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

Eiji Kawamoto,Phyoe Kyawe Myint,Atsushi Ito,Eun Jeong Park,Motomu Shimaoka,Arong Gaowa,Yuichi Akama,Gideon Obeng,Samuel Darkwah,Takanori Yamaguchi,Hiroshi Imai,Nodoka Nago,Takayuki Okamoto,Michael Gyasi Appiah,Siqingaowa Caidengbate,Ryo Esumi,Asami Masui-Ito

doi:10.3390/biomedicines9020162

Eiji Kawamoto, Phyoe Kyawe Myint + Show 15 more

Open Access

https://doi.org/10.3390/biomedicines9020162

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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 β1 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 β1 integrin of breast cancer cell lines to fibronectin.

Highlights

To investigate whether MDA-MB-231 was inhibited by TMD123-Fc through the binding of β1 integrin to fibronectin, we evaluated binding of the β1 integrin to fibronectin by adding a divalent ion (1 mM Mg2+ and Ca2+ ) to immobilized fibronectin and activating the integrin by applying shear stress to the β1 integrin from human breast cancer-derived cell lines
We found that the binding of fibronectin to β1 integrin was inhibited by TMD123-Fc in a concentration-dependent manner (p < 0.05) (Figure 5a,c)
We examined the inhibitory effect of TM on the binding of α5β1 integrin to fibronectin using human breast cancer-derived cell lines

Summary

Introduction

Thrombomodulin (TM) is considered an important cofactor of the anticoagulation protein C system present in vascular endothelial cells [1]. TM can exert anti-inflammatory and cytoprotective properties and was found to be a key component regulating the binding of vascular endothelial cells to white blood cells [2]. TM is expressed in tumor cells in addition to normal vascular endothelial cells, with its expression in tumor cells suggested to regulate tumor cell invasion and proliferation in certain types of cancer. In esophageal cancer, TM expression in cancer cells in metastases is reduced relative to that in primary tumors [3].

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