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Genes to Cells (2009) 14, 617-626. doi:10.1111/j.1365-2443.2009.01293.x
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Cytoplasmic tail of MT1-MMP regulates macrophage motility independently from its protease activity

 Takeharu Sakamoto and Motoharu Seiki*

Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Membrane type-1 matrix metalloproteinase (MT1-MMP) is a proinvasive protease that regulates various cellular functions as evidenced by myriad defects in different types of cells and tissues in MT1-MMP-deficient (MT1–/–) mice. Here we demonstrate that MT1–/– mice exhibit fewer infiltrating macrophages into sites of inflammation. MT1–/– macrophages exhibited a reduced ability to invade reconstituted basement membrane (Matrigel) and invasion by wild type (WT) macrophages was inhibited by a synthetic MMP inhibitor (BB94) to a level similar to that of MT1–/– cells. The rate of migration of MT1–/– macrophages was also low compared to that of the WT cells and re-expression of MT1-MMP in MT1–/– macrophages reconstituted their migratory activity. Unexpectedly, however, BB94 did not inhibit the migration of WT macrophages. The migration-boosting activity of MT1-MMP is retained in a mutant that lacks most of the extracellular portion including the catalytic and hemopexin-like domains. In contrast, deletion of the cytoplasmic (CP) tail abolished the activity completely. Thus, we have demonstrated that MT1-MMP regulates macrophages via its invasion-promoting protease activity as well as its CP-dependent non-proteolytic activity to boost cell migration.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves multiple proteins in the pericellular milieu and has been characterized as a potent modulator of the cell environment (Egeblad & Werb 2002; Itoh & Seiki 2006). Processing of proteins by MT1-MMP alters their activities and thereby regulates a variety of cellular functions, such as motility, invasion, growth, differentiation, apoptosis and morphology, and so on. Mice deficient in MT1-MMP expression (MT1–/–) display multiple defects reflecting the importance of MT1-MMP in different cell types, such as fibroblasts, muscle cells, endothelial cells, osteoblasts, osteoclasts and adipocytes, and so on (Holmbeck et al. 1999; Zhou et al. 2000; Chun et al. 2006; Ohtake et al. 2006).

A major substrate of MT1-MMP is collagen I, which is the most abundant constituent of the tissue extracellular matrix (ECM). MT1-MMP is unique as a membrane-anchored collagenase and its activity is important for a variety of cell functions that require collagenolysis, such as cell growth, invasion and differentiation (Hotary et al. 2003; Chun et al. 2006). Other MT1-MMP substrates include ECM proteins, membrane proteins, and a variety of other proteins located in the cells’ vicinity (Itoh & Seiki 2006; Overall & Dean 2006). As the biological output of MT1-MMP is believed to be mediated by its protease activity, most MT1-MMP studies have focused on exploring mechanisms regulating the protease activity on the cell surface, identifying important substrates and characterizing the biological consequence of the cleavage of substrate proteins (Itoh & Seiki 2006; Overall & Dean 2006). This line of inquiry is still important for understanding the biological roles of MT1-MMP. Aside from these protease-dependent functions, little is known about non-proteolytic functions MT1-MMP.

In this study, we analyze the recruitment of macrophages to sites of inflammation and demonstrate that infiltration of macrophages into the inflammatory site is significantly disturbed in MT1–/– mice. As macrophage movement into a site of inflammation is a multi-step process that involves many factors and cells, our observation may reflect a complex interplay between different types of cells exhibiting defects caused by MT1-MMP-deficiency. However, a simpler hypothesis is that MT1-MMP might affect macrophage function directly. We analyze the roles of MT1-MMP expressed in macrophages by isolating such cells from wild type (WT) and MT1–/– mice. Although MT1-MMP is required for macrophage invasion acting as a protease, it also promotes the motility of the cells. Interestingly, the motility-stimulating activity of MT1-MMP is independent of the protease activity, but dependent upon the cytoplasmic (CP) tail. Thus, our study uncovers dual functions of MT1-MMP to stimulate macrophage invasion; degradation of the ECM by its protease activity and migration-boosting activity mediated by a CP-dependent mechanism.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Macrophage recruitment to an inflammatory site is disturbed in MT1-MMP-deficient mice

