mesenchymal skeletal cells (MSCs), ERK1/2 phosphorylation induced by ECMeintegrin binding leads to RUNX2 activation and osteoblast differentiation (Ge et al., 2012). FAK also activates Wnt/b-catenin signaling (Sun et al., 2016), making the activation of FAK an essential step for osteogenic differentiation (Tamura et al., 2001). In vivo, deletion of FAK in type I collagen-expressing osteoblasts reduced reparative bone formation in mice (Rajshankar et al., 2017). However, the lack of FAK in osteoblasts may be in part compensated for by proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, in the focal adhesion sites (Kim et al., 2007). One important regulator of integrin-mediated signaling is integrin-linked kinase (ILK), a linker between integrins and the cytoskeleton. In osteoblasts, ILK-dependent phosphorylation of a-nascent polypeptideeassociated complex, a c-JUN transcriptional coactivator, potentiates c-JUN-dependent transcription and osteoblast maturation (Meury et al., 2010). ILK also interacts with Wnt signaling by phosphorylating glycogen synthase kinase-3b, leading to b-catenine lymphoid enhancer factor transcriptional activity and expression of Wnt target genes (Dejaeger et al., 2017). Data indicate that conditional inactivation of ILK in osteoprogenitors impaired bone formation associated with reduced bone morphogenetic protein (BMP)/Smad and Wnt/b-catenin signaling, showing the important role of this linker between integrins and the cytoskeleton in bone formation (Dejaeger et al., 2017). In addition to control of cell differentiation, osteoblast attachment to the ECM is essential for cell survival (Grigoriou et al., 2005; Triplett and Pavalko, 2006). Disruption of RGDeintegrin binding leads to altered osteoblast adhesion in vitro (Gronthos et al., 2001), and conversely integrineECM interaction suppresses cell apoptosis through PI3K activation (Frisch and Ruoslahti, 1997). Thus, integrins play an important role in the control of bone formation through activation of signaling mechanisms regulating both osteoblast differentiation and survival (Fig. 17.1). The specific role of integrins in osteoblast function and fate is now better understood. The b1 integrin is the main adhesion receptor required for adhesion to fibronectin and type I collagen in vitro. In vivo, impairment of b1 integrin in mature osteoblasts led to decreased osteoblast activity, bone formation, and bone mass in growing mice (Zimmerman et al., 2000). Disruption of b1 integrin signaling by overexpression of its cytoplasmic tail resulted in skeletal defects, showing the important role of b1 integrin in osteogenesis (Globus et al., 2005). In support of the role of b1 integrin in bone formation, ablation of the specific b1 integrin regulator ICAP-1 in osteoblasts resulted in defective osteoblast proliferation, differentiation, and function; decreased type I collagen deposition; and delayed bone formation in mice (Bouvard et al., 2007; Brunner et al., 2011). Data showed that conditional b1 integrin deletion in early osteogenic mesenchymal cells or in preosteoblasts caused abnormal skeletal ossification or affected intramembranous and

ECM α

β Integrin

ILK

GSK3 β-Catenin

FAK PI3K ERK1/2

AKT β-Catenin AP-1

Runx2

TCF/LEF

FIGURE 17.1 Role of integrins in osteoblasts. Simplified scheme showing how integrin-mediated signals regulate osteoblast function. Extracellular matrix (ECM)eintegrin interaction via RGD interacts with integrin-linked kinase (ILK) and activates focal adhesion kinase (FAK), extracellular signalregulated kinase 1/2 (ERK1/2), and Wnt/b-catenin signaling, leading to Runx2 expression and osteogenic differentiation, and with phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) to trigger osteoblast survival (see text for more details). AP-1, activator protein 1; GSK3, glycogen synthase kinase3; TCF/LEF, T cell factor/lymphoid enhancer factor.

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endochondral bone formation in young mice. In contrast, osteocalcin-specific b1 integrin deletion had only minor effects on the skeletal phenotype, indicating that the b1 integrin is essential for early stages of osteogenesis in vivo (Shekaran et al., 2014). In addition to b1, other integrins were shown to control osteoblast function and fate. Early in vitro studies showed that the aVb3 integrin is involved in dexamethasone- and BMP-2-induced osteoblast differentiation (Cheng et al., 2000, 2001). More recent data indicate that the aVb3 integrin mediates BMP-2-induced osteoblast differentiation in vitro through activation of the ILK/ERK1/2 signaling pathway (Su et al., 2010). Another integrin, aVb5, was found to mediate osteoblast attachment to vitronectin in vitro (Lai et al., 2000). The aVb1 integrin was reported to promote osteoblast differentiation and to inhibit adipocyte differentiation in MSCs through its interaction with RGD present in osteopontin (Chen et al., 2014). Another integrin, a2b1, a major receptor for collagen type 1, is involved in osteoblast differentiation (Takeuchi et al., 1997) by activating ERK signaling and RUNX2 expression in vitro (Xiao et al., 1998). In vivo, a2 integrin deficiency resulted in altered bone properties in mice (Stange et al., 2013). This integrin was found to play a key role in MSC osteogenic differentiation and survival through activation of Rho-associated protein kinase (ROCK) and FAK signaling (Popov et al., 2011; Sens et al., 2017). The a4b1 integrin, which binds to fibronectin, was also found to be involved in MSC homing and osteoblast differentiation in vivo (Kumar and Ponnazhagan, 2007). Both MSCs and osteoblasts express a5b1 integrin, which interacts with fibronectin and is involved in the differentiation of osteoblast precursor cells (Hamidouche et al., 2009). The downregulation of a5 integrin subunit blunts osteoblast differentiation, while its overexpression promotes osteoblast differentiation in MSCs, an effect that is mediated by FAK and ERK1/2 signaling leading to RUNX2 activation (Hamidouche et al., 2009). a5b1 integrin activation also promotes insulin-like growth factor 2 (IGF-2) expression and signaling in MSCs, which contributes to osteogenic differentiation (Hamidouche et al., 2010). In addition, a5b1 integrin activation cross talks with Wnt signaling. a5b1 integrin priming by a monoclonal antibody, or a high-affinity peptide ligand, leads to increased osteogenic differentiation (Hamidouche et al., 2009) in part through activation of PI3K/protein kinase B (AKT) and Wnt/b-catenin signaling, indicating that Wnt/ b-catenin signaling is involved in osteoblast differentiation mediated by a5b1 integrin (Saidak et al., 2015). In pluripotent cells, other integrins may be involved in osteogenic differentiation. In vitro data indicate that fibrinogen binds to the a9b1 integrin in human embryonic stem cells to induce pluripotent stem cell osteogenic differentiation mediated by SMAD1/5/8 signaling and Runx2 expression (Kidwai et al., 2016). There is ample evidence that integrins are critically involved in the response of osteoblasts and osteocytes to mechanical loading via cytoskeletoneintegrin interactions at focal adhesion sites (Katsumi et al., 2004; Bonewald and Johnson, 2008; Turner et al., 2009) and induction of intracellular signaling mechanisms, including FAK, ERK, ROCK, PI3K/AKT, and Wnt signaling (Pommerenke et al., 2002; Wu et al., 2013; Du et al., 2016; Plotkin et al., 2005; Wang et al., 2007; Lee et al., 2010; Chen and Jacobs, 2013; Uda et al., 2017). Several integrins may be involved in the mechanisms mediating mechanotransduction, depending on the context. As expected from the aforementioned important role of b1 integrin in osteoblast differentiation, b1 integrin expressed by osteogenic cells was found to play a major role in mechanotransduction (Iwaniec et al., 2005; Phillips et al., 2008; Litzenberger et al., 2010). In osteocytes, in addition to the b1 integrin, the aVb3 integrin is required for mechanotransduction (Thi et al., 2013), which involves calcium influx (Miyauchi et al., 2006), IGF-1 expression (Dai et al., 2014), and inhibition of the Wnt inhibitor sclerostin by periostin (Bonnet et al., 2012). In vivo, skeletal unloading in mice decreased aVb3 integrin expression in osteoblasts, resulting in altered activation of IGF-1 signaling (Bikle, 2008). Skeletal unloading also reduced a5b1 integrin expression in osteoblasts and osteocytes, resulting in decreased PI3K signaling and altered bone formation (Dufour et al., 2007). Conversely, mechanical strain activates a5b1 integrin (Yan et al., 2012) and PI3K/AKT signaling in osteoblasts (Watabe et al., 2011). The activation of a5b1 integrin by mechanical stress in osteoblasts also causes opening of connexin 43 hemichannels and the subsequent release of anabolic factors (Batra et al., 2012). Other integrins were found to be involved in mechanotransduction. In vitro, the a2 integrin subunit is upregulated during induction on stiffer matrices and mediates the osteogenic differentiation of MSCs (Shih et al., 2011). However, mechanosensitivity appears to depend on the ligation between a2 or a5 subunit and ECM proteins to induce FAK activation (Seong et al., 2013), suggesting that specific integrin subunits may mediate mechanosensitivity in osteoblasts. Thus, several mechanisms generated by integrineECM interactions in osteoblasts and osteocytes are involved in the bone cell response to mechanotransduction. The essential role of integrins in both bone formation and mechanotransduction suggests that targeting specific integrins may be an efficient way to promote osteogenic differentiation and bone regeneration. Several strategies were used to target integrins. One example is the ectopic expression of the a4 integrin in MSCs (Kumar and Ponnazhagan, 2007). While the injection of modified MSCs into mice led to increased cell homing in bone and their subsequent differentiation into osteoblasts (Guan et al., 2012), the systemic injection of a peptidomimetic ligand (LLP2A) that binds the a4b1 integrin, conjugated to a bisphosphonate to allow bone binding, led to increased osteoblast differentiation and bone formation in mice with established osteopenia (Guan et al., 2012; Yao et al., 2013). Another strategy is to target the a5b1

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integrin (Marie, 2013). Lentiviral-mediated expression of the a5 integrin subunit in human MSCs promoted osteogenic differentiation, bone formation (Hamidouche et al., 2009), and bone repair in critical-size bone defects in mice (Srouji et al., 2012). Moreover, a5 integrin activation by a synthetic cyclic peptide, which activates FAK and ERK1/2eMAPK signaling in osteoprogenitor cells, led to increased osteoblast differentiation and to reduced cell apoptosis in vitro (Fromigué et al., 2012). In vivo, the local injection of an a5 integrin agonist peptide into adult mice increased parietal bone formation, indicating that pharmacological activation of a5 integrin in osteoprogenitor cells is effective in promoting de novo bone formation (Fromigué et al., 2012). An alternative strategy to target integrins may be the use of recombinant NELL-1, a secreted osteoinductive protein that binds to b1 integrin. In vitro, this strategy leads to activation of Wnt/ b-catenin signaling and increased osteoblast differentiation. In vivo, delivery of NELL-1 to ovariectomized mice or osteopenic sheep improved bone mineral density (James et al., 2015). These studies showed that targeting integrins may be a promising approach for promoting bone formation and repair in skeletal disorders.

Osteoclasts Osteoclasts are the multinucleated cells that resorb the bone matrix to promote skeletal modeling and renewal (Soysa and Alles, 2016; Cappariello et al., 2014). They rely on a tight and dynamic mechanism of adhesion to the mineralized matrix, which ensures cell changes indispensable for bone resorption. These are (1) the polarization of the osteoclast and (2) the sealing of the extracellular space between the cell and the bone matrix to be removed, called resorption lacuna (Soysa and Alles, 2016; Cappariello et al., 2014) (Fig. 17.2). The principal integrin involved in the adhesion of osteoclasts to the substrate is the aVb3 receptor. While aV-deleted mice are embryonically lethal, b3-deleted mice showed a phenotype mimicking Glanzmann thrombasthenia (Morgan et al., 2010) and osteopetrosis (Zou and Teitelbaum, 2015; McHugh et al., 2000). Osteopetrosis is caused by osteoclast dysfunction, and these observations revealed the relevance of the aVb3 integrin in osteoclast biology. Specifically, b3-null osteoclasts form normally but are prevented from resorbing bone. Their cytoskeletal array is disrupted, adhesion to substrate is reduced, and cell spreading is impeded (McHugh et al., 2000). These results suggest that the aVb3 integrin is not involved in the molecular mechanisms of osteoclastogenesis, but it rather affects the morphological and molecular changes that make osteoclasts capable of resorbing bone. aVb3 heterodimers are clustered in specific adhesion areas, called podosomes (Marchisio et al., 1984), that are typical of osteoclasts and other highly motile cells, such as monocytes, macrophages (Calle et al., 2006), and invasive cancer cells, in which they are called invadopodia (Eddy et al., 2017). Compared with classical focal adhesions, podosomes and invadopodia are much more dynamic, with a turnover of a few minutes rather than the hours observed in focal adhesions (Georgess et al., 2014). A typical feature of osteoclast podosomes is their clustering in a continuous peripheral annulus, called actin ring

Podosomes Lysosomes

Sealing membrane CI-

H+

Resorption lacuna FIGURE 17.2 Role of integrins in osteoclasts. Cartoon showing a polarized osteoclast sitting on the resorption lacuna. Release of protons and chlorides acidifies the lacuna microenvironment, solubilizing the hydroxyapatite and allowing the degradation of the organic matrix by lysosomal enzymes therein secreted. Adhesion to the mineralized matrix occurs through podosomes located in the sealing membrane, which establish contacts with the extracellular molecules through integrin receptors (see text for more details).

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(Cappariello et al., 2014), representing the sealing zone of the apical membrane. Here, the number of podosomes and the extension of the acting ring are proportional to the activity of the osteoclasts, reaching the maximum development during bone resorption, when multiple actin rings/osteoclast can be observed (Cappariello et al., 2014). For their high dynamism, podosomes and their associated aVb3 integrins change rapidly in terms of number and distribution, contributing not only to the sealing of the resorption lacuna and the subsequent organization of the inner irregular domain of the apical membrane lodging the molecular mechanisms of bone resorption, called the ruffled border, but also to the osteoclast motility on the mineralized matrix during and between the bone resorption cycles (Cappariello et al., 2014; Rucci and Teti, 2016). In osteoclast podosomes, the aVb3 integrin is largely represented and concentrated in a circular podosomal area of membrane adhesion to substrate, whose cytosolic center lodges a core of microfilaments; actin-binding proteins, including a-actinin; and actin-branching proteins, including cortactin, the ARP2/3 complex, and WASP and WASP-interacting protein (Georgess et al., 2014; Rucci and Teti, 2016). The aVb3 integrin circular “rosette” that surrounds this microfilament core is associated with several intracellular adhesion proteins, such as vinculin, talin, paxillin and tensin, that link the integrins to the microfilaments (Saltel et al., 2008; Correia et al., 1999; Evans and Matsudaira, 2006; Marchisio et al., 1988; Akisaka et al., 2001). Importantly, this complex structure includes microfilament-severing proteins (gelsolin and cofilin) (Ory et al., 2008), as well as the small GTP-binding protein RHO (Teitelbaum, 2011) and the guanine exchange factors DOCK5 and VAV3, which provide rapid podosome remodeling indispensable for their dynamism (Purev et al., 2009). This is also ensured by the localization in podosomes of the large GTP-binding protein dynamin, which regulates E3 ubiquitin ligase c-CBL-dependent degradation or receptors and signaling factors (Bruzzaniti et al., 2005). GTPase activity of dynamin is enhanced by GRB2, which also increases WASP-mediated ARP2/3-dependent actin nucleation (Spinardi and Marchisio, 2006). Along with the microfilament-severing function of gelsolin and cofilin, this molecular cascade ensures rapid microfilament turnover and podosome disassembly and reassembly (Luxenburg et al., 2012). Moreover, ubiquitin ligases, including c-CBL and CBL-b (Horne et al., 2005), phosphoinositide kinases such as PI3K (Chellaiah et al., 2001), nonreceptor tyrosine kinases such as c-SRC and PYK2 (Duong et al., 1998; Destaing et al., 2008), ABL and FAK (Ray et al., 2012), and phosphatases, including PTPa, PTPε, and SHP2 (Granot-Attas and Elson, 2008), associate with the aVb3 integrin complex to further regulate osteoclast adhesion and podosome dynamics. Genetic studies have been instrumental in demonstrating the relevance of the aVb3 receptor signaling in osteoclast activity. The analysis of c-Src deletion in mice showed that osteoclasts were not able to polarize and became immotile (Lowe et al., 1993). They exhibited a severe osteopetrotic phenotype, which was observed, in milder form, also in Pyk2and c-Cbl-null mice (Gil-Henn et al., 2007; Tanaka et al., 1996). c-SRC coprecipitates with the aVb3 integrin and through its c-CBL tyrosine phosphorylation activity, promotes substrate ubiquitination and prolonged cell survival (Horne et al., 2005). Furthermore, c-SRC contributes to the metabolism of PYK2 and p130CAS, involved in the organization of the sealing membrane (Nakamura et al., 1998). Therefore, given that c-SRC is upstream of the tyrosine phosphorylation cascade involving PYK2, c-CBL and p130CAS, it is likely that these intracellular signaling molecules act synergistically to determine correct cytoskeletal arrangement, podosome formation, and osteoclast polarization and survival. On the extracellular side, the osteoclast aVb3 integrin binds the RGD sequence of a number of matrix proteins that contribute to osteoclast adhesion and outside-in signaling (Rucci and Teti, 2016). Many proteins are recognized by the aVb3 integrin, including osteopontin, bone sialoprotein 2, vitronectin, fibronectin, and von Willebrand factor (Zou and Teitelbaum, 2010). aVb3 integrin recognizes also collagen type I but only upon molecular remodeling, the collagen type I RGD sequence being lodged in a cryptic domain. In osteoclasts, osteopontin, bone sialoprotein II, synthetic RGD peptides, echistatin (an RGD-disintegrin peptide extracted from the poison of viper venom), and the avb3 integrin activating LM609 monoclonal antibody mobilize intracellular calcium through the aVb3 receptor, albeit with some species specificity. In fact, in rat osteoclasts, aVb3 integrin activation triggers intracellular calcium transients and cell retraction (Shankar et al., 1993), while in chicken osteoclasts it induces a calmodulin/Ca2þ-ATPase-dependent calcium efflux and intracellular reduction (Miyauchi et al., 1991). The molecular mechanisms underlying these opposite events have not been investigated, leaving this question still open. Finally, in mouse osteoclasts, osteopontin induces RGD-dependent nuclear translocation of nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis (Tanabe et al., 2011), whose activation is triggered by calcium oscillations (Negishi-Koga and Takayanagi, 2009). The aVb3 integrin is relatively infrequent in the organism, and osteoclasts are among the cells mostly enriched in these receptors. The aVb3 integrin has been considered a pharmacological target to combat cancer-induced osteolytic lesions, osteoporosis, and rheumatoid arthritis (Desgrosellier and Cheresh, 2010; Schneider et al., 2011; Tian et al., 2015). aVb3 integrin targeting molecules include cyclic peptides carrying the aVb3 integrin binding site (Auzzas et al., 2010), neutralizing antibodies (Hsu et al., 2007) and nonpeptide antagonists (Hsu et al., 2007; Sheldrake and Patterson, 2014). Several of them have been tested in preclinical studies, and one has been tested in clinical trials to treat cancer (Danhier et al., 2012).

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Other integrin members are also expressed by osteoclasts. aVb1, aMb2, and aVb5 receptors are expressed by osteoclast mononuclear precursors and switch to aVb3 during preosteoclast maturation in polykarya (Inoue et al., 1998). The a2b1 integrin is engaged in the binding to collagen type I. It does not recognize vitronectin, fibronectin, or fibrinogen and promotes migration and fusion of precursors into mature cells (Townsend et al., 1999). However, these integrins have not been investigated any further and their relevance in osteoclast biology is probably underestimated.

Chondrocytes Chondrocytes express a panel of at least seven integrin heterodimers recognizing molecules of the basal lamina and the territorial cartilage matrix. a5b1, aNb3, and aNb5 are fibronectin receptors expressed by chondrocytes, which, along with the a6b1 receptor recognizing laminin, and the a1b1, a2b1 and a10b1 integrins binding collagen, ensure tight chondrocyte interaction with the substrate (Loeser, 2002; Aszodi et al., 2003). The dominant integrin subunit in chondrocytes is the b1 chain (Woods et al., 2007a). Consequently, conditional chondrocyte knockout (KO) of b1 integrin resulted in a severe cartilage phenotype (Woods et al., 2007a). Typical chondrodysplasia malformations were noticed in these mice, with shorter long bones and reduced growth plate hypertrophic zone. No vascularization of the primary ossification centers was observed, with vessel present only in the periosteum. The growth plate appeared broadened, the chondrocyte columnar array was disrupted, and the hypertrophic zone mineralization was reduced and patchy (Woods et al., 2007a). Given the broad presence of the b1 chain in at least five chondrocyte integrin receptors, such a severe cartilage phenotype is not surprising. Furthermore, a similar outcome was observed in conditional Il1-null chondrocytes in mice (Grashoff et al., 2003; Terpstra et al., 2003). The expression of chondrocyte b1 integrins is regulated by mechanical forces. Tension forces stimulate their expression with a mechanism that appears to be mediated by FAK, and this effect leads to inhibition of chondrogenesis, especially through a2b1 and a5b1 receptors (Takahashi et al., 2003; Onodera et al., 2005). This result is rather surprising and while it is confirmed by the observation that plating mesenchymal stem cells on integrin-activating RGD peptides results in their reduced commitment to chondrogenesis (Connelly et al., 2007), it is contradicted by the discovery that inhibition of b1 integrin activity reduces chondrogenesis and cartilage production (Shakibaei, 1998). These results suggest that integrins have dual roles in cartilage development, inhibiting or inducing chondrogenesis in unlike contexts and in response to different environmental regulations. Functionally, Wang and Kirsch (Wang and Kirsch, 2006) showed that b1 and b5 integrins promote cell survival, while their deletion or blockage by neutralizing antibodies reduces hypertrophic differentiation (Hirsch et al., 1997). Proliferative and hypertrophic chondrocytes of the growth plate express the highest levels of a5b1 receptors, and neutralization of this integrin results in impairment of chondrocyte proliferation (Enomoto-Iwamoto et al., 1997). The a5b1 integrin is also important for the development of the appendicular skeleton and joints. For instance, premature formation of prehypertrophic chondrocyte and joint fusion were observed when a5b1 integrin was misexpressed (Garciadiego-Cázares et al., 2004). Finally, deletion of a10b1 integrin caused dysfunctions of the growth plate, inducing growth retardation (Bengtsson et al., 2005), with increased apoptosis and altered chondrocyte morphology. Instead deletion of the a1 chain caused osteoarthritis but not growth defects (Zemmyo et al., 2003). In addition to integrins, the adhesion of chondrocytes to the cartilage matrix is ensured by other types of molecules. The discoidin domain receptor, Ddr2, is highly expressed by articular chondrocytes in osteoarthritis, and its conditional deficiency in growth plate chondrocytes was associated with decreased chondrocyte proliferation and subsequent dwarfism (Labrador et al., 2001). Annexin V instead mediates adhesion to the N telopeptide of collagen II in articular cartilage and growth plate, where it appears to regulate apoptosis and matrix mineralization (Wang and Kirsch, 2006; Jennings et al., 2001; Kirsch, 2005). Finally, CD44, an important membrane glycoprotein receptor recognizing collagens and hyaluronic acid, increases during chondrogenesis and contributes to the organization of the territorial matrix and the regulation of chondrocyte survival (Nicoll et al., 2002; Knudson, 2003).

Role of cadherins in bone cells Osteoblasts and osteocytes Cadherins are transmembrane glycoproteins that mediate calcium-dependent cellecell adhesion through homophilic interactions of their extracellular domains (Gumbiner, 2005). The intracellular domain of cadherins interacts with cytoskeletal proteins at adherens junction sites and with signaling molecules such as vinculin, a- and b-catenin, and other molecules involved in cellular signaling processes (Nelson and Nusse, 2004). Osteoblasts express Cadh1 (E-cadherin), Cadh2 (N-cadherin), Cadh3 (P-cadherin), and Cadh11 (“osteoblast cadherin”) (Cheng et al., 1998; Goomer et al., 1998;

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Ferrari et al., 2000; Lemonnier et al., 2000; Kawaguchi et al., 2001a), although the expression of these cadherins changes with the stage of osteoblast differentiation. Cadh2 expression initially increases during osteogenic differentiation in vitro but declines thereafter (Ferrari et al., 2000; Greenbaum et al., 2012), whereas Cadh11 is upregulated throughout the osteoblast differentiation program (Kawaguchi et al., 2001b; Shin et al., 2000). In vivo, Cadh2 is expressed in lining cells but not in osteocytes (Shin et al., 2000). In vitro and in vivo studies suggest that cadherins may control precursor cell lineage determination (Marie et al., 2014a). Both Cadh2 and Cadh11 are downregulated with commitment of mesenchymal progenitors to adipogenesis in vitro (Kawaguchi et al., 2001b; Shin et al., 2000). Adult Cadh11-KO mice showed increased number of adipogenic precursors and adipogenic differentiation in vitro, suggesting that Cadh11 blocks adipogenesis (Di Benedetto et al., 2010). However, deletion of Cadh11 does not result in a major skeletal defect in adult mice (Di Benedetto et al., 2010) and causes only modest osteopenia in young mice (Castro et al., 2004), presumably due to compensation by Cadh2. Indeed, ablation of one Cadh2 allele in Cadh11-null mice resulted in severe osteopenia associated with reduced bone strength (Di Benedetto et al., 2010). Consistent with a role for Cadh2 in osteogenesis, overexpression of a dominant-negative Cadh2 mutant in mature osteoblasts using the osteocalcin promoter resulted in reduced osteoblast differentiation, bone formation, and bone mass (Castro et al., 2004). However, Cadh2 haploinsufficiency, or conditional deletion of Cadh2, in osteoblasts led to increased differentiation of bone marrow stromal cells in vitro, suggesting that Cadh2 may inhibit terminal osteoblast differentiation. Thus, although Cadh2 and Cadh11 contribute to early stages of osteoblast differentiation, Cadh2, but not Cadh11, downregulation is required for terminal differentiation. How do cadherins control osteoblast differentiation and fate? As they behave as adhesive molecules, cadherins can control osteoblast precursor cell fate in part through cellecell adhesion (Haÿ et al., 2000). Blocking Cadh2-mediated cellecell adhesion using specific peptides or antibody reduces osteoblast differentiation in vitro (Ferrari et al., 2000; Haÿ et al., 2000). However, overexpression of Cadh2 in osteoblasts driven by the Col1A1 promoter reduced osteoblast activity and peak bone mass, indicating that Cadh2 controls bone formation through other mechanisms than cellecell adhesion (Haÿ et al., 2009a). Cadherins are known modulators of intracellular signaling related to Wnt signaling (Heuberger and Birchmeier, 2010). Cadherins can bind to b-catenin, which leads to increased stabilization of the adhesion structure. Conversely, the release of b-catenin from cadherins destabilizes the adhesion complex and allows cell mobility (Gottardi and Gumbiner, 2001). In addition, cellecell adhesion mediated by cadherins promotes b-catenin phosphorylation and inactivation of Wnt/b-catenin signaling (Lilien and Balsamo, 2005). Furthermore, cadherins can sequestrate b-catenin at the cell membrane, resulting in decreased b-catenin pool availability for nuclear translocation (Gottardi and Gumbiner, 2001). In osteoblasts, several interactions between cadherins and Wnt signaling molecules were shown to control osteogenic differentiation (Lilien and Balsamo, 2005). The decreased b-catenin abundance at cellecell contacts induced by deletion of Cadh2 or Cadh11 led to decreased cellecell adhesion with a negative effect on osteoblastogenesis (Di Benedetto et al., 2010). In addition to this mechanism, Cadh2 can bind to low-density lipoprotein receptorerelated protein (LRP) 5 or LRP6 via Axin, leading to LRP5/6 sequestration at the cell membrane, reduced LRP5/6 availability, and decreased Wnt/b-catenin signaling (Haÿ et al., 2009a). Cadh2/Axin/LRP5/6 interaction in osteoblasts also reduces ERK1/2 and PI3K/AKT signaling, resulting in decreased osteoblast proliferation, differentiation, and survival (Marie et al., 2014a). Furthermore, Cadh2/LRP5/6 interaction in osteogenic cells causes decreased endogenous Wnt3a expression, which contributes to reduced Wnt signaling and osteoblastogenesis (Marie et al., 2014a). The resulting effect of the negative interaction of Cadh2 overexpression on Wnt signaling in osteogenic cells is to decrease bone formation and delay peak bone mass in young mice (Haÿ et al., 2009a). Consistent with the finding that Cadh2 negatively regulates Wnt/b-catenin signaling in osteoblasts (Marie et al., 2014a; Haÿ et al., 2009a), Cadh2 was found to restrain the bone anabolic action of intermittent parathyroid hormone (iPTH). In vitro, the ablation of Cadh2 in osteogenic cells results in increased LRP6/ PTHR1 interaction and enhanced iPTH-induced protein kinase-dependent Wnt/b-catenin signaling. In mice, conditional Cadh2 deletion in osteoprogenitor cells led to a greater than normal osteoblast activity and bone mass in response to iPTH, indicating that Cadh2eLRP6 interaction restrains PTH-induced b-catenin signaling (Haÿ et al., 2009b). Consistently, overexpression of Cadh2 blunted the suppressive effect of PTH on sclerostin/SOST expression in vitro and in vivo, further indicating that Cadh2 expression influences the anabolic effect of iPTH in mice (Revollo et al., 2015). However, the influence of Cadh2 on Wnt signaling and osteogenic cell commitment and differentiation varies with aging. While Cadh2 overexpression in osteoblasts decreased osteoblast differentiation and increased bone marrow adipocyte differentiation in young mice, this phenotype was fully reversed with aging (Yang et al., 2016), which is consistent with the downregulation of Cadh2 during osteoblast maturation. This phenotype was linked to reversal with age of endogenous Wnt5a and Wnt10b signals that are key factors controlling osteogenic cell lineage commitment (Yang et al., 2016). In addition to aging, the negative effect of Cadh2 on Wnt/b-catenin signaling depends on the osteogenic cell differentiation stage. Conditional deletion of Cadh2 in osteoprogenitors at embryonic and perinatal age was detrimental to bone accrual, whereas loss of Cadh2 in osteolineage cells in adult mice favored bone formation (Haÿ et al., 2014), indicating that Wnt/b-catenin

