Role of N-cadherin in bone formation

Authors


Abstract

Cell–cell adhesion mediated by cadherins is essential for the function of bone forming cells during osteogenesis. Here, the evidence that N-cadherin is an important regulator of osteoblast differentiation and osteogenesis is reviewed. Osteoblasts express a limited number of cadherins, including the classic N-cadherin. The expression profile of N-cadherin in osteoblasts during bone formation in vivo and in vitro suggests a role of this molecule in osteogenesis. Functional studies using neutralizing antibodies or antisense oligonucleotides indicate that N-cadherin is involved in the control the expression of osteoblast marker gene expression and differentiation. Cleavage of N-cadherin during osteoblast apoptosis also suggests a role of N-cadherin-mediated-cell–cell adhesion in osteoblast survival. Hormonal and local factors that regulate osteoblast function also regulate N-cadherin expression and subsequent cell–cell adhesion associated with osteoblast differentiation or survival. Signaling mechanisms involved in N-cadherin-mediated cell–cell adhesion and osteoblast gene expression have also been identified. Alterations of N-cadherin expression are associated with abnormal osteoblast differentiation and osteogenesis in pathological conditions. These findings indicate that N-cadherin plays a role in normal and pathological bone formation and provide some insight into the process involved in N-cadherin-mediated cell–cell adhesion and differentiation in osteoblasts. J. Cell. Physiol. 190: 297–305, 2002. © 2002 Wiley-Liss, Inc.

The skeleton is a unique tissue providing support and mineral balance for the organism. It is formed during growth and is maintained during the adult life by continual renewal of the matrix. This renewal is ensured by osteoclasts which resorb the calcified matrix and osteoblasts that are responsible for bone formation and new bone matrix synthesis. Multiple cell–cell and cell–matrix interactions occur during the process of bone formation. Therefore, the determination of cellular and molecular mechanisms involved in these interactions, notably cell–cell interactions, is fundamental in understanding the biology and pathology of bone formation.

A limited number of cell–cell adhesion molecules are expressed by skeletal cells, and some were found to be involved in osteogenesis. This review summarizes the importance of cadherin-mediated-cell–cell interactions in osteoblast biology and the evidence obtained in my and other laboratories that the cell–cell adhesion molecule N-cadherin plays an essential role in normal and pathological bone formation.

OSTEOBLAST BIOLOGY

Bone formation is a complex process involving the commitment of osteoprogenitor cells and their progressive differentiation into mature functional osteoblasts. Osteoblasts originate from undifferentiated mesenchymal stem cells that give rise to cartilage, bone or adipocytes under induction by systemic or local factors (Triffitt, 1996). Stem cells located in the bone marrow stroma can differentiate into osteoblasts, chondroblasts or adipocytes under the control of transcription factors. Marrow stromal cells are committed to the osteoblast lineage by induction of the transcription factor Cbfa1/Runx2 (Ducy and Karsenty, 1998) and possibly other factors (Marie, 2001). When proliferating osteoprogenitors become pre-osteoblasts, cell growth declines and there is a progressive expression of markers of differentiation by post-mitotic osteoblasts (Stein et al., 1990; Aubin and Liu, 1996; Marie, 1999). The sequence of osteogenic differentiation is characterized by the expression of alkaline phosphatase (ALP), an early marker of osteoblast phenotype, associated with the synthesis and deposition of type I collagen and non collagenous bone matrix proteins and increased expression of osteocalcin and bone sialoprotein at the onset of mineralization. Mature osteoblasts that are forming the bone matrix are cuboidal cells lining the bone surface that develop extensive cell–cell contacts. Once the bone matrix synthesis has been deposited, most osteoblasts become flattened lining cells. However, a fraction of cells loose cell–cell junctions and become embedded within the matrix to become osteocytes. Remaining osteoblasts loose both adherence to the matrix and cell–cell junctions and undergo apoptosis, a process believed to play a role in the control of bone formation (Jilka et al., 1998). The control of osteogenesis depends in part on cell–cell and cell–matrix interactions as well as external regulators. Among the various hormones known to regulate osteoblasts, estrogens, parathyroid hormone (PTH), glucocorticoids, and vitamin D are likely to be the more important. Moreover, a large number of local factors also influence osteoblast function. Among them, most important are the Bone Morphogenetic Proteins (BMPs), Transforming Growth factor-β (TGF-β), Insulin like growth factors (IGFs), and Fibroblast Growth Factors (FGFs) which regulate cell proliferation and differentiation (Canalis et al., 1993) through specific signaling pathways (Quarles and Siddhanti, 1996). As discussed below, these agents may modulate osteoblast function by modulating N-cadherin-mediated cell–cell adhesion in osteoblasts.