MT1-MMP is expressed by many types of normal and tumor cells (Sato et al. 1994; Hotary et al. 2000; Seiki & Yana 2003) including monocytes/macrophages (Shankavaram et al. 2001; Matias-Roman et al. 2005). To evaluate the functions of MT1-MMP in recruitment of macrophages to a site of inflammation, we subjected Mt1-mmp–/– (MT1–/–; Ohtake et al. 2006) and Mt1-mmp+/+ (WT) mice to TPA-induced acute ear inflammation (Nakadate et al. 1985; Cramer et al. 2003). The genotypes of the mice are schematically indicated in Fig. 1A and were confirmed by PCR as shown in Fig. 1B. Expression of MT1-MMP mRNA in fibroblasts isolated from the different mouse strains is presented in Fig. 1C. In our inflammation model, one ear is treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), whereas the other is treated with acetone (mock) as illustrated in Fig. 1D. Treated ears were harvested 24 h later for analysis of macrophage infiltration into the tissue.


Figure 1
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  		Figure 1  Experimental design using Mt1-mmp-deficient mice. (A) Illustration of Mt1-mmp genome and its targeted disruption in MT1–/– mice. Exons 1–5 encoding the catalytic domain of MT1-MMP are substituted with LacZ coding sequences in MT1–/– mice. LacZ is expressed as a fusion with a nuclear localizing signal (NLS) using the promoter of the Mt1-mmp locus. The PGK-gpt/neomycin-resistance gene cassette is in the reverse orientation. (B) The genotype of each littermate was confirmed by genomic PCR using site-specific primer sets. The following sequences were used: pWTF, 5'-tgaggtggaaaacacgaccag-3'; pWTR, 5'-atgatggcggagggatcgttag-3'; and pNeo, 5'-acctgcgtgcaatccatcttg-3'. The PWTF/pWTR set amplifies a 160-bp fragment corresponding to the wild-type allele, whereas pNeo/pWTR produces a 401-bp fragment from the recombined allele (arrows). (C) Northern blot analysis of MT1-MMP mRNA detects transcripts in WT and heterozygous fibroblasts but not in knockout homozygote. (D) Schematic illustration of the TPA-induced inflammatory assay.

 
The tissue sections were stained for macrophages using anti-F4/80 antibody and for leukocytes using anti-CD45 antibody (Fig. 2A). The number of infiltrating macrophages and leukocytes was counted and is presented in Fig. 2B. In WT mice, the number of macrophages recruited to the TPA-treated ear was greater than the number recruited to the mock-treated ear (Fig. 2A,B, F4/80 in WT). However, fewer macrophages were recruited to the TPA-treated ears of MT1–/– mice compared to that observed in WT mice (Fig. 2A,B, F4/80 in MT1–/–). The number of macrophages observed in the mock-treated ears did not differ between the two mouse strains (Fig. 2A,B, F4/80/mock). There was no difference in the recruitment of leukocytes to the sites of inflammation in MT1–/– and WT mice (Fig. 2A,B, CD45/TPA).


Figure 2
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  		Figure 2  MT1–/– macrophages less infiltrate into the inflammation site. (A) Infiltration of macrophages and leukocytes into the TPA- or vehicle (mock)-treated ear tissues of WT and MT1–/– mice. The tissue sections were subjected to immunohistochemical analysis. Macrophages were detected by anti-F4/80 antibody and leukocytes were detected by anti-CD45 antibody. Bar = 200 µm. (B) Six fields (40 000 µm2) of each section in Fig. 2A were randomly chosen and immunostaining-positive cells were counted. Mean ± SD (n = 6). **P < 0.01 (Student's t-test).