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Cadh2

Cadh2

β-Catenin sequestration

LRP5/6

Axin

Free β-Catenin ERK1/2 and PI3K/AKT signaling Wnt target genes

Osteoblast differentiation and survival

Bone formation, bone mass FIGURE 17.3 Role of cadherins in osteoblasts. Cadherin 2 (Cadh2) can interact with b-catenin at the cell surface, resulting in b-catenin sequestration, decreased free b-catenin, and reduced Wnt signaling. Cadh2 can also interact with the Wnt coreceptors low-density lipoprotein receptorerelated protein 5 (LRP5) and LRP6, resulting in reduction of Wnt/b-catenin signaling and other signaling pathways that control osteoblast function and survival, bone formation, and bone mass (see text for more details). ERK1/2, extracellular signal-regulated kinase 1/2; PI3K/AKT, phosphatidylinositol 3-kinase/protein kinase B.

signaling, bone formation, and bone mass are directly influenced by the level of expression of Cadh2 at all stages of the osteoblast lineage. Cadherins also modulate Wnt/b-catenin signaling induced in response to mechanotransduction in bone cells. Mechanical stimulation induced by oscillatory fluid flow in osteoblastic cells leads to reduced Cadh2/b-catenin binding at the cell membrane, resulting in b-catenin nuclear translocation and osteogenic differentiation (Fontana et al., 2017). In addition to the impact on Wnt signaling, Cadh2 interacts with PI3K signaling in osteoblastic cells. Cadh2mediated adherens junctions activate the PI3K signaling cascade in osteoblasts, which contributes to osteoblast differentiation and osteogenesis in the perichondrium (Arnsdorf et al., 2009). Conversely, Cadh2 overexpression in osteoblasts reduced Wnt-dependent PI3K/AKT signaling, resulting in decreased osteoblast survival in mice (Marie et al., 2014a; Haÿ et al., 2009a). Thus, cadherins control bone formation through multiple mechanisms that are directly or indirectly linked to Wnt/b-catenin signaling (Fig. 17.3). Based on the finding that Cadh2 interacts with LRP5/6 to reduce b-catenin/Wnt signaling and osteoblast function (Haÿ et al., 2009a), therapeutic approaches targeting Cadh2/LRP5/6 interaction were developed for promoting bone formation (Guntur et al., 2012). In vitro, deletion of the Cadh2 domain interacting with Axin and LRP5/6 leads to promotion of Wnt/ b-catenin signaling and osteoblast differentiation (Fiorino and Harrison, 2016). Moreover, disruption of the Cadh2/LRP5/6 interaction using a competitor peptide that binds to the Cadh2/Axin-interacting domain of LRP5/6 results in enhanced Wnt/ b-catenin signaling and osteoblast function in vitro, and increased calvaria bone formation in mice (Marie and Haÿ, 2013). In senescent osteopenic mice, blocking the Cadh2/LRP5/6 interaction led to increased endogenous Wnt5a and Wnt10b expression, osteogenic differentiation, bone formation, and bone mass (Haÿ et al., 2012), suggesting a therapeutic approach targeting the Cadh2/LRP5/6 interaction for promoting bone formation in the aging skeleton.

Osteoclasts Osteoclasts have been investigated for cadherin expression, especially to understand the mechanisms underlying the fusion process of mononuclear precursors into polykarya (Mbalaviele et al., 1995). Osteoclasts express Cadh1, while they are negative for Cadh2 and Cadh3. Interestingly, Cadh1 expression peaks at the time of preosteoclast fusion, a process that is largely inhibited by Cadh1-neutralizing antibodies. Cadh1 inhibition blocks migration of osteoclast precursors, which is essential for mononuclear cell clustering and fusion of their plasma membranes (Fiorino and Harrison, 2016). In contrast, neutralization of Cadh1 fails to affect proliferation of precursors or adhesion to substrate, confirming a specific role of this cell adhesion molecule in the maturation of polykarya (Mbalaviele et al., 1995). In osteoclast precursors, Cadh1 is localized in areas of membrane protrusions (Fiorino and Harrison, 2016) that are distributed throughout the entire cell surface, but its

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expression dramatically declines in mature multinucleated osteoclasts. Cadh1 engagement in osteoclast precursors activates gene expression, and several osteoclast genes are under the control of the Cadh1 pathway. Blocking Cadh1 function, before the preosteoclasts fuse into polykarya, retards the expression of the osteoclast transcription factor NFATc1, the fusion protein dendritic cell-specific transmembrane protein, and the osteoclast enzymes cathepsin K and tartrate-resistant acid phosphatase. Since these genes are essential for osteoclast multinucleation and maturation, these observations highlight the important role of Cadh1 in the late stage of osteoclastogenesis. Conversely, overexpression of Cadh1 in osteoclast precursors brought forward the activation of NFATc1, which translocates to the nucleus earlier than in control cells (Fiorino and Harrison, 2016). Given that the studies on osteoclast cadherins are scanty, we have no specific clues on the signals induced by these molecules during osteoclastogenesis. However, Cadh1 is implicated in the maturation of a number of other macrophage subtypes. For instance, it is involved in the differentiation and motility of Langerhans cells and regulates the activity of dendritic cells via cooperation of the Wnt/b-catenin signaling (Van den Bossche et al., 2012). Because of the tight interaction of Cadh1 with b-catenin and the role of Wnt/b-catenin signaling in osteoclast formation (Kobayashi et al., 2015), it is likely that these two pathways cooperate for the regulation of osteoclastogenesis as well. Another cadherin isoform identified in osteoclasts is Cadh6/2 (Mbalaviele et al., 1998). Dominant negative or antisense Cadh6/2 prevents osteoclast precursors from interacting with ST2 cells, known to support osteoclastogenesis, suggesting this cell adhesion molecule is involved in heterotypic interaction between these two cell types, leading to full maturation of the osteoclasts (Mbalaviele et al., 1998). Unfortunately, there are no other data supporting this observation; therefore the underlying molecular mechanisms remain elusive.

Chondrocytes The most prominent cadherin in chondrocytes is Cadh2. It is strongly expressed during mesenchymal condensation, which allows the formation of the primordial tissue from which limb cartilage originates (Oberlender and Tuan, 1994). This process can be impaired using Cadh2 blocking antibody, which prevents mesenchymal condensation and subsequent chondrogenesis. Cadh2 was upregulated in an in vitro model of chondrocyte differentiation. It was detected in prechondrogenic cells and transiently increased during cell aggregation, disappearing in hypertrophic chondrocytes (Tavella et al., 1994). Cadh2 appears to be essential for cellular condensation (Woodward and Tuan, 1999). Using high-density micromass cultures, Woodward and Tuan (Woodward and Tuan, 1999) induced chick limb mesenchymal cell condensation by ion cross-links promoted by poly-L-lysine. This condition unveiled a time-dependent increase in Cadh2, while its neutralization by a specific antibody inhibited the effect of poly-L-lysine on chondrogenesis. Forced overexpression of Cadh2 induced precartilage cellular condensation and enhanced chondrogenesis in vitro, with a mechanism that required both extracellular and intracellular domains of Cadh2 (Delise and Tuan, 2002). Cadh2 also appears to be a target for the antichondrogenic effect of retinoic acid. Retinoic acid inhibits the progression of condensed precartilage tissue to cartilage nodules. This progression occurs upon suppression of Cadh2 expression, which is prevented by retinoic acid along with the downregulation of the associated a- and b-catenins. This effect of retinoic acid is blocked by cytochalasin D, a molecule that disrupts the microfilaments implicated in the Cadh2 adhesion function (Cho et al., 2003). Another important regulator of chondrogenesis that requires Cadh2 is transforming growth factor b (TGFb). TGFb induces chondrogenesis through activation of MAPKs, especially p38 and ERK1. These signaling kinases transiently upregulate Cadh2 to allow cellular condensation, followed by its downregulation to induce progression toward chondrogenesis (Tuli et al., 2003). Cellular condensation is also regulated by the small GTP-binding protein RAC1, which triggers upregulation of Cadh2 as well (Woods et al., 2007b). TGFb also induces the expression of Wnt7. Chondrogenesis is regulated by the Wnt pathway and Wnt7a is a lead chondrogenic signaling t that acts in concert with Cadh2. Using the chick limb mesenchymal cell micromass cultures, Tufan et al. (Tufan et al., 2002) investigated the role of Chfz-1 and Chfz-7, which are members of the Wnt pathway encoded by the Frizzled genes. While CHFZ-1 surrounded the nascent cartilage rudiment, CHFZ-7 was expressed in the area of cell condensation, with progressive downregulation toward the peripheral area. This pattern of expression was similar to the expression of Cadh2, and misexpression of Chfz-7 impaired chondrogenesis at the early stage of formation of the precartilage aggregates. Notably, Cadh2 expression was downregulated by Chfz-7 misexpression, suggesting a functional link between Cadh2 and the Wnt pathway during mesenchymal condensation and subsequent chondrogenesis (Tufan et al., 2002). To induce chondrogenesis, Cadh2 must be cleaved by ADAM10. Cadh2 cleavage mutants failed to induce cartilage formation, impeding the organization of cartilage aggregates as well as the synthesis of proteoglycans. Furthermore, overexpression of these mutants downregulated type II collagen, aggrecan, and type X collagen (Nakazora et al., 2010), confirming a pivotal role for enzymatic cleavage in the activation of cartilage matrix production. Another factor involved in chondrogenesis that affects Cadh2 is the C-type natriuretic peptide. Treatment of micromass cultures of chick limb

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mesenchymal cells with this factor upregulated Cadh2 along with collagen type X and glycosaminoglycans, ending up with enhanced chondrogenesis (Alan and Tufan, 2008). Despite these striking observations, Cadh2-null mice have no skeletal alterations. Given the redundant functions of cadherins, it was hypothesized that, in this mouse model, Cadh2 deficiency is compensated for by the activity of Cadh11 in cartilage development. Cadh11 is strongly upregulated in response to chondrogenic conditions and is expressed in normal and osteoarthritic articular cartilage (Stokes et al., 2002). In the growth plate, Cadh11 is expressed in the late hypertrophic zone. Knockdown of Cadh11 inhibited the formation of calcified nodules in a growth plateederived chondrocyte cell line (Matsusaki et al., 2006). The function of Cadh11 has been investigated during the differentiation of mesenchymal stem cells. Kii et al. (Kii et al., 2004) observed that teratomas derived from embryonic stem cells transfected with Cadh11 formed preferentially bone and cartilage. Using tridimensional hanging drop cultures, it was noticed that Cadh11-transfected cells formed sheetlike aggregates, as opposed to Cadh2overexpressing cells, which formed spherical structures, suggesting independent and nonoverlapping functions of Cadh11 and Cadh2 in this context (Kii et al., 2004).

Roles of other attachment molecules in bone cells Syndecans Cell surface proteoglycans are composed of a membrane-associated core protein to which glycosaminoglycan (heparan sulfate [HS] or chondroitin sulfate) chains are covalently attached. HS chains can bind several proteins, including growth factors, signaling proteins, membrane receptors, and ECM proteins (Bishop et al., 2007; Bernfield et al., 1999), thus allowing regulation of the availability and function of signaling proteins (Mitsou et al., 2017; Billings and Pacifici, 2015). Notably, binding of extracellular ligands on HS chains increases the probability for ligands to interact with their highaffinity receptors (Billings and Pacifici, 2015). In the growth plate, HS proteoglycans (HSPGs) bind members of the hedgehog, BMP, fibroblast growth factor (FGF), and Wnt protein families (Huegel et al., 2013). Syndecans are an HSPG family composed of four members (syndecan-1 to -4) (Dews and Mackenzie, 2007). In bone, syndecan-1 is expressed transiently during mesenchymal condensation, syndecan-2 is expressed by mesenchymal cells and persists during bone development and osteoblast differentiation, syndecan-3 is mainly expressed in cartilage, whereas syndecan-4 expression is more ubiquitous (David et al., 1993). In osteoblasts, syndecan-2 is strongly associated with FGF/FGFRs (Molténi et al., 1999a; Song et al., 2007) and granulocyte/macrophage colony-stimulating factor (Modrowski et al., 2000), and contributes to the activity of these ligands. Syndecan-2 expression is regulated by Runx2, Wnts, FGF, and TGFb in osteoblastic cells (Teplyuk et al., 2009; Worapamorn et al., 2002; Dieudonné et al., 2010). BMP-2 increases the synthesis of syndecans (Gutierrez et al., 2006) and interacts with HS complexes to regulate osteoblast differentiation in mesenchymal stromal cells (Manton et al., 2007). In osteogenic cells, syndecan-2 acts as a coreceptor for FGFRs, which is essential for the response to FGF2 (Molténi et al., 1999b; Steinfeld et al., 1996). FGF binding to HS chains results in growth factor dimerization and formation of a tertiary complex with FGFR (Matsuo and Kimura-Yoshida, 2013). Syndecans also interact with Wnt molecules via HS chains to modulate Wnt signaling (Baeg et al., 2001). This leads to the modulation of Wnt molecule concentration at the cell surface, which stabilizes the signaling activity (Fuerer et al., 2010). In bone, syndecan-2 controls the extracellular availability of Wnt effectors and modulates intracellular signals linked to Wnt signaling (Mansouri et al., 2015). Transgenic mice overexpressing syndecan-2 in osteoblasts showed decreased osteogenesis associated with increased mesenchymal osteoprogenitor cell apoptosis. This phenotype results from inhibition of Wnt/b-catenin signaling and decreased production of Wnt ligands, supporting a role of syndecan-2/Wnt signaling interaction in the control of osteoblastogenesis in vivo (Mansouri et al., 2017). In addition to interacting with signaling factors that regulate osteoblasts, syndecans interact with fibronectin to facilitate cell adhesion through fibronectinetransglutaminase complexes induced by syndecan-4-dependent activation of protein kinase Ca (Wang et al., 2010). This complex supports osteoblast adhesion and rescues from cell death by anoikis in a syndecan- and b1 integrin-dependent manner (Wang et al., 2011). Thus, syndecans control osteoblast adhesion and response to exogenous factors by interacting with both ECM and signaling factors, resulting in the modulation of intracellular factors controlling cell fate.

