Cbl-mediated ubiquitination of α5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation
Cbl-mediated ubiquitination of α5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation
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DOI：
10.1242/jcs.01679
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发表时间：
2005-03-15
影响因子：
4
通讯作者：
Marie, PJ
中科院分区：
文献类型：
--
作者：
Kaabeche, K;Guenou, H;Marie, PJ
成纤维细胞生长因子受体信号转导是调控成骨细胞功能的重要机制。为了深入了解成骨细胞中FGF受体-2（FGFR 2）信号转导的调节作用，我们研究了整合素介导的表达活化FGFR 2的人颅骨成骨细胞的附着和细胞存活。FGFR 2活化减少成骨细胞在纤连蛋白上的附着。这与体内人颅骨成骨细胞中正常表达的α 5整合素亚基的表达减少有关。用一种有效的蛋白酶体抑制剂lactacystin治疗，恢复了FGFR 2突变型破骨细胞中α 5整联蛋白的水平。免疫沉淀分析表明，α 5整联蛋白与E3泛素连接酶Cbl和泛素相互作用。免疫细胞化学显示，α 5整合素与FGFR 2和Cbl共定位在膜皱褶区域的前缘。用缺乏Cbl-泛素相互作用所需的RING结构域的70 Z-Cbl突变体或用消除Cbl磷酸酪氨酸结合结构域的结合能力的G306 E Cbl突变体转染恢复了α 5，整联蛋白水平。这表明Cbll介导的遍在化在FGFR 2激活诱导的α 5整联蛋白蛋白酶体降解中发挥重要作用。在FGFR 2突变成骨细胞中，α 5整合素表达减少与Bax/Bel-2比值增加以及caspase-9和-3活性增加相关。在FGFR 2突变成骨细胞中，α 5整联蛋白的强制表达挽救了细胞附着并校正了Bax/Bcl-2比率和半胱天冬酶-3和半胱天冬酶-9活性。我们发现，由FGFR 2激活诱导的Cbl募集通过蛋白酶体触发α 5整合素降解，这导致成骨细胞对纤连蛋白的附着减少和半胱天冬酶依赖性细胞凋亡。这鉴定了α 5整联蛋白亚基在诱导成骨细胞中由FGFR 2活化触发的细胞凋亡中的功能作用，并揭示了Cbl依赖性机制参与了α 5整联蛋白降解诱导的细胞凋亡的协调调节。
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 alpha 5 integrin subunit normally expressed in human calvarial osteoblasts in vivo. Treatment with lactacystin, a potent inhibitor of proteasome, restored alpha 5 integrin levels in FGFR2 mutant osteohlasts. Immunoprecipitation analysis showed that alpha 5 integrin interacts with both the E3 ubiquitin ligase Cbl and ubiquitin. Immunocytochemistry revealed that alpha 5 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 alpha 5, integrin levels. This suggests that Cbl-mediated ubiquitination plays an essential role in a5 integrin proteasome degradation induced by FGFR2 activation. Reduced alpha 5 integrin expression was associated with an increased Bax/Bel-2 ratio and increased caspase-9 and -3 activities in FGFR2 mutant osteoblasts. Forced expression of alpha 5 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 alpha 5 integrin degradation by the proteasome, which results in reduced osteoblast attachment on fibronectin and caspase-dependent apoptosis. This identifies a functional role of the alpha 5 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 alpha 5 integrin degradation.