title: Cbl-mediated ubiquitination of alpha5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation.
abstract: 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 alpha5 integrin subunit normally expressed in human calvarial osteoblasts in vivo. Treatment with lactacystin, a potent inhibitor of proteasome, restored alpha5 integrin levels in FGFR2 mutant osteoblasts. Immunoprecipitation analysis showed that alpha5 integrin interacts with both the E3 ubiquitin ligase Cbl and ubiquitin. Immunocytochemistry revealed that alpha5 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 alpha5 integrin levels. This suggests that Cbl-mediated ubiquitination plays an essential role in alpha5 integrin proteasome degradation induced by FGFR2 activation. Reduced alpha5 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 alpha5 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 alpha5 integrin degradation by the proteasome, which results in reduced osteoblast attachment on fibronectin and caspase-dependent apoptosis. This identifies a functional role of the alpha5 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 alpha5 integrin degradation.
authors.affiliation: INSERM U 606, Lariboisière Hospital, 2 rue Ambroise Paré, 75010 Paris, Université Paris 7, Paris, France.
authors.forename: Karim
authors.initials: K
authors.lastname: Kaabeche
authors.forename: Hind
authors.initials: H
authors.lastname: Guenou
authors.forename: Daniel
authors.initials: D
authors.lastname: Bouvard
authors.forename: Nadège
authors.initials: N
authors.lastname: Didelot
authors.forename: Antoine
authors.initials: A
authors.lastname: Listrat
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Marie
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000168:Acrocephalosyndactylia / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D039482:Integrin alpha5 / Q000737:chemistry / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009154:Mutation; D010006:Osteoblasts / Q000378:metabolism*; D019000:Phosphotyrosine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D012333:RNA, Messenger / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012886:Skull / Q000196:embryology / Q000473:pathology; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D005353:Fibronectins; D039482:Integrin alpha5; D061988:Proteasome Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D025801:Ubiquitin; D051028:bcl-2-Associated X Protein; D005973:Glutamine; C067713:lactacystin; D019000:Phosphotyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C496353:FGFR2 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine