Fibroblast growth factor signaling controlling osteoblast differentiation

Gene 316 (2003) 23 – 32 www.elsevier.com/locate/gene

Review

Fibroblast growth factor signaling controlling osteoblast differentiation P.J. Marie * Laboratory of Osteoblast Biology and Pathology, INSERM U 349 affiliated to CNRS, Lariboisie`re Hospital., 2 rue Ambroise Pare´, 75475 Paris Cedex 10, France Received 19 March 2003; received in revised form 20 March 2003; accepted 13 June 2003 Received by A.J. van Wijnen

Abstract Fibroblast growth factors (FGFs) play important roles in skeletal development and postnatal osteogenesis. FGF signaling controls bone formation by regulating the expression of various genes involved in osteoprogenitor cell replication, osteoblast differentiation and apoptosis. Recent genetic manipulation of FGF expression in mice and studies of the phenotype induced by gain-of-function mutations in FGF receptors in humans revealed the important role of FGF signaling in osteoblast function and differentiation. Additionally, cell biology studies allowed to identify some signaling pathways that are involved in the control of FGF actions in osteoblasts. This led to a better understanding of the functional role of FGF signaling in the control of gene expression in osteoblasts. The elucidation of molecular mechanisms by which FGF signaling promotes osteoblast gene expression and differentiation may help to find novel molecular targets and develop new therapeutic approaches to promote bone formation in human bone disorders. D 2003 Elsevier B.V. All rights reserved. Keywords: FGF; Signaling; Gene; Osteogenesis

1. Introduction The formation of the skeleton is a complex process that involves skeletal patterning during development, bone growth and remodeling during the postnatal life. Bone formation throughout the life is governed by osteoblasts, the bone-forming cell. The rate of bone formation is dependent on the commitment and replication of osteoprogenitor cells, their differentiation into functional osteoblasts and the life span of mature osteoblasts (Marie, 1998; Aubin and Triffitt, 2002). Recent analysis revealed the complexity of the cellular and molecular regulation of osteoblast differentiation by transcription factors during skeletal devel-

Abbreviations: FGFs, fibroblast growth factors; FGFR, FGF receptors; HSPGs, heparan sulfate proteoglycans; ALP, alkaline phosphatase; Col I, collagen type I; OP, osteopontin; BSP, bone sialoprotein; ON, osteonectin; IGF-I, insulin-like growth factor-I; TGF-h, transforming growth factor h; IGF, insulin growth factor; HGF, hepatocyte growth factor; BMP, bone morphogenetic protein; IL6, interleukin 6; bHLH, basic helix loop helix; MAP, mitogen activating protein. * Tel.: +33-1-49-95-63-89; fax: +33-1-49-95-84-52. E-mail address: [email protected] (P.J. Marie). 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00748-0

opment and postnatal life (Stein et al., 1996; Karsenty and Wagner, 2002). Cells are committed to the osteoblast lineage by induction of the transcription factor Runx2/ PEBP2alphaA/CBFA1/AML3 (Banerjee et al., 1997; Ducy and Karsenty, 1998). Other transcription factors such as Osx, Dlx5, Msx2, fos and Twist control Cbfa1/Runx2 function or act downstream to regulate osteoblast gene expression (Nakashima et al., 2002; Karsenty and Wagner, 2002). The coordinate action of these transcription factors results in the expression of several genes that are characteristics of osteoblast differentiation and function, such as alkaline phosphatase (ALP), type I collagen (Col I), osteopontin (OP), osteonectin (ON), bone sialoprotein (BSP) and osteocalcin (OC) that are expressed sequentially during the process of osteogenesis (Stein et al., 1996; Marie, 2001; Aubin and Triffitt, 2001). Osteoblast differentiation genes are regulated by the actions of systemic and local signaling factors. Among these factors are fibroblast growth factors (FGFs), a family of polypeptides that control the proliferation and differentiation of various cell types (Jaye et al., 1992; Basilico and Moscatelli, 1992). The important effects of FGFs on the control of differentiation genes in osteoblasts have been

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recently recognized (Marie et al., 2000, 2002; Hurley et al., 2001). This was mainly due to studies of genetic models in mice and of human mutations affecting FGF signaling (Ornitz and Marie, 2002; Bonaventure and El Ghouzzi, 2003). Furthermore, cell biology studies identified some FGF signaling pathways and molecular mechanisms that are involved in gene regulation by FGF signaling in osteoblasts. This review summarizes the recent development of the functional role of FGF signaling in the control of gene expression in osteoblasts.