CELL–CELL ADHESION MEDIATED BY CADHERINS

Cell–cell adhesion is controlled by cell adhesion molecules (CAM) (Takeichi, 1991; Aplin et al., 1998; Potter et al., 1999). Cadherins belong to a CAM superfamily expressed in various tissues. Several cadherins have been identified and cloned. Based on their structural features, cadherins have been classified as classic cadherins type I and II, desmosomal cadherins, protocadherins, and other related cadherins (Potter et al., 1999). Classic type I cadherins include neural (N-), epithelial (E-), placental (P-), and R-cadherin (cadherin-4). These cadherins are single chain transmembrane glycoproteins that mediate homophilic, calcium-dependent cell–cell adhesion (Takeichi, 1991). Classic type I cadherins exhibit a conserved His-Ala-Val (HAV) motif in the first extracellular domain mediating homophilic interactions between cadherins (Takeichi, 1995). Type II cadherins (cadherin-5 to -12) lack the cell adhesion sequence motif HAV found in the N-terminal part of type I cadherins (Potter et al., 1999).

Cadherins are crucial for cell–cell interactions as their spatiotemporal expression is essential in controlling early stages of morphogenesis during development (Takeichi, 1991; Grunwald, 1993; Larue et al., 1996). Cadherins also play essential roles in the regulation of several physiological processes such as cell migration, proliferation, differentiation, and apoptosis (Gumbiner, 1996; Huber et al., 1996). The functional characteristics and structure-function of cadherins have been extensively characterized (Potter et al., 1999). Cadherins bind to intracellular proteins named catenins and the complex cadherins-catenins modulate a variety of processes including cell–cell adhesion as well as signal transduction and gene transcription. This process is controlled at different levels. Indeed, phosphorylation of cadherins and catenins is known to regulate cadherin-dependent cell adhesion. Furthermore, the expression of cadherins is regulated by various hormones and growth factors that act by activating signal transduction mechanisms as diverse as protein kinase C (PKC), protein kinase A (PKA), src, and others (Grunwald, 1993; Potter et al., 1999; Yagi and Takeichi, 2000). Thus, in many cell types, cadherins are key proteins involved in the control of cell function and regulation, and are controlled by various transduction pathways elicited by hormonal and growth factors.

CELL–CELL ADHESION, N-CADHERIN, AND CHONDROGENESIS

Because cell–cell adhesion is an important process that triggers cell differentiation and deposition of extracellular matrix in mesenchymal tissue (Gumbiner, 1996; Hall and Miyake, 2000) cadherins are expected to play important roles in early steps of skeletal formation. During embryonic skeletal development, N-cadherin is expressed at early stages of skeletal formation and its expression increases markedly during cell condensation during limb bud development (Oberlender and Tuan, 1994a,b; Tsonis et al., 1994). During cartilage development, the early condensation events mediated by cell–cell adhesion involve N-cadherin and N-CAM (Widelitz et al., 1993; De Lise et al., 2000). N-cadherin expression increases during chondrogenic differentiation in vitro and decreases progressively during late stages of chondrogenesis (Oberlender and Tuan, 1994a,b, Tavella et al., 1994; Tsonis et al., 1994; Yoon et al., 2000). Thus, N-cadherin expression appears to be controlled in a time- and space-dependent manner during mesenchymal cell condensation and chondrogenesis. The importance of N-cadherin in cartilage formation is further shown by the finding that perturbation of N-cadherin mediated cell–cell aggregation inhibits both mesenchymal condensation and the process of chondrogenesis (Oberlender and Tuan, 1994a). In addition to N-cadherin, cadherin-11 is also present during mesenchyme condensation and chondrogenesis (Simonneau et al., 1995), suggesting these two molecules may play a role in early stages of skeletal differentiation. Recent data suggest that N-cadherin is a direct target of SOX9, a transcription factor that is essential for chondrocyte differentiation and cartilage formation in rat chondrocytes (Panda et al., 2001), suggesting a molecular pathway by which N-cadherin is controlled during chondrogenesis.

