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Cell Growth & Differentiation Vol. 12, 573-580, November 2001
© 2001 American Association for Cancer Research

Membrane-Type 5 Matrix Metalloproteinase Is Expressed in Differentiated Neurons and Regulates Axonal Growth1

Hiromi Hayashita-Kinoh, Hiroaki Kinoh, Akiko Okada, Kiyoshi Komori, Yoshifumi Itoh, Tadashige Chiba, Masahiro Kajita, Ikuo Yana and Motoharu Seiki2

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


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Expression of membrane-type (MT) 5 matrix metalloproteinase (MMP) in the mouse brain was examined. MT5-MMP was expressed in the cerebrum in embryos, but it declined after birth. In contrast, expression in the cerebellum started to increase postnatally and continued thereafter. The cells expressing MT5-MMP were postmitotic neurons that showed gelatinolytic activities. Specific expression of MT5-MMP was observed in the neurons but not in the glial cells when embryonal mouse carcinoma P19 cells were differentiated in vitro by retinoic acid treatment. Neurons isolated from dorsal root ganglia also expressed MT5-MMP, and it was localized at the edge of growth cone. Proteoglycans inhibit neurite extension and regulate synaptogenesis. The inhibitory effect of the proteoglycans on neurite extension of dorsal root ganglia neurons was effectively eliminated by recombinant MT5-MMP. Thus, MT5-MMP expressed in neurons may play a role in axonal growth that contributes to the regulation of neural network formation.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
During the development of the peripheral nervous system and the CNS,3 interactions between cells and their surrounding ECM are required to generate specific features of tissue architecture. Cell surface molecules and the ECM are known to influence aspects of neurogenesis and nervous system development, including proliferation and migration of cells, differentiation of neuroblasts and glial cells, and guidance and fasciculation of neurites (1 , 2) . Furthermore, by promoting adhesive interactions between differentiating neurons, these molecules could contribute to the recognition of target cells by migrating neurons and thus affect the selective formation of synapses between these cells. The ECM molecules that are particularly implicated in these major steps of nervous system development are the basal membrane components; however, it is not easy to understand the exact roles of each molecule because of their complexity and redundancy. However, laminin appears to promote neuronal cell migration and neurite elongation (3) , and PGs, such as CSPG and HSPG, are rather inhibitory of neurite outgrowth (4 , 5) .

MMPs are a family of zinc-dependent endopeptidases responsible for the degradation of most ECM components (6, 7, 8) . To date, the cDNAs of 21 mammalian MMPs have been identified, and they fall into one of two subgroups, namely, soluble MMPs and MT-MMPs. MT-MMPs are anchored to the plasma membrane via the transmembrane domain (9) or glycosylphosphatidyl inositol moiety at the COOH terminus (10 , 11) , and six MMPs have been assigned to this subgroup. MT1-, MT2-, MT3-, and MT5-MMP (MMP-14, -15, -16, and -24, respectively) are closely related to each other and show almost 70% amino acid sequence identity in their catalytic domains. MT4-MMP (MMP-17) is unique in that it shows only 40% sequence conservation at the same region (12 , 13) . The most recently identified member of this subgroup, MT6-MMP (MMP-25), was most homologous to MT4-MMP and is expressed in leukocytes (14 , 15) . Whereas the soluble MMPs are presumably responsible for ECM degradation over broad areas of tissue, MT-MMPs are likely to participate in the pericellular ECM degradation associated with various cell functions such as proliferation, migration, and invasion. For example, MT1-MMP, which is the most studied MT-MMP molecule, has been detected in malignant tumor cells during cancer progression (9) , endothelial cells during angiogenesis (16, 17, 18) , mesenchymal cells during embryogenesis (19 , 20) , skeletal tissues (18 , 21) , and wound-healing tissues (22) .

MT5-MMP is unique among the MT-MMPs in that it is expressed in a limited range of tissues that includes the brain (23 , 24) and in some brain tumor cell lines (24) . It is noteworthy that MT5-MMP degrades CSPG and dermatin sulfate PG but not laminin (25) . Recently, a study investigating MT5-MMP expression during rat development showed that it was detected mainly in the nervous system (26 , 27) . Thus, MT5-MMP may play specific roles in the development and maintenance of the nervous system, but these roles are not yet clear. In this study, we tried to identify the cells that express MT5-MMP in embryonic and adult murine brains by Northern blotting, in situ hybridization, and immunostaining. We detected MT5-MMP specifically in neuronal cells in the embryo and in adult mouse brain. It was also detected in the DRG neurons cultured in vitro, and localization of MT5-MMP at the growth cone of extending neurite was observed. In the neurite extension assay in vitro, MT5-MMP could effectively eliminate the inhibitory effect of the PGs, suggesting a possible role in neural network formation.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Expression of MT5-MMP mRNA Is Regulated Temporally and Spatially during Brain Development.
To investigate the distribution of MT5-MMP mRNA, Northern blot analysis of adult mouse tissues was performed (Fig. 1A)Citation . MT5-MMP mRNA was predominantly detected in the cerebellum and was almost undetectable in other tissues. Then we monitored MT5-MMP mRNA expression in the various stages of brain development (Fig. 1B)Citation . MT5-MMP mRNA was weakly detected in E14 brain and then increased as development progressed. At E18, the brain was divided into cerebrum and cerebellum, and the separated tissues were further examined. In the cerebrum, expression was found at E18 and continued until birth (P0), but it decreased sharply thereafter (P7) and was almost undetectable at P30. On the other hand, MT5-MMP mRNA levels were low in the cerebellum at E17 and E18. However, expression was induced postnatally (P0, P7, and P30), and it became constitutive in adulthood (Fig. 1A)Citation . The period during which MT5-MMP mRNA is expressed in the cerebrum appears to coincide with neocortex morphogenesis. In contrast, the postnatal expression of MT5-MMP mRNA in the cerebellum is in accord with the time at which immature granular cells in the external germinal layer differentiate, migrate across the molecular layer, and move to the internal granular layer (28 , 29) . The expression in the cerebellum is maintained even after completion of this migration.



