Domain 5 of high molecular weight kininogen inhibits collagen-mediated cancer cell adhesion and invasion in association with α-actinin-4

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Abstract

High molecular weight kininogen (HK) is a plasma glycoprotein with multiple functions, including the regulation of coagulation. We previously demonstrated that domain 5 (D5H), a functional domain of HK, and its derived peptides played an important role in the vitronectin-mediated suppression of cancer cell adhesion and invasion. However, the underlying mechanisms of the D5H-mediated suppressive effects remain to be elucidated. Here, we showed that D5H and its derivatives inhibited the collagen-mediated cell adhesion and invasion of human osteosarcoma MG63 cells. Using purified D5H fused to glutathione-S-transferase (GST) and D5H-derived peptides for column chromatography, an actin-binding protein, α-actinin-4, was identified as a binding protein of D5H with high-affinity for P-5m, a core octapeptide of D5H. Immunofluorescence microscopy demonstrated that D5H co-localized with α-actinin-4 inside MG63 cells. In addition, exogenous GST-D5H added to the culture media was transported into MG63 cells, although GST alone as a control was not. As α-actinin-4 regulates actin polymerization necessary for cell adhesion and is related to the integrin-dependent attachment of cells to the extracellular matrix, our results suggest that D5H may modulate cell adhesion and invasion together with actinin-4.

Highlights

► Collagen-mediated cell adhesion and invasion of MG63 cells was inhibited by D5H. ► Similar effects were observed using D5H-derived peptides. ► α-Actinin-4 is a binding protein of D5H with high-affinity for P-5m. ► D5H was transported into MG63 cells and co-localized with α-actinin-4.

Introduction

Cell adhesion is a fundamental cellular function, and is indispensable for the organization of cell morphology, leading to normal tissue formation and maintenance [1]. In addition to normal cells, the regulation of cell adhesion activity is an important factor for the invasiveness of cancer cells and progression of the tumor itself [2], [3]. Thus, investigation of the molecular mechanisms of cell adhesion and invasion may increase our understanding of the properties of cancer cells, and suggest new approaches for the development of anti-cancer therapeutics.

High molecular weight kininogen (HK) is a plasma glycoprotein and is involved in the regulation of coagulation [4]. It initiates the intrinsic pathway of the blood coagulation cascade by promoting the conversion of factor XII to factor XIIa in cooperation with kallikrein and a negatively charged surface [5]. We previously reported that HK also functions as an inhibitor of cell adhesion [6]. Following this, several studies focused on the effect of HK or its derivatives on cellular functions [7], [8], and demonstrated that domain 5 (D5H), one of the six functional domains of HK, inhibited angiogenesis by inducing apoptosis and down-regulating proliferation and migration of endothelial cells [9], [10], [11].

We also observed that D5H impaired the vitronectin-mediated cell adhesion and invasion in different types of cancer cells by in vitro assay, and that it dramatically suppressed cancer metastasis in an in vivo assay [12], [13]. It was further elucidated that the core amino acid sequence of D5H necessary for such inhibitory effects was Gly484 through Lys491, designated P-5m [12]. However, little is known about the mechanism by which D5H induces the inhibition of cell adhesion and invasion of cancer cells. In addition, since the role of D5H in cell adhesion and invasion was mainly examined using materials coated with vitronectin, it is unclear whether D5H exerts similar functions in other extracellular matrices, such as collagen. Therefore, this study examined the effects of D5H on the collagen-mediated cell adhesion and invasion in human osteosarcoma MG-63 cells, and identified a D5H-binding protein, α-actinin-4, that shows high-affinity binding to P-5m, suggesting that this protein may participate in D5H-induced functions.

Section snippets

Cell culture

Human osteosarcoma MG63 cells were obtained from Riken Cell Bank (Tsukuba, Japan) and were cultured and maintained as monolayers in Minimum essential medium Eagle (MEME, Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) heat inactivated fetal bovine serum, non-essential amino acids solution (Wako, Osaka, Japan) and antibiotics solution (100 unit/ml penicillin, 0.1 mg/ml streptomycin and 0.25 μg/ml amphotericin B; Wako) in a humidified atmosphere of 5% CO2 in air at 37 °C.

Glutathione-S-transferase-fusion protein and peptides

Glutathione-S

D5H inhibits the collagen-mediated cell adhesion and invasion of MG63 cells

We previously reported that GST-D5H inhibited the vitronectin-mediated cell adhesion and invasion of cancer cells [12], [13]. In this study, we examined whether the inhibitory effects of D5H are also observed in MG63 cells using a different extracellular matrix, collagen. GST-D5H significantly inhibited cell adhesion of MG63 cells on the Col-I-coated plates in a concentration-dependent manner, compared with GST (Fig. 1A). Similar results were obtained from the cell invasion assay, where GST-D5H

Discussion

Previously we demonstrated the inhibitory effects of D5H and its derived peptides on the vitronectin-mediated cell adhesion and invasion of cancer cells [12], [13]. Here, we showed similar effects of D5H and its derived peptides using a different extracellular matrix, collagen, although the degree of the inhibition was slightly different between the matrices. Vitronectin is a ligand for most αv integrins, such as αvβ3 and αvβ5 [19], whereas collagen is a ligand for β1 integrins [20], [21]. Our

Acknowledgments

We thank Mrs. Masafumi Suzaki, Noboru Urushiyama and Takefumi Yamamoto for his excellent technical assistance in this study. This study was supported by Grants-in-aid for Scientific Research 〈KAKENHI〉 from Japan Society for the Promotion of Science (JSPS) and The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Mochida Foundation for Medical and Pharmaceutical Research, and Daiichi-Sankyo Foundation of Life Science.

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