2. Bone formation is governed by FGF/FGFR expression FGFs play major roles in skeletal development (Naski and Ornitz, 1998). Bone development is dependent on the expression of members of the FGF family expressed locally during bone formation. In bone, FGF2 transcripts are found in mesenchymal cells and osteoblasts together with FGF9 (Gonzalez et al., 1996; Kim et al., 1998). Postnatally, FGF2 is produced by mature osteoblasts, and is stored in the extracellular matrix (Globus et al., 1989). Another partner, FGF18, is expressed in mesenchymal cells and differentiating osteoblasts during calvarial bone development, and in the perichondrium of developing long bones (Ohbayashi et al., 2002). In addition to be related to FGF expression, the actions of FGFs are dependent on the spatiotemporal pattern of expression of high affinity FGF receptors (FGFR) (Givol and Yayon, 1992; Powers et al., 2000). FGFR1 and FGFR2 are expressed in mesenchymal cells during condensation of mesenchyme prior to deposition of bone matrix at early stages of long bone development, and are also expressed in the cranial suture (Orr-Urtreger et al., 1991; Delezoide et al., 1998). Later in development and in the postnatal life, FGFR1 and FGFR2 are found in pre-osteoblasts and osteoblasts together with FGFR3 (Iseki et al., 1997; Delezoide et al., 1998; Kim et al., 1998; Molte´ni et al., 1999a) (Fig. 1). The coordinate actions of FGFs on gene expression in osteoblasts is not only dependent on FGFR expression, but also on the affinity and specificity of FGF binding to the various alternative splice forms of FGFRs resulting from alternative mRNA splicing and polyadenylation (Ornitz et al., 1996). FGFR1, FGFR2 and FGFR3 exist as two splice variants, IIIb and IIIc. The mesenchymal splice variant of FGFR2 (Fgfr2IIIc) is expressed in early mesenchymal condensates and later in sites of endochondral and intramembranous ossification where it interacts with FGF18 (Eswarakumar et al., 2002). It was recently found that the recessive phenotype of Fgfr2IIIc( / ) mice is characterized initially by decreased expression of Cbfa1/Runx2 and retarded long bone ossification, suggesting that Fgfr2IIIc is a positive regulator of ossification (Eswarakumar et al., 2002). To add to this complex situation, FGFs bind cell surface heparan sulfate proteoglycans (HSPGs) acting as low-affinity co-receptors that interact with FGF binding and signaling (Ornitz, 2000; Schlessinger et al., 2000). Some of

Fig. 1. Schematic representation of the cranial suture showing the distinct expression of FGFs and FGFRs during membranous bone formation. The normal suture is composed of mesenchymal cells that differentiate into osteoprogenitor cells and then in osteoblasts depositing a new bone matrix along the suture edges. FGF2, 9 and 18 transcripts are found in mesenchymal cells and osteoprogenitor cells. FGFR1 and FGFR2 are mainly expressed in osteoprogenitor cells and osteoblasts.

these HSPGs named syndecans are expressed in bone (Solursh et al., 1990) and may affect the osteoblast response to FGF during osteogenesis (Molte´ni et al., 1999b; Modrowski et al., 2000). Because various partners (FGF, FGFR, HSPGs) are involved in FGF signaling, it is likely that subtle changes occurring in the expression or localisation of one of these molecule may induce variable functional effects on FGF signaling and osteoblast gene expression in vivo.

3. FGF signaling controls osteoblast differentiation and apoptosis Based on genetic manipulation in mice, it appears that several FGFs are important factors controlling bone formation. Overexpression of FGF2 in mice induces abnormal long bone formation (Coffin et al., 1995) whereas FGF2 invalidation inhibits it (Montero et al., 2000), pointing to an important role of FGF2 in the control of osteogenesis. Recent data indicate that FGF18 is also an important regulator of bone formation in vivo (Ohbayashi et al., 2002; Liu et al., 2002). Mice lacking FGF18 display delayed ossification and decreased expression of osteogenic markers (Liu et al., 2002). Moreover, FGF18-deficient mice generated by gene targeting show delayed cranial suture closure and long bone ossification (Ohbayashi et al., 2002), further emphasizing the important role of FGF18 in osteogenesis. Other evidence that FGF signaling regulates bone formation in vivo comes from studies showing that FGFs promote osteogenesis in normal and ovariectomized rats (Nagai et al., 1995; Hiroshi et al., 1994; Dunstan et al., 1999; Liang et al., 1999; Power et al., 2002). In long bones, the anabolic effect of FGF on bone formation appears to involve the recruitment of osteoblast precursor cells which then differentiate into osteoblasts (Nakamura et al., 1995;

