Fibroblast growth factor receptor signaling is an important mechanism regulating osteoblast function. To gain an insight into the regulatory role of FGF receptor-2 (FGFR2) signaling in osteoblasts, we investigated integrin-mediated attachment and cell survival in human calvarial osteoblasts expressing activated FGFR2. FGFR2 activation reduced osteoblast attachment on fibronectin. This was associated with reduced expression of the α5 integrin subunit normally expressed in human calvarial osteoblasts in vivo. Treatment with lactacystin, a potent inhibitor of proteasome, restored α5 integrin levels in FGFR2 mutant osteoblasts. Immunoprecipitation analysis showed that α5 integrin interacts with both the E3 ubiquitin ligase Cbl and ubiquitin. Immunocytochemistry revealed that α5 integrin colocalizes with FGFR2 and Cbl at the leading edge in membrane ruffle regions. Transfection with the 70Z-Cbl mutant lacking the RING domain required for Cbl-ubiquitin interaction, or with the G306E Cbl mutant that abolishes the binding ability of Cbl phosphotyrosine-binding domain restored α5 integrin levels. This suggests that Cbl-mediated ubiquitination plays an essential role in α5 integrin proteasome degradation induced by FGFR2 activation. Reduced α5 integrin expression was associated with an increased Bax/Bcl-2 ratio and increased caspase-9 and -3 activities in FGFR2 mutant osteoblasts. Forced expression of α5 integrin rescued cell attachment and corrected both the Bax/Bcl-2 ratio and caspase-3 and caspase-9 activities in FGFR2 mutant osteoblasts. We show that Cbl recruitment induced by FGFR2 activation triggers α5 integrin degradation by the proteasome, which results in reduced osteoblast attachment on fibronectin and caspase-dependent apoptosis. This identifies a functional role of the α5 integrin subunit in the induction of apoptosis triggered by FGFR2 activation in osteoblasts, and reveals that a Cbl-dependent mechanism is involved in the coordinated regulation of cell apoptosis induced by α5 integrin degradation.

Introduction

Osteogenesis is a complex process that involves the differentiation of mesenchymal cells into pre-osteoblasts and osteoblasts that ultimately leads to the synthesis and deposition of bone matrix proteins (Marie, 1998; Aubin and Triffitt, 2002; Karsenty and Wagner, 2002; Lian et al., 2004). Osteoblast differentiation is in part dependent on cell adhesion to bone matrix proteins such as type I collagen and fibronectin (Damsky, 1999; Franceschi, 1999; Zimmerman et al., 2000). Cell adhesion on these matrix molecules is mediated by integrins, a family of type I transmembrane glycoproteins consisting of α and β chains, which recognize and interact with amongst others bone matrix proteins (Plow et al., 2000). Interactions between integrin receptors, fibronectin and type I collagen are known to induce early osteoblast-specific gene expression and differentiation (Lynch et al., 1995; Moursi et al., 1996; Moursi et al., 1997). Although primary osteoblasts were found to express α1, α2, α3, α5, αV, β1, β3 and β5 integrins (Clover et al., 1992; Grzesik and Robey, 1994; Gronthos et al., 1997; Nakayamada et al., 2003), in vivo integrin expression in human osteoblasts is mostly restricted to α3, α5, αV and β1 subunits (Bennett et al., 2001; Hughes et al., 1993). Moreover, osteoblast differentiation appears to be mediated by a select group of integrin receptors that includes α1, α2, α5 and β1 subunits (Moursi et al., 1997; Takeuchi et al., 1997; Xiao et al., 1998; Jikko et al., 1999; Mizuno et al., 2000; Zimmerman et al., 2000). Recent data indicate that integrins mediating cell adhesion to extracellular proteins transduce signals required for cell survival (Grossmann, 2002; Howe et al., 2002; Reginato et al., 2003). Integrin-mediated adhesion to the extracellular matrix therefore protects against apoptosis (Meredith et al., 1993; Ruoslahti and Reed, 1994; Boudreau et al., 1995; Giancotti, 1997). In bone, perturbing the interaction of osteoblasts with fibronectin induces massive apoptosis, suggesting that integrin-mediated cell attachment may control osteoblast survival (Globus et al., 1998). However, the mechanisms involved in the coordinated regulation of cell survival by integrin receptors in osteoblasts are not known. As the osteoblast life span is an important factor controlling membranous and long bone formation (Manolagas, 2000; Fromigué et al., 2004), the elucidation of mechanisms underlying integrin-mediated regulation of osteoblast survival may provide important insights into the regulation of osteogenesis.

Integrin receptors are coupled to growth factor receptors and thereby regulate multiple biological functions (Miranti and Brugge, 2002). Convergence of integrin and growth factor signaling pathways have been described in focal adhesion complexes (Plopper et al., 1995). Integrins were shown to collaborate with growth factors in triggering tyrosine phosphorylation of growth factor receptors such as epidermal growth factor, platelet derived growth factor and fibroblast growth factor (FGF) receptors (FGFRs) (Miyamoto et al., 1996). FGFs bind to and activate FGFRs, which are members of a family of tyrosine kinase receptors (Jaye et al., 1992; Schlessinger, 2000). FGFR activation leads to intracellular signaling and activation of genes involved in cell proliferation, migration, differentiation and survival. In bone, FGF/FGFR signaling plays an important role in regulating osteoblasts and osteogenesis (Hurley et al., 2002; Marie, 2003). This is exemplified by the observation in humans that constitutive activation of FGFR2 by genetic mutation activates several signaling pathways that result in increased osteoblast differentiation (Lomri et al., 1998; Lemonnier et al., 2001a; Ornitz and Marie, 2002) and apoptosis (Mansukhani et al., 2000; Lemonnier et al., 2001b). The increased osteoblast gene expression induced by FGFR2 involves increased expression of the adhesion molecule N-cadherin, revealing a link between FGFR2 signaling, cell-cell adhesion and osteoblast gene expression (Lemonnier et al., 2001a). The role of FGFR signaling in integrin expression and cell attachment in osteoblasts has however not been characterized.

