From the Center for Molecular Medicine and Genetics and Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48202
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ABSTRACT |
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We cloned a novel matrix metalloproteinase (MMP)
called CMMP from cultured primary chicken embryo fibroblasts. The
cDNA-derived CMMP sequence contains 472 amino acids including a
putative 19-residue signal peptide and a unique cysteine in the
catalytic domain, an insertion in a sequence motif that binds the
structural (noncatalytic) zinc of MMPs. Strikingly, a homologously
inserted cysteine is also found in Xenopus XMMP and human
MMP19, two recently cloned novel members of the MMP family.
Phylogenetic analysis suggest that XMMP and MMP19 represent founding
members of the MMP family, whereas CMMP is related to collagenase MMPs.
Bacterially produced recombinant CMMP (without the amino-terminal
inhibition domain), which was autoproteolyzed at the carboxyl-terminal
domain, digested casein and gelatin. As shown by Northern blotting,
CMMP mRNA of 1.8 kilobase pairs was constitutively expressed in
cultured primary chicken embryo fibroblasts and up-regulated by tumor
necrosis factor- and the phorbol ester
12-O-tetradecanoylphorbol-13-acetate, but it was not
regulated by interleukin-1, basic fibroblast growth factor, or retinoic
acid. CMMP mRNA of 1.8 kb was also detected in the head and body of
8-day-old chicken embryos and dramatically up-regulated in 9-day-old
embryos.
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INTRODUCTION |
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Degradation and remodeling of the extracellular matrix (ECM)1 is thought to play important morphogenic roles during growth and development. ECM degradation is largely controlled by a superfamily of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs). At present, 18 different MMPs have been cloned and characterized (1-8), including a novel MMP that we cloned from the frog Xenopus laevis embryos (9). MMPs share similar domains with distinct structure and function, have wide and often overlapping substrate specificities, and are usually grouped as collagenases, gelatinases, stromelysins, and membrane-type MMPs.
Collagenases are the only enzymes that can cleave the triple helical
regions of interstitial fibrillar collagens such as collagen types I,
II, and III, the most abundant proteins in the body. The cleavage
occurs at one specific peptide bond,
Gly775-Ile776 (Leu776 in the
2(I)-chain) in type I collagen and leads to unfolding and
denaturation of the fibrillar collagens. Gelatinases A and B, also
known as 72- and 92-kDa type IV collagenases, digest denatured collagen
(gelatin) and components of basement membranes, a special type of ECM
of epithelial, endothelial, fat, muscle, and peripheral nerve cells
(10). Stromelysins are three different MMPs with wide substrate
specificity, and stromelysin-1 can degrade almost any ECM component
including cartilage proteoglycans (11). Recently, four new members of
the MMP family called membrane-type MMPs (MT-MMPs) were cloned
(12-17). They are not secreted extracellularly but remain on the cell
surface through their carboxyl-terminal transmembrane domain. Recently,
however, Matsumoto et al. (18) characterized a secreted form
of MT3-MMP encoded by an alternatively spliced mRNA. MT-MMPs
degrade ECM components, proteolytically cleave and activate 72-kDa type
IV collagenase, and therefore may regulate pericellular matrix
degradation at the cell surface (19-21).
MMPs are produced as inactive precursors, which are activated by proteolytic removal of their amino-terminal domain, a 78-178-residue-long propeptide (22-24). Thus, it is striking that stromelysin-1 (MMP3) proteolytically activates at least five other members of the MMP family including interstitial collagenase-1 (MMP1), matrilysin (MMP7), neutrophil collagenase-2 (MMP8), 92-kDa type IV collagenase (MMP9), and collagenase-3 (MMP13), suggesting that stromelysin-1 plays a special "upstream" role in ECM degradation and remodeling (25-29). Recently, Okamoto et al. (30) reported that various bacterial proteinases can also activate MMP1, MMP2, and MMP9.
