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Genes to Cells (2007) 12, 1091-1100. doi:10.1111/j.1365-2443.2007.01110.x
© 2007 Blackwell Publishing or its licensors
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Establishment of an MT4-MMP-deficient mouse strain representing an efficient tracking system for MT4-MMP/MMP-17 expression in vivo using ß-galactosidase

 Akiko Rikimaru1, Kiyoshi Komori1, Takeharu Sakamoto1, Hirotake Ichise2, Nobuaki Yoshida2, Ikuo Yana1 and Motoharu Seiki1,*

1 Division of Cancer Cell Research, and
2 Laboratory of Gene Expression and Regulation, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The biological functions of membrane-type 4 matrix metalloproteinase (MT4-MMP/MMP-17) are poorly understood because of the lack of a sensitive system for tracking its expression in vivo. We established a mutant mouse strain (Mt4-mmp–/–) in which Mt4-mmp was replaced with a reporter gene encoding ß-galactosidase (LacZ). Mt4-mmp–/– mice had normal gestations, and no apparent defects in growth, life span and fertility. Using LacZ as a marker, we were able to monitor the expression and promoter activity of Mt4-mmp for the first time in vivo. The tissue distribution of Mt4-mmp mRNA correlated with LacZ expression, and we showed that Mt4-mmp is expressed primarily in cerebrum, lung, spleen, intestine and uterus. We identified LacZ-positive neurons in the cerebrum, smooth muscle cells in the intestine and uterus, and macrophages located in the lung alveolar or intraperitoneal space. Contrary to the reported role of MT4-MMP as a tumor necrosis factor-{alpha} (TNF-{alpha}) sheddase, the lipopolysaccharide (LPS)-induced release of TNF-{alpha} from Mt4-mmp–/– macrophages was similar to that in wild-type cells, and expression of Mt4-mmp mRNA was repressed following LPS stimulation. Thus, we have established a mutant mouse strain for analyzing the physiological functions of MT4-MMP, which also serves as a sensitive system for monitoring and tracking the expression of MT4-MMP in vivo.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The mammalian matrix metalloproteinase (MMP) family has 23 members, which share a common conserved domain structure (Brinckerhoff & Matrisian 2002; Egeblad & Werb 2002). MMPs function by degrading components of the extracellular matrix (ECM), and collectively, they are responsible for tissue remodeling during embryogenesis, organogenesis, tissue regeneration, wound healing and many diseases (Egeblad & Werb 2002). MT4-MMP/MMP17 is a relatively new member of the MMP family and has been poorly characterized to date (Puente et al. 1996; Kajita et al. 1999). One of the barriers to understanding the function(s) of MT4-MMP/MMP17 is the lack of an efficient system for tracking the expression of MT4-MMP in vivo and in vitro. In addition, the enzyme shows weak proteolytic activity against ECM proteins that are efficiently degraded by other MMPs. Mt4-mmp was identified by homology-based cDNA cloning, and its product was identified as a member of the MT-MMP subgroup based on the presence of a hydrophobic signal sequence for GPI-anchoring at its carboxyl terminus (Itoh et al. 1999; Seiki 2003). Most of the MT-MMPs have a type I transmembrane sequence (MT1-, MT2-, MT3- and MT5-MMP) and display potent proteolytic activity towards ECM components, such as collagens and proteoglycans (Seiki 2003; Itoh & Seiki 2006). The GPI-anchored MT-MMPs, including MT4-MMP and MT6-MMP, are distantly related to the transmembrane-anchored MT-MMPs in amino acid sequence and do not possess clear proteolytic activity towards the ECM substrates of the transmembrane-anchored MT-MMPs (Seiki 2003; Itoh & Seiki 2006).

Although information about the substrates of MT4-MMP is limited, there are some reports of its proteolytic activity, using biochemical or cell culture systems in vitro. MT4-MMP activates a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) in vitro, which indicates that it is involved in cartilage destruction (Gao et al. 2004; Patwari et al. 2005). MT4-MMP has been shown to cleave tumor necrosis factor-{alpha} (TNF-{alpha}) in vitro, and ectopic expression of MT4-MMP in COS-7 results in shedding of the membrane-bound form of TNF-{alpha}, which suggests that MT4-MMP also functions as a TNF-{alpha}-converting enzyme (TACE) (English et al. 2000). MT4-MMP is expressed in monocytic cells (Kajita et al. 1999; English et al. 2000), so MT4-MMP-mediated shedding of TNF-{alpha} may play a role in the inflammation process. Mt4-mmp mRNA has also been detected in brain, ovary, testis and colon (Puente et al. 1996; Grant et al. 1999; Kajita et al. 1999). However, the cells that express Mt4-mmp in these tissues have not been defined.

