©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium Ionophores Decrease Pericellular Gelatinolytic Activity via Inhibition of 92-kDa Gelatinase Expression and Decrease of 72-kDa Gelatinase Activation (*)

(Received for publication, March 20, 1995; and in revised form, May 1, 1995)

Jouko Lohi (1)(§), Jorma Keski-Oja (1) (2)(¶)

From the  (1)Departments of Virology and of (2)Dermatology and Venereology, University of Helsinki, FIN-00290 Helsinki, Finland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To understand the roles of intracellular calcium levels on gelatinase/type IV collagenase expression, we analyzed the effects of calcium ionophores on the expression of 92- and 72-kDa gelatinases (MMP-9 and MMP-2) in human fibrosarcoma cells (HT-1080). Calcium ionophores ionomycin and A23187 reduced the levels of pericellular gelatinolytic activity in both untreated and phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor- (TNF)-stimulated cells as determined by degradation of radiolabeled gelatin. Gelatin zymography and immunoblotting revealed a dose-dependent decrease in the levels of secreted 92-kDa gelatinase, which was paralleled by a decrease of its mRNA. Treatment of cells with thapsigargin caused similar decreases of 92-kDa gelatinase mRNA and protein. The decrease of 92-kDa gelatinase expression was due to lower transcription rate as determined by transfection assays with 92-kDa gelatinase/luciferase construct. The expression of 72-kDa gelatinase was only slightly decreased by ionophores. Treatment of HT-1080 cells with PMA, TNF, or concanavalin A resulted in the conversion of 72-kDa gelatinase proenzyme to its presumed 64- and 62-kDa active forms as determined by gelatin zymography and immunoblotting. Simultaneous treatment with the ionophores or thapsigargin resulted in inhibition of PMA-induced gelatinase activation. The expression of membrane-type matrix metalloproteinase, a potential activator of 72-kDa gelatinase, was not affected by ionophores. The results indicate that calcium ionophores decrease gelatinolysis by repressing both the expression of 92-kDa gelatinase and the activation of the 72-kDa gelatinase.


INTRODUCTION

The family of matrix metalloproteinases (MMPs)()comprises at least nine enzymes that collectively are capable of degrading most if not all components of the extracellular matrix. Different MMPs have varying substrate specifities and divergent regulation of gene expression and proenzyme activation(1, 2, 3) . The expression of the interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), matrilysin (MMP-7), and 92-kDa gelatinase (MMP-9) genes is often coregulated and is enhanced by epidermal growth factor, inflammatory cytokines like TNF and interleukin-1, and the phorbol ester, phorbol 12-myristate 13-acetate (PMA)(1, 4, 5, 6, 7, 8, 9) . The common mediator of these inductions is supposed to be the 12-O-tetradecanoylphorbol-13-acetate-responsive element, which is present in the promoter regions of all above-mentioned MMPs and which binds the AP-1 transcription factor complex. In addition, the NF-B and Sp-1 binding sites in the 92-kDa gelatinase promoter are essential for PMA- or TNF-mediated induction(10) . Glucocorticoids, retinoic acid, and transforming growth factor- are known as repressive factors for these MMPs(11, 12, 13, 14) . The 92-kDa gelatinase is secreted by normal alveolar macrophages, polymorphonuclear leukocytes, osteoclasts and keratinocytes, and invading trophoblasts and by several transformed cell lines, but not by fibroblastic cells(8, 15, 16, 17, 18, 19, 20) . The 72-kDa gelatinase is constitutively expressed in most fibroblastic cells and by some transformed epithelial cells, and its expression is enhanced by transforming growth factor- but not by PMA(14, 21, 22) . The promoter of the 72-kDa gelatinase (MMP-2) gene contains an AP-2 binding site and two Sp-1 sites but no 12-O-tetradecanoylphorbol-13-acetate-responsive element (23) .

