Journal of Cell Science 110, 589-596 (1997) 589 Printed in Great Britain © The Company of Biologists Limited 1997 JCS9564 Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts Takuya Sato1, Maria del Carmen Ovejero1, Peng Hou1, Anne-Marie Heegaard1, Masayoshi Kumegawa2, Niels Tækker Foged1 and Jean-Marie Delaissé1,* 1Department of Basic Research, Center for Clinical & Basic Research, Ballerup Byvej 222, DK-2750 Ballerup, Denmark 2The First Department of Oral Anatomy, Meikai University School of Dentistry, Keyakidai 1-1, Sakado, Saitama 350-02, Japan *Author for correspondence SUMMARY The osteoclasts are the cells responsible for bone resorp- on bone sections showed that MT1-MMP is expressed also tion. Matrix metalloproteinases (MMPs) appear crucial in osteoclasts in vivo. Antibodies recognizing MT1-MMP for this process. To identify possible MMP expression in reacted with specific plasma membrane areas corre- osteoclasts, we amplified osteoclast cDNA fragments sponding to lamellipodia and podosomes involved, respec- having homology with MMP genes, and used them as a tively, in migratory and attachment activities of the osteo- probe to screen a rabbit osteoclast cDNA library. We clasts. These observations highlight how cells might bring obtained a cDNA of 1,972 bp encoding a polypeptide of MT1-MMP into contact with focal points of the extracel- 582 amino acids that showed more than 92% identity to lular matrix, and are compatible with a role of MT1- human, mouse, and rat membrane-type 1 MMP (MT1- MMP in migratory and attachment activities of the osteo- MMP), a cell surface proteinase believed to trigger cancer clast. cell invasion. By northern blotting, MT1-MMP was found to be highly expressed in purified osteoclasts when compared with alveolar macrophages and bone stromal Key words: Osteoclast, MMP-14, MT1-MMP, Membrane proteinase, cells, as well as with various tissues. In situ hybridization Podosome, Bone invasion, Bone resorption INTRODUCTION lagenase clearly stained the resorption compartment (Delaissé et al., 1993). Also several other MMPs have recently been The osteoclasts are the cells responsible for the resorption of reported in osteoclasts (Hill et al., 1994), but it is not estab- the extracellular bone matrix. This role is essential for bone lished what might be the main MMP acting in the resorption morphogenesis, repair and maintenance. In order to fulfil this compartment. It is worth noting that all these proteinases are role, the (pre)osteoclast has to recognize, move, and anchor soluble, and it is believed that the sealing ring surrounding the itself to an appropriate site of the bone surface. Further, it has resorption compartment plays an important role for restricting to establish the resorption compartment, which involves a proteolysis therein. series of characteristic events, such as the formation of a tight Recent observations suggest that bone resorption is also seal all around it, the development of the ruffled border, and determined by proteolytic activities that occur before the the secretion of lytic agents. Thus bone resorption relies on a formation of the resorption compartment, and that are series of distinct osteoclastic activities (reviewed by Baron et required for osteoclast migration and anchorage. Indeed, al., 1993). MMPs proved to be indispensable for the recruitment of Because the removal of organic matrix in the resorption osteoclasts to future resorption sites (Blavier and Delaissé, compartment is a striking feature of bone resorption, much 1995), just as they are for many other cell migration effort has been done to identify the proteinases involved in this processes, such as angiogenesis (Fisher et al., 1994), metas- removal. It seems that only cysteine proteinases and matrix- tasis (Tryggvason et al., 1993), wound healing (Salo et al., metalloproteinases (MMPs) are rate limiting for this process 1994), embryo implantation (Librach et al., 1991) or neurite (Everts et al., 1992; Delaissé and Vaes, 1992). Various cysteine outgrowth (Muir, 1994). The molecular mode of action of proteinases have been implicated, but it appeared recently that MMPs in these processes is, however, still unclear. Another cathepsin K might be the key cysteine proteinase (Tezuka et interesting feature is that both osteoclasts and cancer cells al., 1994a; Li et al., 1995; Saneshige et al., 1995; Bossard et exhibit specialized membrane protrusions, called invadopo- al., 1996). High levels of MMP-9 in osteoclasts were repeat- dia or podosomes, establishing close-contact-like adhesions, edly stressed (Wucherpfennig et al., 1994; Reponen et al., and penetrating into the extracellular matrix (Baron et al., 1994; Tezuka et al., 1994b; Okada et al., 1995a; Blavier and 1993; Marchisio et al., 