 
MT1-MMP promotes macrophage invasion

To analyze effect of the MT1-MMP-deficiencey on macrophage invasion, we isolated cells from the bone marrow of MT1–/– and WT mice, and induced them to differentiate into macrophages. Nearly 100% of the differentiated cells from both mice were stained for F4/80 (Fig. 3A). MT1-MMP protein was expressed in the WT but not the MT1–/– macrophages (Fig. 3B). We next tested the invasive ability of these macrophages using a transwell chamber equipped with a membrane filter coated with a reconstituted basement membrane (Matrigel). Cells were seeded on the top of the gel and allowed to invade the Matrigel. Cells that invaded the gel and migrated to the opposite side of the membrane through the pores were counted as invaded cells (Fig. 3C). The MT1–/– macrophages were much less invasive, as 80% fewer of these cells invaded the Matrigel than did the WT macrophages. Inhibition of MMP in WT macrophages with a synthetic inhibitor of MMPs (BB94) reduced invasion to a level similar to that of the MT1–/– macrophages. Thus, the protease activity of MT1-MMP plays a pivotal role in the invasion of WT macrophages. This is not surprising because MT1-MMP has been reported to augment invasion of many types of cells by degrading barriers of cell movement (Egeblad & Werb 2002; Itoh & Seiki 2006).


Figure 3
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  		Figure 3  Macrophages invade reconstituted basement membrane using the proteolytic activity of MT1-MMP. (A) Bone marrow derived macrophages from WT and MT1–/– mice were isolated and stained with F4/80 antibody (green). Nuclei were counter-stained by Hoechst33342 (blue). (B) MT1-MMP protein in WT and MT1–/– macrophages was detected by immunoblotting. Transferrin receptor (TrfR) served as a control. (C) Invasive activities of WT and MT1–/– macrophages. WT and MT1–/– macrophages were seeded on a Matrigel-coated transwells and MCP-1 was added in the lower chamber as a chemoattractant. Cells that reached the opposite side of the membrane were counted. BB94 (10 µM) was used as an inhibitor of MMP.

 
MT1-MMP enhances macrophage motility independently of its protease activity

We also analyzed migration of macrophages by culturing the cells in the presence of monocyte chemoattractant protein-1 (MCP-1) and monitored them with time-lapse microscope (Chemokinesis assay). The average migratory distance of MT1–/– macrophages during the 30 min period was 58% of that of the WT cells (Fig. 4A). Next, we analyzed the chemotaxis of macrophages towards MCP-1 using a transwell chamber. The number of migrated MT1–/– macrophages was approximately 44% of the number of migrated WT cells (Fig. 4B). Thus, MT1-MMP expressed in WT macrophages appears to promote both the invasion of Matrigel and migration of cells on a 2D matrix. To our surprise, BB94, which inhibited the invasion of WT macrophages into Matrigel, failed to inhibit chemotaxis (Fig. 4B, WT + BB94). Thus, the proteolytic activity of MT1-MMP is not required for promoting macrophage motility. In a previous study, another group reported that the migration of fibroblasts was not affected by MT1-MMP (Belien et al. 1999). We isolated fibroblasts from WT and MT1–/– mice and analyzed their motility using serum as a chemoattractant. Consistently with the previous report, we observed no difference in the migratory activity between fibroblasts from the two strains (Fig. 4C). Thus, the migration-stimulating activity of MT1-MMP appears to be specific to macrophages.


Figure 4
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  		Figure 4  MT1-MMP-deficiency causes reduced migratory response of macrophages to MCP-1. (A) Random motility (chemokinesis) of macrophages (n = 40) was monitored in the presence of 10 nM MCP-1 for 30 min and the distance they moved was measured using IMAGEJ software. Data are plotted as means ± SD. **P < 0.01 (Student's t-test). Chemotaxis of macrophages (B) or MEFs (C) was analyzed using transwells not coated with Matrigel. (D) F-actin in macrophages was visualized by staining the cells with phalloidin-Alexa488. Number of fluorescein-positive cells was counted at the indicated time points after MCP-1 stimulation and the ratio of positive cells is presented. Data are plotted as means ± SD (n = 5).