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Pun et al., 2001). FGF signaling is also an important regulator of cranial osteogenesis. In the calvaria, FGF2 and FGF9 promote osteogenic differentiation (Iseki et al., 1997; Rice et al., 2000), and blocking FGF2 biological activity prevents cranial osteogenesis in vivo (Greenwald et al., 2001; Moore et al., 2002). Very recent data using conditional inactivation of FGF receptor 2 in pre-osteoblasts confirmed that FGFR2 signaling is required for osteoblast proliferation and for the maintenance of osteogenesis (Yu et al., 2003). Therefore, FGF signaling induced by various FGFs appears to regulate cell proliferation and differentiation positively in membranous and long bone osteogenesis (Fig. 2). The anabolic effects of FGF on bone formation are mediated by several cellular mechanisms. FGF2, 4, and 9 and 18 were found to regulate the replication of calvaria osteoblastic cells in culture (Canalis et al., 1988; Iseki et al., 1997; Rice et al., 2000; Shimoaka et al., 2002). In long bone marrow stromal cells, FGF2 promotes cell growth which results in osteoblast differentiation and matrix mineralization (Pitaru et al., 1993; Noff et al., 1989; Martin et al., 1997; Pri-Chen et al., 1998; Walsh et al., 2000). In addition, FGF2 enhances the marrow stromal cell population that is responsive to Bone morphogenetic protein (BMP)-2 and dexamethasone (Locklin et al., 1995; Scutt and Bertram, 1999). In human calvaria osteoblasts, we showed that FGF2 promotes cell growth in immature cells whereas prolonged FGF2 treatment increases matrix mineralization, indicating that the effects of FGF on osteoblast growth depends on the stage of cell maturation (Debiais et al., 1998). The overall effect of sustained treatment with FGF2 is to promote the replication and differentiation of osteoprogenitor cells in vitro, which results in increased number of functional osteoblasts. This effect is consistent with the in vivo situation where FGF2 acts first on osteoblast precursor cell

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replication to increase osteoblast number and bone formation in endosteal bones (Nakamura et al., 1995). There is also evidence that FGF signaling controls genes involved in osteoblast apoptosis. In vitro, FGF signaling protects from apoptotic effects induced by serum starvation (Hill et al., 1997) and inhibits osteoblast death induced by peroxynitrite (Kelpke et al., 2001). Recent data suggest, however, that FGF signaling acts differently on osteoblast apoptosis depending on the stage of differentiation. We recently found that FGF2 exerts a dual effect on human osteoblast apoptosis as it first protects from apoptosis induced by serum starvation, then stimulates apoptosis in vitro (Debiais et al., 2002). In vivo, FGF treatment also induces apoptosis in more differentiated osteoblasts and overexpression of FGF2 signaling in transgenic mice leads to increased apoptosis in mouse calvaria (Mansukhani et al., 2000). Consistently, we found that constitutive FGFR2 activation induces apoptosis in mature human calvaria osteoblasts (Lemonnier et al., 2001a,b). Thus, acute FGF signaling may reduce apoptosis of immature osteoblasts, and thereby enhances the osteoblast population, whereas continuous signaling may promote apoptosis in more mature osteoblasts, therefore limiting the early increase in the osteoblast pool. Because apoptosis is known to control osteoblast life span and bone formation (Manolagas, 2000), the dual effect of FGF signaling on osteoblast apoptosis is likely to be an important mechanism controlling the rate of osteoblast differentiation and osteogenesis in vivo (Fig. 2).

