Ephrin-B3 ligand promotes glioma invasion through activation of Rac1
Ephrin-B3 ligand promotes glioma invasion through activation of Rac1
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DOI：
10.1158/0008-5472.can-05-4211
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发表时间：
2006-09-01
期刊：
影响因子：
11.2
通讯作者：
Berens, Michael E.
中科院分区：
文献类型：
--
作者：
Nakada, Mitsutoshi;Drake, Kelsey L.;Berens, Michael E.
Eph受体酪氨酸激酶参与神经系统发育。Eph配体是一种与Eph受体结合的跨膜蛋白，Eph受体的相互激活会在相互接触的细胞中产生排斥效应。此前，我们发现EphB2在胶质瘤细胞中的过表达增加了细胞的侵袭力。在这里，通过激光捕获显微切割收集的四种胶质瘤细胞系和侵袭性胶质母细胞瘤细胞中，测定了ePhin-B家族成员的表达谱。在4个胶质瘤细胞系中有4个的迁移细胞(1.3~1.7倍)和8个活检标本中的8个(1.2~10.0倍)的侵袭性肿瘤细胞中，ePhrin-B3的mRNA表达上调。在低表达细胞系(U87、T98G)中强制表达eaffin-B3可刺激细胞在体内外的迁移和侵袭，并伴随着eaffin-B3的酪氨酸磷酸化。在高表达细胞系(U251、SNB19)中，ePhrin-B3与rac1共定位于可移动的野生型细胞的片状脂膜。小干扰RNA(SiRNA)基因转染的细胞形态发生明显改变，体内外侵袭能力降低。EphB2/Fc诱导的侵袭力增加与EphB2/Fc诱导的侵袭力增加有关，内源性Ephin-B3表达的缺失抑制了EphB2/Fc诱导的侵袭性增加，提示侵袭力的增加依赖于EPhin-B3的激活。此外，使用rac1-GTP下拉实验，我们发现ePhin-B3与rac1的激活有关。通过siRNA降低rac1的表达可抵消EphB2/Fc增加的侵袭力。在人脑胶质瘤标本中，eaffin-B3的表达和磷酸化与肿瘤分级的增加相关。免疫组织化学染色显示侵袭性胶质母细胞瘤细胞中有强阳性的磷酸化ephin-B和ephin-B3染色。这些数据表明，ePhin-B3的表达和通过rac1的信号转导对胶质瘤的侵袭至关重要。
Eph receptor tyrosine kinases are involved in nervous system development. Eph ligands, termed ephrins, are transmembrane proteins that bind to Eph receptors, the mutual activation of which causes repulsive effects in reciprocally contacting cells. Previously, we showed that overexpression of EphB2 in glioma cells increases cell invasion. Here, expression profiles of ephrin-B family members were determined in four glioma cell lines and in invading glioblastoma cells collected by laser capture microdissection. Ephrin-B3 mRNA was up-regulated in migrating cells of four of four glioma cell lines (1.3- to 1.7-fold) and in invading tumor cells of eight of eight biopsy specimens (1.2- to 10.0-fold). Forced expression of ephrin-B3 in low expressor cell lines (U87, T98G) stimulated cell migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B3. In high expressor cell lines (U251, SNB19), ephrin-B3 colocalized with Rac1 to lamellipodia of motile wild-type cells. Cells transfected with ephrin-B3 small interfering RNA (siRNA) showed significant morphologic change and decreased invasion in vitro and ex vivo. Depletion of endogenous ephrin-B3 expression abrogated the increase of migration and invasion induced by EphB2/Fc, indicating increased invasion is dependent on ephrin-B3 activation. Furthermore, using a Rac1-GTP pull-down assay, we showed that ephrin-B3 is associated with Rac1 activation. Reduction of Rac1 by siRNA negated the increased invasion by addition of EphB2/Fc. In human glioma specimens, ephrin-B3 expression and phosphorylation correlated with increasing tumor grade. Immunohistochemistry revealed robust staining for phosphorylated ephrin-B and ephrin-B3 in invading glioblastoma cells. These data show that ephrin-B3 expression and signaling through Rac1 are critically important to glioma invasion.