The specific aim of this study was to carry out a functional analysis of MT4-MMP by generating a mutant mouse strain that lacks Mt4-mmp and to establish an efficient system for tracking the expression of MT4-MMP in vivo. We generated an MT4-MMP-null mouse strain by substituting part of the mouse Mt4-mmp genomic locus with the bacterial LacZ gene. This system also enabled us to monitor expression of Mt4-mmp by tracking LacZ activity. MT4-MMP-null mice (Mt4-mmp–/–) had no apparent defects in gestation, growth, morphology, fertility and behavior. The tissue distribution of LacZ mRNA in Mt4-mmp–/– mice correlated well with the expression of Mt4-mmp mRNA in wild-type mice. LacZ activity was detected in neurons in the central nervous system, smooth muscle cells (SMCs) throughout the body, and leukocytes in the lung and abdominal cavity. Using macrophages derived from MT4-MMP-null mice, we demonstrated that MT4-MMP does not function as a TACE in macrophages. Thus, we have established and demonstrated the efficacy of an MT4-MMP-null mouse strain for monitoring and tracking Mt4-mmp expression in vivo.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Gene-targeting to generate mutant mice that express LacZ under the control of the Mt4-mmp promoter

To establish a mutant mouse strain in which the expression of LacZ was controlled by the endogenous Mt4-mmp promoter, a genomic sequence containing the initiation codon and part of the first intron of Mt4-mmp was substituted with a DNA cassette containing LacZ and a neomycin-resistant gene (Fig. 1A). In this system, LacZ is expressed as a fusion protein with a nuclear localization signal (NLS) sequence and should localize to the nucleus. Homologous recombination was used to generate embryonic stem (ES) cell clones in which the targeting vector sequences were incorporated into the mouse Mt4-mmp locus. ES cells were isolated and injected into C57BL/6J blastocysts to generate chimeric male founders. MT4-MMP-deficient mice in an inbred congenic genetic background were generated by backcrossing heterozygous mice with C57BL/6J mice for at least 12 generations. Mt4-mmp–/– mice were generated by intercrossing heterozygous mice, and genomic analysis was carried out by Southern blot (Fig. 1B). Expression of Mt4-mmp and LacZ was also analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using RNA from brain tissues (Fig. 1C). Mt4-mmp mRNA was present in tissue from wild-type (Mt4-mmp+/+) and heterozygous (Mt4-mmp+/–) mice, and absent in tissue from Mt4-mmp–/– mice. LacZ mRNA was not detected in Mt4-mmp+/+ mice, but was present in Mt4-mmp+/– and Mt4-mmp–/– mice. The genotypes of the intercrosses followed a Mendelian ratio, and homozygous null mice grew normally, and their appearance, fertility and life span were similar to those of wild-type mice.


Figure 1
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  		Figure 1  Targeted disruption of MT4-MMP gene. (A) Schematic illustration of the targeting strategy for disrupting Mt4-mmp/mmp-17 in mice. Restriction enzyme sites were mapped in the Mt4-mmp gene locus. Thick bars represent the homologous arms of the targeting vector. Probes for detecting homologous recombination are indicated as short thick bars. The targeting vector contained both arms and sequences encoding LacZ fused with an NLS (NLS-lacZ) in the PGK-gpt/neomycin-resistant gene (Stratagene) cassette. Predicted sizes of BamHI fragments before and after homologous recombination are also indicated. (B) Germ-line transmission of the mutant Mt4-mmp locus was confirmed by Southern blot analysis of genomic tail DNA. (C) RT-PCR of Mt4-mmp and LacZ mRNA shows that Mt4-mmp transcripts are present in brain only from Wt or heterozygous mice and LacZ transcripts are present only in KO or heterozygous mice.