Glypicans and perlecan Glypicans are other cell surface proteoglycans expressed in bone. The glycosaminoglycan-bearing perlecan domain I interacts with ligands such as BMP, FGF, hedgehog, and Wnt proteins (Dwivedi et al., 2013) and thereby supports early chondrogenesis (Farach-Carson et al., 2008). In vitro, glypican-3 is involved in osteogenic commitment, as reduced glypican-3 expression leads to decreased Runx2 expression and osteoblast differentiation in murine osteoblastic cells

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(Haupt et al., 2009). In vivo, glypican-KO mice showed decreased trabecular bone mass and delayed endochondral ossification due to reduced osteoclastogenesis, indicating that this HSPG plays a role in bone growth (Viviano et al., 2005). Another HSPG, perlecan, interacts with the ECM, growth factors, and receptors and influences cellular signaling (Whitelock et al., 2008). In bone, perlecan is expressed in cartilage and strongly potentiates chondrogenic differentiation in vitro (French et al., 1999). Perlecan promotes chondrocyte attachment to the matrix (SundarRaj et al., 1995) and is involved in chondrogenic differentiation in vitro (Gomes Jr et al., 2006; French et al., 2002). In vivo, perlecan-KO mice showed defective endochondral ossification due to decreased proliferation of chondrocytes and reduced prehypertrophic zone (Arikawa-Hirasawa et al., 1999). Consistent with a role of perlecan in growth plate development, reduced perlecan secretion resulted in achondroplasia in mice (Rodgers et al., 2007). Mechanistically, perlecan binds to FGF2 by its HS chains and enhances FGF2 binding to FGFR-1 and FGFR-3 receptors in the growth plate (Smith et al., 2007). Perlecan may also be involved in the control of osteoblastogenesis and bone formation (Lowe et al., 2014). In the bone marrow, cellderived ECM that contains perlecan, among other matrix molecules, preserves the ability of mesenchymal stromal cells to differentiate into osteoblasts or adipocytes (Chen et al., 2007). In addition, exogenous perlecan suppresses adipogenic differentiation and promotes osteogenic differentiation of mesenchymal stem cells in vitro (Nakamura et al., 2014). This may be due in part to enhanced interaction with BMP-2, leading to increased BMP-2 bioactivity (Decarlo et al., 2012). At a later stage of osteoblast differentiation, perlecan is localized in the pericellular space of osteocytes in the lacunocanalicular system in cortical bone (Thompson et al., 2011), where it regulates solute transport and mechanosensing within the osteocyte lacunarecanalicular system (Wang et al., 2014). These studies support the notion that, in addition to being involved in cell attachment to the ECM, HSPGs control bone cell functions by interacting with signaling proteins involved in the response to extracellular signals.

CD44 CD44 is a cellular surface adhesion molecule involved in various processes. CD44 binds to hyaluronan (HA), osteopontin, fibronectin, and collagen type I and thereby may regulate bone cell function (Goodison et al., 1999). As noted earlier, CD44 is involved in chondrocyte survival (Nicoll et al., 2002; Knudson, 2003). Osteoclasts express CD44 (Nakamura et al., 1995; Suzuki et al., 2002), and the interaction of CD44 with HA or osteopontin induces intracellular signals in preosteoclasts, leading to osteoclast formation (Spessotto et al., 2002; Chellaiah et al., 2003; Chellaiah and Hruska, 2003). In vitro, osteopontin signals through calcium and NFATc in osteoclasts (Tanabe et al., 2011). Consistently, CD44 deficiency led to inhibition of osteoclast activity and function by downregulating NF-kB/NFATc1-mediated signaling (Li et al., 2015). Moreover, receptor activator of NF-kB ligand (RANKL) induces CD44 expression, and CD44 promotes the activation of RANKLeRANKeNF-kB-mediated signaling during osteoclastogenesis (Li et al., 2015). In vivo, CD44KO mice showed normal trabecular bone volume but increased cortical thickness, suggesting a site-specific effect of CD44 deficiency (Cao et al., 2005). Accordingly, the reduced osteoclastogenesis and osteoclast function induced by CD44 deficiency counteracts the cortical, but not trabecular, bone loss induced by hindlimb unloading in mice (Li et al., 2015). In contrast to osteoclasts, a role of CD44 in osteoblast function is not firmly established. In vitro, galectin-9 binding to CD44 was reported to induce the formation of a CD44/BMP receptor complex, leading to Smad1/5/8 phosphorylation and osteoblast differentiation (Tanikawa et al., 2010). However, CD44 deficiency inhibited osteoclast but not osteoblast function in hindlimb-unloading-induced bone loss in mice (Li et al., 2015), suggesting a role of CD44 in bone resorption rather than in bone formation.

Immunoglobulin superfamily members Neural cell adhesion molecule, a member of the immunoglobulin superfamily, is a cell surface molecule expressed transiently during osteoblast lineage (Haÿ et al., 2000; Tanikawa et al., 2010), and its expression is associated with the osteogenic phenotype (Rundus et al., 1998). Activated leukocyte cell adhesion molecule (ALCAM or CD166) is another immunoglobulin member expressed by osteoblasts. CD166-deficient mice show increased osteoblast differentiation and bone formation with no change in bone resorption, suggesting that CD166 regulates bone formation (Hooker et al., 2015). Osteoblasts also express intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and the cross talk of these molecules induces interleukin-6 (IL-6) secretion by osteoblasts, suggesting that these adhesion molecules transduce activation signals that induce the production of bone-resorbing cytokines (Tanaka et al., 1995). In addition, ICAM-1 and VCAM-1 mediate cellecell adhesion between osteoclastic precursors and bone marrow stromal cells or osteoblasts, which controls osteoclastogenesis. Neutralization of VCAM-1 in bone marrow stromal cells inhibits the formation of osteoclasts in vitro, indicating that VCAM-1 expression by stromal cells is required for osteoclastogenesis

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(Feuerbach and Feyen, 1997). A fraction of osteoblasts that highly express ICAM-1 strongly adhere to osteoclast precursors, resulting in multinuclear osteoclast-like cell formation, indicating that a subpopulation of ICAM-1-expressing osteoblasts controls osteoclastogenesis (Tanaka et al., 2000). Consistent with a role of ICAM-1 in osteoclastogenesis, ICAM-1-mediated cell-to-cell adhesion of osteoblasts and osteoclast precursors was involved in RANKL-dependent osteoclast maturation stimulated by 1,25-dihydroxyvitamin D, PTH, and IL-1a (Okada et al., 2002).

Osteoactivin Osteoactivin is a glycoprotein expressed by osteoblasts and its expression upregulates osteoblast differentiation in vitro (Abdelmagid et al., 2008). Mice with a loss-of-function mutation in Gpnmb, which encodes osteoactivin, showed decreased trabecular bone mass due to reduced osteoblast differentiation, confirming the positive role of osteoactivin in bone formation (Abdelmagid et al., 2014). Osteoactivin is also expressed in osteoclasts and acts as a negative regulator of osteoclast differentiation and survival, but not function (Abdelmagid et al., 2015). Consistent with these findings, transgenic mice overexpressing osteoactivin under the cytomegalovirus promoter showed increased trabecular bone mass and bone formation, and decreased bone resorption (Frara et al., 2016). Mechanistically, osteoactivin can bind to CD44 in osteoclasts, leading to inhibition of ERK phosphorylation and RANKL-induced osteolysis (Sondag et al., 2016). In murine osteoblastic cells, recombinant osteoactivin stimulates cell adhesion and spreading through its binding to aVb1 integrin and HSPGs at the cell surface. This interaction results in FAK and ERK activation and osteoblast differentiation, suggesting a mechanism by which osteoactivin may control osteoblast differentiation (Moussa et al., 2014).

Chondroadherin Chondroadherin belongs to the family of leucine-rich repeat proteins and was identified in the cartilage matrix, where it promotes attachment of chondrocytes (Larsson et al., 1991). Chondroadherin localizes near the cell surface and is highly expressed in the proliferative and hypertrophic zones of the growth plate. It is a small molecule of 38 kDa molecular weight, containing 359 amino acids. Relevant domains of chondroadherin are a putative signal peptide, a cysteine-rich region at the N-terminal tail, 11 leucine-rich repeats, and a double cysteine loop in the C-terminal tail. Among the members of the leucine-rich repeat protein family, chondroadherin exhibits the unique feature of no posttranslational glycosylation (Neam et al., 1994). The very C terminus of the protein includes a heparin-binding consensus sequence that allows its interaction with heparin (Haglund et al., 2013). This heparin-binding domain recognizes cell surface syndecans and triggers ERK1/2 phosphorylation (Haglund et al., 2013). Chondroadherin is also recognized by the a2b1 integrin expressed by chondrocytes (Camper et al., 1997). The a2b1 integrin binding site was identified in the region carrying the amino acid residues 306e318 at the C terminus of the protein. By affinity purification procedure, a2b1 integrin was confirmed to bind the chondroadherin CQLRGLRRWLEAK318 peptide. A longer chondroadherin peptide, spanning amino acid residues 306e326 (CQLRGLRRWEKLAASRPDATC326) was made cyclic and stable through a disulfide bond occurring between the two terminal cysteines and was largely used to investigate the functional role of chondroadherin in vitro and in vivo. The peptide was confirmed to induce cell adhesion and spreading in an a2b1 integrin-dependent manner, activating ERK1/2 phosphorylation (Haglund et al., 2011). Chondroadherin is expressed also by osteoblasts. It was found to be 50% less expressed in bone biopsies of relatively young female osteoporotic patients (ages between 50 and 65 years), and in ovariectomized mice, a model of estrogen deficiency-induced osteoporosis (Capulli et al., 2014). The cyclic CQLRGLRRWEKLAASRPDATC326 peptide was inactive on osteoblasts, but strongly impaired osteoclastogenesis at the late stage of the process. Specifically, the major effect was exerted by the cyclic peptide on migration of osteoclast precursors, which is mandatory for cell clustering and fusion into mature polykarya. The underlying molecular mechanism involved the decreased expression of migfilin and vasodilator-stimulated phosphoprotein (VASP) (Capulli et al., 2014). Migfilin is associated with adhesion sites and binds filamins, VASP, kindling-2 and the transcription factor CSX/NKX2-5, recruiting acting cytoskeleton and promoting cell adhesion, shape modulation, motility and gene expression (Tu et al., 2003). Migfilin inactivation in mice induced a severe osteopenic phenotype (Xiao et al., 2012). However, this effect was mostly due to reduced osteoblast differentiation with a parallel increase in the proosteoclastogenic cytokine RANKL, which exacerbated osteoclast differentiation. VASP is known to induce monomeric actin recruitment to the barbed end of microfilaments, preventing capping and regulating filament bundling (Krause et al., 2003). It is implicated in cell motility, adhesion, and sensory capacity, localizing in the tips of filopodia and in adhesion structures (Tokuo and Ikebe, 2004). In osteoclasts, VASP is associated with the aVb3 integrin and is activated by NO, which promotes osteoclast motility. Consistently, knockdown of Vasp reduced osteoclast migration on substrate (Yaroslavskiy et al., 2005). Interestingly, cyclic chondroadherin downregulated NO synthase (Nos2)

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in osteoclasts, and treatment with an a2b1 integrin blocking antibody increased osteoclast Nos2 expression, suggesting an inhibitory role of the a2b1 integrin on Nos2 triggered by chondroadherin (Capulli et al., 2014). Overall, the results obtained using the cyclic a2b1 integrin-binding domain of chondroadherin suggest that this ECM component affects osteoclastogenesis via transcriptional downregulation of Nos2 and decreased NO, which regulates migfilin and Vasp expression and is required for preosteoclast migration, clustering, and fusion into multinucleated osteoclasts. Cyclic chondroadherin peptide showed also the ability to impair osteoclastogenesis in vivo. Mice injected with cyclic chondroadherin showed enhanced bone mass and reduced osteoclast variables, while osteoblasts and bone formation were not affected (Capulli et al., 2014). This effect was evident both in normal mice and in mice subjected to ovariectomy. In this circumstance, cyclic chondroadherin blocked the increase in serum level of bone resorption biomarkers and prevented bone loss induced by estrogen deficiency, with an improvement of bone quality. These effects were observed in young and old mice and were mimicked by treatment with the NOS2 activity inhibitor (L-N6-(1-iminoethyl)lysine dihydrochloride). Furthermore, cyclic chondroadherin was effective not only in preventative treatment started at the time of ovariectomy, but also in a curative setting, with the treatment started 5 weeks after ovariectomy, a time at which the osteoporotic phenotype was overt (Capulli et al., 2014). Given the positive response of the bone to treatment with cyclic chondroadherin, the peptide was also tested against bone metastasis-induced osteolysis, which is caused by exacerbated osteoclast activity (Rucci et al., 2015). The results of this study complemented the observations on ovariectomized mice, showing a beneficial effect of the peptide in mice injected intracardiacally or orthotopically with osteotropic breast cancer cells. The peptide reduced the in vitro motility of tumor cells and in vivo tumor growth. It also inhibited the process of tumor-induced osteoclast formation, with consequent reduction of bone resorption, development of osteolytic lesions, and cachexia. Interestingly, cyclic chondroadherin synergistically enhanced the antitumoral effect of the chemotherapeutic doxorubicin, which achieved maximal efficacy at half of the effective dose when administered alone (Rucci et al., 2015). The cyclic chondroadherinedoxorubicin treatment affected tumor cells also in vitro, confirming a synergistic impairment of cell motility at lower doses. Taken together, these results demonstrated that chondroadherin is an important regulator of cell migration, exerting this effect by binding to the a2b1 integrin and impairing the late stage of osteoclast formation and the development of metastatic osteolysis.

Conclusion Multiple in vitro and in vivo studies have shown that integrins, cadherins, and several other adhesion molecules control bone cell function and fate during chondrogenesis, osteoblastogenesis, and osteoclastogenesis. Genetic studies in mice confirmed the importance of some of these adhesion molecules in the control of bone resorption and formation in vivo. These effects are mediated by complex interactions of these adhesion molecules with bone matrix proteins or cell surface molecules, leading to the modulation of intracellular signaling pathways controlling bone cell differentiation, function, and survival. Studies have revealed that the signaling pathways mediated by integrins, cadherins, and other cell adhesion molecules can cross talk with Wnt/b-catenin signaling to regulate osteogenic differentiation and mechanotransduction, and with other signaling mechanisms to control osteoclastogenesis. These advances led to a more comprehensive view of the role of these adhesion molecules in the signaling mechanisms controlling bone cell recruitment and function. Future studies will have to confirm that targeting specific adhesion molecules and their downstream signals may have potential therapeutic implications in reducing bone resorption or promoting bone formation in skeletal disorders.

Acknowledgments The authors thank all collaborators who contributed to the work reviewed in this chapter. This work was supported by the Institut National de la Recherche Médicale (Inserm), the Agence Nationale de la Recherche, and the European Commission FP6 and FP7 programs (P.J.M.), and by the Telethon, the Italian Association of Cancer Research, the PRIN-MIUR, and the European Commission FP6, FP7, and H2020 programs (A.T.).

References Abdelmagid, S.M., Barbe, M.F., Rico, M.C., Salihoglu, S., Arango-Hisijara, I., Selim, A.H., et al., 2008. Osteoactivin, an anabolic factor that regulates osteoblast differentiation and function. Exp. Cell Res. 314, 2334e2351. Abdelmagid, S.M., Belcher, J.Y., Moussa, F.M., Lababidi, S.L., Sondag, G.R., Novak, K.M., et al., 2014. Mutation in osteoactivin decreases bone formation in vivo and osteoblast differentiation in vitro. Am. J. Pathol. 184, 697e713. Abdelmagid, S.M., Sondag, G.R., Moussa, F.M., Belcher, J.Y., Yu, B., Stinnett, H., et al., 2015. Mutation in osteoactivin promotes receptor activator of NFkB ligand (RANKL)-mediated osteoclast differentiation and survival but inhibits osteoclast function. J. Biol. Chem. 290, 20128e20146.

