Dataset Information

The Lectin-Like Domain of Thrombomodulin Inhibits ?1 Integrin-Dependent Binding of Human Breast Cancer-Derived Cell Lines to Fibronectin.

ABSTRACT: Thrombomodulin is a molecule with anti-coagulant and anti-inflammatory properties. Recently, thrombomodulin was reported to be able to bind extracellular matrix proteins, such as fibronectin and collagen; however, whether thrombomodulin regulates the binding of human breast cancer-derived cell lines to the extracellular matrix remains unknown. To investigate this, we created an extracellular domain of thrombomodulin, TMD123-Fc, or domain deletion TM-Fc proteins (TM domain 12-Fc, TM domain 23-Fc) and examined their bindings to fibronectin in vitro by ELISA. The lectin-like domain of thrombomodulin was found to be essential for the binding of the extracellular domain of thrombomodulin to fibronectin. Using a V-well cell adhesion assay or flow cytometry analysis with fluorescent beads, we found that both TMD123-Fc and TMD12-Fc inhibited the binding between ?1 integrin of human breast cancer-derived cell lines and fibronectin. Furthermore, TMD123-Fc and TMD12-Fc inhibited the binding of activated integrins to fibronectin under shear stress in the presence of Ca²⁺ and Mg²⁺ but not under strong integrin-activation conditions in the presence of Mg²⁺ without Ca²⁺. This suggests that thrombomodulin Fc fusion protein administered exogenously at a relatively early stage of inflammation may be applied to the development of new therapies that inhibit the binding of ?1 integrin of breast cancer cell lines to fibronectin.

SUBMITTER: Kawamoto E

PROVIDER: S-EPMC7914543 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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The Lectin-Like Domain of Thrombomodulin Inhibits β1 Integrin-Dependent Binding of Human Breast Cancer-Derived Cell Lines to Fibronectin.

Kawamoto Eiji E Nago Nodoka N Okamoto Takayuki T Gaowa Arong A Masui-Ito Asami A Akama Yuichi Y Darkwah Samuel S Appiah Michael Gyasi MG Myint Phyoe Kyawe PK Obeng Gideon G Ito Atsushi A Caidengbate Siqingaowa S Esumi Ryo R Yamaguchi Takanori T Park Eun Jeong EJ Imai Hiroshi H Shimaoka Motomu M

Biomedicines 20210207 2

Thrombomodulin is a molecule with anti-coagulant and anti-inflammatory properties. Recently, thrombomodulin was reported to be able to bind extracellular matrix proteins, such as fibronectin and collagen; however, whether thrombomodulin regulates the binding of human breast cancer-derived cell lines to the extracellular matrix remains unknown. To investigate this, we created an extracellular domain of thrombomodulin, TMD123-Fc, or domain deletion TM-Fc proteins (TM domain 12-Fc, TM domain 23-Fc) ...[more]

PMID: 33562346

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Project description:BackgroundThe blood-spinal cord barrier (BSCB) is composed of a monolayer of endothelium linked with tight junctions and extracellular matrix (ECM)-rich basement membranes and is surrounded by astrocyte foot processes. Endothelial permeability is regulated by interaction between endothelial cells and ECM proteins. Fibronectin (FN) is a principal ECM component of microvessels. Excessive FN deposition disrupts cell-cell adhesion in fibroblasts through β1 integrin ligation. To determine whether excessive FN deposition contributes to the disruption of endothelial integrity, we used an in vitro model of the endothelial monolayer to investigate whether the FN inhibitor pUR4 prevents FN deposition into the subendothelial matrix and attenuates endothelial leakage.MethodsTo correlate the effects of excessive FN accumulation in microvessels on BSCB disruption, spinal nerve ligation-which induces BSCB leakage-was applied, and FN expression in the spinal cord was evaluated through immunohistochemistry and immunoblotting. To elucidate the effects by which pUR4 modulates endothelial permeability, brain-derived endothelial (bEND.3) cells treated with tumor necrosis factor (TNF)-α were used to mimic a leaky BSCB. A bEND.3 monolayer was preincubated with pUR4 before TNF-α treatment. The transendothelial electrical resistance (TEER) measurement and transendothelial permeability assay were applied to assess the endothelial integrity of the bEND.3 monolayer. Immunofluorescence analysis and immunoblotting were performed to evaluate the inhibitory effects of pUR4 on TNF-α-induced FN deposition. To determine the mechanisms underlying pUR4-mediated endothelial permeability, cell morphology, stress fiber formation, myosin light chain (MLC) phosphorylation, and β1 integrin-mediated signaling were evaluated through immunofluorescence analysis and immunoblotting.ResultsExcessive FN was accumulated in the microvessels of the spinal cord after spinal nerve ligation; moreover, pUR4 inhibited TNF-α-induced FN deposition in the bEND.3 monolayer and maintained intact TEER and endothelial permeability. Furthermore, pUR4 reduced cell morphology alteration, actin stress fiber formation, and MLC phosphorylation, thereby attenuating paracellular gap formation. Moreover, pUR4 reduced β1 integrin activation and downstream signaling.ConclusionspUR4 reduces TNF-α-induced β1 integrin activation by depleting ECM FN, leading to a decrease in endothelial hyperpermeability and maintenance of monolayer integrity. These findings suggest therapeutic benefits of pUR4 in pathological vascular leakage treatment.

Dataset Information

The Lectin-Like Domain of Thrombomodulin Inhibits ?1 Integrin-Dependent Binding of Human Breast Cancer-Derived Cell Lines to Fibronectin.

Publications

The Lectin-Like Domain of Thrombomodulin Inhibits β1 Integrin-Dependent Binding of Human Breast Cancer-Derived Cell Lines to Fibronectin.

Similar Datasets

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