 
Tissue distribution of Mt4-mmp and LacZ transcripts

To determine whether the expression of LacZ mRNA in Mt4-mmp–/– mice represented transcription of endogenous Mt4-mmp, LacZ and Mt4-mmp mRNA transcripts were measured by real time RT-PCR, and amount of each mRNA relative to the lung is presented in Fig. 2. Prominent expression of Mt4-mmp and LacZ mRNA in Mt4-mmp+/+ (Wt) and Mt4-mmp–/– (knockout; KO) mice, respectively, was observed in cerebrum, lung and uterus, and intermediate levels of expression were observed in spleen, stomach, intestine, testis and ovary. Expression was low in cerebellum, heart, liver, kidney, ovary and skeletal muscle. These results demonstrated that the expression pattern of LacZ in KO mice roughly paralleled Mt4-mmp expression in Wt mice. The exception was in the testis, where expression of LacZ was extremely high compared to that of Mt4-mmp. Additional experiments are needed to understand why LacZ expression was higher in testis compared to any other tissue examined.


Figure 2
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  		Figure 2  Tissue distribution of transcripts for MT4-MMP and LacZ. Tissues were isolated from 8-week-old Wt and KO mice. Total RNA was extracted, and Mt4-mmp and LacZ mRNA was measured by real time PCR. The amount of mRNA in the various tissues is presented relative to that in the lung. Data represent the averages of three measurements.

 
Expression of MT4-MMP in cerebrum

We also examined MT4-MMP protein expression in Wt and KO mice by Western blot in the cerebrum, where Mt4-mmp mRNA was transcribed most abundantly (Fig. 2). It was difficult to detect MT4-MMP by analyzing tissue lysates directly because of its low protein levels, so total brain lysate was first subjected to immunoprecipitation to concentrate the protein using a polyclonal anti-MT4-MMP antibody and then subjected to Western blot analysis using an anti-MT4-MMP rat monoclonal antibody (Fig. 3A). Several specific bands were detected in tissue from Wt and heterozygous mice, and the corresponding bands in null mice were absent. The two major bands were 55 and 50 kDa, corresponding to the latent (proform) and activated forms of MT4-MMP, respectively, based on their molecular sizes. The faint band of approximately 60 kDa likely corresponded to the precursor form of MT4-MMP, before the addition of the GPI anchor. Some of the faster migrating bands presumably represented degradation products of MT4-MMP. We next prepared sections of brain from 8-week-old KO mice and examined them for LacZ activity using 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) as a substrate. The histology of brains from KO mice was not significantly different from that of Wt mice (data not shown). Blue signals were detected in the cerebrum, including the cortex, hippocampus and caudate putamen, but not in the cerebellum of KO mice (Fig. 3B). Based on their location, the LacZ-positive cells appeared to be neurons. When we co-immunostained the sections using a neurofilament-specific antibody (Fig. 3C), we found that most of the LacZ-positive cells were also positive for neurofilaments. There were also some neurofilament-positive cells that did not express LacZ (Fig. 3D). When we quantified the immunostaining results, we found that the LacZ-positive ratio differed according to the specific layer of the cerebral cortex (Fig. 3E). The ratio of LacZ-positive cells was high in the multiform and external pyramidal layers, and was comparatively low between them. The biological significance of this expression pattern is not clear, and there were no apparent histological differences in this region between Wt and KO mice.


Figure 3
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  		Figure 3  Expression of MT4-MMP and LacZ in mouse brain. (A) Tissue lysates were prepared from adult mouse brains (8 weeks old) and subjected to immunoprecipitation (IP) using rabbit anti-mouse MT4-MMP polyclonal antibodies. Immunoprecipitates were further analyzed by Western blot using rat anti-mouse MT4-MMP monoclonal antibody. Specific bands (closed arrows) from Wt and heterozygous mice correspond to the precursor of the GPI-anchored form (63 kDa), the GPI-anchored latent form (55 kDa) and the GPI-anchored active form (50 kDa) of MT4-MMP. Lower molecular weight bands (open arrows) presumably correspond to degradation products of the enzyme. Actin was used as the loading control. (B) Frozen tissue sections of mouse brain (Mt4-mmp–/–) were subjected to LacZ and eosin staining. AON, anterior olfactory nucleus; Tu, olfactory tubercle; CP, caudate putamen; Hi, hippocampus; SN, substantia nigra; M, motor cortex; S, somatosensory cortex; V, visual cortex. Prominent staining was observed in cerebrum, hippocampus and corpus striatum. Scale bar corresponds to 4 mm. (C) A frozen tissue section from the LacZ-positive cerebrum area was stained for LacZ (blue) and then for neurofilaments (brown). Scale bar corresponds to 0.2 mm. (D) Cells that were positive for LacZ and neurofilaments were counted in four different fields; number of LacZ-positive, neurofilament (NF)-positive or double-positive cells is presented. (E) Tissue sections of cerebrum were stained with either hematoxylin–eosin (HE) (a) or LacZ/HE (b). Enlarged pictures are presented in the middle panels, and number of cell nuclei was counted under the microscope. The number of cells in the indicated microscopic fields is presented versus the corresponding cortex layers.

