A redox-dependent thiol-switch and a Ca2+ binding site within the hinge region hierarchically depend on each other in α7ß1 integrin regulation.

Integrin-mediated cell contacts with the extracellular matrix (ECM) are essential for cellular adhesion, force transmission, and migration. Several effectors, such as divalent cations and redox-active compounds, regulate ligand binding activities of integrins and influence their cellular functions. To study the role of the Ca2+ binding site within the hinge region of the integrin α7 subunit, we genetically abrogated it in the α7hiΔCa mutant. This mutant folded correctly, associated with the ß1 subunit and was exposed on the cell surface, but showed reduced ligand binding and weaker cell adhesion to laminin-111. Thus, it resembles the α7hiΔSS mutant, in which the redox-regulated pair of cysteines, closeby to the Ca2+ binding site within the hinge, was abrogated. Comparing both mutants in adhesion strength and cell migration revealed that both Ca2+ complexation and redox-regulation within the hinge interdepend on each other. Moreover, protein-chemical analyses of soluble integrin ectodomains containing the same α7 hinge mutations suggest that integrin activation via the subunit α hinge is primed by the formation of the cysteine pair-based crosslinkage. Then, this allows Ca2+ complexation within the hinge, which is another essential step for integrin activation and ligand binding. Thus, the α hinge is an allosteric integrin regulation site, in which both effectors, Ca2+ and redox-active compounds, synergistically and hierarchically induce far-ranging conformational changes, such as the extension of the integrin ectodomain, resulting in integrin activation of ECM ligand binding and altered integrin-mediated cell functions.

Extracellular Vesicle Transfer from Endothelial Cells Drives VE-Cadherin Expression in Breast Cancer Cells, Thereby Causing Heterotypic Cell Contacts.

Cadherins mediate cohesive contacts between isotypic cells by homophilic interaction and prevent contact between heterotypic cells. Breast cancer cells neighboring endothelial cells (ECs) atypically express vascular endothelial (VE)-cadherin. To understand this EC-induced VE-cadherin expression in breast cancer cells, MCF7 and MDA-MB-231 cells expressing different endogenous cadherins were co-cultured with ECs and analyzed for VE-cadherin at the transcriptional level and by confocal microscopy, flow cytometry, and immunoblotting. After losing their endogenous cadherins and neo-expression of VE-cadherin, these cells integrated into an EC monolayer without compromising the barrier function instantly. However, they induced the death of nearby ECs. EC-derived extracellular vesicles (EVs) contained soluble and membrane-anchored forms of VE-cadherin. Only the latter was re-utilized by the cancer cells. In a reporter gene assay, EC-adjacent cancer cells also showed a juxtacrine but no paracrine activation of the endogenous VE-cadherin gene. This cadherin switch enabled intimate contact between cancer and endothelial cells in a chicken chorioallantoic membrane tumor model showing vasculogenic mimicry (VM). This EV-mediated, EC-induced cadherin switch in breast cancer cells and the neo-expression of VE-cadherin mechanistically explain the mutual communication in the tumor microenvironment. Hence, it may be a target to tackle VM, which is often found in breast cancers of poor prognosis.

Extracellular Redox Regulation of α7ß Integrin-Mediated Cell Migration Is Signaled via a Dominant Thiol-Switch.

While adhering to extracellular matrix (ECM) proteins, such as laminin-111, cells temporarily produce hydrogen peroxide at adhesion sites. To study the redox regulation of α7ß1 integrin-mediated cell adhesion to laminin-111, a conserved cysteine pair within the α-subunit hinge region was replaced for alanines. The molecular and cellular effects were analyzed by electron and atomic force microscopy, impedance-based migration assays, flow cytometry and live cell imaging. This cysteine pair constitutes a thiol-switch, which redox-dependently governs the equilibrium between an extended and a bent integrin conformation with high and low ligand binding activity, respectively. Hydrogen peroxide oxidizes the cysteines to a disulfide bond, increases ligand binding and promotes cell migration toward laminin-111. Inversely, extracellular thioredoxin-1 reduces the disulfide, thereby decreasing laminin binding. Mutation of this cysteine pair into the non-oxidizable hinge-mutant shows molecular and cellular effects similar to the reduced wild-type integrin, but lacks redox regulation. This proves the existence of a dominant thiol-switch within the α subunit hinge of α7ß1 integrin, which is sufficient to implement activity regulation by extracellular redox agents in a redox-regulatory circuit. Our data reveal a novel and physiologically relevant thiol-based regulatory mechanism of integrin-mediated cell-ECM interactions, which employs short-lived hydrogen peroxide and extracellular thioredoxin-1 as signaling mediators.

The Interaction between Laminin-332 and α3ß1 Integrin Determines Differentiation and Maintenance of CAFs, and Supports Invasion of Pancreatic Duct Adenocarcinoma Cells.

Ranking among the most lethal tumour entities, pancreatic duct adenocarcinoma cells invade neighbouring tissue resulting in high incidence of metastasis. They are supported by tumour stroma fibroblasts which have undergone differentiation into cancer-associated fibroblasts (CAFs). Stiffness of cell substratum, cytokines, such as transforming growth factor-ß (TGF-ß), and stromal matrix proteins, such as laminin-332, are factors which promote CAF differentiation. In a spheroid culture system, differentiation of CAFs was analysed for laminin-332 production, laminin-binding integrin repertoire, adhesion and migration behaviour, and, in heterospheroids, for their interplay with the pancreatic duct adenocarcinoma AsPC-I cells. Our data reveal that CAFs produce laminin-332 thus contributing to its ectopic deposition within the tumour stroma. Moreover, CAF differentiation correlates with an increased expression of α3ß1 integrin, the principal laminin-332-receptor. Beyond its role as novel CAF marker protein, integrin α3ß1 crucially determines differentiation and maintenance of the CAF phenotype, as knock-out of the integrin α3 subunit reversed the CAF differentiated state. AsPC-I cells co-cultured in heterospheroids with integrin α3-deficient CAFs invaded less than from heterospheroids with wild-type CAFs. This study highlights the role of integrin α3ß1 integrin-laminin-332 interaction of CAFs which promotes and sustains differentiation of CAFs and promotes carcinoma invasion.