 
However, it is possible that MT1-MMP affects the process whereby signals are transmitted from MCP-1 to actin, part of the locomotive machinery of cells. To eliminate this possible explanation, we analyzed the polymerization of actin after stimulation of the cells with MCP-1. The polymerized actin was visualized using phallodin-Alexa488. MCP-1 induced actin polymerization in both WT and MT1–/– macrophages with a similar time course and to a similar extent (Fig. 4D).

Mutant MT1-MMP lacking protease activity enhances macrophage migration

To confirm that MT1-MMP has a motility-boosting activity independent from its protease activity, we expressed FLAG-tagged MT1-MMP (MT1F) or its mutant into MT1–/– macrophages. Expression of MT1F in the cells increased migratory activity, whereas expression of the control LacZ did not (Fig. 5A, LacZ and MT1F). As we observed with WT macrophages, BB94 could not inhibit the migration induced by the expression of MT1F (Fig. 5A, MT1F + BB94).


Figure 5
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  		Figure 5  MT1-MMP promotes chemotaxis of macrophages but not via its protease activity. (A) Expression of MT1-MMP promotes chemotactic response of MT1–/– macrophages towards MCP-1. FLAG-tagged MT1-MMP (MT1F) or its protease mutant with a E/A substitution (E/A) were stably expressed in MT1–/– macrophages using a lentivirus vector. Expression of LacZ was used as a control. Chemotaxis was analyzed using transwells not coated with Matrigel. Mean ± SD (n = 5). **P < 0.01 (Student's t-test). (B) Expression of MT1-MMP and its derivatives in the cells was confirmed by RT-PCR. (C) Proteolytic activity of MT1-MMP derivatives. MT1F or the E/A mutant were expressed in HEK293 cells together with MMP-2. Culture media were collected and subjected to gelatin zymography. MT1F generated processed MMP-2 but E/A mutant did not. MT1-MMP and actin in the cells were detected by immunoblotting as in the lower panels.

 
To confirm further that the motility-boosting activity of MT1-MMP is independent of its protease activity, we expressed a mutant MT1F lacking the protease activity by virtue of a 375E->A (E/A) substitution at a catalytically important site (Rozanov et al. 2001). Expression of this mutant was monitored by RT-PCR (Fig. 5B) and lack of the protease activity was confirmed by assay of the well-known activity of MT1-MMP to cleave MMP-2 for its activation (Fig. 5C). For the protease assay, either MT1F or MT1F E/A mutant was expressed in HEK293 cells together with MMP-2 and the culture media were analyzed by gelatin zymography (Fig. 5C). Both intact (proMMP-2) and a cleaved MMP-2 by MT1-MMP were detected in the media of the cells expressing MT1F, whereas expression of the E/A mutant failed to generate the processed MMP-2. This E/A mutant nevertheless stimulated the motile response of MT1–/– cells as effectively as MT1F did (Fig. 5A, E/A). Thus, MT1-MMP augments macrophage motility, but this effect is not dependent upon its protease activity.

Domain analysis of MT1-MMP for the migration boosting activity

To identify the protein domains responsible for boosting the migration of MT1–/– macrophages, we prepared various deletion mutants of MT1-MMP (Fig. 6A). Each construct was expressed in MT1–/– macrophages (Fig. 6B) and the effect of this expression on cell motility was analyzed with a chemotaxis assay (Fig. 6C). The expression of MT1F augmented chemotaxis and deletion constructs lacking either the catalytic domain (dCAT) or the C-terminal hemopexin-like domain (dHPX) retained this migration-boosting activity (Fig. 6C, dCAT and dHPX). However, deletion of the cytoplasmic (CP) tail from MT1F (dCP) abolished the migration-boosting activity (Fig. 6C, dCP) despite its efficient display on the cell surface in the previous studies (Jiang et al. 2001; Uekita et al. 2001). However, a deletion mutant containing the CP tail anchored to the plasma membrane via the transmembrane domain (TM-CP) retained full activity to augment chemotaxis (Fig. 6C, TM-CP). Thus, the stimulation of macrophage motility by MT1-MMP is not dependent upon its protease activity but instead appears to be mediated by sequences within the CP tail. The CP tail is reported to bind several cellular proteins (Labrecque et al. 2004; Itoh & Seiki 2006; Terawaki et al. 2008) and it remains unclear how the migratory response of macrophages is mediated by the CP tail.


