Activation of αvβ3 Integrin Alters Fibronectin Fibril Formation in Human Trabecular Meshwork Cells in a ROCK-Independent Manner
Activation of αvβ3 Integrin Alters Fibronectin Fibril Formation in Human Trabecular Meshwork Cells in a ROCK-Independent Manner
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DOI：
10.1167/iovs.19-27171
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发表时间：
2019-09-01
影响因子：
4.4
通讯作者：
Peters, Donna M.
中科院分区：
文献类型：
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作者：
Filla, Mark S.;Faralli, Jennifer A.;Peters, Donna M.
目的。纤维连接蛋白纤维化是一种整合素介导的过程，可能与原发性开角型青光眼（POAG）的发病有关。在这里，我们研究了α v β 3整合素对永生化TM-1细胞和人小梁网（HTM）细胞纤维形成的影响。产生过表达野生型β 3 （WT β 3）或组成型活性β 3 （CA β 3）整合素亚基的TM-1细胞。对照细胞用空载体（EV）转导。采用脱氧胆酸（DOC）单层提取、免疫荧光显微镜和细胞上western分析来测定纤维连接蛋白纤维形成水平和纤维连接蛋白纤维组成（EDA+和EDB+纤维连接蛋白）和构象。采用荧光活化细胞分选（FACS）测定α v β 3和α 5 β 1整合素水平。用西仑吉肽和表达WT β 3或CA β 3整合素亚基的腺病毒载体检测α v β 3整合素在HTM细胞中的作用。使用纤维连接蛋白结合肽FUD、β 1整合素功能阻断抗体13和Rho激酶（ROCK）抑制剂y27632来确定典型α 5 β 1整合素介导的途径在纤维形成中的作用。在TM-1和HTM细胞中，α v β 3整合素的激活增强了纤维连接蛋白向doc不溶性原纤维的组装。纤维连接蛋白原纤维的形成依赖于α 5 β 1整合素，可被FUD抑制。然而，Y27632不影响纤维形成。CA - 3细胞组装的原纤维也含有高水平的EDA+和EDB+纤维连接蛋白，以及被拉伸的纤维连接蛋白。α v β 3整合素信号通过β 1整合素/ rock独立机制改变纤维连接蛋白原纤维的沉积和结构。因此，α v β 3整合素可能在改变POAG中纤维连接蛋白基质的功能中发挥重要作用。
PURPOSE. Fibronectin fibrillogenesis is an integrin-mediated process that may contribute to the pathogenesis of primary open-angle glaucoma (POAG). Here, we examined the effects of alpha v beta 3 integrins on fibrillogenesis in immortalized TM-1 cells and human trabecular meshwork (HTM) cells.METHODS. TM-1 cells overexpressing wild-type beta 3 (WT beta 3) or constitutively active beta 3 (CA beta 3) integrin subunits were generated. Control cells were transduced with an empty vector (EV). Deoxycholic acid (DOC) extraction of monolayers, immunofluorescence microscopy, and On-cell western analyses were used to determine levels of fibronectin fibrillogenesis and fibronectin fibril composition (EDA+ and EDB+ fibronectins) and conformation. alpha v beta 3 and alpha 5 beta 1 Integrin levels were determined using fluorescence-activated cell sorting (FACS). Cilengitide and an adenovirus vector expressing WT beta 3 or CA beta 3 integrin subunits were used to examine the role of alpha v beta 3 integrin in HTM cells. The role of the canonical alpha 5 beta 1 integrin-mediated pathway in fibrillogenesis was determined using the fibronectin-binding peptide FUD, the beta 1 integrin function-blocking antibody 13, and the Rho kinase (ROCK) inhibitor Y27632.RESULTS. Activation of alpha v beta 3 integrin enhanced the assembly of fibronectin into DOC-insoluble fibrils in both TM-1 and HTM cells. The formation of fibronectin fibrils was dependent on alpha 5 beta 1 integrin and could be inhibited by FUD. However, fibrillogenesis was unaffected by Y27632. Fibrils assembled by CA beta 3 cells also contained high levels of EDA+ and EDB+ fibronectin and fibronectin that was stretched.CONCLUSIONS. alpha v beta 3 Integrin signaling altered the deposition and structure of fibronectin fibrils using a beta 1 integrin/ROCK-independent mechanism. Thus, alpha v beta 3 integrins could play a significant role in altering the function of fibronectin matrices in POAG.