4. FGF signaling controls osteoblast gene expression FGF signaling regulates the expression of multiple genes that characterize the osteoblast phenotype, albeit these effects appear to be complex. In osteoblastic cell

Fig. 2. Regulation of osteoblast gene expression by FGF signaling during the course of osteogenesis. Bone formation is characterized by the replication of mesenchymal cells and the differentiation of osteoprogenitor cells into mature osteoblast, and ends with osteoblast apoptosis. FGF acts through FGFR (dashed arrows) to control several genes involved in osteoblast commitment, differentiation and apoptosis.

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cultures, FGF signaling was found to inhibit the expression of ALP and Col I, to upregulate OP and BSP, and to modulate ON gene expression in osteoblastic cells (Rodan et al., 1989; Hurley et al., 1993; Delany and Canalis, 1998a,b; Fang et al., 2001; Shimizu-Sasaki et al., 2001; Kalajzic et al., 2003). The effects of FGF signaling on OC expression are more complex and depend on the cell type. Indeed, FGF1 inhibits OC gene expression in rat calvaria cells (Tang et al., 1996), but enhances its expression in bovine bone cells (Schedlich et al., 1994). These effects may be related to direct effects of FGF signaling mediated by transcription factors, and indirect effects mediated by soluble factors or cell – matrix interactions. A direct regulation has been reported in mouse calvaria cells, where FGF-2 and cAMP have synergistic effects on OC expression by acting on specific elements in the OC promoter (Schedlich et al., 1994; Boudreaux and Towler, 1996; Newberry et al., 1996). Another direct effector may be mediated by Cbfa1/Runx2. Indeed, FGF2, FGF4 and FGF8, or transfection with a vector expressing a mutant FGFR2 that is constitutively activated in the presence of FGF ligand, was recently found to stimulate Cbfa1/Runx2 expression in osteoblasts (Zhang et al., 2002; Kim et al., 2003). Furthermore, FGF2 treatment was shown to increase the binding of Runx2 to Runx2-binding consensus sequence in the OC promoter and to activate the transactivation function (Kim et al., 2003), indicating that Cbfa1/Runx2 is a target gene for FGF signaling. This provides an important molecular mechanism by which FGF/FGFR signaling directly activates the expression of osteoblast genes that are dependent on Cbfa1/Runx2 expression (Fig. 2). FGF signaling also controls osteoblasts indirectly through the expression of genes that are involved in the function of osteoblasts. For example, FGF signaling downregulates the expression of connexin-43, a functional protein involved in gap junction-mediated communication (ShiokawaSawada et al., 1997) and upregulates the expression of Ncadherin, a cell membrane protein involved in cell– cell adhesion in osteoblasts (Debiais et al., 2001). Moreover, FGF-2 increases the sodium-dependent phosphate transport in mouse calvaria cells, which is required for matrix calcification (Suzuki et al., 2000). The control of these genes by FGF signaling may thus contribute indirectly to the regulation of osteoblast phenotype. FGFs interact with other growth factor signaling pathways and may thereby regulate osteoblast function (Table 1). For example, FGF2 upregulates TGFh expression in osteoblasts in vitro (Noda and Vogel, 1989) and this effect mediates some of the anabolic action of FGF2 on bone in vivo (Nakamura et al., 1995). Conversely, TGFh regulates FGF2 and FGFR expression in osteoblastic cells (Sobue et al., 2002). Additionally, FGF2 increases insulin-like growth factor-I (IGF-I) gene expression in vitro (Zhang et al., 2002) and in vivo (Power et al., 2002) and inhibits the synthesis of IGF binding protein-5, an important inhibitory protein of

Table 1 Gene regulation by FGF/FGFR signaling in bone Transcription factors

Matrix and cell proteins

Growth factors

Matrix degradation

Apoptotic proteins

AP-1 Cbfa1/Runx2

Col I ON, OP

MMP1 Collagenase-3

Bax IL-1

Twist

BSP, OC N-cadherin Connexin-43 Noggin

TGFh IGF1, IGFBP5 VEGF HGF

TIMPs Stromelysin MMP1

Fas

IGFs in bone cells (Canalis and Gabbitas, 1995; Hurley et al., 1995a,b). Moreover, FGFs increase VEGF (Saadeh et al., 2000; Tokuda et al., 2000) and hepatocyte growth factor (HGF) (Blanquaert et al., 1999) which are both mitogenic factors for osteoprogenitor cells. If confirmed in vivo, these actions of FGF signaling on growth factor expression may mediate some of the effects of FGF on osteoblasts and bone formation. In addition to TGF-h, IGF, HGF and VEGF, FGF interact with BMPs to control osteogenesis in vivo. For example, FGF acts synergistically with BMP4 to promote osteogenesis in rats (Kubota et al., 2002). Also, very recent data indicate that FGF2 and FGFR2 inhibit the expression of the BMP antagonist noggin in the patent cranial suture, resulting in increased BMP4 activity and suture fusion (Warren et al., 2003). This indicates that FGF signaling can control cranial suture fusion indirectly through BMP signaling. It is therefore likely that the biological activities of FGFs in bone depend not only on the balance between FGF and FGFR expression, but also on the presence of other signaling molecules, including BMPs. In addition to control the expression of genes associated with bone formation, FGFs regulate genes that are involved in matrix degradation. FGF signaling increases the expression by osteoblastic cells of IL6 and prostaglandin E2 that are important cytokines involved in bone resorption (Kawaguchi et al., 1995; Hurley et al., 1996). Moreover, FGF signaling transcriptionally increases the expression of interstitial collagenase (Varghese et al., 1995; Hurley et al., 1996; Tang et al., 1996) by acting on specific elements in the collagenase promoter (Newberry et al., 1997). Consistently, FGF2 stimulates collagenase-3 gene transcription through an effect on AP-1 site on the promoter (Varghese et al., 2000), and induces the expression of tissue inhibitors of metalloproteinases (TIMP) 1 and 3 (Varghese et al., 1995) and stromelysin-3 transcription (Delany and Canalis, 1998a,b). These data suggest that FGF2 may modulate bone matrix proteolyse by regulating collagenase expression and activity (Table 1). Whether the molecular regulation of these genes may involve Cbfa1/Runx2 expression or phosphorylation induced by FGF (Xiao et al., 2000) is an interesting hypothesis which remains to be investigated in osteoblasts. Overall, activation of FGF signaling is able to regulate genes involved at all steps of osteogenesis. This

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Fig. 3. Histological aspect of the normal human fetal suture and Apert coronal suture showing the effect of the activating Ser252Trp FGFR-2 mutation on osteoblasts function. The mutation induces premature differentiation of pre-osteoblasts (pre-Ob) into osteoblasts (Ob), resulting in increased de novo matrix depositions (Osteoid) and premature ossification of the suture (5-Am-thick sections stained with von Goldner trichrome; original magnification:  125).

array of genes may participate in regulating cell progression from osteoprogenitor cell to the end of the osteoblast life (Fig. 3).

5. FGFR activation in human craniosynostosis Recent genetic studies revealed the important role of FGFR signaling in the control of osteoblast gene expression and bone formation in vivo (Ornitz and Marie, 2002; Bonaventure and El Ghouzzi, 2003). Several gain-of-function FGFR mutations have been found to induce premature ossification of the cranial sutures (craniosynostosis) causing Apert, Crouzon and other syndromes (Muenke and Schell, 1995; Webster and Donoghue, 1997; Wilkie, 1997). Functional studies have shown that most mutations in FGFR1 and FGFR2 induce constitutive activation of the receptor (Neilson and Friesel, 1995, 1996; Robertson et al., 1998; Plotnikov et al., 2000). In Apert syndrome, FGFR2 mutations enhance receptor occupancy by FGF ligands or prolongation of the duration of receptor signaling (Park et al., 1995; Anderson et al., 1998). In Crouzon syndrome, the C342Y mutation in FGFR2 results in activation of FGFR2 signaling and decreased binding of FGF2 to the receptor (Mangasarian et al., 1997). These gain-of-receptor function mutations involving FGF signaling induce several functional consequences on osteoblast gene expression. We found that activation of FGFR2 signaling in Apert syndrome results in increased expression of osteoblast differentiation genes such as ALP, Col I, OP, OC, as well as N-cadherin and cell –cell agregation, resulting in increased bone formation (Lomri et al., 1998; Lemonnier et al., 2000; Marie et al., 2002) (Fig. 3). FGFR2 downregulation induced by FGFR2 activation could also contribute to the premature osteoblast differentiation induced by FGFR2 mutations in Apert and Crouzon syndromes (Bresnick and Schendel, 1995; Lemonnier et al.,