 
Expression of MT4-MMP in smooth muscle cells (SMC)

Mt4-mmp and LacZ mRNAs were also highly expressed in the uterus. Sections of the uterus from Wt and KO mice were obtained and subjected to hematoxylin and eosin staining. A macroscopic image of a section from a Wt uterus is depicted in Fig. 4A. There were no apparent structural differences between the smooth muscle layers (SMLs) of sections from Wt and KO mice (data not shown). A representative image of a section from a KO mouse is presented in Fig. 4B. SMCs were abundant in the outer longitudinal layer (M1), based on the number of cell nuclei, and were less abundant in the inner circular layer (M2) (Fig. 4B). LacZ signals were detected primarily in the M1 layer (Fig. 4C) and coincided with the location of SMCs. SMLs were also positive for smooth muscle actin (SMA) (data not shown).


Figure 4
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  		Figure 4  Expression of MT4-MMP in smooth muscle tissue. (A) Frozen tissue sections of uterus isolated from 8-week-old homozygous mice were stained for HE. SMLs composed of an outer longitudinal layer (M1) and inner circular layer (M2) are indicated. Scale bar = 0.1 mm. (B and C) Enlarged pictures of SMLs. Tissues were stained for either HE (B) or LacZ/HE (C). Note that M1 layer is enriched in SMCs, and LacZ signals mostly overlap with the nuclei of the cells in the M1 layer. (D–F) Tissue sections of (D) small intestine, (E) large intestine and (F) aorta were subjected to LacZ and HE staining. Note that the SMLs are positive for LacZ. Scale bar = 0.1 mm. (G) Whole intestine or enriched SML from whole intestine was obtained from a Wt mouse (8 weeks old). MT4-MMP was immunoprecipitated from tissue lysate as in Fig. 3 and then analyzed by Western blot using anti-MT4-MMP. Total tissue lysate (Input) was also analyzed by Western blot using either anti-SMA or anti-actin antibody. In each lane, an equal amount of protein was loaded. Latent (55 kDa) and active forms (50 kDa) of MT4-MMP are indicated by closed arrows. (H) Tissue distributions of Mt4-mmp and SMA mRNA expressions were compared. Total RNA was extracted from the indicated tissues, and the amount of mRNA was measured by real time RT-PCR. The amount of mRNA relative to that in lung is presented. Data represent the averages of three measurements. Note that tissues that express SMA also express Mt4-mmp.

 
Other tissues containing SMLs, such as the small intestine (Fig. 4D), large intestine (Fig. 4E) and aorta (Fig. 4F), were also examined for the presence of LacZ. Similar to the uterus, the nuclei of the SMCs in these tissues were also positive for LacZ.

To confirm the expression of MT4-MMP protein in SMCs, we prepared tissue lysates from whole mouse intestine and mucosa-eliminated tissue (Fig. 4G, Whole and SML, respectively). Samples containing equal amounts of protein were subjected to immunoprecipitation using an anti-MT4-MMP polyclonal antibody, and then analyzed by Western blot (Fig. 4G). MT4-MMP was detected predominantly in the SML-enriched tissue from Wt mice, and it was absent in the sample from KO mice (data not shown). These results confirmed that MT4-MMP is expressed in SMCs.

To determine whether other tissues that contained smooth muscle components also expressed Mt4-mmp, we surveyed the tissue distribution of SMA mRNA transcripts and compared it to that of Mt4-mmp (Fig. 4H). All the tissues that expressed SMA mRNA (lung, stomach, intestine, testis, ovary and uterus) also expressed Mt4-mmp mRNA at a comparable level. These results suggested that Mt4-mmp is expressed in SMCs throughout the mouse body.

Tissue macrophages express Mt4-mmp

In addition to the SMCs in vessels, macrophage-like cells in the lung also appeared to express LacZ (Fig. 5A). To examine this further, we isolated tissue macrophages from the lung and the abdominal cavities. The cells were stained for LacZ and F4/80, a macrophage-specific antigen (a member of the epidermal growth factor-transmembrane 7 family), and a representative result showing alveolar macrophages is presented in Fig. 5B. Isolated macrophages were double positive for F4/80 and LacZ, which indicated that alveolar macrophages express Mt4-mmp. This result was consistent with previous observations that monocyte/macrophage cell lines express Mt4-mmp mRNA (Kajita et al. 1999; English et al. 2000).


