The Lectin-Like Domain of Thrombomodulin Inhibits β1 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 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.

The Spike Glycoprotein of SARS-CoV-2 Binds to β1 Integrins Expressed on the Surface of Lung Epithelial Cells

“…A flow cytometric examination confirmed the absence of β1 integrin in the KO cells (Figure 5A). S1 binding of β1-KO cells was completely abolished, compared to that of β1-control cells (i.e., mock transfectants) [16] (Figure 5B), even though β1-KO cells retain αV integrin expression (Figure 5A). These results have revealed that β1 integrins can support binding to S1 protein independently of αV integrins.…”

“…To further validate the specific interaction of β1 integrin with S1 protein, we utilized a β1 integrin knockout (KO) clone of a human breast cancer cell line (MDA-MB-231) that we previously established using CRISPR/Cas9 [16] as a model cell. A flow cytometric examination confirmed the absence of β1 integrin in the KO cells (Figure 5A).…”

“…As S2-Fc protein is a scarce reagent for us, we have studied it only with QG cells, which bound significantly better to S1-Fc protein than to S2-Fc protein (Figure 4 These results confirmed the specific binding of QG-56, 11-18, and THP-1 cells to S1 p tein. To further validate the specific interaction of β1 integrin with S1 protein, we utiliz a β1 integrin knockout (KO) clone of a human breast cancer cell line (MDA-MB-231) t we previously established using CRISPR/Cas9 [16] as a model cell. A flow cytometric amination confirmed the absence of β1 integrin in the KO cells (Figure 5A).…”

“…The MLO-A5 cell line (mouse osteocytes) was obtained from Kerafast (Boston, MA, USA) and grown in MEM alpha (Thermo Fisher Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, USA), 5% calf serum (Biowest, Riverside, MO, USA), and penicillin/streptomycin (Nacalai, Kyoto, Japan). A human monocytic cell line (THP-1) was purchased from ATCC (Manassas, VA, USA) and human lung epithelial cell lines (11)(12)(13)(14)(15)(16)(17)(18) were kindly provided by Dr. Yoshihiro Miyahara (Mie University Medical School, Mie, Japan). The human breast cancer line MDA-MB-231 was from ATCC.…”

“…β1-control (Scr.) and β1-KO clones of MDA-MB-231 generated in our laboratory [16] were used to validate the effect of β1 integrin on cell adhesion to S1 protein. Cells were grown in RPMI-1640 (Nacalai) containing 10% FBS (Equitech-Bio) and penicillin/streptomycin (Nacalai).…”