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First published online 22 February 2005
doi: 10.1242/jcs.01679

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Cbl-mediated ubiquitination of 5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation

¹ INSERM U 606, Lariboisière Hospital, 2 rue Ambroise Paré, 75010 Paris, Université Paris 7, Paris, France
² LEDAC, UMR CNRS/UJF 5538, Institut Albert Bonniot, Faculté de Médecine, 38706 La Tronche CEDEX, France

View larger version (24K): [in a new window]   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.

View larger version (71K): [in a new window]   Fig. 2. Expression levels of 5, v and ß1 integrins in FGFR2 mutant and control (wild type) osteoblasts. (A) Cells were subjected to western blot analysis using specific antibodies against 5, v and ß1 integrins. Equal loading was confirmed by detecting levels of ß-actin. (B) Expression of transcripts for 5, v and ß1 integrins was determined by RT-PCR analysis and GAPDH was used as an internal control. (C) Immunohistochemical analysis of 5 integrin subunit in a normal fetal human coronal suture in vivo. 5 integrin subunit immunoreactivity was localized in osteoblasts (arrows) along the bone matrix. Control sections incubated with IgG showed no specific staining (original magnification, x125).

View larger version (39K): [in a new window]   Fig. 3. The 5 integrin subunit interacts with FGFR2 in osteoblasts. Cell lysates from control and FGFR2 mutant osteoblast cells were immunoprecipitated (IP) with 5 integrin (A) or FGFR2 antibodies (B), resolved by SDS-PAGE and blotted with anti-FGFR2 or anti-5 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-5 integrin subunit antibody (green) or anti FGFR2 polyclonal antibody (red). Note the colocalization (yellow) of the 5 integrin subunit and FGFR2 in membrane ruffle regions (arrows). Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 4. Cbl interacts with 5 integrin subunit and ubiquitin and is involved in 5 integrin proteasome degradation induced by FGFR2 activation. (A) FGFR2 mutant cells were treated with lactacystin for 24 hours, cell lysates were immunoprecipitated (IP) with 5 integrin antibody, resolved with SDS-PAGE and blotted with anti-5 integrin. (B) Cell lysates from control and FGFR2 mutant osteoblasts were immunoprecipitated (IP) with Cbl antibody, resolved by SDS-PAGE and blotted with anti-5 integrin antibody. (C) Cell lysates from control and FGFR2 mutant osteoblasts were immunoprecipitated (IP) with anti-5 integrin, resolved by SDS-PAGE and blotted with anti-ubiquitin antibody.

View larger version (47K): [in a new window]   Fig. 5. Colocalization of 5 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-5 integrin subunit (green) or anti-Cbl (red). Note the colocalization of the 5 integrin subunit with Cbl in membrane ruffle regions (yellow). Bar, 10 µm.

View larger version (48K): [in a new window]   Fig. 6. Cbl-mediated 5 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 5 integrin antibody, resolved by SDS-PAGE and blotted with anti-5 integrin.

View larger version (32K): [in a new window]   Fig. 7. Transfection with 5 integrin plasmid rescues osteoblast attachment on fibronectin in FGFR2 mutant osteoblasts. (A) FGFR2 mutant osteoblasts were transfected with 5 plasmid (pcDNA-5) or the empty vector (pcDNA). After 24 hours, cell lysates were immunoprecipitated (IP) with anti-5 integrin, resolved by SDS-PAGE and blotted with 5 integrin antibody. (B) FGFR2 mutant osteoblasts transfected with 5 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).

View larger version (38K): [in a new window]   Fig. 8. Restoration of 5 integrin levels abolishes caspase-dependent apoptosis induced by FGFR2 activation in osteoblasts. FGFR2 mutant osteoblasts were transfected with the empty vector (pcDNA) or the 5 integrin plasmid (pcDNA-5) 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).