A major step in the regulation of pericellular matrix metalloproteinase activity is the activation of latent proenzyme by cleavage of a 10-kDa amino-terminal fragment. Interstitial collagenase, stromelysin, and 92-kDa gelatinase are activated by soluble enzymes like plasmin, stromelysin, and chymase(3, 24, 25, 26, 27, 28) . However, activation of 72-kDa gelatinase by these enzymes is inefficient and seems to depend on the action of a cell membrane-bound activator (see (3) and (29) -32). The 72-kDa gelatinase processing activity is induced by phorbol esters and concanavalin A and inhibited by retinoic acid(33, 34) . The 72-kDa gelatinase activator is found in purified plasma membrane fractions of various tumor cells, and its action is inhibited by inhibitors of metalloproteinases like 1,10-phenanthroline, EDTA, and tissue inhibitor of metalloproteinases-2 (TIMP-2), suggesting that the activator would be a metalloproteinase(29, 30) . Recently, Sato et al.(31) cloned a cDNA with homology to other matrix metalloproteinases and a transmembrane domain at the COOH terminus and named it membrane-type matrix metalloproteinase (MT-MMP). Overexpression of the MT-MMP in COS-1 and Sf-9 cells induces proteolytic processing of exogenous 72-kDa gelatinase to the active 64- and 62-kDa forms, resembling those seen in PMA- or concanavalin A-treated HT-1080 cells. In fact, MT-MMP seems to be a functional cell surface receptor for 72-kDa gelatinase, and TIMP-2 is needed for this interaction(35) .

Numerous signal transduction pathways utilize ionized calcium as a second messenger. Signal transduction through tyrosine kinase and G-protein-linked receptors converges to the formation of diacylglycerol and inositol trisphosphate by the activation of phospholipase C isoforms bound to the plasma membrane. Binding of inositol trisphosphate to its receptor in the endoplasmic reticulum and other intracellular Ca storage compartments causes then release of ionized calcium to cytoplasm and a transient increase in the intracellular calcium level. Changes in intracellular calcium concentration control numerous cellular processes, including cell growth, differentiation, and transformation(36, 37, 38, 39) . Interstitial collagenase and stromelysin-1 genes are induced by an increase in the intracellular calcium level, and treatment of rabbit synovial fibroblasts with calcium ionophores results in endogenous activation of interstitial collagenase(40, 41) . Treatment of HT-1080 fibrosarcoma cells with carboxy amido-triazole, an inhibitor of receptor-mediated calcium influx, reduces the levels of both latent and activated forms of 72-kDa gelatinase, although an initial increase in the level of 62-kDa activated form is seen(42) . In the present study we have analyzed the effects of calcium ionophores and thapsigargin, an inhibitor of endoplasmic reticulum Ca-ATPase, on the expression and proteolytic processing of the 92-kDa (MMP-9) and 72-kDa gelatinases (MMP-2) in human fibrosarcoma cells (HT-1080). We found that calcium ionophores reduced the basal and PMA-inducible pericellular gelatinolytic activities by inhibiting 92-kDa gelatinase expression and 72-kDa gelatinase activation. The present work describes a novel role for intracellular calcium signaling as a regulator of pericellular gelatinolytic activity.


MATERIALS AND METHODS

Reagents

PMA, calcium ionophore A23187, ionomycin, cycloheximide, actinomycin D, concanavalin A, and thapsigargin were purchased from Sigma. Human recombinant TNF was obtained from Boehringer Mannheim GmbH (Mannheim, Germany).

Cell Cultures

Human fibrosarcoma HT-1080 cells (CCL-121, American Type Culture Collection, Rockville, MD) were cultivated in Eagle's minimal essential medium (MEM) containing 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 100 IU/ml penicillin, and 50 µg/ml streptomycin. The cultures were incubated at 37 °C in a humidified 5% CO atmosphere until confluence. Before experiments, cultures were washed twice with serum-free medium and then incubated under serum-free conditions for 6 h. All experiments were carried out under serum-free conditions. To assay the toxicity of treatments, general protein synthesis was measured by metabolic labeling followed by trichloroacetic acid precipitation. Confluent cultures of HT-1080 cells were incubated under serum-free conditions for 6 h followed by incubation with PMA (4 nM) and ionomycin (50-500 nM) for 12 h. L-[4,5-H]Leucine (2 µCi/ml) (Amersham, Buckinghamshire, United Kingdom) was then added and incubation was continued for 12 h. Conditioned medium was then collected and clarified by centrifugation, and secreted proteins were precipitated by adding bovine serum albumin (3 mg/ml final) and trichloroacetic acid (10% final concentration) followed by incubation on ice for 30 min and centrifugation. Protein pellet was solubilized in 0.3 N NaOH, and radioactivity was measured by liquid scintillation counter.

