Kaabeche Karim, Guenou Hind, Bouvard Daniel, Didelot Nadège, Listrat Antoine, Marie Pierre J
INSERM U 606, Lariboisière Hospital, 2 rue Ambroise Paré, 75010 Paris, Université Paris 7, Paris, France.
J Cell Sci. 2005 Mar 15;118(Pt 6):1223-32. doi: 10.1242/jcs.01679. Epub 2005 Feb 22.
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.
成纤维细胞生长因子受体信号传导是调节成骨细胞功能的重要机制。为深入了解成纤维细胞生长因子受体2（FGFR2）信号传导在成骨细胞中的调节作用，我们研究了表达活化FGFR2的人颅骨成骨细胞中整合素介导的黏附及细胞存活情况。FGFR2激活减少了成骨细胞在纤连蛋白上的黏附。这与体内人颅骨成骨细胞中正常表达的α5整合素亚基表达减少有关。用蛋白酶体的强效抑制剂乳胞素处理可恢复FGFR2突变成骨细胞中的α5整合素水平。免疫沉淀分析表明，α5整合素与E3泛素连接酶Cbl及泛素相互作用。免疫细胞化学显示，α5整合素与FGFR2和Cbl在膜皱褶区域的前沿共定位。用缺乏Cbl-泛素相互作用所需RING结构域的70Z-Cbl突变体或消除Cbl磷酸酪氨酸结合结构域结合能力的G306E Cbl突变体转染可恢复α5整合素水平。这表明Cbl介导的泛素化在FGFR2激活诱导的α5整合素蛋白酶体降解中起重要作用。α5整合素表达减少与FGFR2突变成骨细胞中Bax/Bcl-2比值增加及caspase-9和-3活性增加有关。α5整合素的强制表达挽救了细胞黏附，并纠正了FGFR2突变成骨细胞中的Bax/Bcl-2比值及caspase-3和caspase-9活性。我们发现，FGFR2激活诱导的Cbl募集触发蛋白酶体介导的α5整合素降解，这导致成骨细胞在纤连蛋白上的黏附减少及caspase依赖性凋亡。这确定了α5整合素亚基在成骨细胞中FGFR2激活触发的凋亡诱导中的功能作用，并揭示了一种Cbl依赖性机制参与α5整合素降解诱导的细胞凋亡的协调调节。