2001a,b). In contrast, activating FGFR2 mutations were found to reduce osteoblast differentiation in murine osteoblastic cells in vivo, possibly as a result of the increased cell death induced by the mutations (Mansukhani et al., 2000). Interestingly, recent data indicate that the activating P250R FGFR1 mutation increases expression of Cbfa1/Runx2 in osteoblasts (Zhou et al., 2000), which may provide a molecular mechanism for the premature osteoblast differentiation and cranial suture ossification induced by this mutation. Some of the effects of FGFR gain-of-function mutations on osteoblast gene expression can be mimicked by ectopic FGF signaling in normal osteoblasts. For example, exogenous FGF2 increases N-cadherin expression in human calvaria osteoblasts (Debiais et al., 2001) and downregulates FGFR2 transcripts in the mouse suture (Iseki et al., 1997), which, in the long term, leads to accelerate suture closure by altering the balance between cell proliferation and differentiation (Iseki et al., 1997; Kim et al., 1998). It is thus likely that accelerated cranial bone formation induced by activation of FGFR signaling results in part from increased osteoblast differentiation induced by the expression of Cbfa1/Runx2 and downstream genes (Fig. 4). In addition, recent data indicate that Twist, a bHLH transcription factor, interferes with FGF signaling during cranial suture formation. Mutations in the Twist gene induce Twist haploinsufficiency and premature cranial fusion in mice and humans in the Saethre– Chotzen syndrome (El Ghouzzi et al., 1997; Howard et al., 1997). We found that Twist haploinsufficiency results in altered osteoblast differentiation in human calvaria cells (Yousfi et al., 2001). This may perhaps result, in part, from alteration in FGF signaling because the pattern of FGFR expression is altered in sutures of heterozygous Twist-null mice (Rice et al., 2000). Moreover, Twist was recently found to inhibit osteoblast differentiation by downregulating FGFR3 expression (Funato et al., 2001). Thus, several transcription factors may interfere with FGF signaling to control osteoblast differentiation.

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Fig. 4. Mechanisms by which activation of FGFR2 signaling in Apert syndrome controls human osteoblast differentiation and apoptosis. The Ser252Trp FGFR-2 increases expression of osteoblast differentiation genes such as ALP, Col I, OP, OC, as well as N-cadherin and cell – cell agregation, resulting in increased bone formation. In mature osteoblasts, PKC activation induced by FGF signaling also induces overexpression of IL-1 and Fas, activation of caspase-8 and increased Bax/Bcl-2 levels, resulting in apoptosis.

Activation of FGFR signaling also affect osteoblast apoptosis. Apert and Crouzon FGFR2 mutations promote apoptosis in mature mouse and human osteoblasts or osteocytes (Mansukhani et al., 2000; Lemonnier et al., 2001a,b). This involves FGF signaling because this effect is mimicked by FGF treatment or FGF2 overexpression in transgenic mice (Mansukhani et al., 2000). In Apert syndrome, we showed that FGF signaling induces overexpression of IL-1 and Fas, activation of caspase-8 and increased Bax/Bcl-2 levels (Lemonnier et al., 2001a,b), which provides a mechanism by which FGF/FGFR signaling promotes osteoblast apoptosis (Fig. 4). Because apoptosis is an important mechanism by which osteoblastic cells are cleared during cranial suture formation, it can be postulated that this process is a necessary event compensating for the accelerated osteoblast differentiation induced by FGFR mutations.

6. FGF signaling pathways in osteoblasts Recent advances have been made in the identification of signaling pathways that are involved in FGF signaling in osteoblasts. FGF binding to FGFR leads to FGFR dimerization, phosphorylation of intrinsic tyrosine residues, which leads to activation of several signal transduction pathways (Szebenyi and Fallon, 1999). In osteoblasts, FGF-2 activates MAP kinase signal transduction including mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinases (Erks), p38 MAP kinase and protein kinase C (PKC) (Newberry et al., 1997; Mansukhani et al., 2000; Debiais et al., 2001). Some of these pathways were found to mediate effects of FGF signaling on osteoblast gene regulation. For example,