1984; Lakkakorpi and Väänänen, Delaissé, 1995) and antibodies recognizing interstitial col- 1991; Teti et al., 1991; Aubin, 1992). Podosomes of cancer 590 T. Sato and others cells were shown to degrade extracellular matrix molecules Sweden). Single strand cDNA was synthesized from mRNA by and, accordingly, various proteolytic activities were localized using a cDNA synthesis kit (Pharmacia). cDNA fragments were at their surface (Chen et al., 1984; Kelly et al., 1994). It was amplified by polymerase chain reaction (PCR) using degenerate also suggested that podosomes of osteoclasts bear proteinases primers corresponding to the conserved amino acid sequences in the on their surface (Teti et al., 1991; Aubin, 1992). However, cysteine switch (PRCGVPD) and in the catalytic domain neither such proteinases, nor any membrane proteinase have (GDXHFDXXE). The reactions were cycled 45 times through the following steps: 1 minute at 94°C, 1 minute at 55°C, 1 minute at ever been reported in osteoclasts. 72°C. The PCR products were purified after agarose gel elec- The recent discovery of 4 MMPs with a transmembrane trophoresis. The purified cDNA was cloned into a pCRII vector domain (membrane-type MMPs, MT-MMPs) shows promise (Invitrogen, San Diego, CA) according to the instruction manual. for understanding how cell surface proteolysis is involved in The nucleotide sequences were determined from both strands by the invasive processes (Sato et al., 1994a; Will and Hinzmann, dideoxy chain-termination method. Analysis of the sequence data 1995; Takino et al., 1995; Puente et al., 1996). One of these, was performed as described (Devereux et al., 1984). The cDNA MT1-MMP (also called MMP-14), is overexpressed in insert contained in one of the clones shared 90% identity to a portion malignant tumour tissue, is localized on the surface of the (nt 271-648) of human MT1-MMP, and was used as a probe for cancer cells (Sato et al., 1994a), is able to degrade extracellu- screening of a rabbit osteoclast cDNA library. lar matrix molecules either directly (Pei and Weiss, 1996; Imai Isolation of MMP cDNA from osteoclast cDNA library et al., 1996) or indirectly (Sato et al., 1994a; Knaüper et al., A rabbit osteoclast cDNA library (Tezuka et al., 1994b) was screened 1996), and has been considered the trigger of cancer cell with the cDNA probe radiolabeled by using a multiprime DNA invasion (Sato et al., 1994a). labeling system (Amersham International plc., Buckinghamshire, To identify possible MT-MMPs expressed by osteoclasts, we England) and [α-32P]dCTP, as described elsewhere (Tezuka et al., used homology screening for MMPs in a rabbit osteoclast 1992). Isolated clones were converted to the plasmid form according cDNA library. We report here the isolation and the character- to the instruction manual (Stratagene, La Jolla, CA). The nucleotide istics of a cDNA encoding rabbit MT1-MMP, and demonstrate sequences were determined from both strands and the sequence data its expression in osteoclasts. Moreover, immunolocalizations were analyzed as described above. were consistent with an association of MT1-MMP to Amplification of cDNA 3′-ends podosomes and to lamellipodia of osteoclasts, giving insight in cDNA synthesized from rabbit osteoclast poly(A)+ RNA was the way invading cells direct proteolytic activity to specific amplified with the poly(dT) primer 5′-TAGAATTCTTTTTTTT- points of the extracellular matrix. TTTTTTTTTTTT-3′ and a 17 residue primer derived from the rabbit MT1-MMP sequence (1,803-1,819 in Fig. 1) by PCR. A cDNA fragment of 170 bp was cloned and sequenced as described MATERIALS AND METHODS above. Cells and organs for RNA preparation Northern blotting Osteoclasts were purified from long bones of 10-day-old rabbits Total RNA (5 µg) isolated from various organs and cells was blotted (Statens Seruminstitut, Copenhagen, Denmark) as described on nylon membranes after formaldehyde agarose gel electrophoresis, (Tezuka et al., 1992; Sato et al., 1994b; for a detailed protocol, see and hybridized with radioactive probes. A fragment of rabbit MT1- Helfrich et al., 1994). Briefly, the long bones were minced, the bone MMP cDNA (397-783 in Fig. 1), of human MT1-MMP (Sato et al., fragments were agitated, and the cells that were released (‘unfrac- 1994a, nucleotides 1,647-2,889, a kind gift from Drs H. Sato and M. tionated bone cells’) were cultured in plastic dishes in alpha-MEM Seiki, Kanazawa University) and a synthetic oligonucleotide corre- supplemented with 5% FBS. After overnight cultivation, the dishes sponding to 28 S ribosomal RNA were used as probes. cDNA probes were rinsed with phosphate buffered saline (PBS) and treated with were radiolabeled with a multiprime DNA labeling system pronase E (0.001%) and EDTA (0.02%) for 10 minutes, at 37°C.