Abstract
Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as “integrin signals”, but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced by integrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition of ROCK and myosin II activity, i.e. the reactions occurred independently of intracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching of the adherent cells induced a robust phosphorylation only of ERK1/2 and the phosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via α5β1 or αvβ3 integrins were detected. The importance of mitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signals induced by cyclic cell stretching, it abolished the activation of AKT and reduced the actin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that “integrin signals” are composed of separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and extracellular ROS had specific and distinct effects on the integrin signals induced by cell attachment and mechanical stretching.
Highlights
Integrins are transmembrane cell-matrix receptors able to act as signaling mechano-sensors by linking extracellular ligands to actin filaments in adhesion sites. [1,2,3]
Cells were seeded into wells coated with FN or the non-adhesive polymer Pluronic (PL), and in order to compare signals generated by avb3 and a5b1 and integrins after binding to FN, we used the fibroblast-like cell lines GD25 and GD25b1
Since myosin light chain (MLC) S19 can be phosphorylated by other kinases than ROCK, primarily by MLC kinase, the effect by Y27632 on MLC pS19 levels may partly be ascribed to the increased myosin phosphatase targeting subunit 1 (MYPT1) activity
Summary
Integrins are transmembrane cell-matrix receptors able to act as signaling mechano-sensors by linking extracellular ligands to actin filaments in adhesion sites. [1,2,3]. Integrins are transmembrane cell-matrix receptors able to act as signaling mechano-sensors by linking extracellular ligands to actin filaments in adhesion sites. Several integrin adhesion siteassociated proteins can change conformation upon mechanical stimulation and thereby expose cryptic binding or phosphorylation sites, three prototype examples being talin, p130CAS, and fibronectin (FN) [4,5,6]. Integrins generate signals upon mere clustering [8], and ligand binding induces structural changes in integrins that may trigger additional signals [8,9]. ROS derived from mitochondria [10,11] as well as from NADPH oxidases (NOXes) and 5-lipoxygenase (5-LOX) [12,13] were reported to have important roles in integrin-mediated attachment, spreading, and the associated changes in the cytoskeleton. Taddei et al [11] proposed that mitochondrial ROS are involved in an early phase during cell attachment, whereas 5-LOX would mainly be responsible for the later phase of ROS generation
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