The ubiquitin ligase Cbl acts as an adaptor protein that is phosphorylated and recruited to activated receptor tyrosine kinases (reviewed by Sanjay et al., 2001). Cbl downregulates receptor tyrosine kinases by mediating ubiquitination resulting in proteasome-mediated degradation of these molecules after ligand binding. The mechanism by which Cbl negatively regulates these molecules involves Cbl recruitment, which allows polyubiquitination of activated receptors such as EGF, PDGF and FGFR (Joazeiro et al., 1999; Levkowitz et al., 1999; Yokouchi et al., 1999). This involves the phosphotyrosine-binding (PTB) domain of Cbl, which binds to the activated receptor, and the RING domain that is required for ubiquitination (Yokouchi et al., 1999; Thien and Langdon, 2001). Cbl is also known to interact with receptor tyrosine kinase associated proteins such as Src proteins, and to regulate Src activity (Andoniou et al., 2000; Sanjay et al., 2001). We recently showed that constitutive FGFR2 activation in human osteoblasts results in Cbl-mediated Src protein ubiquitination by the proteasome, which contributes to the increased osteoblast differentiation induced by FGFR2 activation (Kaabeche et al., 2004). The role of Cbl in integrin regulation in osteoblasts is at present unknown.

In the present study, we show that FGFR2 associates with Cbl and the α5 integrin subunit to promote Cbl-mediated ubiquitination of α5, resulting in reduced cell attachment and osteoblast apoptosis. This establishes for the first time a functional link between FGFR2 signaling and Cbl-mediated integrin expression resulting in altered cell attachment and apoptosis in osteoblasts.

Materials and Methods

Tissue and cell cultures

Cranial coronal sutures were obtained from a 27-week-old Apert fetus bearing the FGFR2 Ser252Trp mutation, the most frequent mutation in Apert syndrome, and from an age-matched fetus with no evidence of bone disease (kindly provided by A.-L. Delezoide, Hopital R. Debré, Paris, France) in accordance with the French Ethical Committee recommendations. Normal (wild-type) and FGFR2 mutant human calvarial cells obtained by collagenase digestion from coronal sutures were immortalized by transfection with the origin defective large-T antigen of the SV-40 oncogene as previously described (Lomri et al., 1998). FGFR2 mutant cells display increased expression of osteoblast marker genes and increased in vitro osteogenic capacity compared to control cells, a phenotype which is similar to the pathological features observed in fetal Apert coronal sutures in vivo (Lemonnier et al., 2001a). All cells were cultured in Dulbecco's modified essential medium (DMEM) supplemented with glutamine (292 mg/l), 10% heat inactivated fetal calf serum (FCS), and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin).

Cell adhesion

For the dose-dependent attachment assay on fibronectin, a titration curve of human fibronectin was used at concentrations ranging from 0 to 0.15 μg/ml coated on 96-well plates. The non-adhesive surface was blocked using 3% BSA in PBS for 1 hour at room temperature. FGFR2 mutant and control cells (50,000 cells) were allowed to adhere for 1 hour at 37°C in DMEM serum-free medium. Wells were extensively washed in PBS and stained with Crystal Violet. Dye was homogenized using acetic acid (0.1%) and OD at 570 nm was measured using a plate reader. For the time-course attachment assay, FGFR2 mutant and control cells were plated at 100,000 cells/well in six-well plates coated with fibronectin or type I collagen (BioCat Cellware, Beckton Dickinson, Le Pont de Claix, France) and cultured for 30, 60, 120 or 240 minutes in DMEM with 10% FCS. Adherent cells were collected using trypsin/EDTA (0.1%) and counted using a Coulter counter ZM (Beckman-Coulter).

Plasmids and transfection

FGFR2 mutant and control cells plated at 2500 cells/cm2 the day before transfection were co-transfected with the plasmid (2.5 μg/cm2 dish) and pSV-β-galactosidase (50 ng β-gal) control vector (Promega) in DMEM with 1% FCS. Cells were incubated with empty vector (pcDNA3) or α5 integrin, 70Z-Cbl or Cbl-G306E vectors and Exgen 500 (Euromedex) according to the manufacturer's directions. Efficiency of transfection was controlled by determination of β-gal activity (β-gal reporter gene assay, Roche). The number of β-gal+ cells was counted 24 hours post-transfection. For the cell adhesion assay after α5 integrin transfection, FGFR2 mutant cells plated on fibronectin-coated plates were transfected with the α5 plasmid or the empty vector and cultured for 48 hours before counting as described above.

Inhibition of proteasome

To determine whether α5 integrin subunit is degraded by the proteasome, FGFR2 mutant cells were treated with 10 μM lactacystin (Calbiochem), a specific proteasome inhibitor that binds covalently to the active-site N-terminal threonine residue in proteasome β-subunits (Fenteany and Schreiber, 1998). The cells were treated for 24 hours with lactacystine or solvent and α5 protein levels were determined by immunoprecipitation and western blot analysis.

RT-PCR analysis

The expression of integrin transcripts was examined in FGFR2 mutant and control cells by reverse-transcription polymerase chain reaction (RT-PCR) analysis. Confluent cells were washed with PBS, and total cellular RNA was extracted using the Extract-All reagent (Eurobio, France) according to the manufacturer's protocol. 3 μg total RNA were reverse transcribed at 37°C for 1 hour. cDNA samples were amplified (30 cycles for integrins, 23 cycles for GAPDH) using specific primers (Table 1). Southern blots were performed by running aliquots of amplified cDNAs on 1% agarose gels followed by transfer onto nylon membranes (Appligene-Oncor) according to the manufacturer's protocol. Hybridization of blots was carried out overnight at 50°C with [γ-32P]ATP-labelled internal antisense primers. Membranes were then washed and exposed to Kodak X-ray films at –80°C with intensifying screens and the signal for each gene was related to that of GAPDH.