CELL–CELL ADHESION, N-CADHERIN, AND OSTEOGENESIS

Cell–cell adhesion mediated by cadherins plays also an important role in osteogenesis. During the early stages of membranous osteogenesis, mesenchymal cell condensation is required for the condensation of the mesenchyme which precedes ossification (Hall and Miyake, 2000). At these stages as well as during bone remodeling, bone formation involves the development of multiple cell–cell contacts and cell–matrix interactions (Bennett et al., 2001). These interactions are mediated by proteins such as cadherins which ensure cell–cell adhesion, connexins which are involved in cell–cell communications through gap junctions, and integrins involved in cell-matrix adhesion (Fig. 1). These complex cell–cell and cell–matrix interactions are required for osteoblast adhesion, communication, and gene expression during early stages of cell differentiation (Civitelli, 1995; Globus et al., 1995; Franceschi, 1999).

Figure 1.

The osteoblast differentiation process is associated with the progressive development of multiple interactions including cell–cell adherence, cell–cell communications, and cell–matrix attachment mediated by cadherins, gap-junctions and integrins, respectively.

In vivo, several types of cell adhesion molecules were found to be expressed by osteoblasts. Chicken and human calvaria osteoblasts express the classical members of the cadherin family, N-cadherin, E-cadherin, and N-CAM (Lee and Chuong, 1992; Lemonnier et al., 2000). In the fetal human cranial suture, mesenchymal cells, preosteoblasts and osteoblasts, express N-CAM whereas pre-osteoblasts and osteoblasts express mainly E-cadherin and N-cadherin (Fig. 2). N-cadherin is also expressed transiently in osteoblasts during postnatal long bone formation (Lee and Chuong, 1992). Moreover, N-cadherin expression persists in periosteal cells in adult rat tibia, as visualized by in situ hybridization (Ferrari et al., 2000). Thus, N-cadherin is expressed at all stages of bone formation, although at various level of expression.

Figure 2.

Immunohistochemistry of part of a fetal human cranial suture showing strong expression of N-cadherin (b), E-cadherin (f), and N-CAM in pre-osteoblasts (ob) and mature osteoblasts (ob) during osteogenesis. (b: bone; m: mesenchymal cells; a,e,f show negative immunochistochemical controls. (Original magnification: 125 ×).

In vitro, the expression of CAMs and cadherins in skeletal cells varies with the cell type or the cell origin (Table 1). Osteoclasts were found to express E-cadherin, but not P- and N-cadherin. E-cadherin appears to be required for the fusion of mononuclear osteoclast precursors since synthetic peptides containing the cell adhesion recognition sequence of cadherins decreased osteoclast formation, which suggests that E-cadherin is involved in the generation of multinucleated osteoclasts (Mbalaviele et al., 1995). Cadherin-6, a type II cadherin, is expressed by mouse stromal cells and is required for osteoclast formation in a mouse coculture model of osteoclastogenesis, suggesting a critical role of cadherin-6 isoforms in the heterotopic interactions between cells of the osteoclast lineage and cells of the osteoblast lineage which are required for osteoclast differentiation (Mbalaviele et al., 1998). These interactions may contribute to the generation of multinucleated osteoclasts and thereby to the physiological control of bone remodeling.

Table 1. Regulation of N-cadherin expression and signaling pathways involved in osteoblasts
AgentRegulationSignaling pathway
  1. −, inhibition; +, activation; U, unknown.

PTH+PKA
Dexamethasone−/+U
1,25(OH)2DU
TNFαU
IL1U
BMP-2+PKC
Phorbol ester+PKC
TGF-2+PKC, src

Osteoblastic cells express several cadherins in vitro. Cadherin-4, a type I cadherin, has been identified in human trabecular bone osteoblasts and bone marrow stromal cells (Cheng et al., 1998). N-cadherin was found to be expressed by mouse and human calvaria osteoblasts (Tsutsumimoto et al., 1999; Lemonnier et al., 2000; Luegmayr et al., 2000; Wennberg et al., 2000) and human trabecular bone cells (Cheng et al., 1998). E-cadherin was found expressed in human bone marrow stromal cells (Turel and Rao, 1998), murine calvaria cells, rat osteosarcoma cells (Babich and Foti, 1994; Tsutsumimoto et al., 1999), and human calvaria cells (Haÿ et al., 2000; Lemonnier et al., 2000). In these cells, the expression of N- and E-cadherins is localized in cell membranes, consistent with a role of these membrane molecules in cell–cell adhesion. Cadherin-11/OB has been described in murine osteoblastic cell lines, osteoblast precursor cell lines and calvaria osteoblasts (Okazaki et al., 1994, Tsutsumimoto et al., 1999), and in all human cell lines tested (Cheng et al., 1998). Rabbit bone marrow cells and bone cells also express cadherin-11 mRNA (Goomer et al., 1998). Interestingly, a splice variant form of human cadherin-11 is expressed in bone, where it could enhance the calcium-dependent adhesion of the intact form of cadherin-11 during osteoblast differentiation (Kawaguchi et al., 2001). Although a variety of cadherins are expressed in osteoblasts depending on the cell type, species, origin, and stage of differentiation; cadherin-11 and N-cadherin appear to be the more abundant cadherins expressed in osteoblastic cells in vitro.