Assays for Gelatinolytic Activity

To assay the pericellular gelatinolytic activity of cultured cells, tissue culture wells were coated with radiolabeled gelatin. Briefly, 1 µl (60 nCi) of N-[propionate-2,3-H]propionylated rat type I collagen (0.19 mCi/mg; DuPont NEN) was diluted with 60 µl of 1 mg/ml unlabeled type I gelatin and heated at 80 °C for 30 min. Gelatin was then diluted with MEM, applied onto culture dishes, and allowed to attach for 2 h. Unbound radioactivity was removed by extensive washing with MEM, and HT-1080 cells were seeded on the dish. One day later the cultures were washed with serum-free MEM for 6 h, after which fresh serum-free MEM was changed, supplemented with PMA (4 nM), ionomycin (500 nM), TNF (10 ng/ml), and concanavalin A (50 µg/ml), and incubation was continued for 30 h. Subsequently, 500-µl aliquots of the conditioned medium were analyzed by liquid scintillation counter to measure the release of H-labeled peptides.

To analyze the approximate molecular weights of the gelatinolytic proteins, conditioned medium from HT-1080 cells was assayed by gelatin zymography essentially as described(16, 43) . Samples (15 µl) of conditioned medium were dissolved in nonreducing Laemmli sample buffer and separated by electrophoresis using discontinuous 3.5:7% polyacrylamide gels containing 1 mg/ml gelatin(44) . After electrophoresis the gels were washed twice with 50 mM Tris-HCl buffer, pH 7.6, containing 5 mM CaCl, 1 µM ZnCl, 2.5% Triton X-100 (v/v) for 15 min to remove SDS, followed by a brief rinsing in washing buffer without Triton X-100. The gels were then incubated at 37 °C for 1-2 days in 50 mM Tris-HCl buffer containing 5 mM CaCl, 1 µM ZnCl, 1% Triton X-100, 0.02% NaN, pH 7.6. The digestion was terminated by 10% acetic acid followed by staining with Coomassie Brilliant Blue R-250 and destaining with 10% acetic acid, 10% methanol. Zones of enzymatic activity were seen as negatively stained bands.

Immunoblotting Assay for 72- and 92-kDa Gelatinases

For the immunoblotting analysis, confluent cultures of HT-1080 cells were treated with PMA (4 nM) and ionomycin (500 nM) under serum free conditions. After 24-h incubation the conditioned medium was harvested, clarified by centrifugation, and an aliquot (15 µl) was dissolved in Laemmli sample buffer and subjected to 4-15% gradient SDS-PAGE under nonreducing conditions. Proteins were electrophoretically transferred to nitrocellulose (Schleicher & Schuell, Dassel, Germany) using a semi-dry blotting apparatus at 0.8 mA/cm for 1 h. Membranes were saturated with 5% milk in TBST (10 mM Tris-HCl buffer, pH 8.0, containing 150 mM NaCl, and 0.05% Tween 20) and incubated with monoclonal antibodies against human 72- and 92-kDa gelatinases (clones 42-5D11 and 7-11C, respectively, Oncogene Science, Inc., Cambridge, MA) (1 and 2 µg/ml in TBST, respectively). After several washes with the same buffer, the bound antibodies were detected using biotinylated anti-mouse IgG antibodies and peroxidase-conjugated streptavidin (Dakopatts, Copenhagen, Denmark) and enhanced chemiluminescence Western blotting detection system (Amersham International PIC, Amersham, UK) as described(45) .