Figure 5
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  		Figure 5  Expression of MT4-MMP in macrophages. (A) Frozen tissue sections of lung isolated from 8-week-old homozygous mice were stained for LacZ and eosin. Note that SMLs and macrophage-like cells are positive for LacZ staining. Scale bar = 0.1 mm. (B) Alveolar macrophages were isolated from an MT4-MMP-null mouse and subjected to LacZ staining (left) and F4/80 immunostaining (right). Scale bar = 0.1 mm. Arrows indicate typical double-positive cells.

 
Role of MT4-MMP in TNF-{alpha} shedding

Previously it was reported that MT4-MMP exhibited TACE-like activity when expressed in COS-7 cells (English et al. 2000). Since monocyte/macrophages express MT4-MMP, it has been proposed that MT4-MMP plays a role in TNF-{alpha} shedding. We took advantage of our MT4-MMP-null mice to examine whether MT4-MMP plays a role in TNF-{alpha} shedding in macrophages. Macrophages were isolated from the peritoneum of Wt and KO mice, and the expression of mRNA transcripts for MT4-MMP and TNF-{alpha} was assessed by real time RT-PCR (Fig. 6A,B). The expression of Mt4-mmp mRNA decreased dramatically at 8 h after stimulation with lipopolysaccharide (LPS) (Fig. 6A) and increased gradually thereafter (data not shown). The expression levels of LacZ mRNA in KO cells also decreased, similar to Mt4-mmp mRNA in Wt cells. Thus, LPS stimulation, which is known to induce shedding of TNF-{alpha}, transiently down-regulated the expression of MT4-MMP.


Figure 6
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  		Figure 6  Roles of MT4-MMP in TNF-{alpha} shedding in macrophages. (A) Intraperitoneal macrophages were cultured in the presence of LPS for 8 h. Mt4-mmp mRNA from Wt cells and LacZ mRNA from Mt4-mmp–/– (KO) cells were measured by real time RT-PCR before (–) and after (+) LPS stimulation. The level of mRNAs relative to that of untreated Wt cells is indicated. (B) Induction of TNF-{alpha} mRNA measured by real time RT-PCR. The amount of mRNAs relative to that of Wt cells treated with LPS is indicated. (C) Secreted TNF-{alpha} in the culture medium was measured by ELISA. Secretion of TNF-{alpha} was greatly enhanced by LPS treatment of macrophages of both genotypes. Secretion of TNF-{alpha} was inhibited by a synthetic MMP inhibitor, BB94 (10 µM).

 
In contrast to Mt4-mmp mRNA levels, the level of TNF-{alpha} mRNA increased markedly 8 h after LPS stimulation (Fig. 6B). The rate of induction of TNF-{alpha} mRNA expression in MT4-MMP-null cells was similar to Wt cells. Upon LPS treatment of Wt cells, the concentration of TNF-{alpha} in the culture medium increased (Fig. 6C), and this was also observed in LPS-treated MT4-MMP-null macrophages. These results indicated that while MT4-MMP has TACE-like activity in vitro, it is not involved in TNF-{alpha} shedding in macrophages. TNF-{alpha} shedding was inhibited by a synthetic MMP inhibitor, BB94, indicating that another metalloproteinase, presumably TACE, plays a major role in shedding in macrophages.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We established an MT4-MMP-deficient mutant mouse strain carrying the LacZ gene under the control of the intrinsic Mt4-mmp promoter. MT4-MMP-null mice had normal appearance, behavior, life span and fertility. This system enabled us to monitor Mt4-mmp expression in vivo by tracking LacZ activity. The tissue distribution of mRNA for Mt4-mmp and LacZ correlated well throughout the mouse body, with the exception of the testis, where the levels of LacZ mRNA were extremely high compared to Mt4-mmp mRNA. It is possible that there is a tissue-specific repressor element in the first intron of Mt4-mmp genome which is deleted in the testis in mutant mice.