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Akisaka, T., Yoshida, H., Inoue, S., Shimizu, 2001. Organization of cytoskeletal F-actin, G-actin, and gelsolin in the adhesion structures in cultured osteoclast. J. Bone Miner. Res. 16, 1248e1255. Alan, T., Tufan, A.C., 2008. C-type natriuretic peptide regulation of limb mesenchymal chondrogenesis is accompanied by altered N-cadherin and collagen type X-related functions. J. Cell. Biochem. 105, 227e235. Arikawa-Hirasawa, E., Watanabe, H., Takami, H., Hassell, J.R., Yamada, Y., 1999. Perlecan is essential for cartilage and cephalic development. Nat. Genet. 23, 354e358. Arnsdorf, E.J., Tummala, P., Jacobs, C.R., 2009. Non-canonical Wnt signaling and N-cadherin related beta-catenin signaling play a role in mechanically induced osteogenic cell fate. PLoS One 4, e5388. Aszodi, A., Hunziker, E.B., Brakebusch, C., Fässler, R., 2003. Beta1 integrins regulate chondrocyte rotation, G1 progression, and cytokinesis. Genes Dev. 17, 2465e2479. Auzzas, L., Zanardi, F., Battistini, L., Burreddu, P., Carta, P., Rassu, G., et al., 2010. Targeting alphavbeta3 integrin: design and applications of mono- and multifunctional RGD-based peptides and semipeptides. Curr. Med. Chem. 17, 1255e1299. Baeg, G.H., Lin, X., Khare, N., Baumgartner, S., Perrimon, N., 2001. Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128, 87e94. Batra, N., Burra, S., Siller-Jackson, A.J., Gu, S., Xia, X., Weber, G.F., et al., 2012. Mechanical stress-activated integrin a5b1 induces opening of connexin 43 hemichannels. Proc. Natl. Acad. Sci. U. S. A. 109, 3359e3364. Bengtsson, T., Aszodi, A., Nicolae, C., Hunziker, E.B., Lundgren-Akerlund, E., Fässler, R., 2005. Loss of alpha10beta1 integrin expression leads to moderate dysfunction of growth plate chondrocytes. J. Cell Sci. 118, 929e936. Bennett, J.H., Moffatt, S., Horton, M., 2001. Cell adhesion molecules in human osteoblasts: structure and function. Histol. Histopathol. 16, 603e611. Bernfield, M., Götte, M., Park, P.W., Reizes, O., Fitzgerald, M.L., Lincecum, J., et al., 1999. Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68, 729e777. Bikle, D.D., 2008. Integrins, insulin like growth factors, and the skeletal response to load. Osteoporos. Int. 19, 1237e1246. Billings, P.C., Pacifici, M., 2015. Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries. Connect. Tissue Res. 56, 272e280. Bishop, J.R., Schuksz, M., Esko, J.D., 2007. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446, 1030e1037. Bonewald, L.F., Johnson, M.L., 2008. Osteocytes, mechanosensing and Wnt signaling. Bone 42, 606e615. Bonnet, N., Conway, S.J., Ferrari, S.L., 2012. Regulation of beta catenin signaling and parathyroid hormone anabolic effects in bone by the matricellular protein periostin. Proc. Natl. Acad. Sci. U. S. A. 109, 15048e15053. Bouvard, D., Aszodi, A., Kostka, G., Block, M.R., Albigès-Rizo, C., Fässler, R., 2007. Defective osteoblast function in ICAP-1-deficient mice. Development 134, 2615e2625. Brunner, M., Millon-Frémillon, A., Chevalier, G., Nakchbandi, I.A., Mosher, D., Block, M.R., et al., 2011. Osteoblast mineralization requires beta1 integrin/ICAP-1-dependent fibronectin deposition. J. Cell Biol. 194, 307e322. Bruzzaniti, A., Neff, L., Sanjay, A., Horne, W.C., De Camilli, P., Baron, R., 2005. Dynamin forms a Src kinase-sensitive complex with Cbl and regulates podosomes and osteoclast activity. Mol. Biol. Cell 16, 3301e3313. Calle, Y., Burns, S., Thrasher, A.J., Jones, G.E., 2006. The leukocyte podosome. Eur. J. Cell Biol. 85, 151e157. Campbell, I.D., Humphries, M.J., 2011. Integrin structure, activation, and interactions. Cold Spring Harb Perspect Biol 3 (3). Camper, L., Heinegard, D., Lundgren-Åkerlund, E., 1997. Integrin alpha2beta1 is a receptor for the cartilage matrix protein chondroadherin. J. Cell Biol. 138, 1159e1167. Cao, J.J., Singleton, P.A., Majumdar, S., Boudignon, B., Burghardt, A., Kurimoto, P., et al., 2005. Hyaluronan increases RANKL expression in bone marrow stromal cells through CD44. J. Bone Miner. Res. 20, 30e40. Cappariello, A., Maurizi, A., Veeriah, V., Teti, A., 2014. The great beauty of the osteoclast. Arch. Biochem. Biophys. 558, 70e78. Capulli, M., Olstad, O.K., Önnerfjord, P., Tillgren, V., Muraca, M., Gautvik, K.M., et al., 2014. The C-terminal domain of chondroadherin: a new regulator of osteoclast motility counteracting bone loss. J. Bone Miner. Res. 29, 1833e1846. Castro, C.H., Shin, C.S., Stains, J.P., Cheng, S.L., Sheikh, S., Mbalaviele, G., et al., 2004. Targeted expression of a dominant-negative N-cadherin in vivo delays peak bone mass and increases adipogenesis. J. Cell Sci. 117, 2853e2864. Chellaiah, M.A., Hruska, K.A., 2003. The integrin alpha(v)beta(3) and CD44 regulate the actions of osteopontin on osteoclast motility. Calcif. Tissue Int. 72, 197e205. Chellaiah, M.A., Biswas, R.S., Yuen, D., Alvarez, U.M., Hruska, K.A., 2001. Phosphatidylinositol 3,4,5-trisphosphate directs association of Src homology 2-containing signaling proteins with gelsolin. J. Biol. Chem. 276, 47434e47444. Chellaiah, M.A., Kizer, N., Biswas, R., Alvarez, U., Strauss-Schoenberger, J., Rifas, L., et al., 2003. Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression. Mol. Biol. Cell 14, 173e189. Chen, J.C., Jacobs, C.R., 2013. Mechanically induced osteogenic lineage commitment of stem cells. Stem Cell Res. Ther. 4, 107. Chen, X.D., Dusevich, V., Feng, J.Q., Manolagas, S.C., Jilka, R.L., 2007. Extracellular matrix made by bone marrow cells facilitates expansion of marrow-derived mesenchymal progenitor cells and prevents their differentiation into osteoblasts. J. Bone Miner. Res. 22, 1943e1956. Chen, Q., Shou, P., Zhang, L., Xu, C., Zheng, C., Han, Y., Li, W., et al., 2014. An osteopontin-integrin interaction plays a critical role in directing adipogenesis and osteogenesis by mesenchymal stem cells. Stem Cell. 32, 327e337. Cheng, S.L., Lecanda, F., Davidson, M.K., Warlow, P.M., Zhang, S.F., Zhang, L., et al., 1998. Human osteoblasts express a repertoire of cadherins, which are critical for BMP-2-induced osteogenic differentiation. J. Bone Miner. Res. 13, 633e644.

Integrins and other cell surface attachment molecules of bone cells Chapter | 17

415

Cheng, S.L., Lai, C.F., Fausto, A., Chellaiah, M., Feng, X., McHugh, K.P., et al., 2000. Regulation of alphaVbeta3 and alphaVbeta5 integrins by dexamethasone in normal human osteoblastic cells. J. Cell. Biochem. 77, 265e276. Cheng, S.L., Lai, C.F., Blystone, S.D., Avioli, L.V., 2001. Bone mineralization and osteoblast differentiation are negatively modulated by integrin alpha(v)beta3. J. Bone Miner. Res. 16, 277e288. Cho, S.H., Oh, C.D., Kim, S.J., Kim, I.C., Chun, J.S., 2003. Retinoic acid inhibits chondrogenesis of mesenchymal cells by sustaining expression of Ncadherin and its associated proteins. Cell Biochem 89, 837e847. Clover, J., Dodds, R.A., Gowen, M., 1992. Integrin subunit expression by human osteoblasts and osteoclasts in situ and in culture. J. Cell Sci. 103, 267e271. Connelly, J.T., García, A.J., Levenston, M.E., 2007. Inhibition of in vitro chondrogenesis in RGD-modified three-dimensional alginate gels. Biomaterials 28, 1071e1083. Correia, I., Chu, D., Chou, Y.H., Goldman, R.D., Matsudaira, P., 1999. Integrating the actin and vimentin cytoskeletons. Adhesion dependent formation of fimbrinevimentin complexes in macrophages. J. Cell Biol. 146, 831e842. Dai, Z., Guo, F., Wu, F., Xu, H., Yang, C., Li, J., et al., 2014. Integrin avb3 mediates the synergetic regulation of core-binding factor a1 transcriptional activity by gravity and insulin-like growth factor-1 through phosphoinositide 3-kinase signaling. Bone 69, 126e132. Danhier, F., Le Breton, A., Préat, V., 2012. RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis. Mol. Pharm. 9, 2961e2973. David, G., Bai, X.M., Van der Schueren, B., Marynen, P., Cassiman, J.J., Van den Berghe, H., 1993. Spatial and temporal changes in the expression of fibroglycan (syndecan-2) during mouse embryonic development. Development 119, 841e854. Decarlo, A.A., Belousova, M., Ellis, A.L., Petersen, D., Grenett, H., Hardigan, P., et al., 2012. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis. BMC Biotechnol. 12, 60. Dejaeger, M., Böhm, A.M., Dirckx, N., Devriese, J., Nefyodova, E., Cardoen, R., et al., 2017. Integrin-linked kinase regulates bone formation by controlling cytoskeletal organization and modulating BMP and Wnt signaling in osteoprogenitors. J. Bone Miner. Res. 32, 2087e2102. Delise, A.M., Tuan, R.S., 2002. Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro. Dev. Dynam. 225, 195e204. Desgrosellier, J.S., Cheresh, D.A., 2010. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Canc. 10, 9e22. Destaing, O., Sanjay, A., Itzstein, C., Horne, W.C., Toomre, D., De Camilli, P., et al., 2008. The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts. Mol. Biol. Cell 19, 394e404. Dews, I.C., Mackenzie, K.R., 2007. Transmembrane domains of the syndecan family of growth factor coreceptors display a hierarchy of homotypic and heterotypic interactions. Proc. Natl. Acad. Sci. U. S. A. 104, 20782e20787. Di Benedetto, A., Watkins, M., Grimston, S., Salazar, V., Donsante, C., Mbalaviele, G., et al., 2010. N-cadherin and cadherin 11 modulate postnatal bone growth and osteoblast differentiation by distinct mechanisms. J. Cell Sci. 123, 2640e2648. Dieudonné, F.X., Marion, A., Haÿ, E., Marie, P.J., Modrowski, D., 2010. High Wnt signaling represses the proapoptotic proteoglycan syndecan-2 in osteosarcoma cells. Cancer Res. 70, 5399e5408. Du, J., Zu, Y., Li, J., Du, S., Xu, Y., Zhang, L., et al., 2016. Extracellular matrix stiffness dictates Wnt expression through integrin pathway. Sci. Rep. 6, 20395. Dufour, C., Holy, X., Marie, P.J., 2007. Skeletal unloading induces osteoblast apoptosis and targets alpha5beta1-PI3K-Bcl-2 signaling in rat bone. Exp. Cell Res. 313, 394e403. Duong, L.T., Lakkakorpi, P.T., Nakamura, I., Machwate, M., Nagy, R.M., Rodan, G.A., 1998. PYK2 in osteoclasts is an adhesion kinase, localized in the sealing zone, activated by ligation of alpha(v)beta3 integrin, and phosphorylated by src kinase. J. Clin. Investig. 102, 881e892. Dwivedi, P.P., Lam, N., Powell, B.C., 2013. Boning up on glypicans-Opportunities for new insights into bone biology. Cell Biochem. Funct. 31, 91e114. Eddy, R.J., Weidmann, M.D., Sharma, V.P., Condeelis, J.S., 2017. Tumor cell invadopodia: invasive protrusions that orchestrate metastasis. Trends Cell Biol. 27, 595e607. Enomoto-Iwamoto, M., Iwamoto, M., Nakashima, K., Mukudai, Y., Boettiger, D., Pacifici, M., et al., 1997. Involvement of alpha5beta1 integrin in matrix interactions and proliferation of chondrocytes. J. Bone Miner. Res. 12, 1124e1132. Evans, J.G., Matsudaira, P., 2006. Structure and dynamics of macrophage podosomes. Eur. J. Cell Biol. 85, 145e149. Farach-Carson, M.C., Brown, A.J., Lynam, M., Safran, J.B., Carson, D.D., 2008. A novel peptide sequence in perlecan domain IV supports cell adhesion, spreading and FAK activation. Matrix Biol. 27, 150e160. Ferrari, S.L., Traianedes, K., Thorne, M., Lafage-Proust, M.H., Genever, P., Cecchini, M.G., et al., 2000. A role for N-cadherin in the development of the differentiated osteoblastic phenotype. J. Bone Miner. Res. 15, 198e208. Feuerbach, D., Feyen, J.H., 1997. Expression of the cell-adhesion molecule VCAM-1 by stromal cells is necessary for osteoclastogenesis. FEBS Lett. 402, 21e24. Fiorino, C., Harrison, R.E., 2016. E-cadherin is important for cell differentiation during osteoclastogenesis. Bone 86, 106e118. Fontana, F., Hickman-Brecks, C.L., Salazar, V.S., Revollo, L., Abou-Ezzi, G., Grimston, S.K., et al., 2017. N-cadherin regulation of bone growth and homeostasis is osteolineage stage-specific. J. Bone Miner. Res. 32, 1332e1342. Frara, N., Abdelmagid, S.M., Sondag, G.R., Moussa, F.M., Yingling, V.R., Owen, T.A., et al., 2016. Transgenic expression of osteoactivin/gpnmb enhances bone formation in vivo and osteoprogenitor differentiation ex vivo. J. Cell. Physiol. 231, 72e83. French, M.M., Smith, S.E., Akanbi, K., Sanford, T., Hecht, J., Farach-Carson, M.C., et al., 1999. Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro. J. Cell Biol. 145, 1103e1115.