Table 1.

Primer and probe sequences used for RT-PCR analysis

mRNA Primer Size
α5   Sense   5′ -TCTGCCTCAATGCTTCTGG- 3′   224 bp  
  Antisense   5′ -GTTGAGAGCGATGTGAATCG- 3′   
  Internal   5′ -ACGTTGCTGACTCCATTGG- 3′   
αV   Sense   5′ -CCAAGTTCATTCAGCAAGGC- 3′   130 bp  
  Antisense   5′ -CTTGGCAGACAATCTTCAAGC- 3′   
  Internal   5′ -AAGAATGACACGGTTGCCG- 3′   
β1   Sense   5′ -TGAGATGTGTCAGACCTGCC- 3′   207 bp  
  Antisense   5′ -AACCATGACCTCGTTGTTCC- 3′   
  Internal   5′ -CAGTCGTCAACATCCTTCTCC- 3′   
GAPDH   Sense   5′ -GGGCTGTTTTAACTCTGGT- 3′   702 bp  
  Antisense   5′ -TGGCAGGTTTTTCTAGACGG- 5′   
  Internal   5′ -CATGAGTCCTTCCACGATACC- 3′   
mRNA Primer Size
α5   Sense   5′ -TCTGCCTCAATGCTTCTGG- 3′   224 bp  
  Antisense   5′ -GTTGAGAGCGATGTGAATCG- 3′   
  Internal   5′ -ACGTTGCTGACTCCATTGG- 3′   
αV   Sense   5′ -CCAAGTTCATTCAGCAAGGC- 3′   130 bp  
  Antisense   5′ -CTTGGCAGACAATCTTCAAGC- 3′   
  Internal   5′ -AAGAATGACACGGTTGCCG- 3′   
β1   Sense   5′ -TGAGATGTGTCAGACCTGCC- 3′   207 bp  
  Antisense   5′ -AACCATGACCTCGTTGTTCC- 3′   
  Internal   5′ -CAGTCGTCAACATCCTTCTCC- 3′   
GAPDH   Sense   5′ -GGGCTGTTTTAACTCTGGT- 3′   702 bp  
  Antisense   5′ -TGGCAGGTTTTTCTAGACGG- 5′   
  Internal   5′ -CATGAGTCCTTCCACGATACC- 3′   

Immunohistochemistry, immunoprecipitation and western blot analysis

For immunohistochemistry, coronal sutures obtained from a normal fetus were fixed in formaldehyde, dehydrated in ethanol and embedded in paraffin as described previously (Lemonnier et al., 2000) and used for immunohistochemical detection of the α5 integrin subunit. Non specific sites were saturated using serum (10% FCS) for 24 hours at 4°C, then the sections were incubated with anti-α5 antibody or non-specific goat serum (1:1000), and the signal was amplified and revealed using the golden bead amplification system described previously (Lemonnier et al., 2000).

For immunocytochemistry, FGFR2 mutant and control cells were plated on uncoated glass coverslips overnight in DMEM with 10% FCS and immunostaining was performed essentially as described previously (Fournier et al., 2002) using antibodies against Cbl, FGFR2 and α5 integrin (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000.

Western blot analysis of integrins in confluent FGFR2 mutant and control cells cultured on fibronectin-coated wells was performed. Cell extracts were scraped in ice-cold lysis buffer containing protease inhibitors (Boehringer, Mannheim, Germany). Lysates were clarified by centrifugation at 12,000 g for 10 minutes at 4°C. The protein content of the resulting supernatants was determined by the DC Protein assay (BioRad Laboratories, CA). Proteins were then resolved by 6% SDS-PAGE and transferred onto PVDF Hybond-P membranes (Amersham). The membranes were incubated overnight with 10% blocking solution (Roche) and then with goat anti-α5 or anti-αv, mouse anti-β1 antibodies (Santa Cruz), or β-actin (Sigma) diluted 1:500. Membranes were washed and incubated with specific peroxidase-coupled appropriate secondary antibodies and the signal was visualized using a chemiluminescence detection system (Perbio-Science, Erembodegem, Belgium).

For immunoprecipitation analysis, equal aliquots (300 μg) of protein lysates were immunoprecipitated using 2.5 μg specific antibodies (anti-α5, anti-Cbl or anti-ubiquitin; Santa Cruz) and incubated overnight at 4°C in a rotating device. After 24 hours, 20 μl protein A/G agarose (Santa Cruz) were added and incubated for 1 hour at 4°C. Immunoprecipitates were then collected by centrifugation at 1200 g for 3 minutes, the pellets were washed four times with lysis buffer, and resuspended in 50 μl running buffer. Aliquots were then subjected to electrophoresis as described above, and membranes were reacted with α5, Cbl, ubiquitin, Bax or Bcl-2 antibodies (Santa Cruz). Immunoblots were probed with peroxidase-coupled specific secondary antibodies as indicated, and visualized by enhanced chemiluminescence.

Caspase activity

Caspase-3, -6, -7 and -9 activities were determined essentially as described with minor modifications (Lemonnier et al., 2001b).

Data analysis

The results presented are representative of three to four different experiments. Differences between the mean±s.e.m. were analysed using the statistical package super-ANOVA (Macintosh, Abacus concepts, Berkeley, CA) with a minimal significance of P<0.05.