Figure 6
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  		Figure 6  The cytoplasmic tail of MT1-MMP regulates macrophage chemotaxis. (A) Deletion constructs of MT1-MMP are illustrated. CAT, catalytic domain; HPX, hemopexin-like domain; TM, transmembrane domain; CP, cytoplasmic tail; dCAT, deletion of the catalytic domain; dHPX, deletion of the hemopexin-like domain; dCP, deletion of the cytoplasmic domain; TM-CP, deletion of extracellular domains. All the proteins were expressed as a FLAG-tagged form fused to the N-terminus. (B) MT1-MMP derivatives were expressed in MT1–/– macrophages using a lentivirus vector. Expression of mRNA was confirmed by RT-PCR. (C) MT1–/– macrophages expressing each mutant were subjected to chemotaxis assay. Mean ± SD (n = 5). **P < 0.01 (Student's t-test).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Infiltration of macrophages into sites of TPA-induced inflammation was reduced in MT1–/– mice, whereas the recruitment of leukocytes to such sites was not affected by the lack of MT1-MMP. Recruitment of macrophages to inflammatory sites is a multistep process that can be affected by different types of cells and the tissue environment. As the lack of MT1-MMP affects the functions of many types of cell, our observation may reflect multiple defects in MT1–/– mice. Although we cannot exclude this possibility, we demonstrated that MT1-MMP affects macrophage functions directly. MT1–/– macrophages exhibited reduced invasion activity into Matrigel. Inhibition of MMP activity in WT macrophages using BB94 inhibited invasion to a level similar to that observed in MT1–/– cells. Thus, the ECM-degrading activity of MT1-MMP presumably contributes to the recruitment of macrophages to sites of inflammation in mice by eliminating barriers to macrophage movement.

In addition to the invasion-promoting activity, we also demonstrated that MT1-MMP stimulates the migration of macrophages. We confirmed that the migration-stimulating activity in macrophages did not require protease activity by different approaches, because such a non-proteolytic function of MT1-MMP in macrophages has not been reported to date. First, we showed that the migration of WT macrophages was not inhibited by BB94. Second, we observed that forced expression of MT1-MMP in MT1–/– macrophages boosted their migratory activity, and this effect was not inhibited by BB94. Third, analysis of the migration-boosting activity of MT1-MMP mutants revealed that point mutation at the catalytic site or deletion of the extracellular protease domain did not abolish the migration-boosting activity. However, deletion of the CP tail abolished this activity. Finally, a mutant MT1-MMP comprising solely the transmembrane domain and the CP tail was sufficient to impart the migration-boosting activity.

The migration-stimulating activity of MT1-MMP appeared to be specific to macrophages, because the lack of MT1-MMP did not affect the migration of fibroblasts. Migration of leukocytes to inflammatory sites was also unaffected by the lack of MT1-MMP. However, there are various in vitro studies that report the stimulation of cell migration by MT1-MMP in different types of cells and these effects have been shown to be mediated by the protease activity of MT1-MMP (Kajita et al. 2001; Ueda et al. 2003; Sato et al. 2005; Itoh 2006; Barbolina & Stack 2008). MT1-MMP is expressed in many types of migratory cells, such as macrophages, neural crest cells and malignant cancer cells (Egeblad & Werb 2002; Itoh & Seiki 2006). In these cells, proteolytic modulation of cell surface proteins by MT1-MMP alters cell signaling regulating migration. However, what we observed here is that MT1-MMP stimulates macrophage migration without requiring its protease activity and the CP tail plays a major role.