416 PART | I Basic principles

French, M.M., Gomes Jr., R.R., Timpl, R., Höök, M., Czymmek, K., Farach-Carson, M.C., et al., 2002. Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I. J. Bone Miner. Res. 17, 48e55. Frisch, S.M., Ruoslahti, E., 1997. Integrins and anoikis. Curr. Opin. Cell Biol. 9, 701e706. Fromigué, O., Brun, J., Marty, C., Da Nascimento, S., Sonnet, P., Marie, P.J., 2012. Peptide-based activation of alpha5 integrin for promoting osteogenesis. J. Cell. Biochem. 113, 3029e3038. Fuerer, C., Habib, S.J., Nusse, R., 2010. A study on the interactions between heparan sulfate proteoglycans and Wnt proteins. Dev. Dynam. 239, 184e190. Garciadiego-Cázares, D., Rosales, C., Katoh, M., Chimal-Monroy, J., 2004. Coordination of chondrocyte differentiation and joint formation by alpha5beta1 integrin in the developing appendicular skeleton. Development 131, 4735e4742. Ge, C., Yang, Q., Zhao, G., Yu, H., Kirkwood, K.L., Franceschi, R.T., 2012. Interactions between extracellular signal-regulated kinase 1/2 and P38 MAP kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity. J. Bone Miner. Res. 27, 538e551. Georgess, D., Machuca-Gayet, I., Blangy, A., Jurdic, P., 2014. Podosome organization drives osteoclast-mediated bone resorption. Cell Adhes. Migrat. 8, 191e204. Gil-Henn, H., Destaing, O., Sims, N.A., Aoki, K., Alles, N., Neff, L., et al., 2007. Defective microtubule-dependent podosome organization in osteoclasts leads to increased bone density in Pyk2(-/-) mice. J. Cell Biol. 178, 1053e1064. Globus, R.K., Amblard, D., Nishimura, Y., Iwaniec, U.T., Kim, J.B., Almeida, E.A., et al., 2005. Skeletal phenotype of growing transgenic mice that express a function-perturbing form of beta1 integrin in osteoblasts. Calcif. Tissue Int. 76, 39e49. Gomes Jr., R.R., Joshi, S.S., Farach-Carson, M.C., Carson, D.D., 2006. Ribozyme-mediated perlecan knockdown impairs chondrogenic differentiation of C3H10T1/2 fibroblasts. Differentiation 74, 53e63. Goodison, S., Urquidi, V., Tarin, D., 1999. CD44 cell adhesion molecules. Mol. Pathol. 52, 189e196. Goomer, R.S., Maris, T., Amiel, D., 1998. Age-related changes in the expression of cadherin-11, the mesenchyme specific calcium-dependent cell adhesion molecule. Calcif. Tissue Int. 62, 532e537. Gottardi, C.J., Gumbiner, B.M., 2001. Adhesion signaling: how beta-catenin interacts with its partners. Curr. Biol. 11, R792eR794. Granot-Attas, S., Elson, A., 2008. Protein tyrosine phosphatases in osteoclast differentiation, adhesion, and bone resorption. Eur. J. Cell Biol. 87, 479e490. Grashoff, C., Aszódi, A., Sakai, T., Hunziker, E.B., Fässler, R., 2003. Integrin-linked kinase regulates chondrocyte shape and proliferation. EMBO Rep. 4, 432e438. Greenbaum, A.M., Revollo, L.D., Woloszynek, J.R., Civitelli, R., Link, D.C., 2012. N-cadherin in osteolineage cells is not required for maintenance of hematopoietic stem cells. Blood 120, 295e302. Grigoriou, V., Shapiro, I.M., Cavalcanti-Adam, E.A., Composto, R.J., Ducheyne, P., Adams, C.S., 2005. Apoptosis and survival of osteoblast-like cells are regulated by surface attachment. J. Biol. Chem. 280, 1733e1739. Gronthos, S., Simmons, P.J., Graves, S.E., Robey, P.G., 2001. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix. Bone 28, 174e178. Grzesik, W.J., Robey, P.G., 1994. Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro. J. Bone Miner. Res. 9, 487e496. Grzesik, W.J., 1997. Integrins and bone–cell adhesion and beyond. Arch. Immunol. Ther. Exp. 45, 271e275. Guan, M., Yao, W., Liu, R., Lam, K.S., Nolta, J., Jia, J., et al., 2012. Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass. Nat. Med. 18, 456e462. Gumbiner, B.M., 2005. Regulation of cadherin-mediated adhesion in morphogenesis. Nat. Rev. Mol. Cell Biol. 6, 622e634. Guntur, A.R., Rosen, C.J., Naski, M.C., 2012. N-cadherin adherens junctions mediate osteogenesis through PI3K signaling. Bone 50, 54e62. Gutierrez, J., Osses, N., Brandan, E., 2006. Changes in secreted and cell associated proteoglycan synthesis during conversion of myoblasts to osteoblasts in response to bone morphogenetic protein-2: role of decorin in cell response to BMP-2. J. Cell. Physiol. 206, 58e67. Haglund, L., Tillgren, V., Addis, L., Wenglén, C., Recklies, A., Heinegård, D., 2011. Identification and characterization of the integrin a2b1 binding motif in chondroadherin mediating cell attachment. J. Biol. Chem. 286, 3925e3934. Haglund, L., Tillgren, V., Önnerfjord, P., Heinegård, D., 2013. The C-terminal peptide of chondroadherin modulate cellular activity by selectively binding to heparan sulfate chains. J. Biol. Chem. 288, 995e1008. Hamidouche, Z., Fromigué, O., Ringe, J., Häupl, T., Vaudin, P., Pagès, J.C., et al., 2009. Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis. Proc. Natl. Acad. Sci. U. S. A. 106, 18587e18591. Hamidouche, Z., Fromigué, O., Ringe, J., Häupl, T., Marie, P.J., 2010. Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation. BMC Cell Biol. 11, 44. Haupt, L.M., Murali, S., Mun, F.K., Teplyuk, N., Mei, L.F., Stein, G.S., et al., 2009. The heparan sulfate proteoglycan (HSPG) glypican-3 mediates commitment of MC3T3-E1 cells toward osteogenesis. J. Cell. Physiol. 220, 780e791. Haÿ, E., Lemonnier, J., Modrowski, D., Lomri, A., Lasmoles, F., Marie, P.J., 2000. N- and E-cadherin mediate early human calvaria osteoblast differentiation promoted by bone morphogenetic protein-2. J. Cell. Physiol. 183, 117e128. Haÿ, E., Laplantine, E., Geoffroy, V., Frain, M., Kohler, T., Muller, R., et al., 2009a. N-cadherin interacts with axin and LRP5 to negatively regulate Wnt/ beta-catenin signaling, osteoblast function, and bone formation. Mol. Cell Biol. 29, 953e964. Haÿ, E., Nouraud, A., Marie, P.J., 2009b. N-cadherin negatively regulates osteoblast proliferation and survival by antagonizing Wnt, ERK and PI3K/Akt signalling. PLoS One 4, e8284.

Integrins and other cell surface attachment molecules of bone cells Chapter | 17

417

Haÿ, E., Buczkowski, T., Marty, C., Da Nascimento, S., Sonnet, P., Marie, P.J., 2012. Peptide-based mediated disruption of N-cadherin-LRP5/6 interaction promotes Wnt signaling and bone formation. J. Bone Miner. Res. 27, 1852e1863. Haÿ, E., Dieudonné, F.X., Saidak, Z., Marty, C., Brun, J., Da Nascimento, S., et al., 2014. N-cadherin/Wnt interaction controls bone marrow mesenchymal cell fate and bone mass during aging. J. Cell. Physiol. 229, 1765e1767. Heuberger, J., Birchmeier, W., 2010. Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling. Cold Spring Harb. Perspect. Biol. 2, a002915. Hirsch, M.S., Lunsford, L.E., Trinkaus-Randall, V., Svoboda, K.K., 1997. Chondrocyte survival and differentiation in situ are integrin mediated. Dev. Dynam. 210, 249e263. Hooker, R.A., Chitteti, B.R., Egan, P.H., Cheng, Y.H., Himes, E.R., Meijome, T., et al., 2015. Activated leukocyte cell adhesion molecule (ALCAM or CD166) modulates bone phenotype and hematopoiesis. J. Musculoskelet. Neuronal Interact. 15, 83e94. Horne, W.C., Sanjay, A., Bruzzaniti, A., Baron, R., 2005. The role(s) of Src kinase and Cbl proteins in the regulation of osteoclast differentiation and function. Immunol. Rev. 208, 106e125. Horton, M.A., 2001. Integrin antagonists as inhibitors of bone resorption: implications for treatment. Proc. Nutr. Soc. 60, 275e281. Hsu, A.R., Veeravagu, A., Cai, W., Hou, L.C., Tse, V., Chen, X., 2007. Integrin alpha v beta 3 antagonists for anti-angiogenic cancer treatment. Recent Pat. Anti-Cancer Drug Discov. 2, 143e158. Huegel, J., Sgariglia, F., Enomoto-Iwamoto, M., Koyama, E., Dormans, J.P., Pacifici, M., 2013. Heparan sulfate in skeletal development, growth, and pathology: the case of hereditary multiple exostoses. Dev. Dynam. 242, 1021e1032. Hughes, D.E., Salter, D.M., Dedhar, S., Simpson, R., 1993. Integrin expression in human bone. J. Bone Miner. Res. 8, 527e533. Hultenby, K., Reinholt, F.P., Heinegård, D., 1993. Distribution of integrin subunits on rat metaphyseal osteoclasts and osteoblasts. Eur. J. Cell Biol. 62, 86e93. Inoue, M., Namba, N., Chappel, J., Teitelbaum, S.L., Ross, F.P., 1998. Granulocyte macrophage-colony stimulating factor reciprocally regulates alphavassociated integrins on murine osteoclast precursors. Mol. Endocrinol. 12, 1955e1962. Iwaniec, U.T., Wronski, T.J., Amblard, D., Nishimura, Y., van der Meulen, M.C., Wade, C.E., et al., 2005. Effects of disrupted beta1-integrin function on the skeletal response to short-term hindlimb unloading in mice. J. Appl. Physiol. 98, 690e696. James, A.W., Shen, J., Zhang, X., Asatrian, G., Goyal, R., Kwak, J.H., et al., 2015. NELL-1 in the treatment of osteoporotic bone loss. Nat. Commun. 6, 7362. Jennings, L., Wu, L., King, K.B., Hämmerle, H., Cs-Szabo, G., Mollenhauer, J., 2001. The effects of collagen fragments on the extracellular matrix metabolism of bovine and human chondrocytes. Connect. Tissue Res. 42, 71e86. Jikko, A., Harris, S.E., Chen, D., Mendrick, D.L., Damsky, C.H., 1999. Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2. J. Bone Miner. Res. 14, 1075e1083. Katsumi, A., Orr, A.W., Tzima, E., Schwartz, M.A., 2004. Integrins in mechanotransduction. J. Biol. Chem. 279, 12001e12004. Kawaguchi, J., Kii, I., Sugiyama, Y., Takeshita, S., Kudo, A., 2001a. The transition of cadherin expression in osteoblast differentiation from mesenchymal cells: consistent expression of cadherin-11 in osteoblast lineage. J. Bone Miner. Res. 16, 260e269. Kawaguchi, J., Kii, I., Sugiyama, Y., Takeshita, S., Kudo, A., 2001b. The transition of cadherin expression in osteoblast differentiation from mesenchymal cells: consistent expression of cadherin-11 in osteoblast lineage. J. Bone Miner. Res. 16, 260, 26. Khatiwala, C.B., Kim, P.D., Peyton, S.R., Putnam, A.J., 2009. ECM compliance regulates osteogenesis by influencing MAPK signaling downstream of RhoA and ROCK. J. Bone Miner. Res. 24, 886e898. Kidwai, F., Edwards, J., Zou, L., Kaufman, D.S., 2016. Fibrinogen induces Runx2 activity and osteogenic development from human pluripotent stem cells. Stem Cell. 34, 2079e2089. Kii, I., Amizuka, N., Shimomura, J., Saga, Y., Kudo, A., 2004. Cell-cell interaction mediated by cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage. J. Bone Miner. Res. 19, 1840e1849. Kim, J.B., Leucht, P., Luppen, C.A., Park, Y.J., Beggs, H.E., Damsky, C.H., et al., 2007. Reconciling the roles of FAK in osteoblast differentiation, osteoclast remodeling, and bone regeneration. Bone 41, 39e51. Kirsch, T., 2005. Annexins e their role in cartilage mineralization. Front. Biosci. 10, 576e581. Knudson, C.B., 2003. Hyaluronan and CD44: strategic players for cell-matrix interactions during chondrogenesis and matrix assembly. Birth Defects Res C Embryo Today 69, 174e196. Kobayashi, Y., Uehara, S., Koide, M., Takahashi, N., 2015. The regulation of osteoclast differentiation by Wnt signals. Bonekey Rep. 4, 713. Krause, M., Dent, E.W., Bear, J.E., Loureiro, J.J., Gertler, F.B., 2003. Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu. Rev. Cell Dev. Biol. 19, 541e564. Kumar, S., Ponnazhagan, S., 2007. Bone homing of mesenchymal stem cells by ectopic alpha 4 integrin expression. FASEB J. 21, 3917e3927. Labrador, J.P., Azcoitia, V., Tuckermann, J., Lin, C., Olaso, E., Mañes, S., et al., 2001. The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism. EMBO Rep. 2, 446e452. Lai, C.F., Feng, X., Nishimura, R., Teitelbaum, S.L., Avioli, L.V., Ross, F.P., et al., 2000. Transforming growth factor-beta up-regulates the beta 5 integrin subunit expression via Sp1 and Smad signaling. J. Biol. Chem. 275, 36400e36406. Lai, C.F., Chaudhary, L., Fausto, A., Halstead, L.R., Ory, D.S., Avioli, L.V., et al., 2001. Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells. J. Biol. Chem. 276, 14443e14450. Larsson, T., Sommarin, Y., Paulsson, M., Antonsson, P., Hedbom, E., Wendel, M., et al., 1991. Cartilage matrix proteins. A basic 36-kDa protein with a restricted distribution to cartilage and bone. J. Biol. Chem. 266, 20428e20433.

