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  • Membrane Type 1 Matrix Metalloproteinase (MT1-MMP/ MMP-14) Cleaves and Releases a 22-kDa Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) Fragment from Tumor Cells* Received for publication, July 24, 2006, and in revised form, September 26, 2006 Published, JBC Papers in Press, October 18, 2006, DOI 10.1074/jbc.M606993200

    Nagayasu Egawa‡, Naohiko Koshikawa‡, Taizo Tomari‡, Kazuki Nabeshima§, Toshiaki Isobe¶, and Motoharu Seiki‡1

    From the ‡Division of Cancer Cell Research, Institute of Medical Science, the University of Tokyo, Tokyo 108-8639, the §Department of Pathology, Fukuoka University Hospital 814-0180, Fukuoka 814-0180, and the ¶Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan

    Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is amultifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is report- edly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumorHT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shed- ding was enhanced by phorbol 12-myristate 13-acetate and inhib- ited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic pep- tide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for theMMP inducing activity of EMMPRIN and for cell-cell interac- tions,presumably through its ability toengage inhomophilic inter- actions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP- dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down- regulate its function. At the same time, the released 22-kDa frag- mentmaymediate the expression ofMMPs in tumor tissues.

    The extracellular matrix metalloproteinase inducer (EMMPRIN2; also known as CD147, tumor collagenase-stimu-

    lating factor, basigin, andM6) is amultifunctional glycoprotein that belongs to the immunoglobulin superfamily (1–4). EMMPRIN-null mice are sterile and have defects in spermato- genesis, fertilization, sensory and memory functions, and mixed lymphocyte responses (5–7). However, the exact mech- anisms underlying the observed defects are still largely unknown. The protein backbone of EMMPRIN is 28 kDa, but the

    molecular mass of the glycosylated form varies between 44 and 66 kDa (4). The extracellular portion of EMMPRIN contains two Ig-like motifs and three potential N-glycosylation sites (8). EMMPRIN is expressed at high levels in many types of tumors and stromal cells (9–14), and the N-terminal Ig-like domain, which can form homodimers (15, 16), may modulate cell-cell interactions within tumor tissues or during metastasis. EMMPRIN is released from tumor cells and acts as an inducer of collagenase (MMP-1) expression in the surrounding stroma and tumor cells (17–19). Matrix metalloproteinases (MMPs) are zinc-binding

    endopeptidases responsible for the turnover of many proteins in the extracellular space, including those that compose the extracellularmatrix (ECM), cell adhesionmolecules, cytokines, growth factors, and receptors (20). Most tumor types and the surrounding stromal cells overexpress multiple MMPs, which are important players in promoting tumor growth, invasion, and metastasis (20). EMMPRIN also induces MMP-2, -3, and -9, MT1-MMP, and MT2-MMP in addition to MMP-1 (9, 21–25); only glycosylated EMMPRIN is able to induce these MMPs (26), and theN-terminal Ig-like domain is also indispen- sable for the MMP inducing activity (26), as well as for the homophilic interactions of the protein (26). Therefore, EMMPRIN potentially mediates the excessive production of MMPs in tumor tissue and is expected to act as a modulator of ECM in tumor tissues through the activity of the MMPs that it induces.* This work was supported by a grant-in-aid for scientific research on priorityareas, “Integrative Research toward the Conquest of Cancer,” from the

    Ministry of Education, Culture, Sports, Science and Technology of Japan (to M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi- cate this fact.

    1 To whom correspondence should be addressed. Tel.: 81-3-5449-5255; Fax: 81-3-5449-5414; E-mail: mseiki@ims.u-tokyo.ac.jp.