It is unclear how profoundly the CP-dependent mechanism contributes to the macrophage invasion into Matrigel. The invasive activity of cells is largely correlated to the amount of protease activity on the cell surface. However, the CP tail of MT1-MMP regulates its internalization thereby affecting the amount of the protease on the cell surface (Jiang et al. 2001; Uekita et al. 2001). Thus, it is difficult to distinguish the individual contribution of the protease activity and the effect of the CP tail on cell invasion when using the dCP mutant of MT1-MMP.

The CP-dependent mechanism appears to operate in a cell-type specific manner, at least between macrophages and mouse embryonic fibroblasts (MEFs) and it is an important question how this specificity is substantiated by the molecules in the cells. One possibility is that the CP tail of MT1-MMP plays a role in transmitting receptor-mediated signals elicited by MCP-1. However, treatment of MT1–/– macrophages with MCP-1 induced actin polymerization just as effectively as in the WT cells, arguing against this idea. Another possibility is that a certain cell-type specific protein binds to the CP tail of MT1-MMP and elicits or suppresses cellular mechanisms for migration. Indeed, the CP tail of MT1-MMP can bind several proteins and some of them may play a role in the migratory process (Uekita et al. 2004; Sato et al. 2005; Itoh & Seiki 2006; Barbolina & Stack 2008). Our findings may also have relevance to a recent observation that TIMP-2 binding to MT1-MMP promotes growth and migration of a human tumor cell line MCF-7 (D’Alessio et al. 2008). This effect does not require degradation of the ECM by MT1-MMP and is dependent on the cytoplasmic tail. These questions can be clarified most directly by determining the exact mechanism by which MT1-MMP promotes macrophage migration in a fashion independent of its protease activity and such studies are underway.

A recent study reports apparently contradictory observations to ours (Schneider et al. 2008). In this study, MT1–/– mice were used to analyze the in vivo function of MT1-MMP in atherosclerosis. They found a similar level of macrophage infiltration into the atherosclerotic plaques of either MT1–/– or WT mice at 8 or 16 weeks after commencement of an atherogenic diet. As experimental designs, mouse strains and observed tissues are different, it is not easy to compare the two experiments. However, it should be noted that the interval between stimulus and observation in the two experiments is different. Although we observed the early inflammatory response at 24 h after stimulation, the reported study analyzed the mice at 8 and 16 weeks after the atherogenic diet was initiated. This might be a major factor that explains the different results. Even in our study, a similar number of macrophages were present in the mock-treated tissues of WT and MT1–/– mice (Fig. 2A, mock). It should be noted as well that the lack of MT1-MMP in macrophages reduced invasion and migration, although not completely. Thus, it is plausible that defective recruitment of macrophages to sites of inflammation in MT1–/– mice occurs only during the acute phase of inflammation and may gradually recover to a level similar to that in WT mice during a chronic phase of inflammation.

MT1-MMP plays multiple roles in different types of cell by modulating functions of substrate proteins on the cell surface. In contrast, we uncovered a new function of MT1-MMP that regulates macrophage motility independently from its protease activity. This is the first report that MT1-MMP-deficient macrophages exhibit a dysfunction related to a non-proteolytic function of MT1-MMP and provides a clue to explore new functions of MT1-MMP.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Mt1-mmp gene-targeted mice

A mouse (129SVJ) genomic clone encoding MT1-MMP was used to construct the targeting vector indicated in Fig. 1. This vector can be used to disrupt and substitute exons 1–5, which encode the catalytic domain, with a gene encoding LacZ fused to a nuclear localizing signal (NLS; Kanegae et al. 1995). The PGK-gpt/neomycin-resistance gene cassette (Stratagene) was inserted in the reverse direction, downstream of lacZ. Mt1-mmp+/– mice were obtained and crossed to generate Mt1-mmp–/– mice. Established Mt1-mmp–/– mice display a systemic skeletal malformation and have a short lifespan of approximately 3–4 weeks, consistent with other Mt1-mmp-deficient strains (Holmbeck et al. 1999; Zhou et al. 2000; Ohtake et al. 2006). Mt1-mmp+/– mice were backcrossed against C57BL/6 background mice 12 times. Mice were maintained under specific pathogen–free conditions. Experiments were carried out on 10- to 18-day-old littermates of WT and MT1–/– mice. The experiments were conducted according to the institutional ethical guidelines for animal experiments and the safety guidelines for gene manipulation experiments (The Institute of Medical Science, University of Tokyo).

