Stefan MASURE, Guy NYS, Pierre FITEN, Jo VAN DAMME and Ghislain OPDENAKKER. The Rega Institute ... cDNA, a cosmid clone with the mouse gene was isolated. .... hydroxide (MeHgOH, 10 mM) (Bailey and Davidson, 1976). Synthesis ...
Eur. J . Biochem. 218,129-141 (1993) 0FEBS 1993
Mouse gelatinase B cDNA cloning, regulation of expression and glycosylation in WEHI-3 macrophages and gene organisation Stefan MASURE, Guy NYS, Pierre FITEN, Jo VAN DAMME and Ghislain OPDENAKKER The Rega Institute for Medical Research, University of Leuven, Belgium (Received June 7/August 30, 1993) - EJB 93 0835/1
Gelatinase B is a regulated matrix metalloproteinase with an important role in the remodelling of extracellular matrices and of basement membranes. To study the structure and function of gelatinase B in the mouse, the cDNA was cloned from a macrophage cell line (WEHI-3). Using this cDNA, a cosmid clone with the mouse gene was isolated. The complete gene (8 kbp) was sequenced and compared with the human gene structure. There was 78% similarity at the cDNA level and the exodintron structure of the murine gene was similar to the human counterpart. At the 5’ untranslated side, 1200 bp of the promoter/enhancer region were sequenced and found to contain several transacting-factor-binding sites. The mRNA transcription-initiation site was determined by non-isotopic primer-extension analysis. Polymerase-chain-reaction amplification of cDNAs yielded indirect evidence for a reverse-transcription stop in WEHI-3 cell mRNA. The DNA-derived mouse-protein structure exhibited 82% similarity with the human one. This similarity was functionally reflected by cross-reactivity of the mouse protein with an antiserum against human gelatinase B. The production of murine gelatinase B was studied at the protein level by zymography and at the mRNA level by Northern blot analysis. In WEHI-3 cells the gelatinase B protein is induced by bacterial lipopolysaccharide, phorbol ester, double-stranded RNA and the cytokine interleukin-1. Regulation of activity and structural heterogeneity of gelatinase B in WEHI-3 cells were shown to occur at the gene regulatory level, by expression of the matrix metalloproteinase inhibitor TIMP-1, and by glycosylation of the secreted protein.
Matrix metalloproteinases (MMP) belong to a class of proteolytic enzymes which act together in degrading most components of extracellular matrices and of basement membranes. Physiological processes in which MMP play a role include embryonic growth and development, migration of blood leukocytes into tissues and tissue remodelling (Matrisian, 1992). Elevated levels of certain MMP are believed to be associated with various pathological states such as tumorcell invasion and metastasis and inflammatory processes such as rheumatoid arthritis and multiple sclerosis (Opdenakker and Van Damme, 1992a,b). Correspondence to G. Opdenakker, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium Fax: +32 16 337340. Abbreviations. con A, concanavalin A; FITC, fluorescein isothiocyanate; IL, interleukin; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; PCR, polymerase chain reaction; PIC, polyriboinosinic.polyribocytidylicacid; PMA, 4-P-phorbol 12-myristate 13-acetate; RACE, rapid amplification of cDNA ends; TIMP, tissue inhibitor of metalloproteinases; MeHgOH, methylmercuric hydroxide ; RT, reverse transcription. Enzymes. Gelatinase A (EC 3.4.24.24); gelatinase B (EC 3.4.24.35). Note. The novel nucleotide sequence data published here have been deposited with the EMBL GenBank and DDBT sequence data banks and are available under the accession numbers X72794 and X72795, for the gene and cDNA, respectively.
Three classes of MMP are commonly recognized based on substrate specificity: collagenases, stromelysins and gelatinases. Several members of each class have been purified and characterized and their genes cloned and sequenced, in humans and in some other species (Docherty and Murphy, 1990; Woessner, 1991; Matrisian, 1992). In humans, two types of gelatinases (also called type IV collagenases) of molecular mass 72 kDa and 92 kDa have been purified, characterized and intensively studied. The genes coding for the 72-kDa enzyme form (MMP-2; gelatinase A) and for the related but different 92-kDa form (MMP-9 ; gelatinase B) have been cloned and sequenced at both the cDNA and the genomic level (Collier et al., 1988; Huhtala et al., 1990 a,b; Wilhelm et al., 1989; Huhtala et al., 1991). The 72-kDa form is secreted by various cell types including fibroblasts, epithelial cells and tumor cells (Collier et al., 1988), and is also present in body fluids (Vartio and Baumann, 1989). The 92-kDa enzyme is produced mainly by neutrophils, monocytes, macrophages and several tumor cell lines (Wilhelm et al., 1989; Masure et al., 1990, 1991; Devarajan et al., 1992). Both types of human gelatinases have similar substrate specificities: they can degrade native type IV and V collagen as well as gelatin (degraded collagen). The regulation of the production and activity of the gelatinase enzymes (and the other MMP) obviously is complex and occurs at different levels. Transcriptional control differs markedly between the two gelatinases. The 72-kDa gelatinase is constitutively produced by fibroblasts and other cell
130 types but its mRNA and protein levels can in some instances be increased or decreased by certain cytokines, growth factors or other substances (Collier et al., 1988; Hipps et al., 1991 ; Overall et al., 1991). In contrast, basal production of the 92-kDa gelatinase is usually low or absent in most normal cell types. It can be upregulated in vitro by various cytokines and other inducers in, for instance, leukocytes and to a lesser extent in diploid fibroblasts (Masure et al., 1990, 1991; Opdenakker et al., 1991a,b; Hipps et al., 1991). In vivo, gelatinase B activity varies with disease severity as observed in rheumatoid arthritis synovial fluids (Opdenakker et al., 1991b) and in cerebrospinal fluids of patients with multiple sclerosis and other inflammatory neurological diseases (Gijbels et al., 1992). There exist substantial quantitative and qualitative differences in gelatinase expression between different cell types. For example, normal fibroblasts mainly produce the 72-kDa gelatinase (Collier et al., 1988), whereas neutrophils exclusively produce the 92-kDa form (Masure et al., 1991; Devarajan et al., 1992). Most tumorcell lines and monocytes can produce both (Masure et al., 1990; Devarajan et al., 1992; Houde et al., 1993). The activity of the gelatinases is also controlled at the post-translational level. Both types of gelatinases are secreted by their producer cells in a latent form as inactive zymogens (proenzymes), which can be activated by many natural or synthetic substances through a mechanism common to all MMP, the so-called cysteine switch (Van Wart and Birkedal-Hansen, 1990). Of particular interest is the fact that, physiologically, many of these enzymes act in cascades which might lead to considerable amplifications in activity. In this way, MMP have been found to be post-translationally activatable and linked to the plasminogen-activator/plasmin system (Vaes and Eeckhout, 1975). Glycosylation of gelatinase polypeptides is another type of post-translational modification with a potential regulatory function. Additional extracellular control of gelatinase activity is achieved by complex formation with specific natural protease inhibitors, known as the tissue inhibitors of metalloproteinases (TIMP). Human TJMP-1 and TIMP-2 inhibit all known MMP in their activated form, including both types of gelatinases. However, TIMP-1 also specifically binds to the 92-kDa pro-gelatinase (Wilhelm et al., 1989), whereas TIMP-2 preferentially binds to the 72-kDa pro-gelatinase (Goldberg et al., 1989). Production and secretion of TIMP are also tightly regulated by cytokines such as interleukin-1 (IL-1) and IL-6 (Murphy et al., 1985; Sat0 et al., 1990; Lotz and Guerne, 1991). Gelatin-degrading enzymes with similar characteristics have also been discovered in species other than humans. The cDNA of the murine homologue of the human 72-kDa gelatinase has been cloned from mouse NIH-3T3 cells and shows high similarity with its human counterpart (Reponen et al., 1992). A 95-kDa MMP that degrades type IV and type V collagen has been isolated and characterized from rat mammary carcinoma cells and is probably the rat homologue of the human gelatinase B (Lyons et al., 1991). In mice, elevated secretion of a 95-kDa gelatinase has been correlated with increased metastatic potential of tumor cells (Yamagata et al., 1989). Very recently, the cDNA coding for this enzyme has been cloned from a mouse metastatic tumor cell line that expresses high levels of a 105-kDa gelatinase (Tanaka et al., 1993). In this study, we describe the molecular cloning, complete genomic and partial cDNA sequence analysis, and the regulation of expression of the gene for a 110-kDa gelatinase from mouse macrophages. The tools thus generated will
serve in the further development and study of mouse model systems to investigate the role of gelatinase B in normal and pathological processes.