MMPs are expressed widely during embryogenesis, often in a highly tissue- and cell-specific pattern, suggesting distinct roles for MMPs in growth and development (17, 31-37). In adult life, MMPs are rarely expressed except in rapidly remodeling tissues such as the term placenta, menstrual endometrium, and involuting mammary glands and during wound healing and inflammation (38-42). MMPs are also thought to play a critical role in tumor growth and metastasis (43, 44) and in the progression of other diseases such as arthritis, atherosclerosis, and aneurysm (3, 45-47).
To study the role of ECM degradation and remodeling in early vertebrate development, we are cloning MMPs and tissue inhibitors of metalloproteinases from chicken embryos and cultured cells (17). Here we describe the cloning and characterization of a novel member of the MMP family, called CMMP, from cultured primary chicken embryo fibroblasts. A cDNA-derived CMMP sequence contains 472 amino acids including a putative 19-residue signal peptide and a unique cysteine in the catalytic domain. A homologously placed cysteine is found only in Xenopus XMMP and human MMP19, two recently cloned novel MMPs (5, 8, 9).
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MATERIALS AND METHODS |
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Cells--
Primary chicken embryo fibroblasts were isolated from
skin dissected from the heads of day 7 embryos (17) and cultured for 5 days until confluent and then treated for 16 h with TNF (5 units/ml), TPA (100 ng/ml), basic FGF (10 ng/ml), IL-1 (5 units/ml), or
5 × 107 M retinoic acid (Fig. 6) before
the isolation of RNA as described below.
RT-PCR with Universal MMP Primers-- We synthesized two degenerate inosine-containing oligodeoxynucleotides called universal MMP primers (17). The forward primer (Y35, 20-mer 16-degenerate with five inosines), agCCI(A/C)GITG(C/T)GGI(A/G)(A/T)ICCIGA, and the reverse primer (Y36, 23-mer 24-degenerate with five inosines), gaTGICCIA(A/G/T)(C/T)TC(A/G)TGI(A/G)CIGC IAC cover all codon usage and amino acid permutations in the two most conserved MMP sequences PRCG(N/V)PD and VA(A/V)HE(F/I/L)GH, respectively. The two nucleotides in lowercase letters were added for cloning purposes (see below). In reverse transcription-coupled PCR, these primers are expected to amplify cDNAs from all mRNAs for MMPs except that of matrilysin (MMP7) in which the second conserved sequence is altered (underlined) YAATHELGH (48). Total RNA was isolated from cultured primary chicken embryo fibroblasts treated with TPA (100 ng/ml) for 16 h by a modified acid guanidinium thiocyanate phenol-chloroform method (49, 50), and 1 µg was used for cDNA synthesis by reverse transcription as described (17) followed by PCR using primers Y35 and Y36 and 1 unit of Taq DNA polymerase (Perkin-Elmer). The PCR products were analyzed by 1% agarose gel electrophoresis in 0.5 × Tris borate-EDTA buffer and visualized by EtBr-staining and UV light.
cDNA Cloning and Sequence Analysis-- The PCR products were cloned into the ScrFI site of pCR-Script Direct SK(+) vector (Stratagene) and sequenced using the dideoxynucleotide method (Sequenase kit, version 2.0, DNA sequencing kit, U.S. Biochemical Corp.). Later, cycle sequencing was carried out using Taq DyeDeoxy (Applied Biosystems), and the samples were run on a model 373A DNA Sequencer (Applied Biosystems). Analysis of several clones with a 395-bp cDNA insert confirmed its MMP origin yet revealed significant sequence differences including a unique cysteine (Cys174) in the catalytic domain not found in other MMPs. Subsequently, we found a homologously placed cysteine in XMMP, a novel MMP that we recently cloned from the frog X. laevis embryos (9), and in human MMP19 also called MMP18 (Refs. 5 and 8; see Fig. 3).
