Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin, USA.
Invest Ophthalmol Vis Sci. 2011 Mar 25;52(3):1684-92. doi: 10.1167/iovs.10-6397. Print 2011 Mar.
To determine whether integrin-linked kinase (ILK) controls the organization of the actin cytoskeleton in the trabecular meshwork (TM) by regulating integrin co-signaling.
The cell binding domain and the Heparin II (Hep II) domain of fibronectin were used to activate α5β1 and α4β1 integrin signaling, respectively, in differentiated human TM (HTM) cells. The role of ILK was determined using either ILK small interfering RNA (siRNA) to knockout ILK expression or the ILK inhibitors, KP392 and QLT0267. The knockdown of ILK expression was verified by Western blot analysis. The presence of actin stress fibers and focal adhesions was determined by labeling cultures with phalloidin and anti-talin or ILK antibodies, respectively.
Cell spreading in differentiated HTM cells required ILK, since ILK siRNA and the ILK inhibitors significantly reduced cell spreading, actin polymerization, and the localization of talin and ILK in focal adhesions (FAs). Both cell spreading and the localization of talin and ILK to FAs in differentiated HTM cells could be rescued by inducing α4β1 integrin signaling with a recombinant Hep II domain of fibronectin, even though α4β1 integrins were not found in FAs. In the absence of ILK inhibition, the Hep II domain had minimal effect on α5β1 integrin-mediated spreading.
This study suggests that cooperative α5β1/α4β1 integrin signaling may be regulated by ILK trans-dominantly and that alterations in ILK activity may affect actin cytoskeleton organization and contractility in the TM.
通过调节整合素共信号，确定整合素连接激酶（ILK）是否通过控制小梁网（TM）中肌动蛋白细胞骨架的组织来控制其功能。
分别使用纤连蛋白的细胞结合域和肝素 II（Hep II）域来激活α5β1 和 α4β1 整合素信号。使用 ILK 小干扰 RNA（siRNA）敲除 ILK 表达或使用 ILK 抑制剂 KP392 和 QLT0267 来确定 ILK 的作用。通过 Western blot 分析验证 ILK 表达的敲低。通过用鬼笔环肽和抗塔林或 ILK 抗体分别对培养物进行标记来确定肌动蛋白应力纤维和黏着斑的存在。
在分化的 HTM 细胞中，细胞铺展需要 ILK，因为 ILK siRNA 和 ILK 抑制剂显著降低了细胞铺展、肌动蛋白聚合以及黏着斑（FA）中塔林和 ILK 的定位。即使在 FA 中未发现 α4β1 整合素，用纤连蛋白的重组 Hep II 结构域诱导 α4β1 整合素信号也可以挽救分化的 HTM 细胞中的细胞铺展以及塔林和 ILK 向 FA 的定位。在没有 ILK 抑制的情况下，Hep II 结构域对 α5β1 整合素介导的铺展的影响很小。
本研究表明，协同的α5β1/α4β1 整合素信号可能通过 ILK 反式显性调节，ILK 活性的改变可能影响 TM 中的肌动蛋白细胞骨架组织和收缩性。