EXPERIMENTAL PROCEDURES Materials Restriction endonucleases used were from Boehringer Mannheim or from BRL Life Technologies. [a-32P]dCTPwas from Amersham. Other sources are mentioned in the text.
Cloning of mouse 110-kDa-gelatinasecDNA A commercially available cDNA library from the mouse macrophage cell line WEHI-3 in the A ZAP I1 vector (Stratagene) was used to isolate mouse gelatinase cDNA clones. Plaque screening of the cDNA library was performed by means of standard methods. Briefly, plates containing approximately 1.5X 10' plaque-forming units were grown for 8 h or more and plaques were then transferred to Hybond N nylon membranes (Amersham). Filters were hybridized overnight at 42°C in a hybridization buffer containing 50% formamide. The library was screened (1.5X 10" clones) with the [32P]-dCTP-labelled full-size cDNA coding for the human 92-kDa tumor-cell type IV collagenase. This cDNA was obtained from the p92174.1 plasmid (Wilhelm et al., 1989; a kind gift from Dr G. I. Goldberg and Dr B. Manner) by digestion with XbaI, yielding a 2.3-kb cDNA probe. Filters were washed at low stringency (final wash in 0.2X NaCKit [20X NaCUCit, 3 M NaC1, 0.3 M disodium citrate, pH 7.01, 0.1% SDS at 42OC) and autoradiographed. Positive plaques were isolated and two more cycles of plaque screening and purification were performed to finally isolate one positive plaque. In vivo excision and recircularization of the cloned insert from the 2 ZAP I1 vector with the R-408 helper phage was performed as described by the manufacturer (Stratagene). This resulted in a pBluescript phagemid with a 2.0-kb cDNA insert representing the 3' end of the mRNA for the mouse 110-kDa gelatinase (clone pSM2). After DNA sequence analysis, a 780-bp 5' fragment was obtained from this clone by digestion of the insert with restriction endonucleases TthIIIl and XhoI. The cDNA library was reprobed with this fragment. Since the probe was now 100% similar, hybridization and wash conditions were adapted accordingly. Hybridization was performed without formamide at 65 "C and final washes were with 0.1 X buffer A (20X buffer A, 3 M NaC1, 0.2 M NaH,PO,. H,O, 0.02 M EDTA, pH 7.4), 0.1 % SDS at 65 "C. Two more clones were isolated (out of 1.5X10bscreened) and had the same 2.0-kb insert. As this commercial library apparently did not contain any full-size cDNA clones for the mouse gelatinase B, further attempts were made to isolate cDNA fragments containing the putative 400 missing 5' base pairs. First, another cDNA library was constructed in Uni ZAP XR (Stratagene) using mRNA purified from cells optimally induced for gelatinase expression with bacterial lipopolysaccharide (LPS from Escherichia coli strain 0111 :B4; 1 pg/ml). Approximately 2 X lo6 clones from this library were then screened using the 5' end 780-bp fragment of our original incomplete cDNA clone. Since no clones extending at the 5' end could be isolated, several other approaches were then tried. Total RNA or poly(A)-rich RNA made from WEHI-3 cell cultures, optimally induced for gelatinase expression (1 pg/ml LPS), were
131 used as a template in first-strand-synthesis reactions. Prior to reverse transcription of mRNA, the RNA was denatured to remove secondary structures at elevated temperatures (65 70°C) or by addition of the denaturing agent methylmercuric hydroxide (MeHgOH, 10 mM) (Bailey and Davidson, 1976). Synthesis of first strand cDNA was performed using either oligo(dT) or a 25-base oligonucleotide specific for the mouse gelatinase DNA as a primer. This specific primer was an antisense oligonucleotide annealing about 200 bp downstream from the 5' end of our first isolated cDNA clone and contained two mismatches to create a XbaI restriction site (MAS1, S'-CGTCTACjATGGGCATCTCCCTGAAC-3'; Xbd site is underlined, mismatches are double underlined). Reverse transcription was carried out using different enzymes at different temperatures: M-MuLV reverse transcriptase (Stratagene) at 37 "C, TetZ thermostable reverse transcriptase/DNA polymerase (Amersham) at 55 -7O"C, Super-. script reverse transcriptase (BRL Life Technologies) at 45 55 "C or Retrotherm thermostable reverse transcriptase/DNA polymerase (Epicentre Technologies) at 70- 80 "C. The cDNA products of different combinations of these protocols were subcloned in the pUC19 plasmid vector and the presence of gelatinase cDNA sequences in all libraries was determined by a polymerase chain reaction (PCR) using the specific primer MAS1 and a second primer annealing to the vector DNA near the inserted cDNA sequences (ssDNAfonv or ssDNAbackw). The 5' RACE (rapid amplification of cDNA ends) method for the amplification of partially known cDNA sequences (Frohman et al., 1988) was also performed according to the instructions of the manufacturer (BRL Life Technologies) using cDNA pools obtained from different combinations of the protocols described above.
Genomic cloning of the mouse 110-kDa-gelatinasegene A mouse genomic liver DNA library in the cosmid pWE15 (Stratagene) was screened with the mouse gelatinase cDNA probe (780-bp TthIIIl -XhaI fragment) according to the instructions of the manufacturer. In the primary screening, approximately 60 positive clones were detected out of lo6 clones screened. 10 positive clones were further purified in a secondary screening. Cosmid DNA preparation, restriction digestion with PstI or XbaI and gel electrophoresis analysis yielded three different cosmid clones. One of these clones, MMG1, was further analyzed. Cosmid DNA from this clone was prepared by alkaline lysis and column purification (Qiagen) and was fragmented by digestion with several different restriction endonucleases. Restriction fragments containing gelatinase DNA sequences were detected by hybridization of Southern blots made from this fragmented cosmid DNA with [cx-32P]dCTP-labelled mouse gelatinase probes obtained from the cDNA clone pSM2. Hybridizing fragments were then subcloned in the plasmid vector pUC19 and partially sequenced. The complete DNA sequence of the gelatinase gene was determined by subcloning of smaller restriction fragments from these primary subclones into pUC19 and subsequent sequence analysis.
Sequence analysis of mouse 110-kDa-gelatinaseDNA The cDNA and genomic DNA sequences of the cloned mouse gelatinase were determined using the dideoxynucleotide termination method (Sanger et al., 1977) on an Automated Laser Fluorescence analysis system (A. L. F. DNA Sequencer, Pharmacia). Sequencing was completed on both
strands using T7 DNA polymerase (Pharmacia) and fluorescein isothiocyanate(F1TC)-labelled M13 universal primers or specific custom-made FITC-labelled oligonucleotides (Eurogentec or Pharmacia). Sequencing data were analyzed using the PUGENE software package.
Determination of the transcription-initiation site by fluorescent primer-extension analysis To determine the start site for transcription on the mouse gelatinase B U A , primer-extension analysis was performed. The classical isotopic technology was adapted to a non-isotopic fluorescent system compatible with automated DNA sequencing. Total RNA was prepared from WEHI-3 cell cultures treated with 10 yg/ml LPS for 24 h. Poly(A)rich RNA was isolated by chromatography on oligo(dT)-cellulose (Pharmacia). 4.5 pg template poly(A)-rich RNA was then combined with 20 pmol FITC-labelled 20-nucleotide antisense primer annealing approximately 100 nucleotides downstream of the ATG translation-initiation codon of the mouse gelatinase B gene (primer MMGPEX, 5'-GGAAGACCACAAAAGTCGGC-3'). The RNA/primer mixture was precipitated with ethanol and the pellet resuspended in 30 yl hybridization buffer (40 mM Pipes, pH 6.4, 1mM EDTA, 0.4M NaC1, 80% formamide). The sample was heated at 85 "C for 10 min to denature the RNA then slowly cooled to 42 "C. Primer annealing was performed for 15 h at 42 "C. The sample was then again ethanol precipitated and resuspended in water. The primers were extended with reverse transcriptase in a total volume of 50 p1 in the presence of 20 mM Tris/HCl pH 8.4, 50 mM KC1,2.5 mM MgCl,, 100 yg/ml bovine serum albumin, 2 mM of each dNTP, 10 mM dithiothreitol, 200 U human placental RNAse inhibitor (Amersham) and 400 U Superscript reverse transcriptase (BRL Life Technologies). The reaction was incubated for 1 h at 42°C and was then stopped by incubation at 55°C for 5 min. The RNA was digested by addition of 4 U RNAse H (BRL Life Technologies) and incubation for 10 min at 55 "C. After phenolkhloroform extraction and ethanol precipitation, the fluorescent extension product was analysed on a sequencing gel along with sequencing reactions from a genomic mouse gelatinase B subclone prepared using the same oligonucleotide primer as used in the primer-extension assay. The hardware consisted of the A. L. F. DNA sequencer and the software was the A. L. F. Manager (Pharmacia).