Results

To investigate the effect of FGFR2 activation on osteoblast attachment, we first determined the number of attached FGFR2 mutant osteoblasts and control (wild-type) cells after plating on plastic dishes coated with type I collagen or fibronectin, two bone matrix proteins expressed during bone development and involved in osteoblast differentiation (Damsky, 1999). Attachment of FGFR2 mutant cells was about 20% lower than attachment of control cells when cultured on fibronectin (Fig. 1A). Similar results were found on collagen-coated dishes (Fig. 1B). The reduced adhesion of FGFR2 mutant osteoblasts on type I collagen may result as a consequence of the presence of fibronectin in the serum. Indeed, cell adhesion of FGFR2 mutant cells was also lower than that of control cells when cultured on plastic substrate in the presence of 10% FCS (data not shown). FGFR2 mutant cells showed lower attachment on fibronectin at concentrations up to 0.08 μg/ml, compared to control cells (Fig. 1C). These results demonstrate that constitutive activation of FGFR2 in mutant osteoblasts results in decreased cell attachment on fibronectin in vitro.

Fig. 1.

FGFR2 activation reduces osteoblast attachment on bone matrix proteins. FGFR2 mutant and control (wild-type) cells were cultured on fibronectin-coated dishes (A) or type I collagen-coated dishes (B) for the indicated times, or on fibronectin at the indicated concentration for 1 hour (C), and the number of attached cells was recorded. * Indicates a significant difference compared to number in control cells (P<0.05). The data are the mean±s.e.m. (n=3-6) and are representative of three separate experiments.

Fig. 1.

FGFR2 activation reduces osteoblast attachment on bone matrix proteins. FGFR2 mutant and control (wild-type) cells were cultured on fibronectin-coated dishes (A) or type I collagen-coated dishes (B) for the indicated times, or on fibronectin at the indicated concentration for 1 hour (C), and the number of attached cells was recorded. * Indicates a significant difference compared to number in control cells (P<0.05). The data are the mean±s.e.m. (n=3-6) and are representative of three separate experiments.

As cell-matrix interactions are mediated by integrins, we investigated whether the reduced cell attachment induced by FGFR2 activation in osteoblasts was associated with alteration of specific integrins. We restricted analysis to the most important integrins (α5, αV, β1) expressed in vivo in human osteoblasts and that mediate cell adhesion on fibronectin or type I collagen. Analysis at the translational level by western blotting showed that α5 protein levels were lower in FGFR2 mutants compared to control cells (Fig. 2A). In contrast, αv and β1 protein levels were higher in mutant cells compared to control cells cultured in basal culture conditions, indicating that FGFR2 activation induces a specific decrease in α5 integrin subunit. Analyses carried out at the transcriptional (reverse transcription-polymerase chain reaction) level showed that mRNA levels for α5, αv and β1 integrin subunits did not differ markedly in FGFR2 mutant compared to control cells (Fig. 2B), suggesting that a post-transcriptional mechanism is responsible for the reduced α5 protein levels induced by FGFR2 activation. To validate the in vivo relevance of these in vitro data, we examined expression of the α5 integrin subunit by immunohistochemical analysis of the cranial suture. Immunoreactivity of α5 integrin was detected in osteoblasts along the bone matrix in the cranial suture, illustrating the in vivo expression of this integrin subunit in normal human cranial bone suture (Fig. 2C). Control sections showed no specific staining. These results show that the α5 integrin subunit is expressed by osteoblasts in vitro and in vivo in human calvarial osteoblasts and that FGFR2 activation reduces α5 expression at the protein, but not the RNA level in these cells.

Fig. 2.

Expression levels of α5, αv and β1 integrins in FGFR2 mutant and control (wild type) osteoblasts. (A) Cells were subjected to western blot analysis using specific antibodies against α5, αv and β1 integrins. Equal loading was confirmed by detecting levels of β-actin. (B) Expression of transcripts for α5, αv and β1 integrins was determined by RT-PCR analysis and GAPDH was used as an internal control. (C) Immunohistochemical analysis of α5 integrin subunit in a normal fetal human coronal suture in vivo. α5 integrin subunit immunoreactivity was localized in osteoblasts (arrows) along the bone matrix. Control sections incubated with IgG showed no specific staining (original magnification, ×125).

Fig. 2.

Expression levels of α5, αv and β1 integrins in FGFR2 mutant and control (wild type) osteoblasts. (A) Cells were subjected to western blot analysis using specific antibodies against α5, αv and β1 integrins. Equal loading was confirmed by detecting levels of β-actin. (B) Expression of transcripts for α5, αv and β1 integrins was determined by RT-PCR analysis and GAPDH was used as an internal control. (C) Immunohistochemical analysis of α5 integrin subunit in a normal fetal human coronal suture in vivo. α5 integrin subunit immunoreactivity was localized in osteoblasts (arrows) along the bone matrix. Control sections incubated with IgG showed no specific staining (original magnification, ×125).

As human calvarial osteoblasts express FGFR2 in vitro and in vivo (Lemonnier et al., 2000; Kaabeche et al., 2004) and FGFR2 activation reduces α5 integrin subunit expression (Fig. 2A), we investigated whether FGFR2 might interact with α5 integrin in mutant osteoblasts. Immunoprecipitation analysis showed that α5 integrin co-immunoprecipitated with FGFR2 in control and FGFR2 mutant osteoblasts cultured on fibronectin (Fig. 3A). FGFR2 coimmunoprecipitated with α5 integrin in control and FGFR2 mutant osteoblasts, confirming the interaction between the two proteins (Fig. 3B). Both FGFR2 levels and α5 integrin levels were reduced in mutant cells, confirming the reduced α5 integrin expression (Fig. 2A) and the reduced FGFR2 expression in mutant osteoblasts (Kaabeche et al., 2004). To confirm that FGFR2 interacts with α5 integrin in osteoblasts at the cellular level, we performed an immunocytochemical analysis and found that FGFR2 mutant osteoblasts displayed lower adhesion and reduced cell spreading compared to control cells (Fig. 3C). Consistent with the biochemical results, the immunocytochemical analysis showed decreased α5 integrin levels in mutant cells. Moreover, we found that α5 integrin colocalized with FGFR2 at the leading edge of the cell in membrane ruffle regions (Fig. 3C), confirming the immunoprecipitation analysis. Overall, these data strongly indicate that α5 integrin subunit interacts with FGFR2 in human calvarial osteoblasts.