ROLE OF N-CADHERIN IN OSTEOBLAST DIFFERENTIATION

Several lines of evidence indicate that N-cadherin influences osteoblast differentiation at different stages of the osteoblast lineage. A recent analysis indicate that the expression of N-cadherin in mesenchymal cells varies with cell differentiation towards the osteogenic, myogenic, or adipogenic pathway. In these cells, N-cadherin mRNA levels increase during osteogenic and myogenic differentiation, and decrease during adipogenic differentiation (Shin et al., 2000). This is however not specific for N-cadherin, since cadherin-11 follows the same pattern (Shin et al., 2000; Kawaguchi et al., 2001a). It is likely that the two main homophilic cell–cell adhesion molecules N-cadherin and cadherin-11 which are expressed in osteoblasts may have complementary roles during osteoblast differentiation (Kawaguchi et al., 2001). However, experimental studies do not allow to conclude on the respective role of N-cadherin and cadherin-11 during the early and late stages of osteoblast differentiation. Therefore, the contribution of N-cadherin and cadherin-11 in osteogenesis in vivo has yet to be determined. Unfortunately, the N-cadherin null mutation is lethal (Radice et al., 1997), which does not allow to study the role of this molecule at different stages of osteogenesis in vivo. In contrast, recent data indicate that cadherin-11 null mutant mouse show reduced bone density in calvaria and long bone metaphysis, suggesting that cadherin-11 regulates osteoblast differentiation, although the precise mechanisms remain unknown (Kawaguchi et al., 2001b).

Several in vitro experiments provided evidence indicating that N-cadherin plays a role in osteoblast differentiation. This is first illustrated by the in vitro model of osteogenesis in which osteoblastic cells derived from periodontal ligament form multilayers in culture, allowing the formation of bone-like nodules. In these conditions, N-cadherin, but not E-cadherin or P-cadherin, is expressed. Furthermore, N-cadherin mRNA levels increase at the stages of nodule formation and mineralization, suggesting a role of this molecule in osteoblast differentiation and bone formation in vitro (Lin et al., 1999). Secondly, N-cadherin mRNA expression increases concomitantly with osteoblast differentiation in vitro, as evaluated by changes in specific gene expression (ALP, osteocalcin) (Ferrari et al., 2000). The functional role of N-cadherin in osteoblast differentiation and osteogenesis was also established using different methods such as HAV peptide perturbation (Cheng et al., 1998; Ferrari et al., 2000), neutralizing antibodies (Oberlender and Tuan, 1994b; Haÿ et al., 2000), antisense oligonucleotides (Haÿ et al., 2000), and transfection with dominant negative N-cadherin (Cheng et al., 1998; Ferrari et al., 2000). For example, treatment of trabecular bone cells with a synthetic peptide containing the HAV motif from the extracellular domain of type I cadherins inhibits the establishment of cell–cell contacts and down-regulates ALP expression in osteoblast cultures (Ferrari et al., 2000). This suggests that N-cadherin-mediated cell–cell interactions are important for the function of osteoblasts. Furthermore, a truncated N-cadherin mutant with dominant negative action was found to reduce cell–cell adhesion as well as the expression of bone matrix proteins (bone sialoprotein, osteocalcin, type I collagen), ALP and matrix mineralization in cultured osteoblasts (Cheng et al., 2000; Ferrari et al., 2000). In human calvaria osteoblasts, we demonstrated that inhibition of cell–cell adhesion mediated by N-cadherin using specific neutralizing antibodies results in reduced expression of ALP, osteocalcin and type I collagen expression induced by BMP-2, showing that N-cadherin-mediated cell–cell adhesion is essential for the expression of osteoblast gene expression (Haÿ et al., 2000). Overall, these studies indicate a role of N-cadherin in osteoblast differentiation and function.