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Fig. 1. Expression of MT-MMP mRNAs in mouse tissues by Northern blot analysis. A, total RNA samples isolated from tissues of C57BL/6J mice were analyzed by Northern blotting using a 32P-labeled MT5-MMP cDNA probe. The amount of RNA sample loaded was ascertained by ethidium bromide staining of 28S rRNA. B, total RNA extracted from mouse brain at the indicated day of gestation (E days) or postnatally (P days). Cerebrum and cerebellum were separated from E18, P0, P7, and P30 brains and studied by Northern blotting.

 
Histological Detection of MT5-MMP in the Developing Nervous System.
To characterize temporal and special regulation of MT5-MMP expression over the course of mouse development, in situ hybridization and immunohistochemical analyses were carried out using sagittal sections of the embryo at E15.5 and whole brain at P14 and P60 (Fig. 2)Citation . At E15.5, MT5-MMP signals by immunostaining were not strong enough to demonstrate the sites of expression in the whole section (Fig. 2, A and B)Citation , but closer examination revealed significant levels of signals in the tissues that contain neurons, such as the ventricular zone of the cerebrum (Fig. 2, C and D)Citation , the eye (data not shown), and DRGs (Fig. 2, E and F)Citation .



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Fig. 2. Localization of MT5-MMP in the murine nervous system. Whole sections of mouse E15.5 embryos (A-F) and brain sections of P14 juveniles (G-J) and P60 adults (K-T) were examined by immunohistochemistry and in situ hybridization. A, C, E, G, K, O, and R were stained with H&E, and B, D, F, J, Q, and T were immunostained with antimouse MT5-MMP rabbit polyclonal antibody. H, I, L, M, P, and S were hybridized with digoxygenin-labeled MT5-MMP antisense RNA probes (H, L, P, and S) or sense RNA probe (I and M). The boxed areas in A and K were magnified and are shown in C and D and O and R, respectively. drg, dorsal root ganglion; lu, lung; he, heart; li, liver; ob, olfactory bulb, rmt, rostral migratory tract; svz, subventricular zone; co, cortex; dg, dentate gyrus; ce, cerebellum. Bars, 500 (A-C and G-M) and 100 µm (O-T).

 
In the brain at P14 (Fig. 2, G-J)Citation , both MT5-MMP mRNA (Fig. 2H)Citation and the protein (Fig. 2J)Citation were detected in the granular layers in the cerebellum, the olfactory bulb, the dentate gyrus, the subventricular zone of the lateral ventricle, and the rostral migratory tract that continuously supplies granule cells and periglomerular cells from the subventricular zone to the olfactory bulb. Significant signals were also detected in the cortex (Fig. 2H)Citation . Hybridization signals were very low in the tissue when sense probe was used (Fig. 2I)Citation . Thus, detection of MT5-MMP mRNA was specific and in accord with the result of immunostaining.

In the adult brain at P60 (Fig. 2, K–T)Citation , strong MT5-MMP mRNA signals were detected in the granular layer of the cerebellum (Fig. 2, L and S)Citation and in the dentate gyrus of the cerebrum (Fig. 2, L and P)Citation . The presence of MT5-MMP protein was also detected by immunostaining at the corresponding sites (Fig. 2, Q and T)Citation . Thus, MT5-MMP seems to be expressed specifically in neurons, although it is regulated temporally, depending on the cells.

Specific Expression of MT5-MMP in the Differentiated Neurons in Vitro.
Because MT5-MMP was detected in neuronal tissues, we examined whether the differentiation of P19 (an embryonic mouse carcinoma cell line) cells into neurons can turn on MT5-MMP expression. P19 cells differentiate into neuronal and glial cells when the cells are treated with RA. Differentiated neurons can be enriched by culturing the RA-treated cells in the presence of Ara-C, which inhibits DNA synthesis and kills the proliferating glial and undifferentiated cells. Differentiation of neuronal cells was monitored by measuring neurofilament mRNA, whereas differentiation of glial cells was assessed by measuring the mRNA for GFAP. As shown in Fig. 3ACitation , expression of the mRNAs for neurofilament and GFAP was induced by RA treatment. However, the GFAP mRNA level was lower in the Ara-C-treated cell population, indicating that it is expressed in the differentiated glial cells that were killed by Ara-C treatment. In contrast, neurofilament mRNA was enriched in the Ara-C-resistant cell population, indicating that it is expressed in the differentiated neurons. With regard to MT5-MMP mRNA, the pattern of expression is similar to that seen for neurofilament mRNA but not GFAP mRNA, indicating that expression of MT5-MMP mRNA is induced in P19 cells when they differentiate into neurons. Transcripts for other members of the MT-MMP subgroup were also examined using the same blot. Only MT3-MMP mRNA showed an induction pattern similar to that of MT5-MMP, although its levels were low. Expression of MT1-MMP and MT2-MMP mRNA was also induced by RA treatment but was found mainly in the Ara-C-sensitive glial cell population. The expression level of MT4-MMP was low and was not affected by the RA treatment.