Both Mt4-mmp and LacZ were expressed in cerebrum, and MT4-MMP protein was indeed detected in this tissue. The pattern of LacZ staining in the cerebrum indicated that neurons expressed MT4-MMP; we confirmed this by co-staining for neurofilaments. Most of the LacZ-positive cells in the cerebrum were also positive for neurofilaments (Fig. 3C). There was significant expression of LacZ in cortex, hippocampus and caudate putamen. However, not all neuronal cells expressed LacZ, even in cortex layers (Fig. 3E). This was most evident in the cerebellum, where very few cells in the tissue expressed LacZ (Fig. 3B). The tissue architecture of the cerebrum in mutant mice appeared normal, which suggests that the role of neuron-specific MT4-MMP expression may be more important postnatal rather than in developmental stages. LacZ activity was also detected in SMLs in several organs, and MT4-MMP protein was detected in the SMCs of the intestine, along with SMA (Fig. 4G). The tissues that expressed SMA mRNA also expressed Mt4-mmp mRNA at a comparable level, which indicates that MT4-MMP is expressed in SMCs throughout the mouse body (Fig. 4H).

Previously it was reported that MT4-MMP had TACE-like activity, and promoted the release of membrane-bound TNF-{alpha} (English et al. 2000). TACE is a member of the ADAM family and is reportedly the major shedding enzyme for the membrane-bound form of TNF-{alpha} in macrophages (Black et al. 1997; Schlondorff & Blobel 1999). We isolated macrophages from Wt or MT4-MMP-null mice and stimulated them with LPS to induce TNF-{alpha} shedding. TNF-{alpha} was released into the culture medium to a similar extent in cultures of MT4-MMP-positive and MT4-MMP-negative macrophages; thus, the contribution of MT4-MMP to the shedding of TNF-{alpha} appears to be negligible, at least in macrophages. It is interesting to note that the transcription of Mt4-mmp decreased after stimulation of cells with LPS while that of TNF-{alpha} increased dramatically. Thus, the down-regulation of MT4-MMP may be necessary for the cellular response to LPS. Although MT4-MMP appeared to have a minor role in TNF-{alpha} shedding in macrophages, we cannot rule out the possibility that MT4-MMP is involved in TNF-{alpha} shedding in other type of cells, particularly those that lack TACE. The survival rate of LPS-treated mice was not affected by the loss of MT4-MMP, which is expected, since MT4-MMP is not involved in the release of TNF-{alpha} in macrophages (data not shown). In regards to other macrophage functions, phagocytosis, invasion of reconstituted basement membrane, and chemotaxis all appeared to be normal in MT4-MMP-null cells (data not shown).

We also performed blood tests on the different mouse strains to examine various biochemical markers related to the functions of the kidney and liver (data not shown). Although most of the results were not significantly different, urea nitrogen levels were slightly but significantly higher in KO mice (1.25-fold, P < 0.05) compared to Wt, which suggests a possible defect in glomerular filtration function. Alanine aminotransferase levels were also higher in KO mice (1.6-fold, P < 0.01), suggesting that there was increased tissue damage in these mice. A detailed analysis of these observations is currently underway in our laboratory.

It has also been reported that MT4-MMP is a processing enzyme for ADAMTS-4 and ADAMTS-5, which function as aggrecanases (Gao et al. 2004; Patwari et al. 2005). Thus, it has been suggested that MT4-MMP plays a role in cartilage degradation by activating ADAMTS-4 and ADAMTS-5. However, it was recently reported, using tissue from the mice generated in the current study, that MT4-MMP deficiency does not affect ADAMTS-5-mediated aggrecan degradation (Stewart et al. 2006).

In summary, we generated mutant mice that expressed LacZ under the control of the Mt4-mmp promoter, and we used this system to efficiently monitor the endogenous promoter activity of Mt4-mmp in vivo. We identified MT4-MMP-producing cells in the central nervous system and SMLs, as well as macrophages, which were previously shown to express Mt4-mmp. Using MT4-MMP-deficient macrophages, we showed that MT4-MMP is not involved in the shedding of TNF-{alpha}. Although a clear phenotype of the mutant mice has not emerged, an analysis of the mutant mice under various conditions is ongoing in our laboratory.