To determine a full-length nucleotide and amino acid sequence for this novel MMP, called CMMP, we used the following rapid amplification of 5'- and 3'-cDNA ends strategy. For the 5'-end of the mRNA, we used the degenerate primer Y36 and Superscript II RNase H-reverse transcriptase (Life Technologies, Inc.) and constructed a cDNA library by the anchor ligation method as described by Apte and Siebert (51). Briefly, 2 µg of total RNA from primary chicken embryo fibroblasts was reverse transcribed with Y36 and Superscript, and the single-stranded cDNA was purified with a clean-up column (Promega) and ligated overnight to the anchor. 1 µl of the ligation reaction was used for amplification with the anchor primer and Y36 primer to construct a 5'-end cDNA sublibrary for MMPs. The 5'-end cDNA sublibrary was then amplified using the anchor primer and a nested specific primer designed from the chicken CMMP sequence. The specific PCR products were cloned and sequenced to obtain the 5'-end sequence of CMMP. For the 3'-end of the mRNA, the "lock-docking" NNPhylogenetic Tree Analysis-- Unweighted dendrograms were produced using the program MegAlign, version 3.03 for the PowerMacintosh (DNA Star, Madison, WI), using a gap penalty of 10, a gap penalty length of 10, and the CLUSTAL alignment algorithm. In Fig. 4, we used full-length MMP sequences without signal peptides that were predicted using the on-line server.2 A similar dendrogram was also constructed from MMP sequences of the catalytic domain.
Bacterial Expression--
To produce in bacteria E. coli an active recombinant CMMP without the signal peptide
(residues 1-19) and amino-terminal propeptide (residues 20-104), we
used PCR and a forward primer
5'-CGCCATATGTATGGTGTTACTCTACCTGGA-3' containing an
NdeI site (underlined) and a reverse primer
5'-GGAATTCATTAACAGTTAAACCAGCTGTTAC-3' containing an
EcoRI site. The PCR product was cut with NdeI and EcoRI and cloned into pET28a bacterial expression vector
(Novagen). This vector provides the amino-terminal His tag (six
histidines) for CMMP. Similarly, to produce an active recombinant human
collagenase-1 (MMP1) without the signal and propeptide, we used PCR and
a forward primer 5'-CGCCATATGTTTGTCCTCACTGAGGGGAAC-3'
containing an NdeI site and a reverse primer
5'-CAGCTAGCGGCCGCTCAATTTTTCCTGCAGTTGAA-3' containing
an NotI site and cloned the PCR product into pET28a bacterial expression vector. These CMMP and MMP1 constructs were confirmed by nucleotide sequencing and transfected into the
BL21(DE3)pLysS E. coli bacterial host (Novagen). Expression
of the constructs was induced by
isopropyl-1-thio--D-galactopyranoside, and the His-tagged CMMP and MMP1 were isolated from inclusion bodies (55), purified using nickel affinity chromatography as described in the
manufacturer's protocol, and characterized by SDS-polyacrylamide gel
electrophoresis and Coomassie Blue staining. In contrast to MMP1, which
remained a full-length protein, CMMP appeared in SDS-gel electrophoresis as a major band of 29 kDa, and three minor bands of
smaller size. This is significantly smaller than the calculated molecular weight of 42,190 (374 residues including six histidines), indicating autoproteolysis and carboxyl-terminal truncation of the
recombinant CMMP. This autoproteolysis occurs during the refolding step
in the isolation protocol (55), since in the inclusion bodies CMMP was
a full-length 39-kDa polypeptide.
Zymography-- Casein and gelatin zymography was performed as described (20). In substrate digestion analysis, 10 ng of bacterially produced recombinant CMMP or MMP1 was incubated with 15 µg of casein or gelatin for 1 h at 37 °C or with 15 µg of rat tail acid-soluble fibrillar type I collagen (Sigma) for 1 h at room temperature. The digestion products were analyzed by SDS-gel electrophoresis and staining with Coomassie Blue. In control reactions, EDTA (20 mM) was used to inhibit MMP activity.