Fig. 3.

The α5 integrin subunit interacts with FGFR2 in osteoblasts. Cell lysates from control and FGFR2 mutant osteoblast cells were immunoprecipitated (IP) with α5 integrin (A) or FGFR2 antibodies (B), resolved by SDS-PAGE and blotted with anti-FGFR2 or anti-α5 integrin antibodies. (C) Control and FGFR2 mutant osteoblasts cultured on glass coverslips overnight in DMEM containing 10% FCS were fixed and stained with an anti-α5 integrin subunit antibody (green) or anti FGFR2 polyclonal antibody (red). Note the colocalization (yellow) of the α5 integrin subunit and FGFR2 in membrane ruffle regions (arrows). Bar, 10 μm.

Fig. 3.

The α5 integrin subunit interacts with FGFR2 in osteoblasts. Cell lysates from control and FGFR2 mutant osteoblast cells were immunoprecipitated (IP) with α5 integrin (A) or FGFR2 antibodies (B), resolved by SDS-PAGE and blotted with anti-FGFR2 or anti-α5 integrin antibodies. (C) Control and FGFR2 mutant osteoblasts cultured on glass coverslips overnight in DMEM containing 10% FCS were fixed and stained with an anti-α5 integrin subunit antibody (green) or anti FGFR2 polyclonal antibody (red). Note the colocalization (yellow) of the α5 integrin subunit and FGFR2 in membrane ruffle regions (arrows). Bar, 10 μm.

As α5 integrin mRNA levels are unchanged in FGFR2 mutant osteoblasts (Fig. 2B), we postulated that the reduced expression of the integrin might result from protein downregulation induced by FGFR2 activation. Ubiquitin-dependent proteasome degradation is an important process involved in protein downregulation. We therefore investigated whether the decreased α5 integrin levels in FGFR2 mutant cells may result from ubiquitin-mediated proteasome degradation. Treatment with the specific proteasome inhibitor lactacystin (10 μM) increased α5 integrin to normal levels in mutant osteoblasts and actually restored normal α5 integrin levels (Fig. 4A). This strongly suggests that the decreased expression of α5 integrin induced by FGFR2 activation results from increased proteasome-mediated degradation. The E3 ubiquitin ligase Cbl is known to interact with several molecules such as Src and tyrosine kinase receptors, resulting in their ubiquitination and proteasome degradation (Sanjay et al., 2001). Our recent data showed that FGFR2 activation induces Cbl recruitment, which contributes to Fyn, Lyn and FGFR2 degradation in osteoblasts (Kaabeche et al., 2004). We therefore hypothesized that Cbl recruitment induced by FGFR2 activation might also mediate α5 integrin proteasome degradation in mutant osteoblasts. As shown in Fig. 4B, immunoprecipitation analysis revealed that the ubiquitin ligase Cbl immunoprecipitated with α5 integrin in mutant cells, indicating an interaction between the two proteins. Activation of Cbl results in recruitment of ubiquitin, resulting in proteasome degradation. We therefore investigated whether α5 integrin can interact with ubiquitin in mutant osteoblasts and found that α5 integrin immunoprecipitates with ubiquitin, indicating that ubiquitin is associated with α5 integrin in this context (Fig. 4C). Moreover, the level of α5 integrin associated with ubiquitin was higher in FGFR2 mutant cells compared to control cells, suggesting increased ubiquitin-α5 integrin recruitment in mutant osteoblasts. To confirm the interaction between α5 integrin and Cbl, we performed an immunocytochemical analysis. We found that Cbl colocalized with α5 integrin at the leading edge of the cells, further indicating interactions between the two proteins (Fig. 5). Overall, these results show that α5 integrin subunit interacts with both FGFR2 and the ubiquitin ligase Cbl and that the α5 integrin subunit is targeted to proteasome degradation once associated with Cbl in mutant osteoblasts.

Fig. 4.

Cbl interacts with α5 integrin subunit and ubiquitin and is involved in α5 integrin proteasome degradation induced by FGFR2 activation. (A) FGFR2 mutant cells were treated with lactacystin for 24 hours, cell lysates were immunoprecipitated (IP) with α5 integrin antibody, resolved with SDS-PAGE and blotted with anti-α5 integrin. (B) Cell lysates from control and FGFR2 mutant osteoblasts were immunoprecipitated (IP) with Cbl antibody, resolved by SDS-PAGE and blotted with anti-α5 integrin antibody. (C) Cell lysates from control and FGFR2 mutant osteoblasts were immunoprecipitated (IP) with anti-α5 integrin, resolved by SDS-PAGE and blotted with anti-ubiquitin antibody.

Fig. 4.

Cbl interacts with α5 integrin subunit and ubiquitin and is involved in α5 integrin proteasome degradation induced by FGFR2 activation. (A) FGFR2 mutant cells were treated with lactacystin for 24 hours, cell lysates were immunoprecipitated (IP) with α5 integrin antibody, resolved with SDS-PAGE and blotted with anti-α5 integrin. (B) Cell lysates from control and FGFR2 mutant osteoblasts were immunoprecipitated (IP) with Cbl antibody, resolved by SDS-PAGE and blotted with anti-α5 integrin antibody. (C) Cell lysates from control and FGFR2 mutant osteoblasts were immunoprecipitated (IP) with anti-α5 integrin, resolved by SDS-PAGE and blotted with anti-ubiquitin antibody.

