Fibronectin (FN) assembly into an insoluble fibrillar matrix is a crucial step in many cell responses to extracellular matrix (ECM) properties, especially with regards to the integrin-related mechanosensitive signaling pathway. We have previously reported that the silencing of expression of integrin-linked kinase (ILK) in human intestinal epithelial crypt (HIEC) cells causes significant reductions in proliferation and spreading through concomitantly acquired impairment of soluble FN deposition. These defects in ILK-depleted cells are rescued by growth on exogenous FN. In the present study we investigated the contribution of ILK in the fibrillogenesis of FN and its relation to integrin-actin axis signaling and organization. We show that de novo fibrillogenesis of endogenous soluble FN is ILK-dependent. This function seemingly induces the assembly of an ECM that supports increased cytoskeletal tension and the development of a fully spread contractile cell phenotype. We observed that HIEC cell adhesion to exogenous FN or collagen-I (Col-I) is sufficient to restore fibrillogenesis of endogenous FN in ILK-depleted cells. We also found that optimal engagement of the Ras homolog gene family member A (RhoA) GTPase/Rho-associated kinase (ROCK-1, ROCK-2)/myosin light chain (MLC) pathway, actin ventral stress fiber formation, and integrin adhesion complex (IAC) maturation rely primarily upon the cell’s capacity to execute FN fibrillogenesis, independent of any significant ILK input. Lastly, we confirm the integrin α5β1 as the main integrin responsible for FN assembly, although in ILK-depleted cells αV-class integrins expression is needed to allow the rescue of FN fibrillogenesis on exogenous substrate. Our study demonstrates that ILK specifically induces the initiation of FN fibrillogenesis during cell spreading, which promotes RhoA/ROCK-dependent cell contractility and maturation of the integrin-actin axis structures. However, the fibrillogenesis process and its downstream effect on RhoA signaling, cell contractility and spreading are ILK-independent in human intestinal epithelial crypt cells.

中文翻译：

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ILK 通过在扩散过程中诱导内源性可溶性纤连蛋白的原纤维形成，支持 RhoA/ROCK 介导的人肠上皮隐窝细胞的收缩性


纤连蛋白 (FN) 组装成不溶性纤维状基质是许多细胞对细胞外基质 (ECM) 特性作出反应的关键步骤，特别是在整合素相关的机械敏感信号通路方面。我们之前曾报道，人肠上皮隐窝 (HIEC) 细胞中整合素连接激酶 (ILK) 表达的沉默会通过伴随的可溶性 FN 沉积受损而导致增殖和扩散显着减少。 ILK 耗尽的细胞中的这些缺陷可通过外源 FN 的生长来修复。在本研究中，我们研究了 ILK 在 FN 纤维形成中的贡献及其与整合素肌动蛋白轴信号传导和组织的关系。我们发现内源性可溶性 FN 的从头原纤维形成是 ILK 依赖性的。这一功能似乎诱导了 ECM 的组装，支持细胞骨架张力的增加和完全扩散的收缩细胞表型的发展。我们观察到，HIEC 细胞与外源 FN 或胶原蛋白 I (Col-I) 的粘附足以恢复 ILK 耗尽细胞中内源 FN 的纤维形成。我们还发现 Ras 同源基因家族成员 A (RhoA) GTPase/Rho 相关激酶 (ROCK-1、ROCK-2)/肌球蛋白轻链 (MLC) 通路、肌动蛋白腹侧应力纤维形成和整合素粘附的最佳结合复合物 (IAC) 的成熟主要依赖于细胞执行 FN 纤维生成的能力，与任何重要的 ILK 输入无关。最后，我们确认整合素α5β1是负责FN组装的主要整合素，尽管在ILK耗尽的细胞中需要αV类整合素表达来拯救外源性底物上的FN纤维形成。 我们的研究表明，ILK 在细胞扩散过程中特异性诱导 FN 纤维形成的启动，从而促进 RhoA/ROCK 依赖性细胞收缩性和整合素-肌动蛋白轴结构的成熟。然而，在人肠上皮隐窝细胞中，原纤维形成过程及其对 RhoA 信号传导、细胞收缩性和扩散的下游影响是不依赖于 ILK 的。