Northern Hybridization--
Total RNA (30 µg) was isolated
from cultured chicken embryo fibroblasts by the acid-phenol method (49,
50) and run on a 1% agarose-formaldehyde gel in 0.5× MOPS buffer. For
the experiments, the cell cultures were treated with TPA (100 ng/ml),
FGF (10 ng/ml), IL-1 (5 units/ml), all-trans-retinoic
acid (5 × 10
7 M), or TNF (5 units/ml)
for 16 h before RNA extraction. To isolate total RNA from chicken
embryos, the head and body were dissected from 8- and 9-day-old
embryos, frozen in liquid nitrogen, and pulverized to fine powder using
a pestle and mortar. After lysis in 4.5 M guanidinium
isothiocyanate solution, the subsequent steps were as described (49,
50). The RNA from the agarose gel was blotted in 10 × SSC (1.5 M NaCl, 0.15 M sodium citrate) to Nytran membrane. A 0.4-kb cDNA of CMMP was 32P-labeled by PCR
(56) and hybridized overnight at 65 °C in 5 × SSC, 5 × Denhardt's solution, 0.1 mg/ml denatured salmon sperm DNA, 0.1% SDS.
For control experiments, CMMP probe was first removed in boiling
distilled water containing 0.1% SDS, and the filters were then
hybridized with a 967-bp chicken glyceraldehyde-3-phosphate dehydrogenase probe that was prepared from total RNA and
32P-labeled by PCR using primers
5'-ATGGTGAAAGTCGGAGTCAACGGA-3' (forward) and
5'-TCACTCCTTGGATGCCATGTGGAC-3' (reverse). The membrane was washed at
65 °C in 0.1 × SSC, 0.1% SDS and exposed overnight to Kodak
x-ray film.
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RESULTS AND DISCUSSION |
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Cloning and Sequence Analysis of CMMP-- To isolate cDNA clones for chicken MMPs, we first synthesized two degenerate inosine-containing MMP primers designed from the two most conserved MMP sequences (17). The first sequence PRCG(N/V)PD, containing an unpaired cysteine necessary for the latency of MMPs (57), is at the end of the amino-terminal propeptide that is proteolytically removed from the active MMPs. The second sequence VA(A/V)HE(F/I/L)GH is in the catalytic domain and has two of the three histidines that coordinate the catalytic zinc in MMPs. Using total RNA from cultured and TPA-treated primary chicken embryo fibroblasts, cDNA was synthesized with reverse transcriptase and random hexamer primers followed by PCR amplification using the degenerate MMP primers. As described previously (17), we obtained at least seven distinct PCR products between 350 and 900 bp. Cloning and sequencing of one of these PCR products of 395 bp confirmed that it was derived from an MMP but also revealed significant sequence differences from previously characterized MMPs (see below).
A reverse transcription-coupled PCR strategy of cDNA cloning (see "Materials and Methods") allowed us to determine the 1817-nucleotide and complete 472-amino acid sequence of this novel MMP, which is called CMMP (C for its unique cysteine, see below). The cDNA-deduced CMMP sequence contains a potential 19-residue signal peptide with a characteristic hydropathy profile but no other significant hydrophobic segments, suggesting that CMMP is a secretory protein. CMMP has an amino-terminal propeptide domain of 85 amino acids with the conserved PRCGVPD motif (Fig. 1); deletion of this domain generates a proteolytically active CMMP (Fig. 5). The chicken CMMP sequence has two potential sites for N-linked sugars, Asn116 and Asn123, both in the catalytic domain, and five cysteines. Cys97, Cys286, and Cys472 (CMMP numbering) are conserved in all MMPs, whereas Cys174 and Cys388 characterize CMMP (see below). Excluding the signal peptide and any posttranslational modifications, CMMP has a calculated molecular weight of 51,047.