The mechanisms by which N-cadherin controls osteoblast marker genes remain, however, unclear. N-cadherin-mediated cell–cell contacts may subsequently induce immediate and early intracellular signaling events leading to gene expression (Barth et al., 1997; Knudsen et al., 1998). N-cadherin itself may also participate in some signal transduction events involved in osteoblast differentiation (Aplin et al., 1998; Getsios et al., 1998; Knudsen et al., 1998) although N-cadherin phosphorylation appears unlikely (Daniel and Reynolds, 1997). It is also possible that increased cell–cell adhesion mediated by N-cadherin may lead to increased cell–cell communication and to coordinated gene expression via gap junctions in osteoblasts because N-cadherin was found to be associated with connexin43-mediated gap junctional communications and possibly osteogenesis (Rundus et al., 1998). Finally, cross-talks between N-cadherin and integrins (Aplin et al., 1998; Monier-Gavelle and Duband, 1997) or other cadherins may be involved in signal transduction and osteoblast differentiation gene expression and this merits further analysis. Although the expression and regulation of N-cadherin associated with osteoblast differentiation and function strongly suggests a role of this molecule in bone formation, it is likely that the induction of osteoblast differentiation and osteogenesis does not depend entirely on N-cadherin function and that other cell–cell adhesion molecules may be involved in osteoblast differentiation.

In addition to play a role in osteoblast differentiation, N-cadherin may also be involved in osteoblast behavior. At the end of the formation period, some osteoblasts embed themselves in the matrix and become osteocytes. In this condition, the loss of cell–cell contacts is associated with decreased cadherin expression in vitro (Kawaguchi et al., 2001a) and in vivo (Ferrari et al., 2000). Therefore, a decrease in N-cadherin expression in osteoblasts leading to loss of cell–cell contacts may contribute to the change from the osteoblast to the osteocyte phenotype. Loss of cell–cell contact also occurs during osteoblast apoptosis. Recent evidence suggests that N-cadherin-mediated cell–cell adhesion regulate the apoptotic process in osteoblasts (Hunter et al., 2001). Indeed, in osteoblast undergoing apoptosis, proteolytic cleavage of N-cadherin is associated with activation of the effector caspase-3, a crucial step in the apoptotic process. The decreased expression of N-cadherin in apoptotic osteoblasts result in part from cleavage of the cytoplasmic domain by caspase-3, and extracellular domain of N-cadherin possibly by metalloproteases. Moreover, disruption of N-cadherin-mediated cell–cell adhesion using blocking antibody activates caspase-3 and induce apoptosis in human osteoblasts, indicating that N-cadherin-mediated cell–cell adhesion prevents osteoblast survival (Hunter et al., 2001). Thus, N-cadherin may be involved in the control of osteoblast behavior into osteocytes and dying cells (Fig. 3).

Figure 3.

Proposed role of N-cadherin during chondroblast, osteoblast, and adipocyte differentiation. Changes in N-cadherin expression in mesenchymal cell are associated with parallel changes in cell–cell adhesion and differentiation into adipocytes, chondrocytes or osteoblasts. During bone formation, high N-cadherin expression in mature osteoblasts is associated with increased cell–cell interactions whereas osteocytes or apoptotic osteoblasts show reduced N-cadherin expression and loss of cell–cell adhesion.

N-CADHERIN REGULATION IN OSTEOBLASTS

A number of hormonal and local factors were found to regulate cadherins in osteoblastic cells. Babich and Foti (1994) were the first to show that parathyroid hormone (PTH), an hormone that is known to induce osteoblast retraction and cytoskeletal disorganization in osteoblasts (Lomri and Marie, 1988), also reduces osteoblast cell–cell adhesion and causes E-like cadherin internalization in the cytosol. Moreover, PTH was reported to stimulate bone formation in vivo and to increase osteocalcin mRNA expression together with N-cadherin in osteoblasts (Suva et al., 1994), suggesting a role of N-cadherin in early and late stages of osteoblast differentiation controlled by PTH. In contrast to the effect of PTH, 1,25(OH)2 vitamin D3 was found to down-regulate N-cadherin in murine osteoblastic cells (Luegmayr et al., 2000). This effect appears to be specific for N-cadherin as cadherin-11 was not affected. This may be tissue specific because the expression of N-cadherin is increased by 1,25-(OH)2 D3 in chick limb bud mesenchymal cells undergoing chondrogenesis (Tsonis et al., 1994).