Cell culture

Bone marrow-derived macrophages were obtained as described previously (Celada et al. 1984; Kobayashi et al. 2002). In brief, bone marrow was obtained from the tibia, femur and humerus via flushing with PBS. Collected cells were then cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 20% FBS and 30% L929-conditioned medium, after which they were grown at 37 °C in humidified 5% CO2 for 7 days. The macrophages were rendered quiescent by culturing them overnight in medium lacking the L929-conditioned medium. Mouse embryonic fibroblasts (MEFs) were prepared as described previously (Taniwaki et al. 2007) and cultured in DMEM supplemented with 10% FBS. HEK293 cells were purchased from ATCC and cultured in DMEM high glucose (Sigma) supplemented with 10% FBS.

Plasmids

Expression constructs for mutant MT1-MMP were prepared using a PCR-based method. The domains used for construction of the mutants were CAT (112–284 aa), HPX (319–507 aa), CP (562–581 aa) and the extracellular domain (112–507 aa). Sequences encoding the FLAG peptide tags were inserted immediately downstream of the furin cleavage site, as it is exposed at the N-terminus following proteolytic processing. These proteins were expressed in cells using lentivirus (pLenti6) vectors (Invitrogen) or pcDNA3.1 (Invitrogen) vectors.

TPA-induced ear inflammation assay

TPA (1.5 µg in 10 µL acetone) was spotted onto the left ear, as reported previously (Cramer et al. 2003). The right ear was mock-treated with acetone alone. Ear tissues were dissected 24 h later, embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Japan), and frozen with liquid N2 for histological analysis.

Immunohistochemistry

Frozen sections (10 µm) were fixed in 4% paraformaldehyde/PBS for 5 min and incubated in 0.03% H2O2/PBS for 15 min. Blocking was carried out in 5% goat serum and 3% BSA in PBS for 1 h at room temperature. Sections were then incubated with anti-F4/80 (BMA Biomedicals) or anti-CD45 (Beckman Coulter) antibodies at 4 °C overnight. After washing with PBS, sections were incubated with Histofine HRP conjugated anti-rat IgG antibody (Nichirei) for 30 min and developed chromogenically in DAB solution.

Immunostaining

Cells were fixed with 4% paraformaldehyde for 5 min. After blocking in PBS containing 5% goat serum and 3% BSA, cells were incubated with rat anti-F4/80 (BMA Biomedicals) or control IgG (Sigma) for 1 h, then washed three times and incubated for 1 h with anti-rat IgG Alexa488 conjugate (Invitrogen). Cells were counter-stained with Hoechst33342, washed five times with PBS, mounted and observed by CCD microscopy.

RNA isolation, reverse transcription and real-time PCR

Total RNA was isolated from macrophages using TRIZol (Invitrogen) and subjected to reverse transcription (RT) using SuperscriptII (Invitrogen) and random primers.

Invasion and migration assays

Matrigel invasion and transwell migration assays were carried out as described previously (Ueda et al. 2003; Nonaka et al. 2005). Briefly, transwells with 8 µm pore size filters (Corning) covered with or without matrigel (Becton Dickinson) were inserted into 24-well plates. DMEM (500 µL) containing 10 ng/mL MCP-1 (R&D Systems; macrophages) or 10% FBS (MEFs) was added to the lower chamber, whereas a 200-µL cell suspension (2 x 105 cells) was placed in the upper chamber. The plates were incubated at 37 °C in a 5% CO2 atmosphere for 2 and 6 h, for the migration and invasion assays, respectively. Cells in the lower chamber were then stained with Giemsa solution and counted.