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A Unique Cysteine in the Catalytic Domain of CMMP-- There is a unique cysteine in the catalytic domain of CMMP (Fig. 1, circled). This is interesting because previously no other MMPs were found to have cysteine in the catalytic domain. MMP2 and MMP9, however, contain a fibronectin-like domain of 176 residues with 12 cysteines, inserted into the catalytic domain in front of the active site zinc-binding motif. Strikingly, a homologously placed cysteine is also found in Xenopus XMMP and human MMP19, two recently cloned novel members of the MMP family (Fig. 3). XMMP has 604 amino acids including a putative signal peptide of 22 residues, and interestingly, the XMMP gene is transiently expressed during early Xenopus development. It is first activated in gastrula and neurula embryos, and then down-regulated in pre-tail bud embryos (9). Human MMP19 has 508 amino acids and is mainly expressed in placenta, lung, pancreas, ovaries, spleen, and intestine (8). Of note is that Cossins et al. (5) also cloned a novel human MMP (identical to MMP19) and called it MMP18. This is confusing, since Stolov et al. (6) had previously cloned Xenopus collagenase-4, which they named MMP18. So far, we have no evidence for human or mouse homologs of CMMP (see below) and therefore do not suggest any number for CMMP. Indeed, we suggest that the future numbering of novel MMPs should follow the discovery and characterization of human MMPs.
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Phylogenetic Tree of MMPs-- The chicken CMMP sequence appears significantly diverged from other MMPs but is most related to the human collagenases (MMP1, MMP8) with an overall amino acid identity of 52%. This suggests that CMMP is not a homolog of previously characterized MMPs but represents a novel member of the MMP family. In comparison, chicken 72-kDa type IV collagenase (MMP2) and MT3-MMP are 84 and 89% identical with their human homologs, respectively (17, 66).
Interestingly, in a phylogenetic tree of MMPs (Fig. 4), XMMP lies at the very bottom, providing an outlier from which the branching and MMP evolution begins. MMP19 appears along one of the earliest branching points together with stromelysin-3 (MMP11) and MT4-MMP, whereas CMMP together with MMP1 and MMP8 form the top of the tree. Similar evolutionary relationships were also evident by constructing the phylogenetic tree from the catalytic domains of MMPs (not shown). With respect to the conserved unique cysteine, this scenario implies that it was present in an ancestral MMP, which gave rise to XMMP, and then was lost in further MMP evolution and again inserted into MMP19 and CMMP. One wonders what might be the selective pressure to maintain the unique cysteine in the catalytic domain of these three MMPs.
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Characterization of CMMP Activity-- To study the proteolytic activity of CMMP, we produced in E. coli bacteria a recombinant form of CMMP without the 85-residue amino-terminal inhibitory propeptide. For protein purification purposes, six histidines (His tag) were added to the amino-terminal end of CMMP, which begins with the sequence 86YGVTL (Fig. 1). After isolation and purification by nickel affinity chromatography, the His-tagged CMMP migrated in SDS-gel electrophoresis as a major band of 29 kDa and three minor bands of smaller size (not shown). This is significantly smaller than the calculated molecular weight 42,190 (374 residues including six histidines), indicating autoproteolysis and carboxyl-terminal truncation of the recombinant CMMP. We made a similar His-tagged recombinant construct of the human collagenase-1 (MMP1) without the 80-residue amino-terminal propeptide. It begins with the sequence 81FVLTE. In SDS-gel electrophoresis, it migrated as a major band of 42 kDa (not shown), which agrees with the calculated molecular weight of 43,465 (376 residues including six histidines).
As shown in (Fig. 5), the bacteria produced recombinant CMMP-digested gelatin and casein with an efficiency comparable with that of the bacteria-produced human MMP1. However, the two enzymes produced different digestion products from gelatin, indicating different substrate specificities of CMMP and MMP1 (Fig. 5B). Both enzymes were inhibited by 20 mM EDTA, a characteristic feature of MMPs. MMP1 produced the characteristic three-quarter and one-quarter cleavage products from native fibrillar type I collagen, whereas CMMP was unable to cleave type I collagen (Fig. 5E). However, because of its partial carboxyl-terminal truncation, which would abolish collagenolytic activity, if any, these data are not conclusive and must await the purification and analysis of full-length CMMP.
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CMMP mRNA Expression-- In Northern blotting (Fig. 6), CMMP appeared as a single mRNA of 1.8 kb, which was constitutively expressed in cultured primary chicken embryo fibroblasts. Its level was significantly up-regulated in cells treated with TNF or the phorbol ester TPA, whereas basic FGF, IL-1, or retinoic acid had little or no effect. This suggests a different mechanism of gene regulation of CMMP from other MMPs, since, in general, retinoic acid inhibits and TNF, TPA, IL-1, and basic FGF induce MMP gene expression in several types of cells in culture.