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Fig. 3. Neuron-specific expression of MT5-MMP in differentiating P19 embryonic carcinoma cells. A, P19 cells were treated with RA to induce differentiation into neuronal and glial cells. Differentiated neuronal cells were enriched from the resulting population that also contained proliferating glial cells and undifferentiated cells by killing the latter cells with Ara-C. Total RNA samples were extracted and analyzed by Northern blotting using the indicated probes. B, immunostaining was performed for the indicated proteins. Confocal microscope images are shown for MT5-MMP (a), neurofilament (b), and a merging (c) of images a and b. Light-field image is indicated in d. Bar, 100 µm.

 
During differentiation of the RA-treated P19 cells, at least two types of cells were morphologically distinguishable (Fig. 3B, d)Citation . One type consisted of small round cells with extended fibers that were not observed in the original P19 cell population (data not shown). The other cell type was composed of fibroblastic cells. The round cells reacted with antineurofilament antibody, especially at their extended fibers (Fig. 3B, b)Citation , indicating that these cells are the differentiated neurons. MT5-MMP was detected in these neurofilament-positive cells but not in the fibroblastic cells (Fig. 3, a and c)Citation . Thus, it is clear that the expression of MT5-MMP is specifically induced in the cells according to the differentiation into neurons.

Gelatinolytic Activity of the Cells Expressing MT5-MMP in Brain.
MT5-MMP degrades gelatin and PGs, such as CSPG and dermatin sulfate PG (25) . To examine whether the cells in the brain that express MT5-MMP show gelatinolytic activity, we performed in situ zymography using FITC-labeled gelatin, whose fluorescence is quenched intramolecularly and can be activated by digestion. In the NB mouse, proliferating granule cell precursors were found in the external germinal layer of cerebellum (Fig. 4A)Citation , but neither MT5-MMP expression (Fig. 4B)Citation nor gelatinolytic activity (Fig. 4C)Citation was observed there. Expression of MT5-MMP became evident in the cells that were migrating to the granule layer at P9 (Fig. 4, D and E)Citation , and these cells showed gelatinolytic activity (Fig. 4F)Citation . In the adult mouse, expression of MT5-MMP was maintained constitutively in the Purkinje cells and granule cells (Fig. 4, G and H)Citation , and these cells also showed gelatinolytic activity (Fig. 4I)Citation . The gelatinolytic activity detected in the brain is thought to reflect MMP activity because synthetic MMP inhibitor, BB-94, abolished the activity almost completely (Fig. 4J)Citation , whereas the serine proteinase inhibitor, pefabloc, did not (Fig. 4K)Citation . Thus, the cells expressing MT5-MMP in brain showed good correlation to those have gelatinolytic activities.



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Fig. 4. Colocalization of MT5-MMP expression and gelatinolytic activity in serial sections of the developing murine cerebellum. Tissue sections were prepared from NB, 9-day-old (P9), and 3-month-old (P90) mice. Gelatinolytic activity was monitored by fluorescence generated by DQ-gelatin after its cleavage by MT5-MMP. A, D, and G show the sections stained with H&E. B, E, and H show the location of MT5-MMP on the sections as detected by immunohistochemistry. C, F, and I-K show the pattern of DQ-gelatin degradation on the sections. In J and K, the sections were treated with the MT5-MMP inhibitor BB-94 or with the serine protease inhibitor pefabloc, respectively. Bars, 50 µm.

 
MT5-MMP Localizes at the Growth Cone of Neurons and May Regulate Neurite Outgrowth.
Recent studies have demonstrated potential roles of MMPs in neurite extension (5 , 30) . To understand the role of MT5-MMP in neurons, the subcellular localization of the enzyme was first examined using isolated DRG from a NB mouse (Fig. 5)Citation . The DRG cells cultured on a coverglass coated with laminin expressed neurofilament as demonstrated by immunostaining and extended neurites (Fig. 5B)Citation . MT5-MMP was detected in the same cells localizing at the cell body and growing neurite, including the edge of the growth cone (Fig. 5, A and B)Citation . The intense MT5-MMP signals along with veil-rich regions and filopodia at the leading edge suggest that MT5-MMP could be involved in growth cone invasion and neurite extension by degrading the surrounding ECM.



