PDF(Pubmed)
Abstract：
The integrin αvβ6 is expressed at low levels in most normal healthy tissue but is very often upregulated in a disease context including cancer and fibrosis. Integrins use endocytosis and trafficking as a means of regulating their surface expression and thus their functions, however little is known of how this process is regulated in the context of αvβ6. As αvβ6 is a major target for the development of therapeutics in cancer and fibrosis, understanding these dynamics is critical in the development of αvβ6-targeted therapies. Following development of a flow cytometry-based assay to measure ligand (A20FMDV2 or LAP)-bound αvβ6 endocytosis, an siRNA screen was performed to identify which genes were responsible for internalising αvβ6. These data identified 15 genes (DNM2, CBLB, DNM3, CBL, EEA1, CLTC, ARFGAP3, CAV1, CYTH2, CAV3, CAV2, IQSEC1, AP2M1, TSG101) which significantly decreased endocytosis, predominantly within dynamin-dependent pathways. Inhibition of these dynamin-dependent pathways significantly reduced αvβ6-dependent migration (αvβ6-specific migration was 547 ± 128 under control conditions, reduced to 225 ± 73 with clathrin inhibition, and 280 ± 51 with caveolin inhibition). Colocalization studies of αvβ6 with endosome markers revealed that up to 6 h post-internalisation of ligand, αvβ6 remains in Rab11-positive endosomes in a perinuclear location, with no evidence of αvβ6 degradation up to 48 h post exposure to A20FMDV2. Additionally, 60% of ligand-bound αvβ6 was recycled back to the surface by 6 h. With studies ongoing using conjugated A20FMDV2 to therapeutically target αvβ6 in cancer and fibrosis, these data have important implications. Binding of A20FMDV2 seemingly removes much of the αvβ6 from the cell membrane, and upon its recycling, a large fraction appears to still be in the ligand-bound state. While these results are observed with A20FMDV2, these data will be of value in the design of αvβ6-specific therapeutics and potentially the types of therapeutic load.
摘要：
整联蛋白αvβ6在大多数正常健康组织中以低水平表达,但在包括癌症和纤维化的疾病背景下经常上调。整合素使用胞吞和运输作为调节其表面表达的手段，从而调节其功能，然而,对于该过程在αvβ6的背景下是如何调节的知之甚少。由于αvβ6是癌症和纤维化治疗发展的主要目标，理解这些动力学对于αvβ6靶向治疗的发展至关重要。在开发基于流式细胞术的测定以测量配体（A20FMDV2或LAP）结合的αvβ6内吞作用之后，进行siRNA筛选以鉴定哪些基因负责内化αvβ6.这些数据确定了15个基因(DNM2，CBLB，DNM3,CBL,EEA1,CLTC,ARFGAP3、CAV1、CYTH2、CAV3、CAV2、IQSEC1、AP2M1、TSG101)显著降低胞吞作用，主要在动态蛋白依赖性途径内。抑制这些动态蛋白依赖性途径显着降低了αvβ6依赖性迁移（在对照条件下，αvβ6特异性迁移为547±128，抑制网格蛋白降低到225±73，和280±51，具有小窝蛋白抑制）。αvβ6与内体标记物的共定位研究表明，配体内化后长达6小时，αvβ6保留在核周围位置的Rab11阳性内体中，在暴露于A20FMDV2后48小时内没有αvβ6降解的证据。此外,6小时后，60%的配体结合的αvβ6被循环回表面。正在进行的研究使用缀合的A20FMDV2在癌症和纤维化中治疗靶向αvβ6，这些数据具有重要意义。A20FMDV2的结合似乎从细胞膜上去除大部分αvβ6，在回收利用时，大部分似乎仍处于配体结合状态。虽然用A20FMDV2观察到这些结果,但是这些数据在αvβ6特异性治疗剂的设计和潜在的治疗负荷的类型中将是有价值的。