Fig. 5.

Colocalization of α5 integrin subunit and Cbl in osteoblasts. Control and FGFR2 mutant osteoblasts were cultured overnight on glass coverslips overnight in DMEM containing 10% FCS. After fixation, the cells were stained with an anti-α5 integrin subunit (green) or anti-Cbl (red). Note the colocalization of the α5 integrin subunit with Cbl in membrane ruffle regions (yellow). Bar, 10 μm.

Fig. 5.

Colocalization of α5 integrin subunit and Cbl in osteoblasts. Control and FGFR2 mutant osteoblasts were cultured overnight on glass coverslips overnight in DMEM containing 10% FCS. After fixation, the cells were stained with an anti-α5 integrin subunit (green) or anti-Cbl (red). Note the colocalization of the α5 integrin subunit with Cbl in membrane ruffle regions (yellow). Bar, 10 μm.

The RING finger of Cbl directs recruitment of the ubiquitin system and is important for protein ubiquitination induced by tyrosine kinase receptors (Waterman et al., 1999; Yokouchi et al., 1999). To determine whether Cbl-mediated α5 integrin ubiquitination in FGFR2 mutant osteoblasts is dependent on the RING domain of Cbl, FGFR2 mutant cells were transfected with 70Z-Cbl in which the RING finger is disrupted (Yokouchi et al., 2001) and changes in α5 integrin protein levels were determined. Transfection of FGFR2 mutant osteoblasts with 70Z-Cbl resulted in increased α5 integrin protein levels in mutant cells, which were restored to control levels, as revealed by immunoprecipitation analysis (Fig. 6). This reflects the inhibitory effect of 70Z-Cbl on ubiquitin recruitment and proteasome degradation of the α5 integrin. Moreover, this shows that the increased Cbl-dependent α5 integrin degradation induced by FGFR2 activation requires the presence of the RING finger domain of Cbl. The PTB domain in the N-terminal half of Cbl is another domain of Cbl that binds to activated tyrosine kinase receptors. We investigated the role of the Cbl PTB domain in Cbl-mediated α5 integrin degradation induced by FGFR2 activation in osteoblasts. Transfection of FGFR2 mutant cells with the Cbl G306E mutant in which a point mutation abolishes the binding ability of the Cbl PTB domain to the activated receptor increased α5 integrin protein levels in FGFR2 mutant cells (Fig. 6). This indicates that Cbl-mediated ubiquitination of α5 integrin is dependent on interaction with the PTB domain of Cbl which interacts with the activated FGFR2.

Fig. 6.

Cbl-mediated α5 integrin subunit proteasome degradation induced by FGFR2 activation requires the RING and PTB domains of Cbl. FGFR2 mutant osteoblasts were transfected with the 70Z-Cbl mutant, which lacks the RING domain required for Cbl-ubiquitin interaction, with the G306E Cbl mutant that inactivates the PTB domain of Cbl G306E, or with the empty vector (pcDNA). After 24 hours, cell lysates were immunoprecipitated (IP) with α5 integrin antibody, resolved by SDS-PAGE and blotted with anti-α5 integrin.

Fig. 6.

Cbl-mediated α5 integrin subunit proteasome degradation induced by FGFR2 activation requires the RING and PTB domains of Cbl. FGFR2 mutant osteoblasts were transfected with the 70Z-Cbl mutant, which lacks the RING domain required for Cbl-ubiquitin interaction, with the G306E Cbl mutant that inactivates the PTB domain of Cbl G306E, or with the empty vector (pcDNA). After 24 hours, cell lysates were immunoprecipitated (IP) with α5 integrin antibody, resolved by SDS-PAGE and blotted with anti-α5 integrin.

The above results indicate that FGFR2 activation reduces α5 integrin levels and osteoblast attachment as a consequence of Cbl-dependent degradation by the proteasome. We therefore assessed the functional implication of the observed α5 integrin downregulation induced by FGFR2 activation in osteoblasts. To this aim, FGFR2 mutant cells were transfected with an α5 integrin plasmid in order to rescue α5 integrin expression, and changes in α5 integrin levels and cell attachment on fibronectin were determined. Transfection of FGFR2 mutant osteoblasts with the α5 plasmid resulted in increased α5 integrin levels upon immunoprecipitation analysis (Fig. 7A). Interestingly, forced expression of α5 integrin in FGFR2 mutant cells fully rescued the defective cell attachment on fibronectin (Fig. 7B). This indicates that the reduced osteoblast attachment induced by FGFR2 activation is functionally related to α5 integrin downregulation.

Fig. 7.

Transfection with α5 integrin plasmid rescues osteoblast attachment on fibronectin in FGFR2 mutant osteoblasts. (A) FGFR2 mutant osteoblasts were transfected with α5 plasmid (pcDNA-α5) or the empty vector (pcDNA). After 24 hours, cell lysates were immunoprecipitated (IP) with anti-α5 integrin, resolved by SDS-PAGE and blotted with α5 integrin antibody. (B) FGFR2 mutant osteoblasts transfected with α5 integrin plasmid or empty vector were plated on fibronectin-coated dishes, cultured for 48 hours and the number of adherent cells was counted. Data are the mean±s.e.m. of six wells; a and b indicate a significant difference with control and pcDNA-mutant cells, respectively (P<0.05).

Fig. 7.