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Fig. 5. MT5-MMP is expressed at the growth cone of neurons. Neuronal cells isolated from DRG were cultured on cover glasses coated with poly-L-lysine and 10 µg/ml laminin. A, cells were immunostained with a polyclonal antibody against MT5-MMP. B, cells were immunostained with the antibody against neurofilament. C, merged picture of A and B. D, a high-power field image of the growth cone (boxed area of C).

 
PGs are rich in brain and reported to inhibit neurite extension under the similar culture conditions used here (1 , 5) . Because MT5-MMP can degrade some of these PGs (25) , we tested whether MT5-MMP affects ECM molecules that modulate neurite extension in vitro. DRG cells were cultured on a coverglass coated with different substrata, and axon length was measured after visualization of neurites by immunostaining using antineurofilament antibody (Fig. 6, A-I)Citation . As reported previously (2) , laminin promoted neurite outgrowth (Fig. 6, B and J)Citation , but MT5-MMP itself (data not shown) or treatment of the laminin coat with MT5-MMP (Fig. 6, C and J)Citation did not affect neurite extension. Laminin mixed with either CSPG or HSPG did not promote neurite extension (Fig. 6, D, G, and J)Citation , indicating that these PGs inhibit neurite extension. Then we tested whether MT5-MMP could abolish the inhibitory activity of the PGs. Pretreatment of the CSPG/laminin- or HSPG/laminin-coated coverglass with recombinant MT5-MMP abolished the inhibitory effect of the PGs and promoted neurite extension effectively (Fig. 6, E, H, and J)Citation . The effect of MT5-MMP depends on its proteolytic activity because BB-94 abolished the effect of MT5-MMP completely (Fig. 6, F, I, and J)Citation . These results suggest that MT5-MMP may assist neurons in forming networks by facilitating neurite extension through the degradation of inhibitory ECM components including PGs at the growing neurite edge.



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Fig. 6. MT5-MMP degrades PGs, and this promotes neurite outgrowth. A, neurite growth of isolated DRG neurons was examined in vitro by cultivating the cells on cover glasses coated with laminin (B) or laminin plus CSPGs (D) or HSPGs (G). In other experiments, cells were plated on laminin and PG-coated glass slides that had been pretreated with recombinant MT5-MMP (C, E, F, H, and I). In F and I, MT5-MMP treatment was carried out in the presence of inhibitor BB-94. Extended neurites were visualized by immunostaining for neurofilament (A–I). The length of the neurites was measured under the confocal laser microscope, and average values of 30 cells are indicated by the bars shown in J. Bar, 100 µm. *, P < 0.0001 (Student’s t test).

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
MMPs are a family of zinc-dependent proteinases responsible for ECM turnover. They are thus involved in the regulation of tissue formation and remodeling (31) . For this purpose, expression of MMPs is tightly regulated in a tissue- and cell-specific manner. Some MMPs are expressed in the CNS as well as in other tissues and organs. For example, MMP-2, MMP-3, and MMP-9 and TIMP-1, TIMP-2, TIMP-3 are expressed in the rat cerebellum (32) . MT1-MMP is also expressed in microglia (33) and some brain tumors (34) . However, of all of the MMPs identified thus far, MT5-MMP is unique in that its expression is relatively restricted to brain (see Fig. 1ACitation ), as reported previously (24 , 26 , 27) . In this study, we showed that MT5-MMP is expressed in mouse neurons. We also found that expression of MT5-MMP was induced in P19 cells when the cells differentiate into neurons. Such induction was not observed in the cells differentiating into glial cells. Of the five MT-MMPs examined, only MT5-MMP and MT3-MMP were specifically induced in the differentiated neurons. MT1-MMP and MT2-MMP were expressed mainly in glial cells, whereas the expression level of MT4-MMP in P19 cells was low and was not affected by differentiation.

Although MT5-MMP is expressed in a broad range of neural tissues, as demonstrated in this study and by others (26 , 27) , it seems to be regulated differentially even in neuronal cells. We found that it was particularly expressed in the cerebrum of the developing embryo brain as shown in Fig. 1Citation . After birth, however, the expression in the cerebrum declined sharply. The period during which MT5-MMP is expressed in the cerebrum corresponds to that of neocortex formation. In the cerebellum, however, MT5-MMP expression differed kinetically from that observed in the cerebrum in that it was first observed after birth. MT5-MMP-expressing cells were postmitotic granule cells that were migrating from the external germinal layer to the granular layer after traversing the molecular layer (35) . In the adult mouse, high levels of constitutive expression of MT5-MMP were seen only in limited areas of the brain, such as the cerebellum, olfactory bulb, dentate gyrus, subventricular zone of the lateral ventricle, and rostal migratory tract. Interestingly, both the hippocampus and the cerebellum have been identified as areas that are characterized by high degrees of synaptic remodeling associated with plasticity (36) . Thus, expression of MT5-MMP may be associated with the morphogenic potential of neurons.