Lack of efficient tools for tracking the expression of MT4-MMP in vivo has hampered the identification of cells that express MT4-MMP, and the analysis of its physiological functions. We have developed a valuable new tool for studying the biological functions of MT4-MMP in vivo.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Targeted disruption of Mt4-mmp mouse genomic sequences

A 129 SVJ mouse genomic library (Amplicon Express, Pullman, WA) was screened with a DNA fragment encompassing full-length mouse MT4-MMP. A 4.7-kb fragment, from the EcoRI site to the initiation codon in the first exon, and a 4.2-kb SmaI-SmaI fragment, containing part of the first intron and the second exon, were used as the 5' and 3' arms of the targeting vector, respectively. Following homologous recombination, the targeting vector deleted a 2.5-kb region of MT4-MMP, from the initiation codon in the first exon to part of the first intron. The deleted region was substituted with a sequence containing the LacZ gene, in which the LacZ coding unit was fused in frame to an NLS sequence (Kanegae et al. 1995) and the PGK-gpt/neomycin-resistant gene (Invitrogen, Carlsbad, CA) in the reverse orientation. Electroporation of the targeting vector, selection of mutant (ES) cells at day 14 and injection of blastocysts (C57BL/6J strain) with ES cells were performed as described previously (Gondo et al. 1994).

Southern blot analysis

Germ-line transmission of the mutant Mt4-mmp locus was confirmed by Southern blot. Genomic DNA isolated from mouse tail was digested with BamHI, separated by electrophoresis on a 1% agarose gel and then transferred to a nitrocellulose filter. Non-targeted and targeted alleles were detected using specific probes close to the 5' and 3' arms of the targeting vector, respectively (Fig. 1A).

Detection of mRNA

Total RNA was extracted from tissues of 8-week-old mice or from cultured cells using TRIZOL (Invitrogen). Messenger RNA was detected by RT-PCR. Specific primer sets for the detection of mRNAs were as follows: Mt4-mmp, 5'-GGCAGCTACAGACCCAGGAGGAAC-3' and 5'-GCTCACCGCATACCAGCCAGTCGC-3'; LacZ, 5'-ATCGTGCGGTGGTTGAACTG-3' and 5'-TGCTGACGGTTAACGCCTCG-3'; TNF-{alpha}, 5'-CAAAGGGATGAGAAGTTCCC-3' and 5'-TTTGAGATCCATGCCGTTGG-3'; GAPDH, 5'-CGGTGCTGAGTATGTCGTGGAGTC-3' and 5'-GGACACATTGGGGGTAGGAACAC-3'. After reverse transcription using random primers, samples were subjected to amplification under the following conditions: 95 °C for 2 min and then 40 cycles of 95 °C for 30 s, 65 °C for 30 s and 72 °C for 30 s.

Real-time PCR quantitation

Total RNA was extracted from tissues of 8-week-old mice or from cultured cells using TRIZOL (Invitrogen). The amount of mRNA was measured using the Smart Cycler (TaKaRa, Japan) and SYBR Green I nucleic acid gel stain (Bio Whittaker Molecular Applications, Walkersville, MD). Specific primer sets for the detection of mRNAs were as follows: Mt4-mmp, 5'-CTGGCATCCTAGATGAGGCC-3' and 5'-GAATGTCCGGACCCTCCAAG-3'; LacZ, 5'-ATCGTGCGGTGGTTGAACTG-3' and 5'-TGCTGACGGTTAACGCCTCG-3'; SMA, 5'-CATGGAAAAGATCTGGCACCAC-3' and 5'-CCAGCACAATACCAGTTGTACG-3'; TNF-{alpha}, 5'-CAAAGGGATGAGAAGTTCCC-3' and 5'-TTTGAGATCCATGCCGTTGG-3'; GAPDH, 5'-GTGAAGGTCGGTGTGAACGG-3' and 5'-GCTTCCCATTCTCGGCCTTG-3'. After reverse transcription using random primers, samples were subjected to amplification under the following conditions: 95 °C for 15 s and then 40 cycles of 95 °C for 1 s, 60 °C for 5 s and 72 °C for 6 s for LacZ, SMA, TNF-{alpha} and GAPDH or 95 °C for 15 s and then 40 cycles of 95 °C for 1 s, 62 °C for 5 s and 72 °C for 6 s for Mt4-mmp.

