Abstract
BackgroundFibronectin (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.ResultsWe 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.ConclusionOur 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.
Highlights
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
In the present study, considering the importance of FN for the intestinal crypt ECM/niche [27] and that integrin-linked kinase (ILK) contribution to the regulation of intestinal epithelial cell functions is related to FN deposition or adhesion [26, 28,29,30,31], we further investigated the functional linkage between ILK and FN for the regulation of integrin-actin axis dynamics in the context of human intestinal epithelial crypt (HIEC) cell spreading
Western blot (WB) and immunofluorescence (IF) staining confirmed that like parental HIEC cells [26], subconfluent cells transfected with a control, non-interfering Small interfering ribonucleic acid (siRNA) abundantly expressed ILK (Fig. 1a, silencing siRNA (siCNS)), which
Summary
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. Integrin adhesion complexes (IAC) act as critical physical links between the ECM and the actin-based cytoskeleton (e.g. stress fibers), in addition to constituting functional cellular mechanosensing centers linked to the intracellular signaling network (e.g. RhoGTPases), which in turn direct cell response to ECM properties (e.g. stiffness, molecular composition, and spacing) [1,2,3,4]. Force applied by stress fibers anchor to FA help to sequestrate tensin and integrin α5β1 centripetally to form elongated fibrillar structures [7], constituting the defining step in the formation of FB [5, 8]
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