The expression pattern of MT5-MMP in the brain may indicate that MT5-MMP is required for the migration of neuronal cells and the extension of neurites that is required to form neural networks. The ECM in the brain is rich in various types of PGs and regulates neural network formation by modulating cell migration and neurite outgrowth (1) . CSPG is reported to be expressed in the mesenchyme along the boundary of the neurons and may possibly confine the growth of axons within the nerve fiber bundles. CSPG is also reported to be capable of inhibiting neurite outgrowth in vitro, and it may contribute to axonal regenerative failure after CNS injury (4 , 5) . HSPG is expressed along the nerve pathway, but it has recently been reported that HSPG has little effect on the spreading and migration of ensheathing neuronal cells (37) . Thus, the PGs may be important regulators in the development of the CNS. The core proteins of CSPG and HSPG are potential substrates for MMPs expressed by nerve cells. Indeed, MMP-2 is expressed in chicken DRG neurons and can dissolve CSPG during neurite outgrowth, at least in vitro (38) . In our experiments, efficient neurite outgrowth was observed on laminin-coated glass, and it was inhibited when either CSPG or HSPG was mixed with laminin. However, when the PG coat was pretreated with recombinant MT5-MMP, the inhibitory effect of the PG was specifically eliminated, whereas the stimulatory effect of laminin was not affected. Subcellular localization studies showed that the enzyme is located at the growth cone of neurons, which confirms the notion that it is required to degrade inhibitory PGs during axonal extension. PGs are also known to be reservoirs for factors such as amphoterin and pleiotrophin that promote neurite outgrowth (39) , although some of these factors have also been reported to be inhibitory (40) . Thus, MT5-MMP may also modulate neuronal cell functions by causing the release of these factors from the reservoir.

It is noteworthy that MMP-2 produced by chicken DRG neurons also localized at the growth cone (38) . Because MMP-2 is a soluble enzyme, it has to bind some cell surface molecules to localize there. During activation of pro-MMP-2 by the cells expressing MT1-MMP, pro-MMP-2 binds to MT1-MMP using TIMP-2 as an adaptor (9) . MT5-MMP also activates pro-MMP-2, presumably by forming a complex similar to that of MT1-MMP (25) . Thus, MT5-MMP may also be a device to use MMP-2 activity at the edge of growth cone by binding the enzyme. Although our study does not exclude the possibility that other MT-MMPs are also expressed there and play similar roles, specific expression of MT5-MMP in neurons compared with that of other MT-MMPs may suggest major roles for this enzyme in the pericellular proteolysis associated with nerve cell functions.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cloning and Sequencing.
Random-primed and oligodeoxythymidylic acid-primed cDNA libraries were constructed using polyadenylic acid-containing RNA from a murine E17 brain and cloned into ZAP II (Stratagene, La Jolla, CA). The random-primed cDNA library was screened with a 32P-labeled human MT3-MMP cDNA probe (41) . Positive phages were isolated and subjected to in vivo excision according to the manufacturer’s protocol (Stratagene). Sequencing showed that the isolated inserts were either MT3-MMP cDNAs (GenBank accession number AB021228) or MT5-MMP cDNAs (GenBank accession number AB021226).

Probes and Antibodies.
cDNA probes for mouse MT-MMPs, GFAP, neurofilament, and GAPDH contain the following sequences: (a) nt 1201–1808 of MT1-MMP cDNA (accession number U54984); (b) nt 194–585 of MT2-MMP cDNA (accession number D86332); (c) nt 357–756 of MT3-MMP cDNA (accession number AB021228); (d) nt 944-1286 of MT4-MMP cDNA (accession number AB021224); (e) nt 1132–1729 of MT5-MMP cDNA (accession number AB021226); (f) nt 733-1216 of GFAP cDNA (accession number K01347); (g) nt 652-1338 of neurofilament cDNA (accession number M20480); and (h) nt 310–763 of GAPDH cDNA (accession number M32599).

Rabbit polyclonal antibody was raised against recombinant MT5-MMP expressed in Escherichia coli, and the specificity of this antibody was confirmed by using COS-1 cells transfected with each of the MT-MMP expression plasmids. Western blotting and immunocytostaining assays showed that the antibody reacted only with MT5-MMP (data not shown). This antibody was used at a 1:200 dilution in immunohistochemistry assays. Antineurofilament antibody was purchased from Chemicon International Inc. (Temecula, CA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

RNA Preparation and Northern Blotting.
Total RNA was extracted from various C57BL/6J mouse tissues by the acid guanidinium thiocyanate-phenol-chloroform extraction method (42) . About 20 µg of total RNA were separated by electrophoresis on an agarose gel, blotted onto Hybond N nylon membrane (Amersham Pharmacia, Uppsala, Sweden), and hybridized with 32P-labeled probes. As a control for the sample amounts applied in each of the lanes, GAPDH mRNA was detected simultaneously.

In Situ Hybridization.
In situ hybridization was carried out as described by Kinoh et al. (19) . The cRNA probes were labeled with digoxigenin according to the instructions of the manufacturer (Roche Diagnostics GmbH, Mannheim, Germany). Brains of adult or embryonic mice were dissected and embedded in OCT compound (Tissue-Tek, Torrance, CA) and then quickly frozen in liquid nitrogen. Tissue blocks were cut into 5-µm-thick sections with a cryostat microtome. Sections were incubated overnight at 55°C in the hybridization buffer (0.2 µg/ml digoxigenin-labeled RNA probe, 50% formamide, 5x SSC, 50 µg/ml yeast tRNA, 1% SDS, and 50 µg/ml heparin) and washed under high-stringency conditions. Expression of mRNA was visualized using a DIG detection kit (Roche Diagnostics GmbH).