Dexamethasone, a stimulator of osteoblast differentiation, has distinct effects on N-cadherin expression in vitro, depending on the cell type. Dexamethasone inhibits the expression of N-cadherin and cadherin-11 but up-regulates cadherin 4 in long term culture of bone marrow stromal cells and human trabecular osteoblasts (Lecanda et al., 2000). In contrast, dexamethasone has no effect on N-cadherin mRNA levels in human calvaria osteoblasts (Delannoy et al., 2001). Moreover, a cadherin inhibitor containing HAV which is effective in inhibiting adhesion mediated by type I cadherins only partially prevents dexamethasone-induced stimulation of ALP (Cheng et al., 1998). Thus, cell–cell adhesion does not appear to be critical for dexamethasone induction of ALP in human osteoblastic cells (Lecanda et al., 2000).

Some cytokines are known to regulate N-cadherin in osteoblasts. In murine calvaria osteoblasts, TNFα and IL-1 were found to suppress N-cadherin but not cadherin-11/OB cadherin expression. Because TNFα and IL-1 are potent bone resorbing agents, their effects on N-cadherin expression in osteoblasts and the resulting decreased cell–cell adhesion were suggested to facilitate the adhesion of osteoclasts on the bone matrix surface (Tsutsumimoto et al., 1999). In addition, the TNFα and IL-1-induced decreased N-cadherin expression and cell–cell adhesion may be related to the decreased osteoblast differentiation induced by these cytokines in osteoblasts (Modrowski et al., 1995), as shown by reduced ALP expression in osteoblasts (Tsutsumimoto et al., 1999).

Cell–cell adhesion molecules are also controlled by members of the TGF-β family. In nonskeletal cells, activin (Jiang et al., 1993), TGF-β (Roubin et al., 1990), and BMP-7/OP-1 (Perides et al., 1992) modulate N-CAM expression. In chick limb bud mesenchymal cells, TGF-β increases N-cadherin (Tsonis et al., 1994). In contrast, TGF-β decreases N-cadherin expression during cartilage differentiation (Kawai et al., 1999), sugesting that the effect depends on the stage of cell differentiation. BMP-2 is another important member of the TGF-β family that plays an important role in cartilage and bone formation through its effects on cells of the osteoblastic lineage (Marie, 1997). BMP-2 was found to increase N-cadherin expression during chondrogenic differentiation of murine multipotent mesenchymal cells (Haas and Tuan, 1999). Moreover, a dominant negative N-cadherin mutant dramatically inhibits BMP-2-stimulated chondrogenesis, suggesting that stimulation of chondrogenic differentiation in murine mesenchymal cells requires N-cadherin expression and function (Haas and Tuan, 1999). In osteoblasts, N-cadherin regulation by BMP-2 appears to depend on the type of osteogenesis (membranous vs. endochondral) or the stage of development (neonatal vs. adult age). Neither N-cadherin nor cadherin-11 expression are affected by BMP-2 in human bone marrow stromal cells and human trabecular osteoblastic cells (Cheng et al., 1998). In contrast, we found that rhBMP-2 rapidly promotes N- and E-cadherin mRNA and protein levels in human calvaria osteoblastic cells by a transcriptional effect (Haÿ et al., 2000). The stimulatory effect of rhBMP-2 on N- and E-cadherin expression in human osteoblasts have functional consequences on cell–cell adhesion and osteoblast differentiation, because neutralizing anti-N- and E-cadherin antibodies abolish the rhBMP-2-induced increase in ALP, osteocalcin and Cbfa1/Runx2 expression, indicating an essential role of these cell–cell adhesion molecules in the expression of this osteoblast markers in human calvaria cells (Haÿ et al., 2000). BMP-2-induced ALP activity may also be prevented by a synthetic peptide containing the HAV motif of N-cadherin in human bone marrow cells (Cheng et al., 1998). Moreover, BMP-2-induced expression of osteopontin and bone sialoprotein in murine calvaria cells can be inhibited by a dominant negative cadherin (Cheng et al., 2000). These studies indicate that cell–cell interactions mediated by N-cadherin are essential for osteoblast differentiation promoted by BMP-2.

