Elsevier

Experimental Cell Research

Volume 293, Issue 2, 15 February 2004, Pages 259-266
Experimental Cell Research

Direct cell–cell interaction enhances pro-MMP-2 production and activation in co-culture of laryngeal cancer cells and fibroblasts: involvement of EMMPRIN and MT1-MMP

https://doi.org/10.1016/j.yexcr.2003.10.010Get rights and content

Abstract

Matrix metalloproteinases-2 (MMP-2, gelatinase A) has been regarded as a crucial enzyme for tumor progression, invasion, and metastasis by its capability to degrade the basement membrane components, and its activation process is critical for tumor development. Recently, EMMPRIN/CD147, which is a member of immunoglobulin superfamily, has been reported to be highly expressed in tumor cells and induce production of MMPs from fibroblasts adjacent to the tumor cells. In this study, we demonstrated that production of pro- and active forms of MMP-2 by human dermal fibroblasts was enhanced by the direct cell–cell contact with co-cultured HEp-2 cells derived from a human laryngeal epidermoid carcinoma. The results from immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that HEp-2 cells express a higher level of EMMPRIN but only a low level of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP), whereas fibroblasts express a higher level of MMP-2 and MT1-MMP and a low level of EMMPRIN. In a mixed co-culture with direct cell–cell contacts, co-cultured HEp-2 cells stimulated the pro- and active MMP-2 production from fibroblasts, but not in a separated co-culture through polycarbonate membrane. Production of pro/active MMP-2 and MT1-MMP (activator of pro-MMP-2) in fibroblasts was induced by the addition of membrane fractions prepared from HEp-2 cells to the fibroblast culture, and the induction was suppressed by the EMMPRIN depletion after immunoprecipitation, signifying the participation of EMMPRIN for the induction and activation of MMP-2. Our results suggest an importance of the direct cell–cell interaction involving EMMPRIN rather than humoral factors such as cytokines for pro-MMP-2 production and activation followed by tumor progression, invasion, and metastasis in laryngeal cancer.

Introduction

Degradation of extracellular matrix (ECM) components by matrix metalloproteinases (MMPs) is critical for tumor cell invasion and metastasis [1], [2]. Since disruption of the basement membrane is a crucial step for tumor progression, of MMPs, matrix metalloproteinases-2 (MMP-2) has been noted because of its activity to degrade type IV collagen, a major component of the basement membrane, and membrane type 1-matrix metalloproteinase (MT1-MMP) has been recognized as a key factor for pro-MMP-2 activation [3]. Recent studies have revealed that tumor cells utilize MMPs such as MMP-2 produced by neighboring stromal cells including fibroblasts rather than by tumor cells themselves [4], [5] for tumor progression, invasion, and metastasis, and that tumor cells can stimulate MMP production by stromal cells via soluble factors such as cytokines [6], [7], [8], [9] or through cell–cell interaction mediated by cell adhesion molecules such as EMMPRIN [10].

In addition to several humoral factors such as cytokines affecting the production and activation of ECM-degrading proteases including MMPs in tumor cells and/or stromal cells, EMMPRIN/CD147 has been noted to be highly expressed on tumor cell surface and involved in tumor progression by inducing the production of MMPs including MMP-2 by neighboring stromal cells [11], [12], [13]. EMMPRIN belongs to immunoglobulin superfamily, is referred to also as TCSF [14], basigin [15], neurotherin [16], or M6 antigen [17], and is a transmembrane glycoprotein with two extracellular domains and a short cytoplasmic domain [14], [18]. It has been reported that EMMPRIN is enriched in variety of human carcinomas [19], [20], [21] and contributes to tumor invasion and metastasis by stimulating fibroblasts nearby to secrete an increased amount of MMPs [4], [22]. However, very few attempts have been made to clarify an involvement of EMMPRIN in progression of head and neck cancers.

To explore the mechanism of tumor progression, in this study, we examined an expression of MMPs and EMMPRIN in cultured HEp-2 cells derived from a human laryngeal epidermoid carcinoma, fibroblasts, and their co-culture, and confirmed the importance of direct cell–cell association between tumor cells and fibroblasts and the contribution of EMMPRIN to stimulation of MMP-2 production and activation from fibroblasts, namely, stromal cells.

Section snippets

Cells and cell culture

HEp-2 cells, a human laryngeal carcinoma cell line, were provided by Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). Human fibroblasts were isolated from the dermis of a healthy subject. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 at 37°C. The protocol used in this study for specimen preparation was previously approved

Stimulation of pro-MMP-2 production and activation by co-culture of HEp-2 cells and fibroblasts

It has been reported that the interaction of cancer cells with stromal cells is a pivotal step to produce pro-MMP-2 and/or activate pro-MMP-2 for tumor progression. To explore an involvement of cell–cell interaction in pro-MMP-2 production and/or pro-MMP-2 activation, a cell line HEp-2 derived from a human larynx epidermoid carcinoma was co-cultured with human skin fibroblasts to analyze pro-MMP-2 production and activation. Zymographic analysis of the conditioned media from fibroblast and HEp-2

Discussion

MMPs, a family of zinc-dependent proteinases, are capable to degrade a variety of ECM components, and have various physiological functions in normal tissues [25], [26], whereas the degradation of ECM components by MMPs is a determining step in tumor cell invasion and metastasis [1], [2], [27], [28]. Of MMPs, MMP-2 (gelatinase A) is able to degrade type IV collagen in the basement membrane for tumor progression [1], and therefore pro-MMP-2 expression and activation are important events in many

Acknowledgements

The authors thank Drs. Katsuyuki Imai, Takeya Sato, Da-Ren Wang, and Nobuyo Higashi (Department of Cell Biology and Histology, Akita University School of Medicine) for their valuable discussions. The expert technical support provided by Naosuke Kojima (Department of Cell Biology and Histology, Akita University School of Medicine) was also gratefully appreciated.

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