Immunoprecipitation and Western blot analysis

Mouse tissues were immersed in liquid nitrogen, shattered and then lysed in RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 1% TritonX-100 and 1% deoxycholic acid). Cultured cells were lysed directly in RIPA buffer. For immunoprecipitation, lysates were incubated with rabbit anti-mouse MT4-MMP polyclonal antibodies and Protein G Sepharose 4 Fast Flow (GE Healthcare, Buckinghamshire, UK) at 4 °C overnight. Protein G Sepharose was then collected by centrifugation and washed 3 times with RIPA buffer. The collected beads were re-suspended in loading buffer and boiled for 10 min. Samples were then separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond-ECL, GE Healthcare). The membranes were blocked in PBS-T containing 5% fat-free dry milk and then incubated successively with rat anti-mouse MT4-MMP monoclonal antibody (gift from Dr Ichiro Miki and Dr Satoshi Ohta, Kyowa Co. Ltd, Tokyo, Japan) and an appropriate secondary antibody conjugated to horseradish peroxidase. Proteins were visualized using an ECL system (ECL plus a Western blotting detection system, GE Healthcare).

LacZ and immunostaining in brain and uterus

Frozen sections (10 µm) of 8-week-old mouse tissue were fixed in PBS containing 0.2% glutaraldehyde for 30 s, rinsed in PBS and then incubated in X-gal buffer (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2 and 0.1% X-gal in PBS) at 37 °C for 10 min overnight. Purple signals were observed by microscopy.

After LacZ staining, brain sections were blocked in 0.5% BSA in PBS for 30 min, followed by incubation with primary antibody (Rabbit anti-neurofilament 200 kDa polyclonal antibody, Millipore, Billerica, MA) for 1 h, washed in PBS 3 times and incubated with secondary antibody and enzymatic reagent (HISTOFINE SAB-PO(R) kit, NICHIREI Biosciences, Japan), according to the manufacturer's instructions. After a set of washes in PBS, sections were incubated with DAB substrate (HISTOFINE DAB substrate kit, NICHIREI Biosciences) for 10 min and brown signals were observed by microscope.

After LacZ staining, uterus sections were blocked in 0.5% BSA in PBS for 30 min, followed by incubation with primary antibody (monoclonal anti-{alpha}-SMAclone 1A4 Cy3 conjugate, SIGMA, St. Louis, MO) for 1 h. After a set of washes in PBS, samples were analyzed using a CCD fluorescent microscope equipped with a digital camera (IM70-Cool SNAP, OLYMPUS, Japan).

Isolation of primary murine alveolar and peritoneal macrophages

Mouse lungs were filled and flushed 5 times with 1 mL PBS containing 5 mM EDTA, and alveolar macrophages were collected from the lavage fluid. For peritoneal macrophages, mice were administered 4.05% thioglycollate medium intraperitoneally. After 72 h, induced peritoneal macrophages were isolated from naive male mice by lavaging the peritoneum twice with 5 mL PBS containing 5 mM EDTA. Cells in the lavage fluid were collected by centrifugation, suspended in RPMI 1640 supplemented with 5% fetal bovine serum and transferred to a tissue culture dish. After 2 h, non-adherent cells were removed by extensive washing with PBS and adherent cells were collected (more than 80% of the adherent cells were positive for F4/80 staining). Macrophages were collected from either Wt, Mt4-mmp+/– or KO mice. For stimulation of the cells with LPS (100 ng/mL), peritoneal macrophages were pre-cultured overnight in RPMI 1640 supplemented with 5% fetal bovine serum.

LacZ and F4/80 staining of alveolar macrophage

Alveolar macrophages in six-well plates were fixed in PBS containing 0.2% glutaraldehyde for 30 s, rinsed in PBS and then incubated in X-gal buffer at 37 °C overnight. After LacZ staining, macrophages were blocked in 0.5% BSA in PBS for 30 min, then incubated with anti-F4/80 antibody for 1 h, washed in PBS 3 times and incubated with secondary antibody for 30 min. After a set of washes in PBS, the cells were analyzed using a CCD fluorescent microscope equipped with a digital camera (IM70-Cool SNAP, OLYMPUS).

Detection of TNF-{alpha}

Macrophages were cultured in RPMI 1640 medium overnight before being stimulated with LPS (100 ng/mL). Eight hours after stimulation, culture medium was collected and analyzed by ELISA to detect TNF-{alpha}, using the AssayMax mouse TNF-{alpha} ELISA kit (ASSAY PRO), according to the manufacturer's instructions.


   Acknowledgements
 
We thank Roy Zent for helpful discussions and Chieko Konish for excellent technical assistance. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas "Integrative Research Toward the Conquest of Cancer" from the Ministry of Education, Culture, Sports, Science and Technology of Japan to M.S.


   Footnotes
 
Communicated by: Tadashi Yamamoto

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


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 23 August 2006
Accepted: 19 June 2007





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