Another local regulator of cell–cell adhesion in osteoblasts is FGF-2. FGFs are known to play important roles in the regulation of osteoblastic cell growth and differentiation (Marie et al., 2000). We found that FGF-2 increases N-cadherin but not E-cadherin mRNA levels in human calvaria osteoblasts. The stimulatory effect of FGF-2 on N-cadherin mRNA levels is rapid, transient, and transcriptional. Specific anti-N-cadherin antibodies inhibited the cell aggregation induced by FGF-2, showing that N-cadherin is involved in the promoting effect of FGF-2-on cell–cell adhesion (Debiais et al., 2001). One of the roles of FGF-2 may be to increase N-cadherin expression and cell–cell adhesion in the normal human calvaria during the early steps of mesenchyme condensation. In contrast to BMP-2, the effect of FGF-2 on N-cadherin expression is not associated with increased osteoblast differentiation. This may in part be related to the transient effect of FGF-2 on N-cadherin expression, since stimulation of osteoblast differentiation requires long-term treatment with FGF-2 (Debiais et al., 1998). Multiple signal transduction pathways have been implicated in FGF signaling (Jaye et al., 1992). Binding of FGFs to Fibroblast growth factor receptors (FGFRs) leads to signaling events that trigger gene expression. The analysis of signaling molecules in osteoblasts showed that PKC and src signaling pathways are required for the stimulation of N-cadherin expression, suggesting that N-cadherin is a target gene for PKC (Debiais et al., 2001). The PKC family consists of three classes of isoenzymes known as conventional calcium- and diaglycerol (DAG)-dependent isoforms (Newton, 1995). Conventional and novel PKC isoenzymes bind DAG and are sensitive to phorbol esters. Direct activation of PKC by acute treatment with phorbol ester increases N-cadherin, cell–cell aggregation, and ALP activity in human calvaria osteoblsts which supports a role for PKC in the control of cell–cell adhesion and cell differentiation mediated by N-cadherin in osteoblasts (Delannoy et al., 2001). This effect, together with the previous evidence that BMP-2 (Haÿ et al., 2001) and FGF-2 activates PKC and increases N-cadherin expression (Debiais et al., 2001), suggests a role for PKC in the control of N-cadherin expression and cell–cell adhesion in human osteoblasts (Fig. 4).

Figure 4.

Control of N-cadherin in osteoblasts by local factors. Up-regulation of N-cadherin mRNA or protein levels in osteoblasts involves activation of PKC and results in increased cell–cell adhesion, osteoblast marker gene expression and bone formation, whereas down-regulation of N-cadherin is associated with osteoblast apoptosis.

The available data indicate that the regulation of osteoblast differentiation and survival by hormonal and local factors may be in part dependent on N-cadherin-mediated cell–cell adhesion and expression in osteoblasts. One can speculate that II-1 and TNFα may induce osteoblast apoptosis (Tsuboi et al., 1999; Lemonnier et al., 2001b) in part by down-regulating N-cadherin expression (Hunter et al., 2001). In addition, PTH and BMP-2 may increase bone formation in part by their effects on N-cadherin expression in cells of the osteoblast lineage (Suva et al., 1994; Haÿ et al., 2000). Thus, hormonal and local factors that regulate osteoblast function may act in part by regulating N-cadherin expression or cleavage and subsequent cell–cell adhesion that generates intracellular signals leading to gene expression, osteoblast differentiation or survival (Fig. 4). The molecular mechanisms involved in the regulation of N-cadherin expression in osteoblasts remain unknown. Preliminary data from my laboratory indicates potential sites of regulation in the human N-cadherin promoter (Le Mée and Marie, 2001). Further molecular analysis will determine the molecular mechanisms by which N-cadherin expression is regulated in osteoblasts.

N-CADHERIN AND BONE PATHOLOGY

Besides playing a role in osteoblast biology, there is some evidence that deranged cadherin expression may be associated with disorders of bone formation. Cadherin-mediated cell–cell adhesion is known to play a role in the morphogenesis of cancer cells (Potter et al., 1999). The variable expression of cadherins may regulate the cadherin-mediated adhesion, alter the morphogenetic process, and thereby contribute to change in cell differentiation and the formation of soft and bone tumors (Sato et al., 1999). In bone, the analysis of N-cadherin protein levels revealed decreased N-cadherin expression in primary and metastatic osteosarcomas. Moreover, osteosarcoma cells show focal or weak cytoplasmic distribution of N-cadherin and cadherin-11, instead of being normally associated with the cell surface (Kashima et al., 1999). This suggests that the reduced expression of N-cadherin and the deranged expression of cadherin-11 may play a role in metastasis in osteosarcoma tumors.