Isolation of RNA and Northern Hybridization Analysis

Confluent cultures were washed twice with serum-free medium and then incubated under serum-free conditions for 6 h. Subsequently, PMA (4 nM), TNF (10 ng/ml), concanavalin A (50 µg/ml), calcium ionophores A23187 (10-2000 nM) or ionomycin (10-2000 nM), or thapsigargin (10-2000 nM) were added into the medium as indicated, and incubation was continued for 24 h or as indicated. The cultures were then washed with phosphate-buffered saline (170 mM NaCl, 10 mM sodium phosphate buffer, pH 7.4) and lysed for RNA extraction. RNA was purified by pelleting a guanidinium thiocyanate-N-laurylsarcosyl cell lysate through a CsCl cushion(46) . Total RNA (15 µg) was electrophoresed on a 0.8% formaldehyde-agarose gel and transferred to a Zeta-Probe filter (Bio-Rad) in 20 SSC (1 SSC is 150 mM NaCl, 15 mM sodium citrate buffer, pH 7.0) and immobilized by UV cross-linking. The filters were hybridized to probes labeled with [-P]dCTP (Amersham) by the random priming method (Pharmacia LKB, Uppsala, Sweden). Hybridization was performed in 50% deionized formamide, 7% SDS, 250 mM NaCl, 250 mM NaHPO, 1 mM EDTA at 42 °C for 24 h. Subsequent washing was carried out in 0.2 SSC containing 0.1% SDS at 63 °C. Stripping, when needed, was performed in 0.1 SSC, 0.5% SDS at 100 °C for 10 min. The following human cDNA probes were used: collagenase(47) , 72-kDa gelatinase(21) , and 92-kDa gelatinase(14) . Rat glyceraldehyde-3-phosphate dehydrogenase (48) cDNA was used to standardize the loading of RNAs. For detection of human MT-MMP mRNA, a 699-base pair cDNA probe corresponding to bases 884-1582 of the published MT-MMP sequence (31) was generated by reverse transcription-polymerase chain reaction from HT-1080 cDNA.()

Transient and Stable Transfections of HT-1080 Cells with 92-kDa Gelatinase Promoter-Luciferase Construct

To study the transcription of the 92-kDa gelatinase gene by transient transfection, we constructed a reporter plasmid containing a 2.2-kb fragment of the 5`-flanking region of 92-kDa gelatinase gene (-2161 to +20) linked to luciferase gene in pGL2-Basic vector (Promega Corp., Madison, WI). Promoter activity was then monitored by determining the luciferase activity of transiently transfected HT-1080 cells using a Luciferase Assay System kit according to manufacturer's instructions (Promega). Transfection efficiency was controlled by cotransfecting the promoter/luciferase construct with pCMV control plasmid containing cytomegalovirus promoter linked to the -galactosidase gene (Clontech Laboratories Inc., Palo Alto, CA) and correcting the luciferase activities for -galactosidase activity, which was measured as described(49) .

Transfection was performed using lipofectamine according to manufacturer's instructions (Life Technologies, Inc.). Subconfluent 60-mm cultures of HT-1080 cells were washed twice with serum-free MEM. For each transfection, 6 µg of DNA and 18 µl of lipofectamine were mixed and incubated for 30 min at room temperature. DNA/liposome mixture was then transferred onto cells and incubated for 6 h. Subsequently, the cells were washed with MEM containing 0.2% bovine serum albumin for 30 min, re-washed with MEM, and incubated in MEM overnight. Next day PMA (4 nM) and ionomycin (500 nM) were added, and the incubation was continued for 30 h. At the end of this incubation the cells were lysed in reporter lysis buffer (Promega) and analyzed for luciferase and -galactosidase activities.

For stable transfections, a cDNA containing a 2.1-kb fragment of the 5`-flanking region of 92-kDa gelatinase gene (-2112 to +20) linked to luciferase gene was cloned to pcDNA3 expression vector (Invitrogen Corp.) containing the neomycin resistance gene to substitute the cytomegalovirus promoter of the vector. HT-1080 cells were transfected as described above and selected for 2 weeks in MEM containing 0.7 mg/ml neomycin analogue G418. Maintenance culture of cells was performed using MEM containing 0.25 mg/ml G418. For experiments, confluent cultures of transfected cells were washed with serum-free MEM without G418 extensively for 6 h and treated with PMA or ionomycin for 12 h. Subsequently, the cells were lysed and assayed for luciferase activity as described above.