Cloning of mouse TIMP-1 by reverse transcriptiodPCR (RTFCR) To obtain a cDNA probe for mouse TIMP-1, L-929 mouse fibroblasts in culture (approximately 4 X lo7 cells) were treated for 17 h with 4 ng/ml of semi-purified murine IL-6 (Van D a m e et al., 1991), an IL-6 concentration known to induce substantial TIMP levels in human fibroblast cultures (Sato et al., 1990, Lotz and Guerne, 1991). Subsequently, total RNA was prepared from this culture as described previously (Opdenakker et al., 1982). 5 yg total RNA from the IL-6-treated L-929 culture was then reverse transcribed with RAV-2 reverse transcriptase (Amersham) in the presence of oligo(dT) and random hexanucleotide primers and of human placental RNAse inhibitor (Amersham). This first-strand-cDNA synthesis product was utilized in a PCR reaction using two synthetic 26-nucleotide primers (Eurogentec) based on the known cDNA sequence of murine TIMP-1 (Edwards et al., 1986) with one or two mismatches at the
132 5' end to create a XbaI restriction site. The sense primer MMTIMPFOR, 5'-GGCTICTAGAGACACACCAGAGATAC-3', was located just 5' of the translation-start site. The antisense primer MMTIMPREV, 5'-GGAAATsTAGACAGTGTTCAGGCTTC-3', was located just 3' of the translation-stop codon (XbaI site is underlined, mismatches are double underlined). PCR amplifications were performed in a thermal DNA cycler (PHC-2, Techne) using the following conditions; 25 pmol of each primer were combined with first strand cDNA, amplification buffer (50mM KC1, 1OmM TrisMC1, pH 8.3, 1.5 mM MgC12, 0.01% gelatin) and 250 pM of dNTP in 50 p1. After 5 min denaturation at 95 "C, 2.5 U native Taq polymerase (Perkin Elmer Cetus) were added and 60 cycles of PCR were performed (95 "C for 45 s/ 60°C for 55 s/72"C for 45 s), followed by a final 5-min extension at 72°C. The PCR product was then extracted twice with chloroform and ethanol precipitated. After XbaI restriction digestion, the sample was electrophoresed in 1.2% agarose and the 688-bp TIMP band was excised from the gel, eluted and subcloned in an XbaI-digested pUC19 plasmid vector. The complete cDNA sequence of one subclone (pMMTIMP1) was determined and corresponded exactly to the published murine TIMP-1 cDNA sequence (Edwards et al., 1986; base pairs -22 to +664). The 688-bp XbaI fragment of this clone was further used as a cDNA probe in Northern blot experiments.
Northern blot analysis For the determination of tissue-specific expression of mouse gelatinase B and TIMP-1 mRNAs, a Northern blot made from purified poly(A)-rich RNA from different organs or tissues (2 pg RNA/tissue) from BALB/c mice (Clontech) was hybridized using the mouse gelatinase B and TIMP-1 cDNA probes. First, the blot was hybridized at 65°C for approximately 20 h in a buffer containing 5X buffer A, 1OX Denhardt's reagent, 2% SDS and lOOpg/ml herring sperm DNA. The 780-bp TthIIIl -XhoI fragment of plasmid pSM2 (see above) was used as the mouse gelatinase B probe. After hybridization of the [a-32P]dCTP-labelledprobe, filters were washed at room temperature for 3 x 1 5 min with 2X NaCY Cit, 0.05% SDS and at 50°C for 2 x 2 0 min with 0.1 X NaCV Cit, 0.1% SDS. Filters were finally exposed to preflashed Curix RP I1 film (Agfa Gevaert) with two intensifying screens at -70°C for approximately 3 days. The Northern blot was subsequently hybridized under the same conditions with the 688-bp XbaI fragment of plasmid pMMTIMPl (see above). In addition to the equivalent loading of poly(A)-rich RNA from the different tissues (2 pgAane), another control for the quality and quantity of the mRNA preparation was performed. The blot was hybridized with the human P-actin cDNA probe (encoding a house-keeping gene). We detected P-actin transcripts of 2.0 kb and 1.6 kb and the tissue-specific abundancies were identical to those described (Lamballe et al., 1991; and data not shown).
Induction of cells for the production of gelatinases Mouse macrophage WEHI-3 cells were cultured in 75-cm2 culture flasks in Dulbecco's minimal essential medium containing 10% fetal calf serum. Confluent cells were washed with NaCllP, (137mM NaC1, 8 m M Na,HPO,, 1.5 mM KH2P04, 2.5 mM KCl, pH 7.2) and refed with serum-free medium supplemented with 2% human serum albumin and with one of several inducers : IL-1P, 4-P-phorbol
12-myristate 13-acetate (PMA), bacterial LPS (from E. coli strain 0111 :B4), double-stranded RNA (PIC; polyriboinosinic.polyribocytidylic acid), and concanavalin A (con A) (Van Damme et al., 1991). After incubation, the conditioned medium was harvested and gelatinase activity was determined by zymography.
Detection of gelatinase activity Gelatinase activity in cell culture supernatants was detected by SDSPAGE zymography using gelatin (0.1%) as substrate, as described previously (Masure et al., 1990). Briefly, microliter samples of serum-free cell culture supernatants were electrophoresed without prior denaturation in gelatin-containing acrylamide gels. Gels were then washed to remove SDS and to reactivate the gelatinases. After an overnight incubation to develop enzyme activity, the gels were stained with Coomassie blue. Gelatinase activity was detected as unstained bands on a blue background.
Immunoprecipitation of gelatinases The gelatinase activity present in the supernatants from murine WEHI-3 cell cultures was immunoprecipitated using two different rabbit polyclonal gelatinase antisera directed against human tumor cell gelatinases and against human neutrophil gelatinase. The latter was prepared by immunization of a rabbit with purified human neutrophil gelatinase (Masure et al., 1991). As positive controls, gelatinases in the supernatants from human THP-1 myelomonocytic leukemia cells were immunoprecipitated with the two antisera. Immunoprecipitations were performed as described previously (Masure et al., 1991). Briefly, gelatinases were reacted with the antibody and the immune complexes precipitated by binding to protein A on previously treated Staphybcoccus cells. After extensive washing of the precipitates, the complexed molecules were dissociated with SDS and separated by SDSPAGE. Reactivated gelatinase activity was determined by zymography as described above.