On the other hand, there is genetic and molecular evidence that N-cadherin overexpression plays a role in premature membranous osteogenesis in humans induced by activating mutations in FGFR-2. FGFR-2 mutations in Apert syndrome induce abnormal ossification of cranial sutures (craniosynostosis) (Muenke et al., 1995) which results from activation of the osteoblast differentiation pathway, as evidenced by overexpression of ALP, type I collagen, and osteocalcin in the affected suture (Lomri et al., 1998; Lemonnier et al., 2000). We found that this phenotype induced by Apert FGFR-2 mutations is associated with increased N- and E-cadherin expression in pre-osteoblasts and osteoblasts in vivo and in vitro (Lemonnier et al., 2001a; Lomri et al., 2001). Moreover, the increased N-cadherin and E-cadherin mRNA and protein levels in mutant osteoblasts cause increased cell–cell adhesion without changes in α-and β-catenins. Further studies using specific neutralizing anti-N-cadherin or antisense oligonucleotides demonstrated that N-cadherin is specifically implicated in the activation of osteoblast differentiation and osteogenesis in Apert craniosynostosis. Functional analysis of the signaling pathways revealed that the activation of N-cadherin expression and osteoblast phenotype is mediated by PKCα activation in mutant osteoblasts (Lemonnier et al., 2001a), confirming that PKC plays a role in the regulation of N-cadherin in osteoblasts (Fig. 4). In contrast to these findings in osteoblasts, inhibition of PKC in chondrocytes results in sustained expression of N-cadherin (Yoon et al., 2000). Thus, although PKC signaling coupled with the regulation of N-cadherin appears to play a key role in chondrogenic and osteoblastic differentiation, distinct regulatory mechanisms appear to be involved in the expression of this cell adhesion molecule in chondroblasts and osteoblasts.

The evidence described above that N-cadherin is involved in osteoblast differentiation may have several implications in the treatment of bone loss associated with aging. It is known that bone formation is insufficient to compensate for the increased bone resorption occuring during aging or in postmenopausal conditions (Rodan and Martin, 2000). Osteoblast population decreases with age whereas the adipogenesis in the bone marow stroma increases (Nuttall and Gimble, 2000). Recent data indicate that cadherin-11 expression in bone decreases with age in association with reduced bone density (Goomer et al., 1998). Although N-cadherin increases during osteoblast differentiation and decreases during adipogenesis in mesenchymal cells (Shin et al., 2000), it is unknown whether N-cadherin expression in bone decreases with age. It can be postulated that increasing N-cadherin expression in osteoblast progenitors may result in increased osteoblast differentiation. The associated increase in cell–cell adhesion may also lead to reduce osteoblast apoptosis and therefore increased bone formation during aging. Although N-cadherin may be a target for drug therapy to increase locally bone formation during bone repair or regeneration, this remains to be tested in vivo.

CONCLUSION AND PERSPECTIVES

The available evidence suggest that N-cadherin plays an important role in osteoblast differentiation and osteogenesis. In addition to the in vivo data showing that N-cadherin expression is associated with osteoblast differentiation and osteogenesis, there is also in vitro evidence for an implication of N-cadherin in osteoblast gene expression and function in normal and pathological conditions. However, several questions remain to be addressed on the role of N-cadherin in osteogenesis. First, the molecular mechanisms involved in N-cadherin expression in osteoblasts remain unknown. The identification of these mechanisms may help in delineating pharmacological ways to enhance N-cadherin specifically in cells of the osteoblastic lineage. Targeted deletion or overexpression of N-cadherin in mice using promoters characteristic of early or late stages of osteoblast differentiation may allow to conclude on the role of N-cadherin during the various stages of osteogenesis in vivo. Second, the mechanisms by which N-cadherin expression may control osteoblast marker genes are unclear. In the future, the identification of the signaling and molecular pathways that are involved in the control of cell–cell adhesion mediated by N-cadherin will be of paramount importance to understand the mechanisms involved in skeletal cells. It is expected that future molecular and genetic analyses will provide more information on the role of N-cadherin in the differentiation of osteoblasts and other bone cells in vitro and in vivo.

Acknowledgements

The author thanks the present and past members of his laboratory (F. Debiais, Ph. Delannoy, E. Hay, F. Lasmoles, S. Le Mée, J. Lemonnier, A. Lomri, and D. Modrowski) who contributed significantly to the studies reviewed in this paper.

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