Transfection with α5 integrin plasmid rescues osteoblast attachment on fibronectin in FGFR2 mutant osteoblasts. (A) FGFR2 mutant osteoblasts were transfected with α5 plasmid (pcDNA-α5) or the empty vector (pcDNA). After 24 hours, cell lysates were immunoprecipitated (IP) with anti-α5 integrin, resolved by SDS-PAGE and blotted with α5 integrin antibody. (B) FGFR2 mutant osteoblasts transfected with α5 integrin plasmid or empty vector were plated on fibronectin-coated dishes, cultured for 48 hours and the number of adherent cells was counted. Data are the mean±s.e.m. of six wells; a and b indicate a significant difference with control and pcDNA-mutant cells, respectively (P<0.05).

Alteration of integrin-mediated cell attachment is associated with apoptosis in anchorage-dependent cells (Frisch and Screaton, 2001). Our previous data indicate that FGFR2 activation results in caspase-dependent apoptosis in human calvarial osteoblasts (Lemonnier et al., 2001b). We therefore investigated the implication of α5 integrin in caspase-dependent apoptosis induced by FGFR2 activation. To this goal, we examined the changes in Bax, a pro-apoptotic protein that is known to be involved in caspase-dependent apoptosis (Reed et al., 1996). Western blot analysis after immunoprecipitation showed a marked increase in Bax levels in FGFR2 mutant cells compared to control cells (Fig. 8A). Transfection of FGFR2 mutant osteoblasts with α5 integrin plasmid abolished the increased Bax levels in mutant cells, indicating that restoration of α5 integrin levels corrected the abnormal Bax levels in FGFR2 mutant osteoblasts. We also looked at Bcl-2 levels that are known to be involved in the protection against apoptosis mediated by α5β1 integrin in other cells (Zhang et al., 1995). Bcl-2 levels were slightly decreased in FGFR2 mutant osteoblasts compared to control cells, and forced expression of α5 integrin rescued Bcl-2 protein levels in mutant cells (Fig. 8B). These results indicate that the decreased α5 integrin expression can largely account for the alteration in Bax/Bcl-2 levels induced by FGFR2 activation in osteoblasts.

Fig. 8.

Restoration of α5 integrin levels abolishes caspase-dependent apoptosis induced by FGFR2 activation in osteoblasts. FGFR2 mutant osteoblasts were transfected with the empty vector (pcDNA) or the α5 integrin plasmid (pcDNA-α5) and cultured for 48 hours in medium with 1% FCS. Cell lysates were immunoprecipitated (IP) with Bax or Bcl-2 antibodies, resolved by SDS-PAGE and blotted with anti-Bax (A) or anti-Bcl-2 (B). Caspase-9 activity (C) and caspase-3 activity (D) were determined in FGFR2 mutant and control osteoblasts and the levels were corrected for protein content. Data are the mean±s.e.m. of three wells; a and b indicate a significant difference with control and pcDNA-mutant cells, respectively (P<0.05).

Fig. 8.

Restoration of α5 integrin levels abolishes caspase-dependent apoptosis induced by FGFR2 activation in osteoblasts. FGFR2 mutant osteoblasts were transfected with the empty vector (pcDNA) or the α5 integrin plasmid (pcDNA-α5) and cultured for 48 hours in medium with 1% FCS. Cell lysates were immunoprecipitated (IP) with Bax or Bcl-2 antibodies, resolved by SDS-PAGE and blotted with anti-Bax (A) or anti-Bcl-2 (B). Caspase-9 activity (C) and caspase-3 activity (D) were determined in FGFR2 mutant and control osteoblasts and the levels were corrected for protein content. Data are the mean±s.e.m. of three wells; a and b indicate a significant difference with control and pcDNA-mutant cells, respectively (P<0.05).

To further investigate the downstream mechanisms by which decreased α5 integrin expression may mediate apoptosis in mutant osteoblasts, we performed biochemical analyses to determine caspase-9 activity that is altered by changes in Bax/Bcl-2 ratio during apoptosis (Reed et al., 1996). Caspase-9 activity was increased in FGFR2 mutant osteoblasts compared to control cells, and transfection with the α5 integrin plasmid decreased caspase-9 activity which was restored to normal levels (Fig. 8C). To confirm the implication of α5 integrin in osteoblast apoptosis induced by FGFR2 activation, we investigated the changes in caspase-3, a key caspase involved in DNA fragmentation that is activated by caspase-9 (Reed et al., 1996). Caspase-3 activity was increased in FGFR2 mutant cells and transfection with the α5 integrin plasmid abolished the increased caspase-3 activity induced by FGFR2 activation (Fig. 8D). We conclude that rescue of α5 integrin expression in FGFR2 mutant osteoblasts restores cell attachment, corrects Bax/Bcl-2 to normal levels and abolishes the increased caspase-9 and caspase-3 activity induced by FGFR2 activation. This supports a functional role for α5 integrin downregulation in cell detachment and apoptosis induced by FGFR2 activation in osteoblasts.

Discussion

Although FGFR signaling plays an important role in osteoblast proliferation, differentiation and survival, the role of FGFR signaling in integrin expression and osteoblast attachment remains unknown. In this study, we investigated the role of FGFR2 in integrin-mediated osteoblast attachment and survival and the mechanisms involved in this effect. We used a cellular model characterized by an activating FGFR2 mutation that promotes apoptosis in osteoblasts (Manshukani et al., 2000; Lemonnier et al., 2001b). Using this model, we showed that FGFR2 activation reduces osteoblast attachment on fibronectin and type I collagen, indicating that activation of FGFR2 controls osteoblast adhesion on these bone matrix proteins. The finding that constitutive FGFR2 activation reduces cell attachment suggests a possible alteration of cell-matrix interaction mediated by integrins. Consistent with the reported expression in human osteoblasts in vitro and in vivo, we found that human calvarial osteoblasts express α5, αv and β1 integrin subunits. Recent data indicate that α5β1 integrin is mainly responsible for osteoblast adhesion on fibronectin (Nesti et al., 2002). Interestingly, we found that FGFR2 activation specifically induced downregulation of α5 integrin, which supports a key role for this integrin subunit in the alteration of osteoblast attachment on fibronectin induced by FGFR2 activation. This role of α5 integrin may not be restricted to fibronectin because fibronectin is known to play an integral role in osteoblast interaction with type I collagen (Weiss and Reddi, 1981).