RESULTS

Calcium Ionophores Inhibit the Gelatinolytic Activity of HT-1080 Cells

Cultured human fibrosarcoma cells (HT-1080) were used as a model to study the effects of elevated intracellular calcium levels on the pericellular gelatinolytic activity. HT-1080 cells were seeded on culture plates coated with denatured H-labeled rat type I collagen (gelatin). After overnight attachment and 6-h incubation in serum-free medium, the cultures were treated with the calcium ionophore ionomycin together with PMA for 30 h, and the radioactivity released in the conditioned medium was measured by a scintillation counter (Fig. 1A). Basal level of released radioactivity (including both enzymatic and non-enzymatic release) was increased about 3-fold by 4 nM PMA. Simultaneous treatment of cells with 50 nM ionomycin led to a notable decrease in the released radioactivity, and 500 nM ionomycin completely blocked the gelatinolysis-inducing activity of PMA.


Figure 1: Calcium ionophores and thapsigargin decrease pericellular gelatinolytic activity and secretion of 92-kDa gelatinase and inhibit the activation of 72-kDa gelatinase in PMA-treated fibrosarcoma cells. A, degradation of substratum bound H-labeled gelatin. HT-1080 cells were seeded on [H]gelatin-coated tissue culture plates and incubated for 18 h (see ``Materials and Methods''). Subsequently, the cultures were washed, incubated with serum-free medium for 6 h, washed again, and incubated with PMA and ionomycin as shown on the figure for 30 h. Conditioned medium was then collected, clarified by centrifugation, and the amount of released radioactivity was measured. The values represent the mean of three independent experiments. Standard deviation is indicated by error bars. B, gelatin zymography. Confluent cultures of HT-1080 cells were treated with A23187, ionomycin, or thapsigargin and PMA under serum-free conditions for 24 h as shown on the figure (see ``Materials and Methods''). Aliquots of conditioned medium (15 µl) were analyzed by gelatin zymography using 6% SDS-PAGE. C, immunoblotting of 72- and 92-kDa gelatinases. Confluent cultures of HT-1080 cells were treated with PMA (4 nM) and ionomycin (500 nM) for 24 h. Aliquots (15 µl) of conditioned medium were analyzed by 4-15% gradient SDS-PAGE under nonreducing conditions followed by immunoblotting using monoclonal antibodies against 72- and 92-kDa gelatinases. Note the inhibition of PMA-induced 72-kDa gelatinase processing and decrease of 92-kDa gelatinase expression by ionomycin.



To study the molecular forms of gelatinolytic enzymes responsible for the increased gelatinolytic activity of PMA-treated cells, and the effect of calcium ionophores on their secretion, conditioned medium of HT-1080 cells was analyzed by gelatin zymography. Untreated cells secreted mainly 72-kDa (pro)gelatinase, whereas only very low levels of 92-kDa gelatinase were observed (Fig. 1B). Treatment of the cells with PMA resulted in enhancement of 92-kDa gelatinase secretion and partial conversion/proteolytic processing of 72-kDa gelatinase proenzyme to the presumably activated 64- and 62-kDa forms of 72-kDa gelatinase(33) . Cotreatment with ionomycin or another calcium ionophore A23187 resulted in the disappearance of 92-, 64-, and 62-kDa gelatinolytic protein bands, suggesting that calcium ionophores inhibit both the PMA-induced secretion of 92-kDa gelatinase and activation of 72-kDa gelatinase. The changes in the gelatinolytic activities were paralleled by a decrease in the level of 92-kDa gelatinase and by inhibition of the PMA-induced processing of 72-kDa gelatinase secreted into the conditioned medium as determined by immunoblotting (Fig. 1C). Treatment of cells with ionomycin alone caused also a moderate decrease in the level of secreted 72-kDa gelatinase. Thapsigargin, an inhibitor of endomembrane Ca-ATPase, is another chemical capable of increasing intracellular calcium levels. Accordingly, treatment of HT-1080 cells with thapsigargin and PMA caused a dose-dependent decrease of 92-kDa gelatinase expression and inhibition of proteolytic processing/activation of 72-kDa gelatinase (Fig. 1B). Metabolic labeling of PMA- and ionomycin-treated cells with [H]leucine followed by trichloroacetic acid precipitation of secreted macromolecules indicated a slight decrease (20%) in general protein synthesis at maximal doses tested (data not shown), but the decrease was much lower than the decrease of gelatinolytic activity.