    2 The abbreviations used are: EMMPRIN, extracellular matrix metalloprotein- ase inducer; MMP, matrix metalloproteinase; CM, conditioned medium; ECM, extracellular matrix; HPLC, high performance liquid chromatography;

    MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; mAb, monoclonal antibody; MT-MMP, membrane-type MMP; pAb, poly- clonal antibody; PMA, phorbol 12-myristate 13-acetate; siRNA, small inter- fering RNA; TCL, total cell lysate; TIMP, tissue inhibitor of metalloprotein- ase; Dox, doxycycline; MS/MS, tandem mass spectrometry; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorter; PBS, phos- phate-buffered saline.

    THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 49, pp. 37576 –37585, December 8, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

    37576 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 49 • DECEMBER 8, 2006

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  • According to the literature, EMMPRIN is released from tumor cells in at least two different ways. A significant amount of intact EMMPRIN is released from tumor cells (27, 28), pos- sibly contained within membrane vesicles (microvesicles) that some tumor cells release upon stimulation (28). The other pathway is proteolytic shedding. In addition to inducing MMPs, EMMPRIN is likely to be cleaved and shed byMMPs or other metalloproteinases, because the shedding of EMMPRIN is inhibited by zinc chelators (25, 29). Supporting this, MMP-1 andMMP-2 can cleave EMMPRIN at the membrane-proximal region, at least in vitro (29). The proteolytic shedding of EMMPRIN from the cell surface is not only important for reg- ulating EMMPRIN function at the cell surface but is also a mechanism to release a soluble MMP inducer. However, the details of the proteolytic processing of EMMPRIN, such as the responsible proteinases, cleavage sites, and stimulants, is very limited. In this context, MT1-MMP (MMP-14) is an interesting can-

    didate for the EMMPRIN shedding as an integral membrane protease responsible for pericellular proteolysis (30, 31). MT1- MMP is expressed in human tumors, and its potential sub- strates are ECM proteins, such as collagens, fibronectin, lami- nins, vitronectin, and aggrecan (30, 31). Other functional proteins that can be cleaved byMT1-MMP include pro-MMPs, CD44, the integrin �v chain, low density lipoprotein receptor- related protein, interleukin 8, and pro-tumor necrosis factor (32–37). Thus, the proteolytic activity ofMT1-MMP is a potent modulator of the pericellular environment and promotes tumor growth and invasion, particularly in collagen-rich envi- ronments (38). Our knowledge about the physiological sub- strates of MT1-MMP is still fragmentary, but proteins that can be cleaved byMT1-MMP are often co-purified with it from cell lysates, and EMMPRIN is among the proteins we identified as co-purifying withMT1-MMP.3 Thus, we were interested in the possibility that MT1-MMP cleaves and releases EMMPRIN from tumor cells. In this study, we characterized the proteolytic shedding of

    EMMPRIN by MT1-MMP for the first time. Two of the three human tumor cell lines tested released a small 22-kDa fragment of EMMPRIN. The shedding was enhanced by treatment of the cellswith phorbol 12-myristate 13-acetate (PMA) and inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 but not by TIMP-1, indicating the possible involvement of MT-MMPs. Indeed, expression, knockdown, and in vitro cleavage studies supported the involvement of MT1-MMP in EMMPRIN pro- teolysis. Our results raise the interesting possibility that MT1- MMP may trigger the expression of MMPs in tumor tissues.

    EXPERIMENTAL PROCEDURES

    Cells and Culture Conditions—The human fibrosarcoma cell line HT1080, human epidermoid carcinoma cell line A431, and humanmelanoma cell lineA375were obtained from theAmer- ican Type Culture Collection (ATCC, Manassas, VA), and the African green monkey kidney cell line COS-7 was obtained from Health Science Research Resources Bank (Osaka, Japan).

    The gastric cancer cell line TMK-1 was a gift from Prof. E. Tahara (Hiroshima University, Hiroshima, Japan). Human pri- mary fibroblast cells were obtained from nonlesional dermis around nodular fasciitis according to the guideline for clinical sample handling in Fukuoka University Hospital. A431, HT1080, COS-7, and human normal fibroblast cells were cul- tured in Dulbecco’s modified Eagle’s medium (Sigma) contai