Immunohistochemical Analysis.
Serial sections were fixed with fresh 4% paraformaldehyde in PBS and blocked by incubation with 5% normal goat serum and 5% bovine serum albumin in PBS. The primary antibody was then added and left for 1 h at room temperature. After washing three times with PBS, the sections were incubated with Alexa 488-conjugated goat antirabbit IgG (Molecular Probe, Engene, OR) for 1 h at room temperature. The immunostained sections were analyzed by confocal laser microscopy (Bio-Rad Laboratories, Hercules, CA).

Differentiation of P19 Cells.
P19 embryonic carcinoma cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in bicarbonate-buffered {alpha}-modified Eagle’s medium supplemented with 10% (v/v) FCS in humidified 5% CO2/air at 37°C. Neural differentiation was induced by culturing the cells on noncoated culture dishes for 4 days in the presence of 10-6 M RA (all trans-RA; Sigma-Aldrich Co., St. Louis, MO). This caused the cells to form aggregates termed embryoid bodies. The cells were then replaced on culture dishes and incubated for 2 more days without RA (43) . Undifferentiated cells and differentiated glial cells were removed by adding 10 µM Ara-C (Sigma-Aldrich Co.) to the culture (44) .

In Situ Zymography.
Gelatin degradation activity was detected by using FITC-labeled DQ gelatin (Molecular Probe) as a substrate. DQ-gelatin fluorescence is quenched intramolecularly, and it is activated by proteolytic degradation. Frozen sections were washed briefly with PBS, overlaid with 50 µg/ml DQ-gelatin in the reaction buffer [0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl2, and 0.2 mM NaN3 (pH 7.6)], and incubated overnight at 37°C in a humidified chamber. The samples were then analyzed by confocal laser microscopy (Bio-Rad Laboratories) without fixation. EDTA (100 µM) or 50 µM BB-94 (gift from Dr. Brown, British Biotec) was used to inhibit MMP activity. Pefabloc (5 mM; Roche Diagnostics GmbH), a serine proteinase inhibitor, was used as a control.

Neurite Extension Assays.
Neurite extension assays were performed as described previously (45 , 46) , with some modifications. Briefly, DRG were obtained from a NB mouse. Other nonneuronal tissues were removed carefully, and DRGs were collected in ice-cold PBS. The DRGs were then treated with trypsin for 10 min, and the cells were collected by a brief centrifugation. The cells were resuspended in PBS, and 5 x 104 cells were cultured for 24 h on coated coverglasses in the presence of 10 µM Ara-C. After 3 days of culture, the cells were fixed with 4% paraformaldehyde in PBS for 1 h, and extended neurites were visualized by immunostaining for neurofilament. Neurite length was measured during confocal laser microscopy (Bio-Rad Laboratories). Coated coverglasses were prepared as follows: poly-L-lysine (20 µg/ml) was coated onto coverglasses, dried for 1 h at room temperature, and washed three times with water. The coverglasses were then coated with 10 µg/ml laminin, laminin plus 10 µg/ml CSPG, or laminin plus 10 µg/ml HSPG.


    Footnotes
 
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 indicate this fact.

1 Supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

2 To whom requests for reprints should be addressed, at Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan. Phone: 81-3-5449-5255; Fax: 81-3-5449-5414; E-mail: mseiki{at}ims.u-tokyo.ac.jp Back

3 The abbreviations used are: CNS, central nervous system; ECM, extracellular matrix; MT, membrane-type; MMP, matrix metalloproteinase; DRG, dorsal root ganglia; PG, proteoglycan; CSPG, chondroitin sulfate proteoglycan; HSPG, heparan sulfate proteoglycan; E, embryonic day; P, postnatal day; RA, retinoic acid; Ara-C, 1-ß’62-D-arabinofuranosylcytosine; GFAP, glial fibrillary acidic protein; NB, newborn; TIMP, tissue inhibitor of metalloproteinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; nt, nucleotide. Back