418 PART | I Basic principles

Lee, Y.S., Chuong, C.M., 1992. Adhesion molecules in skeletogenesis: I. Transient expression of neural cell adhesion molecules (NCAM) in osteoblasts during endochondral and intramembranous ossification. J. Bone Miner. Res. 7, 1435e1446. Lee, D.Y., Li, Y.S., Chang, S.F., Zhou, J., Ho, H.M., Chiu, J.J., et al., 2010. Oscillatory flow-induced proliferation of osteoblast-like cells is mediated by alphavbeta3 and beta1 integrins through synergistic interactions of focal adhesion kinase and Shc with phosphatidylinositol 3-kinase and the Akt/ mTOR/p70S6K pathway. J. Biol. Chem. 285, 30e42. Lemonnier, J., Delannoy, P., Hott, M., Lomri, A., Modrowski, D., Marie, P.J., 2000. The Ser252Trp fibroblast growth factor receptor-2 (FGFR-2) mutation induces PKC-independent downregulation of FGFR-2 associated with premature calvaria osteoblast differentiation. Exp. Cell Res. 256, 158e167. Li, Y., Zhong, G., Sun, W., Zhao, C., Zhang, P., Song, J., et al., 2015. CD44 deficiency inhibits unloading-induced cortical bone loss through downregulation of osteoclast activity. Sci. Rep. 5, 16124. Lilien, J., Balsamo, J., 2005. The regulation of cadherin-mediated adhesion by tyrosine phosphorylation/dephosphorylation of beta-catenin. Curr. Opin. Cell Biol. 17, 459e465. Litzenberger, J.B., Kim, J.B., Tummala, P., Jacobs, C.R., 2010. Beta1 integrins mediate mechanosensitive signaling pathways in osteocytes. Calcif. Tissue Int. 86, 325e332. Loeser, R.F., 2002. Integrins and cell signaling in chondrocytes. Biorheology 39, 119e124. Lowe, C., Yoneda, T., Boyce, B.F., Chen, H., Mundy, G.R., Soriano, P., 1993. Osteopetrosis in Src-deficient mice is due to an autonomous defect of osteoclasts. Proc. Natl. Acad. Sci. U. S. A. 90, 4485e4489. Lowe, D.A., Lepori-Bui, N., Fomin, P.V., Sloofman, L.G., Zhou, X., Farach-Carson, M.C., et al., 2014. Deficiency in perlecan/HSPG2 during bone development enhances osteogenesis and decreases quality of adult bone in mice. Calcif. Tissue Int. 95, 29e38. Luxenburg, C., Winograd-Katz, S., Addadi, L., Geiger, B., 2012. Involvement of actin polymerization in podosome dynamics. J. Cell Sci. 125, 1666e1672. Mansouri, R., Haÿ, E., Marie, P.J., Modrowski, D., 2015. Role of syndecan-2 in osteoblast biology and pathology. Bonekey Rep. 4, 666. Mansouri, R., Jouan, Y., Haÿ, E., Blin-Wakkach, C., Frain, M., Ostertag, A., et al., 2017. Osteoblastic heparan sulfate glycosaminoglycans control bone remodeling by regulating Wnt signaling and the crosstalk between bone surface and marrow cells. Cell Death Dis. 8, e2902. Manton, K.J., Leong, D.F., Cool, S.M., Nurcombe, V., 2007. Disruption of heparan and chondroitin sulfate signaling enhances mesenchymal stem cellderived osteogenic differentiation via bone morphogenetic protein signaling pathways. Stem Cell. 25 (11), 2845e2854. Marchisio, P.C., Cirillo, D., Naldini, L., Primavera, M.V., Teti, A., Zambonin-Zallone, A., 1984. Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures. J. Cell Biol. 99, 1696e1705. Marchisio, P.C., Bergui, L., Corbascio, G.C., Cremona, O., D’Urso, N., Schena, M., et al., 1988. Vinculin, talin, and integrins are localized at specific adhesion sites of malignant B lymphocytes. Blood 66, 830e838. Marie, P.J., Haÿ, E., 2013. Cadherins and Wnt signalling: a functional link controlling bone formation. Bonekey Rep. 2, 330. Marie, P.J., Haÿ, E., Saidak, Z., 2014a. Integrin and cadherin signaling in bone : role and potential therapeutic targets. Trends Endocrinol. Metabol. 25, 567e575. Marie, P.J., Haÿ, E., Modrowski, D., Revollo, L., Mbalaviele, G., Civitelli, R., 2014b. Cadherin-mediated cell-cell adhesion and signaling in the skeleton. Calcif. Tissue Int. 94, 46e54. Marie, P.J., 2013. Targeting integrins to promote bone formation and repair. Nat. Rev. Endocrinol. 9, 288e295. Matsuo, I., Kimura-Yoshida, C., 2013. Extracellular modulation of Fibroblast Growth Factor signaling through heparan sulfate proteoglycans in mammalian development. Curr. Opin. Genet. Dev. 23, 399e407. Matsusaki, T., Aoyama, T., Nishijo, K., Okamoto, T., Nakayama, T., Nakamura, T., et al., 2006. Expression of the cadherin-11 gene is a discriminative factor between articular and growth plate chondrocytes. Osteoarthritis Cartilage 14, 353e366. Mbalaviele, G., Chen, H., Boyce, B.F., Mundy, G.R., Yoneda, T., 1995. The role of cadherin in the generation of multinucleated osteoclasts from mononuclear precursors in murine marrow. J. Clin. Investig. 95, 2757e2765. Mbalaviele, G., Nishimura, R., Myoi, A., Niewolna, M., Reddy, S.V., Chen, D., et al., 1998. Cadherin-6 mediates the heterotypic interactions between the hemopoietic osteoclast cell lineage and stromal cells in a murine model of osteoclast differentiation. J. Cell Biol. 141, 1467e1476. McHugh, K.P., Hodivala-Dilke, K., Zheng, M.H., Namba, N., Lam, J., Novack, D., et al., 2000. Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts. J. Clin. Investig. 105, 433e440. Meury, T., Akhouayri, O., Jafarov, T., Mandic, V., St-Arnaud, R., 2010. Nuclear alpha NAC influences bone matrix mineralization and osteoblast maturation in vivo. Mol. Cell Biol. 30, 43e53. Mitsou, I., Multhaupt, H.A.B., Couchman, J.R., 2017. Proteoglycans, ion channels and cell-matrix adhesion. Biochem. J. 474, 1965e1979. Miyauchi, A., Alvarez, J., Greenfield, E.M., Teti, A., Grano, M., Colucci, S., et al., 1991. Recognition of osteopontin and related peptides by an alpha v beta 3 integrin stimulates immediate cell signals in osteoclasts. J. Biol. Chem. 266, 20369e20374. Miyauchi, A., Gotoh, M., Kamioka, H., Notoya, K., Sekiya, H., Takagi, Y., et al., 2006. AlphaVbeta3 integrin ligands enhance volume-sensitive calcium influx in mechanically stretched osteocytes. J. Bone Miner. Metab. 24, 498e504. Mizuno, M., Fujisawa, R., Kuboki, Y., 2000. Type I collagen-induced osteoblastic differentiation of bone-marrow cells mediated by collagen-alpha2beta1 integrin interaction. J. Cell. Physiol. 184, 207e213. Modrowski, D., Baslé, M., Lomri, A., Marie, P.J., 2000. Syndecan-2 is involved in the mitogenic activity and signaling of granulocyte-macrophage colony-stimulating factor in osteoblasts. J. Biol. Chem. 275, 9178e9185.

Integrins and other cell surface attachment molecules of bone cells Chapter | 17

419

Molténi, A., Modrowski, D., Hott, M., Marie, P.J., 1999a. Differential expression of fibroblast growth factor receptor-1, -2, and -3 and syndecan-1, -2, and -4 in neonatal rat mandibular condyle and calvaria during osteogenic differentiation in vitro. Bone 24, 337e347. Molténi, A., Modrowski, D., Hott, M., Marie, P.J., 1999b. Alterations of matrix- and cell-associated proteoglycans inhibit osteogenesis and growth response to fibroblast growth factor-2 in cultured rat mandibular condyle and calvaria. Cell Tissue Res. 295, 523e536. Morgan, E.A., Schneider, J.G., Baroni, T.E., Uluçkan, O., Heller, E., Hurchla, M.A., et al., 2010. Dissection of platelet and myeloid cell defects by conditional targeting of the beta3-integrin subunit. FASEB J. 24, 1117e1127. Moursi, A.M., Globus, R.K., Damsky, C.H., 1997. Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro. J. Cell Sci. 110, 2187e2196. Moussa, F.M., Hisijara, I.A., Sondag, G.R., Scott, E.M., Frara, N., Abdelmagid, S.M., et al., 2014. Osteoactivin promotes osteoblast adhesion through HSPG and avb1 integrin. J. Cell. Biochem. 115, 1243e1253. Nakamura, H., Kenmotsu, S., Sakai, H., Ozawa, H., 1995. Localization of CD44, the hyaluronate receptor, on the plasma membrane of osteocytes and osteoclasts in rat tibiae. Cell Tissue Res. 280, 225e233. Nakamura, I., Jimi, E., Duong, L.T., Sasaki, T., Takahashi, N., Rodan, G.A., et al., 1998. Tyrosine phosphorylation of p130Cas is involved in actin organization in osteoclasts. J. Biol. Chem. 273, 11144e11149. Nakamura, R., Nakamura, F., Fukunaga, S., 2014. Contrasting effect of perlecan on adipogenic and osteogenic differentiation of mesenchymal stem cells in vitro. Anim. Sci. J. 85, 262e270. Nakazora, S., Matsumine, A., Iino, T., Hasegawa, M., Kinoshita, A., Uemura, K., et al., 2010. The cleavage of N-cadherin is essential for chondrocyte differentiation. Biochem. Biophys. Res. Commun. 400, 493e499. Neam, P.J., Sommarin, Y., Boynton, R.E., Heinegård, D., 1994. The structure of a 38-kDa leucine-rich protein (chondroadherin) isolated from bovine cartilage. J. Biol. Chem. 269, 21547e21554. Negishi-Koga, T., Takayanagi, H., 2009. Ca2þ-NFATc1 signaling is an essential axis of osteoclast differentiation. Immunol. Rev. 231, 241e256. Nelson, W.J., Nusse, R., 2004. Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303, 1483e1487. Nicoll, S.B., Barak, O., Csóka, A.B., Bhatnagar, R.S., Stern, R., 2002. Hyaluronidases and CD44 undergo differential modulation during chondrogenesis. Biochem. Biophys. Res. Commun. 292, 819e825. Oberlender, S.A., Tuan, R.S., 1994. Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis. Development 120, 177e187. Okada, Y., Morimoto, I., Ura, K., Watanabe, K., Eto, S., Kumegawa, M., et al., 2002. Cell-to-Cell adhesion via intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 pathway is involved in 1alpha,25(OH)2D3, PTH and IL-1alpha-induced osteoclast differentiation and bone resorption. Endocr. J. 49, 483e495. Onodera, K., Takahashi, I., Sasano, Y., Bae, J.W., Mitani, H., Kagayama, M., et al., 2005. Stepwise mechanical stretching inhibits chondrogenesis through cell-matrix adhesion mediated by integrins in embryonic rat limb-bud mesenchymal cells. Eur. J. Cell Biol. 84, 45e58. Ory, S., Brazier, H., Pawlak, G., Blangy, A., 2008. Rho-GTPases I osteoclasts: orchestrators of podosome arrangement. Eur. J. Cell Biol. 87, 469e477. Phillips, J.A., Almeida, E.A., Hill, E.L., Aguirre, J.I., Rivera, M.F., Nachbandi, I., et al., 2008. Role for beta1 integrins in cortical osteocytes during acute musculoskeletal disuse. Matrix Biol. 27, 609e618. Plotkin, L.I., Mathov, I., Aguirre, J.I., Parfitt, A.M., Manolagas, S.C., Bellido, T., 2005. Mechanical stimulation prevents osteocyte apoptosis: requirement of integrins, Src kinases, and ERKs. Am. J. Physiol. Cell Physiol. 289, C633eC643. Pommerenke, H., Schmidt, C., Dürr, F., Nebe, B., Lüthen, F., Muller, P., et al., 2002. The mode of mechanical integrin stressing controls intracellular signaling in osteoblasts. J. Bone Miner. Res. 17, 603e611. Popov, C., Radic, T., Haasters, F., Prall, W.C., Aszodi, A., Gullberg, D., Schieker, M., et al., 2011. Integrins alpha2beta1 and alpha11beta1 regulate the survival of mesenchymal stem cells on collagen I. Cell Death Dis. 2, e186. Puleo, D.A., Bizios, R., 1991. RGDS tetrapeptide binds to osteoblasts and inhibits fibronectin-mediated adhesion. Bone 12, 271e276. Purev, E., Neff, L., Horne, W.C., Baron, R., 2009. c-Cbl and Cbl-b act redundantly to protect osteoclasts from apoptosis and to displace HDAC6 from beta-tubulin, stabilizing microtubules and podosomes. Mol. Biol. Cell 20, 4021e4030. Rajshankar, D., Wang, Y., McCulloch, C.A., 2017. Osteogenesis requires FAK-dependent collagen synthesis by fibroblasts and osteoblasts. FASEB J. 31, 937e953. Ray, B.J., Thomas, K., Huang, C.S., Gutknecht, M.F., Botchwey, E.A., Bouton, A.H., 2012. Regulation of osteoclast structure and function by FAK family kinases. J. Leukoc. Biol. 92, 1021e1028. Revollo, L., Kading, J., Jeong, S.Y., Li, J., Salazar, V., Mbalaviele, G., et al., 2015. N-cadherin restrains PTH activation of Lrp6/b-catenin signaling and osteoanabolic action. J. Bone Miner. Res. 30, 274e285. Rodgers, K.D., Sasaki, T., Aszodi, A., Jacenko, O., 2007. Reduced perlecan in mice results in chondrodysplasia resembling Schwartz-Jampel syndrome. Hum. Mol. Genet. 16, 515e528. Rucci, N., Teti, A., 2016. The "love-hate" relationship between osteoclasts and bone matrix. Matrix Biol. 52e54, 176e190. Rucci, N., Capulli, M., Olstad, O.K., Önnerfjord, P., Tillgren, V., Gautvik, K.M., Heinegård, D., Teti, A., 2015. The a2b1 binding domain of chondroadherin inhibits breast cancer-induced bone metastases and impairs primary tumour growth: a preclinical study. Cancer Lett. 358, 67e75. Rundus, V.R., Marshall, G.B., Parker, S.B., Bales, E.S., Hertzberg, E.L., Minkoff, R., 1998. Association of cell and substrate adhesion molecules with connexin43 during intramembranous bone formation. Histochem. J. 30, 879e896. Saidak, Z., Le Henaff, C., Azzi, S., Marty, C., Da Nascimento, S., Sonnet, P., et al., 2015. Wnt/b-catenin signaling mediates osteoblast differentiation triggered by peptide-induced a5b1 integrin priming in mesenchymal skeletal cells. J. Biol. Chem. 290, 6903e6912.