Erk signaling pathway by FGF-2 stimulates AP-1 expression and mediates its mitogenic effect and effects on procollagen, OP, OC and VEGF expression in osteoblasts (Chaudhary and Avioli, 2000; Xiao et al., 2002; Kim et al., 2003; Tokuda et al., 2000; Shimoaka et al., 2002). Erk activation also mediates the FGF-stimulated phosphorylation and transcriptional activity of Cbfa1/Runx2 (Xiao et al., 2002) which implicates an important role for the Erk pathway in FGF signaling controlling osteoblast differentiation (Kim et al., 2003). Another MAPK, the p38 MAP kinase is involved in the increased IL-6 synthesis induced by FGF-2 in osteoblasts (Kozawa et al., 1999). In contrast, the transcriptional activation of the matrix metalloproteinase MMP1 occurs independently of MAP kinase phosphorylation (Newberry et al., 1997), indicating that other pathways are involved in the actions of FGF signaling in osteoblasts. Indeed, it was found that the PKC pathway mediates some of the FGF actions in osteoblasts. For example, FGF2 activates PKC activity and this pathway is implicated in the increased N-cadherin expression in human osteoblasts (Debiais et al., 2001) and in sodium-dependent phosphate transport induced by FGF2 in murine osteoblasts (Suzuki et al., 2000). Further evidence that PKC plays a major role in FGF signaling is the finding that the S252W FGFR2 mutation increases protein PKC phosphorylation (Fragale et al., 1999), PKC mRNA expression (Lomri et al., 2001) and PKC activity in human osteoblasts (Lemonnier et al., 2001a,b). The recent finding that the PKC pathway is involved in the FGF/FGFR-stimulated expression of Cbfa1/Runx2 (Kim et al., 2003) further emphasizes the important role of PKC in FGF signaling. This is supported by our finding that the increased osteoblast differentiation and premature apoptosis induced by FGFR2

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osteoprogenitor cell proliferation and osteoblast differentiation needs to be addressed. In addition, we need to better identify the role of signal transduction pathways that are involved in osteoblast differentiation and survival induced by FGF signaling. It will also be of crucial importance to further identify the interactions between FGF and other growth factor signaling pathways, such as BMPs, during osteogenesis. Finally, the transcriptional regulation of osteoblast genes by FGF signaling requires further investigation. In particular, the role of transcription factors other than Cbfa1/Runx2, such as Twist, that interfere with FGF signaling during osteoblast differentiation and apoptosis warrants further investigation. In the future, determining the mechanisms by which FGF signaling promotes osteogenesis may help to find novel molecular targets for anabolic effectors and perhaps to develop new anabolic therapeutic approaches to promote bone formation or repair in human bone disorders. Fig. 5. FGF signaling pathways regulating gene expression in osteoblasts. FGF binding to FGFR leads to FGFR dimerization and activation of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinases (Erks), p38 MAP kinase and protein kinase C (PKC) which mediate the effects of FGF signaling on the expression of transcription factors and downstream osteoblast differentiation genes.

activation in human osteoblasts is in part mediated by PKC activation (Lemonnier et al., 2001a,b; Marie et al., 2002) (Fig. 5). Other kinases, such as src proteins, may be potential substrates for FGF receptors mediating the control of osteoblast differentiation (Debiais et al., 2001), albeit the precise role of this pathway in FGF signaling remains to be determined. It is likely that gene expression induced by FGF signaling during osteogenesis is controlled by distinct pathways activated at different stages of osteoblast maturation. Additionally, multiple cross-talks and interactions between these pathways may be involved in the control of osteoblast function by FGF signaling.

7. Conclusions and perspectives Significant progress has been made in the recent years in the role of FGF signaling in osteoblast differentiation and bone formation. Studies of mouse models and human genetic mutations affecting FGFRs have been useful to elucidate part of the mechanisms of action of FGF signaling in the control of gene expression during osteoblast proliferation, differentiation and apoptosis. These advances have led to a more comprehensive view of essential signaling molecules involved in FGF signaling controlling osteoblasts during bone formation. Despite these advances on the role of FGF signaling in osteoblast gene expression, several points need to be addressed for a better understanding of the control of osteogenesis by FGF signaling. For example, the precise role of the distinct FGFR isoforms in the control of

Acknowledgements The authors wish to thank present and past members of the group (F. Debiais, Ph. Delannoy, J. Lemonnier, A. Lomri, D. Modrowski, A. Molte´ni) for their contribution to the work of the author presented in this review paper.

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