Glycosylation of murine 110-kDa gelatinase To determine whether the mouse gelatinase is glycosylated, a sample (serum-free cell culture supernatant from WEHI-3 cells treated with 1 pg/ml LPS for 48 h) was treated with N-glycosidase or 0-glycosidase (Boehringer Mannheim). 20 p1 cell culture supernatant was incubated in buffer (50 mM NaH2P04, 50 mM Na,HP04, 20 mM EDTA, 0.1% SDS and 1% Triton X-100 at pH7.0) in the presence or absence of either 0-glycosidase (endo-a-N-acetylgalactosaminidase; 0.1 mu), N-glycosidase F (PNGase F; 40 mu) or a combination of both. Samples were incubated for 6 h at 30°C. After incubation, SDSPAGE loading buffer (0.125 M TrisMCl, pH 6.8, 4% SDS, 0.1 % bromophenol blue, 0.25 M sucrose) was added and gelatinase activity was determined by zymography as described. Mouse gelatinase B from the conditioned medium of LPS-treated WEHI-3 cell cultures was purified by affinity chromatography on gelatin-Sepharose (Masure et al., 1991). Aliquots of this purified gelatinase B preparation were then treated with sialidase alone (Oxford Glycosystems; 5 mu for 6 h at 37°C) or with the combination of sialidase (5 mu) and 0-glycosidase (endo-a-acetylgalactosaminidase,Oxford Glycosystems; 1 m u for 6 h at 37°C). Treated samples to-
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AGTTCCTGAATCATAWGCAAGCCCATTACAGGGCCCCTTCCTTACTGCCCGCACGTGGCCAGCCCTGCCTGCAACGCTGGACTCCGCCTTTGAGGATCC K F L N H R G S P L Q G P F L T A R T U P A L P A T L D S A F E D P
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GCAGACCAAGAGGGTTTTCTTCTTCTCTGGACGTCMATGTGGGTGTACACAGGCMGACCGTGCTGGGCCCCAGGAGTCTGGATMGTTGGGTCTAGGC
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AGGTGGATCCCCAGAGCGTCATTCGCGTGGATMG~GTTCTCTGGTGTGCCCTGGAACTCACAC~CATCTTCCAGTACCMGAC~GCCTATTTCTG 2100
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GAGGGMAGGTGCTAGC~GGCCAGGTACAGACTGGTGATCTCTTCTAGA~CTGG~G~GTGGAGGCAGGCAGGGCTCTCTCTGCCCACCGTCCTTTC 2400
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GAGGTGMCCAGGTGGACGACGTGGGCTACGTGACCTACGAC
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TTGTTGMCTCTTTCT~CACGGATCCCCAACCTT-: __
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Fig. 1. cDNA and genomic DNA coding sequences of the mouse 110-kDa gelatinase and deduced amino acid sequence. The nucleotide sequence of the partial clone pSM2 (cDNA) is shown together with the exon sequences derived from the genomic clone MMGl (gDNA). Nucleotide position 1 corresponds to the transcription-start site as determined by primer-extension analysis. The vertical line at nucleotide position 421 marks the 5' end of the partial cDNA clone. From this position onwards, the sequence is only shown once except for the differences between cDNA and gDNA (boxed). The predicted amino acid sequence is shown under the corresponding DNA sequence. Differences between the cDNA-derived (AAcDNA) and gDNA-derived (AAgDNA) amino acid sequence are also enclosed in the boxes. The highly conserved cysteine-switch region (PRCGVPDV) and putative zinc-binding region (VAAHEFGHALG) are underlined. The three potential N-glycosylation sites and the putative polyadenylation signal (AATAAA) are double-underlined.
gether with appropriate controls were then analyzed on zymography to detect eventual changes in molecular mass.
RESULTS Cloning of the mouse gelatinase B cDNA Screening of a mouse macrophage cDNA library from WEHI-3 cells with the human 92-kDa-gelatinase cDNA
probe resulted in the isolation of one partial cDNA clone (pSM2) containing a 2019-bp insert. The nucleotide sequence of this cDNA clone and the deduced protein sequence form parts of Fig. 1 in conjunction with the complete gelatinase coding sequence obtained by merging the exon sequences from the genomic DNA. The first 1794 bp of the partial cDNA sequence code for the last 598 amino acids of
134 the mouse 110-kDa gelatinase. Comparison of our partial cDNA sequence with the gelatinase B cDNA sequence from a mouse tumor-cell line published after completion of our work (Tanaka et al., 1993) shows 99.7% identity (4 nucleotide differences). Downstream of the coding region, there is a 224-bp 3’ untranslated sequence including the translationstop codon TGA and one polyadenylation signal (AATAAA), and a poly(A)-tail of 80 nucleotides. Comparison of the human and partial mouse cDNA sequences showed 77% identity and suggested that the mouse gelatinase cDNA should theoretically at least be 400 bp longer at the 5’ terminus. Further screening of the same cDNA library, now with a 100% similar probe, led to the isolation of two additional clones. However, by sequence analysis these were found to be of exactly the same size as the original pSM2 clone and to start at exactly the same nucleotide position. Construction of another cDNA library starting from WEHI-3 cells optimally induced for gelatinase expression with LPS and screening of Fig. 2. Presence of a reverse transcription stop in the mRNA for 2x10” recombinant clones of this library with the 100% sim- the mouse gelatinase. Electrophoretic analysis of 5 ~1 of products ilar probe did not result in isolation of clones containing the from PCR-reactions performed on different DNAs using primers missing 5‘ sequence. This led us to postulate the existence of MASl and ssDNAbackw. The arrowhead marks the 360-bp stop a reverse transcription stop in the gelatinase B WEHI-3 band. DNAs used were, lane 1, phage DNA from LPS-induced mRNA preparation that causes premature termination of re- WEHI-3 phage cDNA library (10 ng template); lane 2 , 123 bp DNA ladder (5 Bg); lane 3, cDNA made with primer MASl using mRNA verse transcription during first-strand synthesis. Since this from LPS-induced WEHI-3 cells and ligated into pUC19; lane 4, transcription stop could be due to the presence of secondary control plasmid DNA from clone pSM2 (10 ng template); lane 5, structures in the mRNA molecules, we tried various methods negative control (no template DNA). to denature the mRNA prior to and during first-strand synthesis. More than 10 optimized protocols were tested to reverse transcribe the missing 400 bp using total RNA or purified clones. One of these (MMGl), containing the complete mupoly(A)-rich RNA, different primers [oligo(dT) or a 25-nu- rine gelatinase B gene (all 13 exons, the intron sequences cleotide primer (MAS1) specific for the mouse gelatinase and extended parts of the 5’ and 3’ untranslated regions), was sequence and annealing approximately 200 bp downstream analyzed in detail and approximately 8.2 kb of the gelatinase of the hypothetical reverse-transcription-stop site], different gene were sequenced starting 1.2 kb upstream of the denaturation conditions (temperature, MeHgOH) and dif- translation-initiation codon in exon 1 and ending 344 bp ferent (thermostable) reverse transcriptases/DNA polymer- downstream of the translation-stop codon in exon 13. ases at different temperatures. Cloning followed by PCR Fig. 3A and B shows the structure and the sequencing stratanalysis or 5’-RACE PCR of obtained cDNA products did egy for the complete murine gelatinase B gene, respectively. not yield any additional sequence information. Fig. 2 shows A restriction map for the sequenced part is depicted in representative examples of PCR-products obtained in such Fig. 3C. The complete genomic sequence included the missexperiments. PCR analysis using two primers, MASl and a ing 5’ coding sequence (exons 1 and 2 and part of exon 3). primer annealing in the vector sequence, almost always led The exonhntron junctions were determined by comparison of to the amplification of a DNA fragment of approximately the mouse genomic sequence with the human genomic 92360 bp (Fig. 2). This PCR band migrated at exactly the same kDa gelatinase sequence (Huhtala et al., 1991) and all follow position as the control band obtained by PCR amplification the AG/GU rule (Breathnach