In other cell types, α5β1 expression is regulated by several molecular mechanisms such as transcriptional activation (Delcommenne and Streuli, 1995), mRNA stability (Xu and Clark, 1996) and translational control (Harwood et al., 1999). In the present study, we show that α5 integrin mRNA levels were unchanged whereas α5 protein levels were decreased in FGFR2 mutant osteoblasts, suggesting that FGFR2 activation results in α5 integrin degradation. Ubiquitin-mediated proteasome degradation is an important mechanism controlling the degradation of many proteins (Hochstrasser, 1995). Our finding that specific inhibition of proteasome activity by lactacystin rescued α5 protein levels strongly indicates that α5 integrin downregulation induced by FGFR2 activation occurs through proteasome degradation. Ubiquitin-dependent degradation of proteins involves the ubiquitination of the target protein followed by its degradation by the proteasome (Ciechanover, 1998). The ubiquitin ligase Cbl plays a major role in protein degradation through the proteasome pathway (Sanjay et al., 2001). Here we show that the α5 integrin subunit interacts with both the ubiquitin ligase Cbl and ubiquitin in FGFR2 mutant osteoblasts, which indicates that FGFR2 activation induces Cbl-mediated α5 integrin recruitment, ubiquitination and subsequent degradation via the proteasome. Our finding that transfection with 70Z-Cbl, which lacks the RING domain required for Cbl interaction with ubiquitin restored α5 integrin protein levels, further indicates that Cbl-mediated ubiquitination plays an essential role in proteasomal degradation of the α5 integrin subunit. Because the G306E Cbl mutant, that inactivates the PTB domain of Cbl, rescued α5 protein expression in FGFR2 mutant cells, it appears that the PTB domain of Cbl is involved in the Cbl-mediated downregulation of the α5 subunit in response to ligand-independent, constitutive activation of FGFR2. These data indicate that constitutive FGFR2 activation in osteoblasts activates the ubiquitin ligase activity of Cbl, resulting in ubiquitination and proteasome degradation of the α5 subunit integrin. This provides a Cbl-dependent mechanism by which the α5 integrin protein is downregulated in response to activation of FGFR2 in osteoblasts.

Loss of cell attachment to the extracellular matrix triggers apoptosis through several signaling mechanisms in anchorage-dependent cells (Frisch and Screaton, 2001). It was therefore of interest to determine if the Cbl-dependent downregulation of α5 integrin and subsequent reduction in cell attachment may contribute functionally to osteoblast apoptosis induced by FGFR2 activation. One mechanism of apoptosis involves alteration of Bax/Bcl-2, which triggers cytochrome c release from the mitochondria and activates caspase-9 (Reed et al., 1996). We found that the decreased expression of α5 integrin subunit in FGFR2 mutant osteoblasts was associated with an increased Bax/Bcl-2 ratio and increased caspase-9 and caspase-3 activities, indicating that the alteration of α5 integrin-mediated cell attachment triggers caspase-dependent apoptosis in osteoblasts. This is consistent with the finding in other cells that the α5β1 integrin supports cell survival on fibronectin by increasing Bcl-2 protein transcription (Zhang et al., 1995; Matter and Ruoslahti, 2001). Our finding that forced expression of α5 integrin restored the Bax/Bcl-2 ratio and corrected caspase-9 and caspase-3 activities in FGFR2 mutant osteoblasts strongly indicates that the selective alteration of α5 integrin induced by FGFR2 activation governs apoptotic signals in osteoblasts through Bax/Bcl-2 and activation of the caspase-9-caspase-3 cascade. Thus, Cbl-mediated ubiquitination of the α5 integrin subunit appears to play a major role in the induction of apoptosis induced by FGFR2 activation in osteoblasts. Phosphatidylinositol 3-kinase (PI3K) was shown to be involved in cell survival mediated by α5β1 expression in epithelial cells (Lee and Juliano, 2000). It is interesting to note that activated Cbl forms complexes with PI3K following integrin-mediated cell adhesion (Ojaniemi et al., 1997; Zell et al., 1998) and is involved in PI3K-dependent cell signaling (Meng and Lowell, 1998; Anzai et al., 1999; Finkelstein and Shimizu, 2000). In bone, we recently showed that FGF2 induces cell survival through PI3K signaling in human calvarial osteoblasts (Debiais et al., 2004). The elucidation of the role of this and other signaling molecules acting downstream of FGFR2-Cbl-α5 integrin to trigger osteoblast apoptosis may have important implications with regard to the control of osteogenesis by FGFR signaling.

In summary, our data indicate that Cbl recruitment induced by FGFR2 activation triggers α5 integrin proteasome degradation, which results in reduced osteoblast attachment on fibronectin- and caspase-dependent apoptosis. This supports a functional role of the α5 integrin subunit in the induction of apoptosis triggered by FGFR2 activation. Furthermore, the data point to a novel Cbl-dependent mechanism involved in the coordinate regulation of cell apoptosis induced by α5 integrin degradation in response to FGFR2 signaling in osteoblasts.

Acknowledgements

The authors wish to thank A. Sanjey and R. Baron (Dept of Cell Biology, Yale University School of Medicine, New Haven, CT) for the gift of Cbl mutant plasmids. K.K. is a recipient of a scholarship from the Ministère de la Recherche et de la Technologie (France). H.G. is a recipient of a scholarship from the Association Rhumatisme et Travail (Centre Viggo-Petersen, Hôpital Lariboisière, Paris, France).

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