420 PART | I Basic principles

Salasznyk, R.M., Klees, R.F., Boskey, A., Plopper, G.E., 2007. Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin-5. J. Cell. Biochem. 100, 499e514. Saltel, F., Chabadel, A., Bonnelye, E., Jurdic, P., 2008. Actin cytoskeletal organisation in osteoclasts: a model to decipher transmigration and matrix degradation. Eur. J. Cell Biol. 87, 459e468. Schaffner, P., Dard, M.M., 2003. Structure and function of RGD peptides involved in bone biology. Cell. Mol. Life Sci. 60, 119e132. Schneider, J.G., Amend, S.R., Weilbaecher, K.N., 2011. Integrins and bone metastasis: integrating tumor cell and stromal cell interactions. Bone 48, 54e65. Sens, C., Huck, K., Pettera, S., Uebel, S., Wabnitz, G., Moser, M., et al., 2017. Fibronectins containing extradomain A or B enhance osteoblast differentiation via distinct integrins. J. Biol. Chem. 292, 7745e7760. Seong, J., Tajik, A., Sun, J., Guan, J.L., Humphries, M.J., Craig, S.E., et al., 2013. Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins. Proc. Natl. Acad. Sci. U. S. A. 110, 19372e19377. Shakibaei, M., 1998. Inhibition of chondrogenesis by integrin antibody in vitro. Exp. Cell Res. 240, 95e106. Shankar, G., Davison, I., Helfrich, M.H., Mason, W.T., Horton, M.A., 1993. Integrin receptor-mediated mobilisation of intranuclear calcium in rat osteoclasts. J. Cell Sci. 105, 61e68. Shekaran, A., Shoemaker, J.T., Kavanaugh, T.E., Lin, A.S., LaPlaca, M.C., Fan, Y., et al., 2014. The effect of conditional inactivation of beta 1 integrins using twist 2 Cre, Osterix Cre and osteocalcin Cre lines on skeletal phenotype. Bone 68, 131e141. Sheldrake, H.M., Patterson, L.H., 2014. Strategies to inhibit tumor associated integrin receptors: rationale for dual and multiantagonists. J. Med. Chem. 57, 6301e6315. Shih, Y.R., Tseng, K.F., Lai, H.Y., Lin, C.H., Lee, O.K., 2011. Matrix stiffness regulation of integrin-mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells. J. Bone Miner. Res. 26, 730e738. Shin, C.S., Lecanda, F., Sheikh, S., Weitzmann, L., Cheng, S.L., Civitelli, R., 2000. Relative abundance of different cadherins defines differentiation of mesenchymal precursors into osteogenic, myogenic, or adipogenic pathways. J. Cell. Biochem. 78, 566e577. Smith, S.M., West, L.A., Govindraj, P., Zhang, X., Ornitz, D.M., Hassell, J.R., 2007. Heparan and chondroitin sulfate on growth plate perlecan mediate binding and delivery of FGF-2 to FGF receptors. Matrix Biol. 26, 175e184. Sondag, G.R., Mbimba, T.S., Moussa, F.M., Novak, K., Yu, B., Jaber, F.A., et al., 2016. Osteoactivin inhibition of osteoclastogenesis is mediated through CD44-ERK signaling. Exp. Mol. Med. 48, e257. Song, S.J., Cool, S.M., Nurcombe, V., 2007. Regulated expression of syndecan-4 in rat calvaria osteoblasts induced by fibroblast growth factor-2. J. Cell. Biochem. 100, 402e411. Soysa, N.S., Alles, N., 2016. Osteoclast function and bone-resorbing activity: an overview. Biochem. Biophys. Res. Commun. 476, 115e120. Spessotto, P., Rossi, F.M., Degan, M., Di Francia, R., Perris, R., Colombatti, A., et al., 2002. Hyaluronan-CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9. J. Cell Biol. 158, 1133e1144. Spinardi, L., Marchisio, P.C., 2006. Podosomes as smart regulators of cellular adhesion. Eur. J. Cell Biol. 85, 191e194. Srouji, S., Ben-David, D., Fromigué, O., Vaudin, P., Kuhn, G., Müller, R., et al., 2012. Lentiviral-mediated integrin a5 expression in human adult mesenchymal stromal cells promotes bone repair in mouse cranial and long-bone defects. Hum. Gene Ther. 23, 167e172. Stange, R., Kronenberg, D., Timmen, M., Everding, J., Hidding, H., Eckes, B., et al., 2013. Age-related bone deterioration is diminished by disrupted collagen sensing in integrin a2b1 deficient mice. Bone 56, 48e54. Steinfeld, R., Van Den Berghe, H., David, G., 1996. Stimulation of fibroblast growth factor receptor-1 occupancy and signaling by cell surface-associated syndecans and glypican. J. Cell Biol. 133, 405e416. Stokes, D.G., Liu, G., Coimbra, I.B., Piera-Velazquez, S., Crowl, R.M., Jiménez, S.A., 2002. Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis. Arthritis Rheum. 46, 404e419. Su, J.L., Chiou, J., Tang, C.H., Zhao, M., Tsai, C.H., Chen, P.S., et al., 2010. CYR61 regulates BMP-2-dependent osteoblast differentiation through the {alpha}v{beta}3 integrin/integrin-linked kinase/ERK pathway. J. Biol. Chem. 285, 31325e31336. Sun, C., Yuan, H., Wang, L., Wei, X., Williams, L., Krebsbach, P.H., et al., 2016. FAK promotes osteoblast progenitor cell proliferation and differentiation by enhancing Wnt signaling. J. Bone Miner. Res. 31, 2227e2238. SundarRaj, N., Fite, D., Ledbetter, S., Chakravarti, S., Hassell, J.R., 1995. Perlecan is a component of cartilage matrix and promotes chondrocyte attachment. J. Cell Sci. 108, 2663e2672. Suzuki, K., Zhu, B., Rittling, S.R., Denhardt, D.T., Goldberg, H.A., McCulloch, C.A., et al., 2002. Colocalization of intracellular osteopontin with CD44 is associated with migration, cell fusion, and resorption in osteoclasts. J. Bone Miner. Res. 17, 1486e1497. Takahashi, I., Onodera, K., Sasano, Y., Mizoguchi, I., Bae, J.W., Mitani, H., et al., 2003. Effect of stretching on gene expression of beta1 integrin and focal adhesion kinase and on chondrogenesis through cell-extracellular matrix interactions. Eur. J. Cell Biol. 82, 182e192. Takeuchi, Y., Suzawa, M., Kikuchi, T., Nishida, E., Fujita, T., Matsumoto, T., 1997. Differentiation and transforming growth factor-beta receptor downregulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells. J. Biol. Chem. 14 (272), 29309e29316. Tamura, Y., Takeuchi, Y., Suzawa, M., Fukumoto, S., Kato, M., Miyazono, K., et al., 2001. Focal adhesion kinase activity is required for bone morphogenetic protein-Smad1 signaling and osteoblastic differentiation in murine MC3T3-E1 cells. J. Bone Miner. Res. 16, 1772e1779. Tanabe, N., Wheal, B.D., Kwon, J., Chen, H.H., Shugg, R.P., Sims, S.M., et al., 2011. Osteopontin signals through calcium and nuclear factor of activated T cells (NFAT) in osteoclasts: a novel RGD-dependent pathway promoting cell survival. J. Biol. Chem. 286, 39871e39881.

Integrins and other cell surface attachment molecules of bone cells Chapter | 17

421

Tanaka, Y., Morimoto, I., Nakano, Y., Okada, Y., Hirota, S., Nomura, S., et al., 1995. Osteoblasts are regulated by the cellular adhesion through ICAM-1 and VCAM-1. J. Bone Miner. Res. 10, 1462e1469. Tanaka, S., Amling, M., Neff, L., Peyman, A., Uhlmann, E., Levy, J.B., et al., 1996. c-Cbl is downstream of c-Src in a signalling pathway necessary for bone resorption. Nature 383, 528e531. Tanaka, Y., Maruo, A., Fujii, K., Nomi, M., Nakamura, T., Eto, S., et al., 2000. Intercellular adhesion molecule 1 discriminates functionally different populations of human osteoblasts: characteristic involvement of cell cycle regulators. J. Bone Miner. Res. 15, 1912e1923. Tanikawa, R., Tanikawa, T., Hirashima, M., Yamauchi, A., Tanaka, Y., 2010. Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway. Biochem. Biophys. Res. Commun. 394, 317e322. Tavella, S., Raffo, P., Tacchetti, C., Cancedda, R., Castagnola, P., 1994. N-CAM and N-cadherin expression during in vitro chondrogenesis. Exp. Cell Res. 215, 354e362. Teitelbaum, S.L., 2011. The osteoclast and its unique cytoskeleton. Ann NY Acad Sci 1240, 14e17. Teplyuk, N.M., Haupt, L.M., Ling, L., Dombrowski, C., Mun, F.K., Nathan, S.S., et al., 2009. The osteogenic transcription factor Runx2 regulates components of the fibroblast growth factor/proteoglycan signaling axis in osteoblasts. J. Cell. Biochem. 107, 144e154. Terpstra, L., Prud’homme, J., Arabian, A., Takeda, S., Karsenty, G., Dedhar, S., et al., 2003. Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes. J. Cell Biol. 162, 139e148. Thi, M.M., Suadicani, S.O., Schaffler, M.B., Weinbaum, S., Spray, D.C., 2013. Mechanosensory responses of osteocytes to physiological forces occur along processes and not cell body and require aVb3 integrin. Proc. Natl. Acad. Sci. U. S. A. 110, 21012e21017. Thompson, W.R., Modla, S., Grindel, B.J., Czymmek, K.J., Kirn-Safran, C.B., Wang, L., et al., 2011. Perlecan/Hspg2 deficiency alters the pericellular space of the lacunocanalicular system surrounding osteocytic processes in cortical bone. J. Bone Miner. Res. 26, 618e629. Tian, J., Zhang, F.J., Lei, G.H., 2015. Role of integrins and their ligands in osteoarthritic cartilage. Rheumatol. Int. 35, 787e798. Tokuo, H., Ikebe, M., 2004. Myosin X transports Mena/VASP to the tip of filopodia. Biochem. Biophys. Res. Commun. 319, 214e220. Townsend, P.A., Villanova, I., Teti, A., Horton, M.A., 1999. Beta1 integrin antisense oligodeoxy-nucleotides: utility in controlling osteoclast function. Eur. J. Cell Biol. 78, 485e496. Triplett, J.W., Pavalko, F.M., 2006. Disruption of alpha-actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis. Am. J. Physiol. Cell Physiol. 291, C909eC921. Tu, S., Wu, S., Shi, X., Chen, K., Wu, C., 2003. Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell 113, 37e47. Tufan, A.C., Daumer, K.M., Tuan, R.S., 2002. Frizzled-7 and limb mesenchymal chondrogenesis: effect of misexpression and involvement of N-cadherin. Dev. Dynam. 223, 241e253. Tuli, R., Tuli, S., Nandi, S., Huang, X., Manner, P.A., Hozack, W.J., et al., 2003. Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk. J. Biol. Chem. 278, 41227e41236. Turner, C.H., Warden, S.J., Bellido, T., Plotkin, L.I., Kumar, N., Jasiuk, I., et al., 2009. Mechanobiology of the skeleton. Sci. Signal. 2, pt.3. Uda, Y., Azab, E., Sun, N., Shi, C., Pajevic, P.D., 2017. Osteocyte mechanobiology. Curr. Osteoporos. Rep. 15, 318e332. Van den Bossche, J., Malissen, B., Mantovani, A., De Baetselier, P., Van Ginderachter, J.A., 2012. Regulation and function of the E-cadherin/catenin complex in cells of the monocyte-macrophage lineage and DCs. Blood 119, 1623e1633. Viviano, B.L., Silverstein, L., Pflederer, C., Paine-Saunders, S., Mills, K., Saunders, S., 2005. Altered hematopoiesis in glypican-3-deficient mice results in decreased osteoclast differentiation and a delay in endochondral ossification. Dev. Biol. 282, 152e162. Wang, W., Kirsch, T., 2006. Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes. J. Biol. Chem. 281, 30848e30856. Wang, Y., McNamara, L.M., Schaffler, M.B., Weinbaum, S., 2007. A model for the role of integrins in flow induced mechanotransduction in osteocytes. Proc. Natl. Acad. Sci. U. S. A. 104, 15941e15946. Wang, Z., Collighan, R.J., Gross, S.R., Danen, E.H., Orend, G., Telci, D., et al., 2010. RGD-independent cell adhesion via a tissue transglutaminasefibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 alpha5beta1 integrin co-signaling. J. Biol. Chem. 285, 40212e40229. Wang, Z., Telci, D., Griffin, M., 2011. Importance of syndecan-4 and syndecan -2 in osteoblast cell adhesion and survival mediated by a tissue transglutaminase-fibronectin complex. Exp. Cell Res. 317, 367e381. Wang, B., Lai, X., Price, C., Thompson, W.R., Li, W., Quabili, T.R., et al., 2014. Perlecan-containing pericellular matrix regulates solute transport and mechanosensing within the osteocyte lacunar-canalicular system. J. Bone Miner. Res. 29, 878e891. Watabe, H., Furuhama, T., Tani-Ishii, N., Mikuni-Takagaki, Y., 2011. Mechanotransduction activates a₅b₁ integrin and PI3K/Akt signaling pathways in mandibular osteoblasts. Exp. Cell Res. 317, 2642e2649. Whitelock, J.M., Melrose, J., Iozzo, R.V., 2008. Diverse cell signaling events modulated by perlecan. Biochemistry 47, 11174e11183. Woods, A., Wang, G., Beier, F., 2007a. Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions. J. Cell. Physiol. 213, 1e8. Woods, A., Wang, G., Dupuis, H., Shao, Z., Beier, F., 2007b. Rac1 signaling stimulates N-cadherin expression, mesenchymal condensation, and chondrogenesis. J. Biol. Chem. 282, 23500e23508. Woodward, W.A., Tuan, R.S., 1999. N-Cadherin expression and signaling in limb mesenchymal chondrogenesis: stimulation by poly-L-lysine. Dev. Genet. 24, 178e187.

422 PART | I Basic principles

Worapamorn, W., Tam, S.P., Li, H., Haase, H.R., Bartold, P.M., 2002. Cytokine regulation of syndecan-1 and -2 gene expression in human periodontal fibroblasts and osteoblasts. J. Periodontal. Res. 37, 273e278. Wu, D., Schaffler, M.B., Weinbaum, S., Spray, D.C., 2013. Matrix-dependent adhesion mediates network responses to physiological stimulation of the osteocyte cell process. Proc. Natl. Acad. Sci. U. S. A. 110, 12096e12101. Xiao, G., Wang, D., Benson, M.D., Karsenty, G., Franceschi, R.T., 1998. Role of the alpha2-integrin in osteoblast-specific gene expression and activation of the Osf2 transcription factor. J. Biol. Chem. 273, 32988e32994. Xiao, G., Cheng, H., Cao, H., Chen, K., Tu, Y., Yu, S., et al., 2012. Critical role of filamin-binding LIM protein 1 (FBLP-1)/migfilin in regulation of bone remodeling. J. Biol. Chem. 287, 21450e21460. Yan, Y.X., Gong, Y.W., Guo, Y., Lv, Q., Guo, C., Zhuang, Y., et al., 2012. Mechanical strain regulates osteoblast proliferation through integrin-mediated ERK activation. PLoS One 7, e35709. Yang, H., Dong, J., Xiong, W., Fang, Z., Guan, H., Li, F., 2016. N-cadherin restrains PTH repressive effects on sclerostin/SOST by regulating LRP6PTH1R interaction. Ann. N. Y. Acad. Sci. 1385, 41e52. Yao, W., Guan, M., Jia, J., Dai, W., Lay, Y.A., Amugongo, S., et al., 2013. Reversing bone loss by directing mesenchymal stem cells to bone. Stem Cell. 31, 2003e2014. Yaroslavskiy, B.B., Zhang, Y., Kalla, S.E., García Palacios, V., Sharrow, A.C., Li, Y., et al., 2005. NO-dependent osteoclast motility: reliance on cGMPdependent protein kinase I and VASP. J. Cell Sci. 118, 5479e5487. Zemmyo, M., Meharra, E.J., Kühn, K., Creighton-Achermann, L., Lotz, M., 2003. Accelerated, aging-dependent development of osteoarthritis in alpha1 integrin-deficient mice. Arthritis Rheum. 48, 2873e2880. Zimmerman, D., Jin, F., Leboy, P., Hardy, S., Damsky, C., 2000. Impaired bone formation in transgenic mice resulting from altered integrin function in osteoblasts. Dev. Biol. 220, 2e15. Zou, W., Teitelbaum, S.L., 2010. Integrins, growth factors, and the osteoclast cytoskeleton. Ann. N. Y. Acad. Sci. 1192, 27e31. Zou, W., Teitelbaum, S.L., 2015. Absence of Dap12 and the avb3 integrin causes severe osteopetrosis. J. Cell Biol. 208, 125e136.