Received for publication 1/24/01. Revision received 9/21/01. Accepted for publication 9/25/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Bovolenta P., Fernaud-Espinosa I. Nervous system proteoglycans as modulators of neurite outgrowth. Prog. Neurobiol., 61: 113-132, 2000.[Medline]
  2. Luckenbill-Edds L. Laminin and the mechanism of neuronal outgrowth. Brain Res. Brain Res. Rev., 23: 1-27, 1997.[Medline]
  3. Powell S. K., Kleinman H. K. Neuronal laminins and their cellular receptors. Int. J. Biochem. Cell Biol., 29: 401-414, 1997.[Medline]
  4. Snow D. M., Lemmon V., Carrino D. A., Caplan A. I., Silver J. Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp. Neurol., 109: 111-130, 1990.[Medline]
  5. Zuo J., Neubauer D., Dyess K., Ferguson T. A., Muir D. Degradation of chondroitin sulfate proteoglycan enhances the neurite-promoting potential of spinal cord tissue. Exp. Neurol., 154: 654-662, 1998.[Medline]
  6. Nagase H., Woessner J. F., Jr. Matrix metalloproteinases. J. Biol. Chem., 274: 21491-21494, 1999.[Free Full Text]
  7. Massova I., Kotra L. P., Fridman R., Mobashery S. Matrix metalloproteinases: structures, evolution, and diversification. FASEB J., 12: 1075-1095, 1998.[Abstract/Free Full Text]
  8. Birkedal-Hansen H. Proteolytic remodeling of extracellular matrix. Curr. Opin. Cell Biol., 7: 728-735, 1995.[Medline]
  9. Seiki M. Membrane-type matrix metalloproteinases. APMIS, 107: 137-143, 1999.[Medline]
  10. Itoh Y., Kajita M., Kinoh H., Mori H., Okada A., Seiki M. Membrane type 4 matrix metalloproteinase (MT4-MMP, MMP-17) is a glycosylphosphatidylinositol-anchored proteinase. J. Biol. Chem., 274: 34260-34266, 1999.[Abstract/Free Full Text]
  11. Kojima S., Itoh Y., Matsumoto S., Masuho Y., Seiki M. Membrane-type 6 matrix metalloproteinase (MT6-MMP, MMP-25) is the second glycosyl-phosphatidyl inositol (GPI)-anchored MMP. FEBS Lett., 480: 142-146, 2000.[Medline]
  12. Puente X. S., Pendas A. M., Llano E., Velasco G., Lopez O. C. Molecular cloning of a novel membrane-type matrix metalloproteinase from a human breast carcinoma. Cancer Res., 56: 944-949, 1996.[Abstract/Free Full Text]
  13. Kajita M., Kinoh H., Ito N., Takamura A., Itoh Y., Okada A., Sato H., Seiki M. Human membrane type-4 matrix metalloproteinase (MT4-MMP) is encoded by a novel major transcript: isolation of complementary DNA clones for human and mouse mt4-mmp transcripts. FEBS Lett., 457: 353-356, 1999.[Medline]
  14. Pei D. Leukolysin/MMP25/MT6-MMP: a novel matrix metalloproteinase specifically expressed in the leukocyte lineage. Cell Res., 9: 291-303, 1999.[Medline]
  15. Velasco G., Cal S., Merlos-Suarez A., Ferrando A. A., Alvarez S., Nakano A., Arribas J., Lopez-Otin C. Human MT6-matrix metalloproteinase: identification, progelatinase A activation, and expression in brain tumors. Cancer Res., 60: 877-882, 2000.[Abstract/Free Full Text]
  16. Hiraoka N., Allen E., Apel I. J., Gyetko M. R., Weiss S. J. Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins. Cell, 95: 365-377, 1998.[Medline]
  17. Haas T. L., Davis S. J., Madri J. A. Three-dimensional type I collagen lattices induce coordinate expression of matrix metalloproteinases MT1-MMP and MMP-2 in microvascular endothelial cells. J. Biol. Chem., 273: 3604-3610, 1998.[Abstract/Free Full Text]
  18. Zhou Z., Apte S. S., Soininen R., Cao R., Baaklini G. Y., Rauser R. W., Wang J., Cao Y., Tryggvason K. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proc. Natl. Acad. Sci. USA, 97: 4052-4057, 2000.[Abstract/Free Full Text]
  19. Kinoh H., Sato H., Tsunezuka Y., Takino T., Kawashima A., Okada Y., Seiki M. MT-MMP, the cell surface activator of proMMP-2 (pro-gelatinase A), is expressed with its substrate in mouse tissue during embryogenesis. J. Cell Sci., 109: 953-959, 1996.[Abstract/Free Full Text]
  20. Apte S. S., Fukai N., Beier D. R., Olsen B. R. The matrix metalloproteinase-14 (MMP-14) gene is structurally distinct from other MMP genes and is co-expressed with the TIMP-2 gene during mouse embryogenesis. J. Biol. Chem., 272: 25511-25517, 1997.[Abstract/Free Full Text]
  21. Holmbeck K., Bianco P., Caterina J., Yamada S., Kromer M., Kuznetsov S. A., Mankani M., Robey P. G., Poole A. R., Pidoux I., Ward J. M., Birkedal-Hansen H. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell, 99: 81-92, 1999.[Medline]
  22. Okada A., Tomasetto C., Lutz Y., Bellocq J. P., Rio M. C., Basset P. Expression of matrix metalloproteinases during rat skin wound healing: evidence that membrane type-1 matrix metalloproteinase is a stromal activator of pro-gelatinase A. J. Cell Biol., 137: 67-77, 1997.[Abstract/Free Full Text]
  23. Pei D. Identification and characterization of the fifth membrane-type matrix metalloproteinase MT5-MMP. J. Biol. Chem., 274: 8925-8932, 1999.[Abstract/Free Full Text]