and Chambon, 1981). The with the same primers on plasmid DNA of the pSM2 clone exodintron junctions obtained by comparison of the mouse (Fig. 2). For the analysis of the presence of 5’-end-extending genomic sequence with the human sequence are confirmed clones in all the differently generated libraries we performed by the cDNA sequences derived from WEHI-3 cells, and PCR reactions on pooled plasmid preparations containing from SN-H tumor cells (Tanaka et al., 1993). Table 1 shows DNA from more than 2XlO’O equivalent clones. The absence the exodintron structure of the mouse gelatinase gene and from these experiments of any PCR products of greater than the size of the corresponding exons and introns of the human 360 bp confirms the presence of a strong reverse-transcrip- gelatinase B gene. The mouse 110-kDa gelatinase gene intion stop in the WEHI-3 cell mRNA encoding the 110-kDa cludes 13 exons (ranging in size from 104 bp for exon 12 to gelatinase. PCR analysis with these same two primers on 334 bp for exon 9) separated by untranslated intron sepurified phage DNA obtained from the uninduced and LPS- quences ranging in size from 101 bp (intron 9-10) to induced cDNA libraries (10 ng template DNA being equiva- 1155 bp (intron 8-9). The exon-deduced coding sequence lent to 2.5X lo8 recombinant phages) also revealed this 360- contains 2190 bp and codes for a protein of 730 amino acids bp band (Fig. 2), confirming that these libraries did not con- with an expected molecular mass of approximately 80.5 kDa tain full-size cDNA clones for the mouse gelatinase. Since for the prepro-enzyme, 78.5 kDa for the pro-enzyme and all attempts to obtain the missing 400 bp from cDNA failed, 68.5 kDa for the activated enzyme. Comparison of the mouse a mouse genomic library was screened to complement the gelatinase coding sequence (first 394 bp deduced from genomic DNA, last 1796 bp from cDNA) with the human 92missing 5’ end sequences by exonic DNA. kDa-gelatinase cDNA sequence shows high similarities beGenomic cloning of the mouse 110-kDa-gelatinaseDNA tween both enzymes: 78% at the DNA level and 82% at the Screening of a mouse cosmid library with the gelatinase protein level (75% identity, 7% similarity). Comparison of cDNA probe resulted in the isolation of several gelatinase the exon sequences derived from genomic DNA with the
135 0
2
1
3
4
5
7
6
9
B
kb
I
Human
r;l
10
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1112
D Fig. 3. Structure, restriction map and sequencing strategy for the mouse gelatinase B gene. (A) Schematic representation of the human and mouse gelatinase B genes. Exons are represented by boxes and are numbered in the S'-to-3' direction. Intron sequences are represented by the lines connecting two exons. Relative exon and intron lengths are to scale. The top line shows a scale in kilobase pairs. (B) Sequencing strategy for the mouse gelatinase B gene. The arrows represent direction and extent of part of the sequenced pUC19 subclones. (C) Restriction map of the mouse gelatinase B gene as deduced from the genomic DNA sequence. The upper part shows the location of EcoRI, HindIII, KpnI, PstI, XbaI and XhoI restriction-endonuclease recognition sites, while the bottom part shows the location of BarnHI, PvuII, Sac1 and TthIIIl sites. (D) Domain structure of the mouse gelatinase. The exons are depicted as boxes and the intervening intron sequences and flanking regions as solid lines. The exon parts coding for the different protein domains typical for gelatinases (Matrisian, 1992) are differently shaded and named under the diagram. Table 1. Exonlintron structure of the murine 110-kDa gelatinase B gene. The nucleotide sequences at the boundaries between introns (lower-case letters) and exons (capital letters) are shown together with the size of the exons and introns in base pairs. The sizes of the corresponding exons and introns of the human gelatinase B gene are indicated between brackets (according to Huhtala et al., 1991). The exons were numbered starting at the 5' end of the niRNA. The exodintron junctions were determined by comparison with the human 92kDa type IV collagenase gene (Huhtala et al., 1991) and the sizes of exons and introns were deduced from the sequence analysis. The ATG translation-initiation codon and the TGA stop codon are underlined. untransl., untranslated. Exon no.
Exon size
Exon-intron junctions
Intron size
bP
1
... 25 untransl.
2 3 4 5 6 7 8 9 10 11 12 13
ccatccacag tcacctccag cctcttgcag ctttctacag gtctcctcag gtctctccag taccttccag ttgtttttag ttcttctcag tcttccgcag cttcctgcag tttcccgtag
G AG TG GA TG GG GT T GA A AC
ATG
AGT CCC
GCA ATC CAC ATC CTC GAC TAC CGT TGG CGT TTC
TTG AAC GAC ACT ACG ACC CTG TCT TGG ATG TTG TAT
TAC CAA GGA CCC TAC GCG AGC GGC TAC CAA GAC AAA GCC
... ... CAG CTG ... ... ATC ACA ... ... GGT GTC ... ... A i U GGC ... ... CCT AGT ... ... CCT ACC ... ... CCA GAC ... ... TAT CTG ... ... TTC AAG ... ... TTC TTC ... ... CGT GTC ... ... CAG TAC ... ... CAG TGC
cDNA sequence obtained from the partial clone pSM2 revealed four differences in nucleotides resulting in three differences at the protein level (Fig. 1). In all three cases, a proline was involved. At position 514 in the protein primary structure, an alanine in the cDNA-derived sequence changes to a proline (GCC to CCC); at position 639 a proline changes to a leucine (CCC to CTC) and at position 711 a proline
GCA GAG
gtagacagat
TAC GCG GTC GAG CGA CAA TAT GAC TCT TGG CAA CCT
gtgagatgtc gtgagaattc gtgagatcct gtgagtatgc gtacctctgc gtgagcgggg gtgaggctgg gtaagcaggg gttagtttgt gtaagagcga gtgagggctg
TG G G A G G G GG G AG G
a
+ 224 untransl.
141+25 (138 + 19) 230 (233) 149 (149) 129 (129) 174 (174) 174 (174) 177 (177) 156 (156) 334 (280) 140 (140) 151 (151) 104 (104) 131+224 (1 16f 194)
437 (870) 218 (420) 277 (270) 109 (220) 180 (180) 350 (325) 105 (11.5) 1155 (700) 101 (113) 269 (240) 104 (96) 1150 (1800)
changes to a histidine (CCT to CAT). One change from T in cDNA to C in genomic DNA (nucleotide position 1987 in Fig. 1) did not result in a change in amino acid sequence. The domain structure of the mouse gelatinase B is shown in Fig. 3D and is very similar to the domain structure of the human gelatinase B. However, the mouse gelatinase B protein contains three stretches with additional amino acids as
136 I2
I1
MSPYPPLLLALLAFGCSSMPYOROPTFWFPKDLKTSNLTDTOLAEAYLYRYGYTR~OMMGEKQSLRPALLMLQKOLSLPOTGELDSOTL~IRTPRC 100
muse
..L ....V.V ..V L ..CF ...R...S.L.L..G..R......R....E.........V.E.R..SK..G....L........E......A....M.....
hunan
13
99 14
GVPDVGRFOTFKG-LKLlDHHNITYUIQNYSEDLPRDMIDDAFARAFAVUGEVAPLTFTRVYGPEADIVIOFGVAEHGDGYPFDGKDGLLAHAFPPGAGVO L E.D H.................AV..........L.SA.T........SRD................................P. 1.
mouse
.... ...... ...
hunan
15
16
hman
GDAHFDDDELUSLGKGWIPTYYGNSNGAPCHFPFTFEGRSYSACTTDGRNDGTPUCSTTADYDKDGKFGFCPSERLYTEHGNGEGKPCVFPFIFEGRSY V RF AD A.....I..............S..L.......N..T.DR...........RD..AD....O.....Q. Q.Y
muse
SACTTKGRSDGYRUCATTANYDQDKLYGFCPTRVDATWGGNSAGELCVFPFVFLGKQYSSCTSDGRRDGRLUCATTSNFDTDKKUGFCPDOGYSLFLVA
mouse
.................. .. .. ..
I8
17
.....D ................R...F......A.S..M.............T....E..T...E..G.............S..................
hunan
19 AHEFGHALGLDHSSVPEALMYPLYSYLEGFPLNKDDlDGlOYLYGRGSKPDPRPPATTT~POPTAPPTMCPTIPPTAYPTVGPTVGPT~PSPGPTSSP M.RFT P H...VN..RH...PRPE.E....T.............V...G...VH.SER..A..............
mouse
.. ..
......................
hunan
110
SPGPTGAPSPGPTAPPTAGSSEASTESLSPADNPCNVDVFDAIAEIQGALHFF~GUYUKFLNHRGSPLOGPFLTARTUPALPATLOSAFEDPOTKRVFF ......P ..A...G.....P.T.T.VP...V.DA...UI.......GNO.YL....K..R.SEG...RP.....I.DK.....RK...V..E.LS.KL..
mouse
hunan
1’1 mouse
I 13
112
mouse
299 299 399 399 499 483
599 581
FSGRQMUYYTGKTVLGPRSLDKLGLGPEVTHVSGLLPRRLGKALLFSKGRVURFDLKSOKVDPOSVIRM)KEFSGVPYNSHDIFOYOD~YFCHGKFFUR V AS R.......AD.AQ.T.A.RSGR..M....GR.L....V.A.M...R.ASE..RM.P...LDT..V...RE.....ODR.Y..