As shown in Fig. 6, CMMP mRNA of 1.8 kb was detected in the head and body of 8-day-old chicken embryos and dramatically up-regulated in the head and body of 9-day-old embryos. In control experiments, we detected similar levels of expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. It is interesting to note that MT3-MMP and 72-kDa type IV collagenase mRNAs are constitutively expressed in the head but up-regulated in the body between days 8 and 9 of chicken embryo development (Ref. 17; data not shown).Why Are There So Many MMPs?-- Recent cloning of four different MT-MMPs (12-17) and five novel members of the MMP family called MMP19, porcine enamelysin, chicken CMMP, Xenopus collagenase-4, and XMMP (Refs. 5-9 and this report) brings the current number of different MMPs to 19. Why are there so many different MMPs? This may reflect their highly tissue- and cell-specific expression pattern, in particular during embryonic growth and development. For example, a recently cloned MMP from porcine called enamelysin is expressed exclusively in the tooth's enamel organ (7). Human collagenase-2 (MMP8) is expressed only in neutrophils and cartilage articular chondrocytes (67, 68). High expression of mRNA for the 92-kDa type IV collagenase and MT1-MMP genes is detected in the cells of osteoclast lineage during development (35-37). Expression of the mouse interstitial collagenase, now known to be collagenase-3 (MMP13) (see Ref. 29), is restricted to skeletal tissues; it is expressed in hypertrophic chondrocytes in the growth plates and in osteoblasts and capillary endothelial cells of developing bones (32, 33). Our in situ mRNA hybridization studies on the chicken MT3-MMP showed that it is expressed at high levels in several embryonic tissues such as neural tube, dorsal root ganglia, respiratory epithelium, and in developing cartilage and muscle (17).
It is also possible that the large number of MMPs reflects the complex molecular details of the ECM in tissues and organs. Indeed, more than 80 genes encode the various ECM components in human, of which 34 encode the collagen family of proteins. However, many MMPs are broad spectrum enzymes with overlapping substrate specificities that probably degrade the same ECM components with comparable specificity and kinetics. Therefore, it is possible that the large number and apparent redundancy of MMPs suggest a combinatorial role of MMPs, perhaps in concert with other extracellular proteinases such as plasmin/plasminogen activators, in development, tissue remodeling, or disease. ![]() |
ACKNOWLEDGEMENTS |
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We thank Michael Hagen for the synthesis of many oligodeoxynucleotides; Amy Sang for the sequence of Xenopus collagenase-4 prior to publication; and Irina Massova, Shahriar Mobashery, Minoru S. H. Ko, and Hideaki Nagase for help and comments on the manuscript. Special thanks are due to Anton Scott Goustin for helpful discussions and for Fig. 4.
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Note Added in Proof |
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We have now found that the bacteria produced and activated full-length CMMP, and XMMP, cleave native fibrillar type I collagen into the characteristic three-quarter and one-quarter products (data not shown).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HD24442.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Wayne State University
School of Medicine, Center for Molecular Medicine and Genetics, 5125 Biological Sciences Bldg., 5047 Gullen Mall, Detroit, MI 48202. Tel.:
313-577-2461; Fax: 313-577-8977; E-mail:
markku{at}cmb.biosci.wayne.edu.
1
The abbreviations used are: ECM, extracellular
matrix; EST, expressed sequence tag; FGF, fibroblast growth factor;
IL-1, interleukin-1; TNF, tumor necrosis factor-
; MMP, matrix
metalloproteinase; MOPS, 4-morpholinepropanesulfonic acid; MT-MMP,
membrane-type MMP; PCR, polymerase chain reaction; TPA,
12-O-tetradecanoylphorbol-13-acetate; bp, base pair(s); kb,
kilobase pair(s).
2 Available on the World Wide Web at http://www.cbs.dtu.dk/services/SignalP/.
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REFERENCES |
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