  24. Llano E., Pendas A. M., Freije J. P., Nakano A., Knauper V., Murphy G., Lopez-Otin C. Identification and characterization of human MT5-MMP, a new membrane-bound activator of progelatinase A overexpressed in brain tumors. Cancer Res., 59: 2570-2576, 1999.[Abstract/Free Full Text]
  25. Wang X., Yi J., Lei J., Pei D. Expression, purification and characterization of recombinant mouse MT5- MMP protein products. FEBS Lett., 462: 261-266, 1999.[Medline]
  26. Sekine-Aizawa Y., Hama E., Watanabe K., Tsubuki S., Kanai-Azuma M., Kanai Y., Arai H., Aizawa H., Iwata N., Saido T. C. Matrix metalloproteinase (MMP) system in brain: identification and characterization of brain-specific MMP highly expressed in cerebellum. Eur. J. Neurosci., 13: 935-948, 2001.[Medline]
  27. Jaworski D. M. Developmental regulation of membrane type-5 matrix metalloproteinase (MT5-MMP) expression in the rat nervous system. Brain Res., 860: 174-177, 2000.[Medline]
  28. Altman J. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol., 145: 353-397, 1972.[Medline]
  29. Sisken B. F., Zwick M., Hyde J. F., Cottrill C. M. Maturation of the central nervous system: comparison of equine and other species. Equine Vet. J., (Suppl.): 31-34, 1993.
  30. Duchossoy Y., Horvat J. C., Stettler O. MMP-related gelatinase activity is strongly induced in scar tissue of injured adult spinal cord and forms pathways for ingrowing neurites. Mol. Cell. Neurosci., 17: 945-956, 2001.[Medline]
  31. Werb Z. ECM and cell surface proteolysis: regulating cellular ecology. Cell, 91: 439-442, 1997.[Medline]
  32. Vaillant C., Didier-Bazes M., Hutter A., Belin M. F., Thomasset N. Spatiotemporal expression patterns of metalloproteinases and their inhibitors in the postnatal developing rat cerebellum. J. Neurosci., 19: 4994-5004, 1999.[Abstract/Free Full Text]
  33. Yamada T., Yoshiyama Y., Sato H., Seiki M., Shinagawa A., Takahashi M. White matter microglia produce membrane-type matrix metalloprotease, an activator of gelatinase A, in human brain tissues. Acta Neuropathol., 90: 421-424, 1995.[Medline]
  34. Yamamoto M., Mohanam S., Sawaya R., Fuller G. N., Seiki M., Sato H., Gokaslan Z. L., Liotta L. A., Nicolson G. L., Rao J. S. Differential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro. Cancer Res., 56: 384-392, 1996.[Abstract/Free Full Text]
  35. Alder J., Cho N. K., Hatten M. E. Embryonic precursor cells from the rhombic lip are specified to a cerebellar granule neuron identity. Neuron, 17: 389-399, 1996.[Medline]
  36. Yuste R., Sur M. Development and plasticity of the cerebral cortex: from molecules to maps. J. Neurobiol., 41: 1-6, 1999.[Medline]
  37. Tisay K. T., Key B. The extracellular matrix modulates olfactory neurite outgrowth on ensheathing cells. J. Neurosci., 19: 9890-9899, 1999.[Abstract/Free Full Text]
  38. Zuo J., Ferguson T. A., Hernandez Y. J., Stetler-Stevenson W. G., Muir D. Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin sulfate proteoglycan. J. Neurosci., 18: 5203-5211, 1998.[Abstract/Free Full Text]
  39. Rauvala H., Huttunen H. J., Fages C., Kaksonen M., Kinnunen T., Imai S., Raulo E., Kilpelainen I. Heparin-binding proteins HB-GAM (pleiotrophin) and amphoterin in the regulation of cell motility. Matrix Biol., 19: 377-387, 2000.[Medline]
  40. Quinn C. C., Gray G. E., Hockfield S. A family of proteins implicated in axon guidance and outgrowth. J. Neurobiol., 41: 158-164, 1999.[Medline]
  41. Takino T., Sato H., Shinagawa A., Seiki M. Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP family. J. Biol. Chem., 270: 23013-23020, 1995.[Abstract/Free Full Text]
  42. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159, 1987.[Medline]
  43. Jones-Villeneuve E. M., Rudnicki M. A., Harris J. F., McBurney M. W. Retinoic acid-induced neural differentiation of embryonal carcinoma cells. Mol. Cell. Biol., 3: 2271-2279, 1983.[Abstract/Free Full Text]
  44. Courtney M. J., Coffey E. T. The mechanism of Ara-C-induced apoptosis of differentiating cerebellar granule neurons. Eur. J. Neurosci., 11: 1073-1084, 1999.[Medline]
  45. Rossino P., Gavazzi I., Timpl R., Aumailley M., Abbadini M., Giancotti F., Silengo L., Marchisio P. C., Tarone G. Nerve growth factor induces increased expression of a laminin-binding integrin in rat pheochromocytoma PC12 cells. Exp. Cell. Res., 189: 100-108, 1990.[Medline]
  46. Oh L. Y., Larsen P. H., Krekoski C. A., Edwards D. R., Donovan F., Werb Z., Yong V. W. Matrix metalloproteinase-9/gelatinase B is required for process outgrowth by oligodendrocytes. J. Neurosci., 19: 8464-8475, 1999.[Abstract/Free Full Text]



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