699 MI1
VSFONEVNKMHEVNOVDDVGYVTYDLLOCP-SRS.L 0.. I....ED
730
..... ..... .....
hunan
199 199
707 .. ........... ..... Fig. 4. Comparison of the human and mouse gelatinase B amino acid sequences. The upper line shows the murine sequence as predicted from the genomic exon DNA sequences. The bottom line shows the amino acids from the human gelatinase B which differ from the murine
hunan
counterpart. Identical residues are represented by a dot. The absence of a corresponding amino acid is indicated by a dash. Amino acid stretches in the mouse protein with no human equivalent are double-underlined. The vertical lines above the mouse sequence mark the positions of the genomic exons from which the amino acid sequence is derived. Numbering is according to the diagram in Fig. 3A.
-1174
gagctcaccagtgagaagcatctaagagaagcttgggagaacacccagctctctctctccggctcacaggtctgttcgttgggaagcacatgaaggtctg
-1074
ggcacacaggaggcttagtcagaacagcttgctgaagacagatcaaggccctgctccaccatggtggcaggcgaggaggatggaaggccgggggctgccg
-
974
gctgttggcaagactgtgccaaagctttcctgagtggagcagggcagggctggaggaggggaagggtccatgacgatctcacagctcgggagaggaaggt
- 874
gtttgccccatccaggtcaccccaaggcttagagccaagaccccagtctcctaatttccaatcacaaacctgacaccatcaactgaggtctcgtgaacac
-
774
tgctgaaagtggtttttctgtgtttcgagagtctcattttatcctcagatcaatatagggacaaaggcttgagcgacaaagggtctgtttttgttcttta
-
674
aacagaagaggaaggatagtgctagcctgagaaggatgaagcttctgcttgctcccacatgtgtgtgtccc~~~catctttccttccc
-
574
IF-KB PEA3 AP-1 caaggagtcagcctgctggagctaggggtttgcccca~gaattccc~aatcctgcctcaaagagcctgctccca~aggccagga~ggaag~ga~tc
. 474
,agactctatcag~gatgagaggatagaacctacagt~tggggatgggctccaggctgcactctggccagggagggggtgtctcagaagccc
-
374
aaggaagaggggtctcgggcctcaggtctcccagtcttttactgggctgatcagtcagggccgtcagacctagggctaggtgaatgccccatcctgcaca
-
274
ccctccttccctttcccacaaagtctgcagtttgcagaaactaaaccctgagttctgtggtttcctgtgggtctgggggtcctgcctgacttggcaatgg
-
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AP- 1 gggactgtgggcagggcataagggagggggtagtgtaaacacacacacacacacacacacacacacacacacacacacacacacacacacg~
-
74
SI cataagcctggagggga&ggtcactgattccgt
7
tttactgcctct~atctctgcaaaggcagcgTTAGCCAGMGCTGCG~TC~TCACCA
+ 27
TGAGTCCCTGGCAGCCCCTGCTCCTGGCTCTCCTGGCTTTCGGCTGCAGCTCTGCTGCCCCTTACCAGCGCCAGCCGACTTTTGTGGJCTTCCCC~GA
+ 127
CCTGAAAACCTCCMCCTCACGGACACCCAGCTGGCAGAG
Fig. 5. Nucleotide sequence of the 5’ end flanking region of the murine 110-kDa-gelatinasegene. The nucleotide sequence of 1.2 kb of the promoter/enhancer region is shown (lower-case letters) together with the sequence of the first exon of the mouse 110-kDa gelatinase gene (capital letters). Position 1 is indicated by an arrowhead and corresponds to the transcription-initiation site as determined by primerextension analysis. The CA-repeat sequence is underlined, a TATA-like motif is double-underlined and several possible transcription-factorbinding sites are boxed; three possible Sp-1 binding motifs, two AP-1 boxes, an AP-2 sequence, a PEA3-motif and an NF-KB motif.
compared to the human protein (Fig. 4). Exon 9 encodes a couple and one series of 16 extra amino acids and exon 13 generates a stretch of seven extra amino acids (Fig. 4). At the carboxy terminus, however, the mouse gelatinase misses two amino acids compared to the human counterpart. The mouse gelatinase, as expected, contains both the cysteineswitch region (PRCGVPDVG) and the putative zinc-binding region (VAAHEFGHALG) (Fig. l), which are conserved throughout the whole MMP family.
Analysis of the 5’ end regulatory region More than 1 kb of the upstream regulatory region of the mouse gelatinase gene were sequenced and are presented in Fig. 5. Analysis of these 1200 bp of the 5’ flanking region
revealed the presence of several consensus sequences for regulatory elements. In close analogy with the promoter region of the human 92-kDa gelatinase gene (Huhtala et al., 1991; Sato and Seiki, 1993), there is a TATA-like motif (TTAAA; positions -23 to - 19), two 12-0-tetradecanoyl-phorbol 13acetate-response elements (TGAGTCA; positions - 82 to -76 and -480 to -474), a PEA3 site (AGGAAGC; position -487 to -481) and one possible K B motif (GGAATTCCCC; positions -536 to -527). The promoter region also contains one possible AP-2 recognition sequence (CCCCAGGC; position -598 to -591) and three possible GC boxes (positions -56 to -51; -459 to -454 and -603 to -598) instead of one present in the human gene. A putative transforming-growth-factor-B-inhibitoryelement found in the human gelatinase-promoter region is absent in the
137 and testis. A weak signal was also present in mFWA from skeletal muscle, but no TIMP-1 mRNA was detectable in tissue from brain, liver and kidney (Fig. 7B).
Regulation of gelatinase B in WEHI-3 macrophages
Fig. 6. Non-isotopic determination of the start site for transcription of the mouse gelatinase B gene. The upper half of the figure shows a part of the sequencing reaction performed on a genomic gelatinase B subclone with FITC-labelled primer MMGPEX. The lower part shows the primer-extension reaction performed on poly(A)-rich RNA with the same primer. Both samples were run on the same gel in adjacent lanes. The transcription-start site is indicated by an arrowhead.
mouse gene. As found in the human homologue, the 5’ flanking region contains a microsatellite segment (50 nucleotides) of alternating C and A residues at positions -136 to -85.
Determination of the transcription-initiation site by fluorescent primer-extension analysis Since no full-size cDNA clones for the mouse gelatinase B could be isolated, the start site for transcription of the mRNA remained elusive. After optimization of the technology, primer-extension reactions on purified poly(A)-rich RNA from LPS-treated WEHI-3 cells using a specific FITClabelled primer and analysis of the reaction products on an automated DNA sequencer revealed the presence of several extension products (Fig. 6). Upstream of the ATG translation-initiation codon, three peaks were reproducibly visible at positions -13, -23 and -25 (positions relative to the A of the ATG translation-start codon). Upstream of the -25 peak, no significant signals were present. Therefore, the 5‘-end peak at -25 was determined as the transcription-initiation site. Compared to the human gelatinase B, the mouse 5’-end untranslated sequence is 6 nucleotides longer (Table 1).
Tissue-specific expression of mRNA for gelatinase B and its natural inhibitor, TIMP-1, in the mouse The presence of gelatinase B and TIMP-1 mRNAs in different murine tissues was determined by Northern blot analysis. Two different mRNA transcripts of approximately 2.5 kb and 3.2 kb were detected with the mouse gelatinase cDNA probe in purified tissue-derived poly(A)-rich RNA from mouse brain (weak signal), spleen and lungs (Fig. 7A). No gelatinase mRNA was detectable in tissue from heart, liver, skeletal muscle, kidney and testis. The size of the smallest transcript (2.5 kb) corresponds to the DNA-deduced size in WEHI-3 mouse cells (Fig. 1). Murine TIMP-1 mRNA was detected with a cDNA probe obtained by RTPCR and appeared as a 0.8-kb band in tissue from heart, spleen, lung
To study the effect of various regulatory substances on the production of the mouse gelatinase B enzyme, WEHI-3 cells were treated with various inducers for 6 h or 16 h and the production of gelatinase B was detected by zymography (Fig. 8). The enzyme activity in cell culture supernatants is elevated after 6 h of treatment with LPS, PMA or con A. After 16 h, con-A-stimulated gelatinase B levelled off to control values, whereas the induction by PMA, LPS, PIC and IL-1 reproducibly remained above control levels. In all tested induction protocols, LPS seemed the most potent inducer of gelatinase B in mouse WEHI-3 cells. The intensity of the lower-molecular-mass band present on the zymographs (representing gelatinase A activity) did not change significantly upon induction and was used as internal control for sample processing.