Immunoblot analysis

Cells were lysed with lysis buffer and centrifuged at 20 000 g for 15 min at 4 °C. The supernatants were collected and protein content was measured using the Bradford assay (Bio-Rad). Lysates were separated by SDS-PAGE, transferred to membrane filters, and subjected to immunoblot analysis using anti-MT1-MMP mouse antibody (Daiichi Fine Chemical), anti-transferrin receptor mouse antibody (Invitrogen) or anti-FLAG epitope M2 antibody (Sigma).

Preparation of lentiviral vectors

Lentiviral vectors carrying cDNAs for expression were constructed using the ViraPowerTM Lentiviral Expression System (Invitrogen). Lentiviruses in the culture medium were recovered from the cleared supernatant following centrifugation of the medium at 1400 g. Supernatants were passed through membrane filters (0.45 µm pore size; Millipore), followed by two rounds of centrifugation at 70 000 g for 2 h at 21 °C. The resulting pellet was resuspended in 200 µL DMEM and the virus titer measured using Hela cells. Infected cells were selected by Blastcidin resistance and counted. Lentivirus vectors were transduced into macrophages at 3 MOI and MT1-MMP expression was detected by RT-PCR using the following specific primers: MT1-MMP sense, 5'-atgtctcccgcccctcgacc-3' and antisense, 5'-acattggccttgatctcagt-3' and β-actin sense, 5'-gccaacacagtgctgtctgg-3' and antisense 5'-atctgctggaaggtggacag-3'.

Chemokinesis assay

Macrophages were seeded into 35 mm glass dishes and cultured overnight. Thirty minutes after the addition of 10 ng/mL MCP-1, images were captured every 1 min using a Leica AS MDW time-lapse system (Leica Microsystems). Cell migration was measured as the sum of the linear distances between centroids using IMAGEJ software (NIH).

Actin polymerization assay

Macrophages were starved for 24 h in serum free DMEM and then stimulated with 10 ng/mL MCP-1. After MCP-1 stimulation, macrophages were fixed with 4% PFA at the indicated time points. Then the cells were stained with phallodin-Alexa488 (Invitrogen), washed with PBS three times, mounted, and observed under a CCD fluorescent microscope.

Gelatin zymography

HEK293 cells were seeded into 12-well plates (5 x 104 cells/well) and transfected with pLenti6 MMP-2 (250 ng/well) vector and pcDNA3.1 vectors (250 ng/well) expressing FLAG-tagged MT1-MMP (MT1F), the 375E->A substituted MT1F mutant (E/A), or none (mock). Twenty-four hours after transfection, cells were cultured in serum-free media for 4 h. The culture media were subjected to gelatin zymography assay as described previously (Sato et al. 1994; Taniwaki et al. 2007). Expression of MT1-MMP in cells was detected by immunoblotting.


   Acknowledgements
 
We thank Ikuo Yana and Yohei Ohtake for helpful discussions, and Akiko Rikimaru, Makoto Nagano, Daisuke Hoshino and Nagayasu Egawa for technical assistance. We also thank Dr Mitsuaki Yoshida for his support to T.S., Dr Roy Zent for helpful discussion, and Dr Robert Whittier for careful checking of the manuscript. This work was supported by the Specific Coordination Fund for Promoting Science to T.S. and by a Grant-in-Aid for Scientific Research on Priority Areas, that is, the ‘Integrative Research toward the Conquest of Cancer,’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to M.S. This work was supported in part by Global COE Program ‘Center of Education and Research for the Advanced Genome-Based Medicine—For personalized medicine and the control of worldwide infectious diseases’, MEXT, Japan.


   Footnotes
 
Communicated by: Tadashi Yamamoto

* Correspondence: mseiki{at}ims.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 14 January 2009
Accepted: 19 February 2009




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