Immunoprecipitation of gelatinases To probe for structural similarities between human gelatinases and their murine counterparts, immunoprecipitation experiments were performed with polyclonal antibodies. Gelatinases, present in the supernatants of LPS-treated WEHI-3 cell cultures (mouse macrophage cells) and of PMA-treated THP-1 cell cultures (human myelomonocytic leukemia cells), were precipitated with polyclonal rabbit antisera directed against human tumor-cell-derived gelatinases or neutrophil gelatinases (Fig. 9). Whereas both antisera precipitate the 96-kDa human THP-1 gelatinase, only the anti-(tumorcell gelatinase) serum precipitated detectable quantities of the murine macrophage-derived counterpart. As shown in Fig. 9 this cross-reactivity, however, is limited compared to immunoprecipitation of human gelatinase activities. Glycosylation of murine 110-kDa gelatinase There are three potential N-glycosylation sites in the deduced amino acid sequence for the mouse gelatinase (Fig. 1): NLT (positions 39-41), NIT (positions 120-122) and NYS (positions 127- 129). All three sites are conserved in the human gelatinase sequence (Wilhelm et al., 1989). To define post-translational modifications and differences between the calculated and observed molecular masses of mouse gelatinase B, enzymic deglycosylations were performed. Fig. 10 shows the results of treating crude supernatants containing mouse gelatinase B with 0-glycosidase or N-glycosidase or both. N-glycosidase caused a shift in apparent molecular mass of the gelatinase on zymography of approximately 10 kDa (from 110kDa to 100 kDa). 0-glycosidase treatment alone had no significant effect on the apparent molecular mass of the enzyme. However, purification of 110-kDa gelatinase B from WEHI-3 cells by affinity chromatography on gelatin-Sepharose and treatment of the purified enzyme with sialidase and 0-glycosidase caused a shift of 10-15 kDa in apparent molecular mass (unpublished results).
DISCUSSION We describe the cDNA and genomic cloning of the murine homologue of the human gelatinase B (92-kDa gela-
138
Fig. 7. Tissue-specific expression of gelatinase B and TIMP-1 mRNA. Hybridisation of a Northern blot containing 2 pg of poly(A)-rich RNA from different murine tissues (H, heart; B, brain; S, spleen; L, lung; LI, liver; M, skeletal muscle; K, kidney; T, testis) with the mouse gelatinase cDNA probe (A) and with the mouse TIMP-1 cDNA probe (B). The 2.5-kb and 3.2-kb gelatinase B mRNA bands and the 0.8-kb TIMP-1 mRNA band are indicated by arrowheads. The integrity of the used mRNA was confirmed by hybridization with a pactin cDNA probe (data not shown).
Fig. 9. Immunoprecipitation of human and mouse gelatinases. Gelatin zymography of immunoprecipitated murine 110-kDa gelaFig. 8. Regulation of gelatinase B protein production in WEHI3 cells. Zymographic analysis of 50 p1 of serum-free samples from WEHI-3 cell cultures treated with various inducers for 6 h or 16 h. Inducers used were: interleukin-lP (IL-1; 10 U/ml), 4-P-phorbol 12myristate 13-acetate (PMA; 100 ng/ml), bacterial lipopolysaccharide (LPS; 10 pg/ml), double-stranded RNA (PIC; 100 pg/ml) and concanavalin A (CON; 10 pglml). The lane marked CO contains 50 p1 supernatant from untreated control cells. Molecular-mass standards (MM) are indicated at the left-hand side (in m a ) . The upper and lower molecular-mass bands represent the activity of the mouse gelatinases B and A, respectively.
tinase) enzyme. This mouse gelatinase B migrated at a relative molecular mass of approximately 110 kDa as measured by zymography. Using different approaches, only partial cDNA clones were obtained, all starting at exactly the same location 394 bp downstream of the AUG translation-initiation codon of the gelatinase mRNA. This suggests the pres-
tinase from WEHI-3 cells and human 92-kDa gelatinase from THP1 cells by polyclonal rabbit antisera directed against human gelatinases. 20 p1 conditioned medium from LPS-treated WEHI-3 cell cultures or 5 pl conditioned medium from PMA-treated THP-1 cell cultures were reacted with rabbit preimmune (P) or polyclonal gelatinase antiserum raised against human neutrophil gelatinases (N) or against human tumor-cell-derived gelatinases (T) as described in Experimental procedures. In the control lanes (C), 5 p1 WEHI-3 conditioned medium or 1 pl THP-1 conditioned medium were run without prior immunoprecipitation. The 110-kDa (WEHI-3) and 96-kDa (THP-1) gelatinase bands are indicated by arrowheads.
ence of a strong reverse-transcription stop in the WEHI-3 mRNA for the mouse gelatinase. Such a stop could be caused by the presence of extended direct or inverted repeats in the sequence leading to the formation of strong secondary structures. Similar problems have been reported during the cDNA cloning of two other MMP, the human 72-kDa type IV collagenase (gelatinase A; Collier et al., 1988; Huhtala et al.,
139
Fig. 10. Glycosylation of mouse 110-kDa gelatinase. Zymographic analysis of 2 0 4 samples of gelatinase-containing cell culture supernatants from WEHI-3 cells treated (+) or not (-) with O-glycosidase (0),N-glycosidase (N) or N-glycosidase and 0-glycosidase (O+N) for 6 h at 30°C. The arrowhead at the right-hand side of the figure marks the 110-kDa mouse gelatinase band. The left lane (MM) contains molecular-mass standards (in ma).
1990b) and the rat 72-kDa type IV collagenase (Marti et al., 1993). These authors also obtained partial cDNA clones, all starting at the same 5’ location. Several hundred 5’-end base pairs representing 5‘ untranslated mRNA, the AUG translation-initiation codon and some DNA coding for part of the signal peptide could not be obtained as cDNA. These authors also suggested the presence of mRNA secondary structures as the cause of this transcription stop. After isolation and sequencing of a genomic clone for the mouse gelatinase B, computational analysis did not reveal the presence of any direct or inverted repeats in the 5’ sequences which could be the cause of strong secondary structures in the mRNA. After completion of this work, cloning of a full-size cDNA from a murine squamous carcinoma cell line (SN-H) which overexpresses gelatinase B was reported (Tanaka et al., 1993). Since these authors did not mention the presence of a reverse-transcription stop, some gelatinase B mRNA molecules seem to escape from such a stop in particular cell lines. We therefore suggest, alternatively, that binding of a protein or other molecule to the mRNA at the site of the stop might result in premature termination of first-strand synthesis in particular cells. The genes for the mouse and human gelatinase B are well conserved since similarities at the DNA and at the protein level are considerable. The exotdintron structure is also completely conserved, with only differences in the length of the introns. Comparison of deduced amino acid sequences for human and mouse gelatinases shows that the murine enzyme is longer, having three stretches of extra amino acids. Two stretches of 2 and 16 extra amino acids respectively are coded for by exon 9 and are located in a region of the enzyme that is unique to this type of gelatinase (Huhtala et al., 1991) and that has a high degree of similarity to the collagen a,(V) chain (the so-called type-V-collagen-like domain of the human 92-kDa type IV collagenase). This protein domain probably plays a role in the interaction of the enzyme with type V collagen, which is one of the possible substrates for gelatinase B (Hibbs et al., 1987). Exon 13 of the mouse gela-
tinase, coding for the last part of the hemopexin-like domain, also encodes seven additional amino acids, but is two codons shorter at its 3’ terminus. In the human gelatinase B, it has been shown that the carboxy-terminal end of the enzyme is indispensable for the formation of the specific complex between pro-gelatinase B and TIMP-1. Removal of eight carboxy-terminal amino acid residues or replacement of the cysteine residue at one of the last positions by another amino acid abolishes formation of this complex (Goldberg et al., 1992; Strongin et al., 1993). It is possible that the absence of two amino acid residues in the murine enzyme has an effect on enzyme-inhibitor interactions. Thus, the presence or absence of certain amino acid stretches in the mouse enzyme could eventually lead to subtle differences between mouse and human gelatinase B in for example enzymehbstrate or enzymehnhibitor interactions. The mouse gelatinase sequence derived from the genomic exons differed from the cDNA sequence at four locations. One point mutation was silent, but the other three led to a change in predicted protein primary structure. These changes were not conservative since in all three cases, a proline was involved. This could possibly lead to substantial changes in protein tertiary structure. The reasons and importance of these polymorphisms are not yet known. One of these point mutations (compared to the genomic DNA sequence) was also present in the gelatinase cDNA sequence from metastatic mouse SN-H cells (Tanaka et al., 1993). The initiation site for transcription of the mouse gelatinase B mRNA was determined by primer-extension analysis. Several extension products were visible on the gel, the most 5‘ situated peak corresponding to a T at position -25 relative to the ATG translation-inititiation codon. This T was thus taken as the start site for transcription of the mouse gelatinase B and adopted for the nucleotide numberings. The extension products seen near the ATG translation-start codon (Fig. 6) or downstream of this codon (data not shown) probably represent premature stop sites of the reverse transcriptase due to, for example, secondary structures or methylation (Calzone et al., 1987). The mRNA for the human gelatinase B had a similar relatively short 5’ end untranslated sequence, starting at position -19. A mouse gelatinase B cDNA sequence derived from a mouse tumor cell cDNA library published after completion of our work (Tanaka et al., 1993) reports a 5’ untranslated sequence of 27 bp. However, the first three nucleotides in this sequence do not match our genomic DNA-derived sequence (CTA instead of GTT in our sequence). The reason for this difference might be a polymorphism or a cDNA cloning artifact. The promoter region of the mouse 110-kDa gelatinase gene resembles the human counterpart. The same regulatory elements (with the exception of the TIE-sequence) are found at roughly the same locations in the 5’ flanking region, suggesting that the mouse enzyme is probably regulated in an analogous way. The AP-1-binding site closest to the transcription start has been shown to be essential for induction of the gene by PMA or tumor-necrosis-factor-a in human tumor cells and cooperates synergistically with the elements for the Sp-1-like and NF-lcB-like factors (Sato and Seiki, 1993). Since all three nuclear-factor-binding sites are conserved in the murine gelatinase gene, PMA inducibility is expected to occur via the same mechanism. The functional role of other regulatory elements present in the mouse and human enzymes remains to be determined although we established inducibility in WEHI-3 cells at the protein level with LPS, PMA, IL-1 and PIC. Northern blot analysis of gela-
140 Devarajan, P., Johnston, J. J., Ginsberg, S. S., Van Wart, H. E. & Berliner, N. (1992) Structure and expression of neutrophil gelatinase cDNA. Identity with type IV collagenase from HT1080 cells, J. Biol. Chem. 267, 25228-25232. Docherty, A. J. P. & Murphy, G. (1990) The tissue metalloproteinase family and the inhibitor TIMP: a study using cDNAs and recombinant proteins, Ann. Rheum. Dis. 49, 469-479. Edwards, D. R., Waterhouse, P., Holman, M. L. & Denhardt, D. T. (1986) A growth-responsive gene (16C8) in normal mouse fibroblasts homologous to a human collagenase inhibitor with erythroid-potentiating activity : evidence for inducible and constitutive transcripts, Nucleic Acids Res. 14, 8863-8878. Frohman, M. A., Dush, M. K. & Martin, G. R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer, Proc. Natl Acad. Sci. USA 85, 8998-9002. Gijbels, K., Masure, S., Carton, H. & Opdenakker, G. (1992) Gelatinase in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological disorders, J. Neuroimmunol. 41, 29-34. Goldberg, G. I., Marmer, B. L., Grant, G. A., Eisen, A. Z., Wilhelm, S. & He, C. (1989) Human 72-kilodalton type IV collagenase forms a complex with a tissue inhibitor of metalloproteases designated TIMP-2, Proc. Natl Acad. Sci. USA 86, 8207-8211. Goldberg, G. I., Strongin, A., Collier, I. E., Genrich, T. & Marmer, B. L. (1992) Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin, J. Biol. Chem. 267, 45834591. Hibbs, M. S., Hoidal, J. R. & Kang, A. H. (1987) Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages, J. Clin. Invest. 80, 1644-1650. Hipps, D. S., Hembry, R. M., Docherty, A. J. P., Reynolds, J. J. & Murphy, G. (1991) Purification and characterisation of human 72-kDa gelatinase (type IV collagenase). Use of immunolocalisation to demonstrate the non-coordinate regulation of the 72kDa and 95-kDa gelatinases by human fibroblasts, Biol. Chem. Hoppe-Seyler 372, 287-296. Houde, M., De Bruyne, G., Bracke, M., Ingelman-Sundberg, M., Skoglund, G., Masure, S., Van Damme, J. & Opdenakker, G. (1993) Differential regulation of gelatinase B and tissue-type plasminogen activator expression in human Bowes melanoma This study was possible thanks to the generous gift of the human cells, Int. J. Cancer 53, 395-400. gelatinase cDNA probe by Dr G. I. Goldberg and Dr B. Marmer (Washington University School of Medicine) and to the financial Huhtala, P., Chow, L. T. & Tryggvason, K. (1990a) Structure of the human type IV collagenase gene, J. Biol. Chem. 265, 11077support of the Cancer Research Fund (CRF) of the Algemene Spaar11082. en Lijfrentekas (A.S.L.K.), Belgium, the National Lottery and the National Fund for Scientific Research (N. F. W. O., Levenslijn Huhtala, P., Eddy, R. L., Fan, Y. S., Byers, M. G., Shows, T. B. & Tryggvason, K. (1990b) Completion of the primary structure of Multiple Sclerose). The authors acknowledge the excellent technical assistance of Erik Beuken, Ann Janssen, Jean-Pierre Lenaerts and the human type IV collagenase preproenzyme and assignment of the gene (CLG4) to the q21 region of chromosome 16, Genomics Paul Proost, as well as helpful discussions with Prof. Dr A. Billiau, Dr G. Froyen and Dr K. Vandenbroeck. G. 0. is a senior research 6,554-559. associate of the N. F. W. 0. Huhtala, P., Tuuttila, A,, Chow, L. T., Lohi, J., Keski-Oja, J. & Tryggvason, K. (1991) Complete structure of the human gene for 92-kDa type IV collagenase. 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tinase mRNA transcripts present in different mouse tissues showed that two different murine gelatinase mRNAs can be transcribed. This is in accordance with the situation in mouse SN-H tumor cells (Tanaka et al., 1993) but contrasts to the human situation, where only one 92-kDa gelatinase mRNA species (of 2.8 kb) is found (Wilhelm et al., 1989). The presence of two different mRNA transcripts for murine gelatinase probably results from the use of different polyadenylation signals (Tanaka et al., 1993). The mouse 110-kDa gelatinase is N-glycosylated and 0glycosylated. N-glycosidase treatment of WEHI-3 cell culture supernatants reduces the apparent molecular mass of the gelatinase by approximately 10 kDa. As in the human counterpart, there are three potential N-glycosylation sites. O-glycosidase treatment alone did not result in a further reduction of the molecular mass of the enzyme, but the combination of sialidase and 0-glycosidase caused a shift of approximately 10 kDa in molecular mass. This shows that mouse gelatinase B from WEHI-3 cells contains N-linked and 0-linked sialylated sugars. These sugars represent about 20 kDa of the intact 110-kDa molecule. It is important to notice that the relative mobilities in substrate zymography gels might vary depending on the substrate used and the protein analyzed. This might explain the differences observed in gelatinase B mobilities when comparing our zymographs with the results of other authors (Tanaka et al., 1993; Yamagata et al., 1989). The glycoform spectra of proteins after expression in various cell types can also vary considerably (Parekh et al., 1989) and might contribute to this effect. Gelatinases have been shown to be important proteases in the tissue remodelling in many physiological and pathological processes. Knowledge of the structure and regulation of these enzymes and their specific inhibitors in the mouse and availability of nucleic acid probes might help in studying the function and importance of gelatinases in animal model systems for human pathologies. This could ultimately lead to beneficial treatments for cancer, rheumatoid arthritis, multiple sclerosis and other debilitating human diseases.
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