Processing of 72-kDa Gelatinase to Lower Molecular Weight Forms by PMA, TNF-, or Concanavalin A Stimulation Is Prevented by Ionomycin

We analyzed next whether ionomycin could also block the effects of other known inducers of 72-kDa gelatinase activation and 92-kDa gelatinase expression. HT-1080 cells were cultivated on [H]gelatin-coated dishes as described above, followed by treatment with PMA, TNF-, or concanavalin A in the presence of ionomycin for 30 h as indicated (Fig. 2A). Scintillation counting of the supernatant fluids showed 2-3-fold increases in the levels of released radioactivity in cultures treated with PMA, TNF-, or concanavalin A, indicating an increase in the pericellular gelatinolytic activity (Fig. 2A). Addition of ionomycin completely prevented this increase. To characterize the regulation of different gelatinases and their activation by the above-mentioned factors, aliquots of conditioned medium of a similar experiment were assayed by gelatin zymography. Accordingly, treatment of the cells with PMA or TNF- resulted in an increase in 92-kDa gelatinase secretion and partial conversion of 72-kDa gelatinase proenzyme to 64- and 62-kDa forms. Treatment of cells with concanavalin A caused almost complete conversion of 72-kDa gelatinase to 64- and 62-kDa forms, but did not affect the secretion of 92-kDa gelatinase. Treatment with ionomycin resulted both in the inhibition of the induction of 92-kDa gelatinase secretion and reduced activation of 72-kDa gelatinase (Fig. 2B).


Figure 2: Ionomycin (iono) inhibits the induction of pericellular gelatinolytic activity and 92-kDa gelatinase secretion and 72-kDa gelatinase activation in PMA, TNF-, and concanavalin A (ConA)-treated cells. A, degradation of substratum bound H-labeled gelatin. HT-1080 cells were seeded on [H]gelatin-coated tissue culture plates and incubated for 18 h and washed (see legend to Fig. 1). The cultures were then incubated with TNF- (10 ng/ml), concanavalin A (50 µg/ml), or PMA (4 nM) in the presence of ionomycin (500 nM) for 30 h as shown on the figure. Conditioned medium was then collected, clarified by centrifugation, and the amount of released radioactivity was measured. The values represent the mean of three independent experiments. Standard deviations are indicated by error bars. B, Gelatin zymography. Confluent cultures of HT-1080 cells were treated with TNF- (10 ng/ml), concanavalin A (50 µg/ml), or PMA (4 nM) and ionomycin (500 nM) under serum-free conditions for 24 h as shown on the figure. Aliquots of conditioned medium were analyzed by gelatin zymography using 6% SDS-PAGE.



Induction of 92-kDa Gelatinase Gene Expression Is Inhibited by Calcium Ionophores and Thapsigargin

To study the effect of elevated intracellular calcium on the expression of 92- and 72-kDa gelatinase mRNAs, HT-1080 cells were treated with PMA and increasing concentrations of ionomycin. At the termination of incubation the cells were lysed, and total RNA was extracted and analyzed by Northern hybridization. In accordance with earlier results (4, 14) PMA increased markedly the mRNA levels of collagenase and 92-kDa gelatinase, but had no effect on the expression of 72-kDa gelatinase (Fig. 3A). Ionomycin (40 nM) reduced the levels of 92-kDa gelatinase mRNA in PMA-treated cells, and at 500 nM it was barely detectable. As expected, the expression of interstitial collagenase gene was induced by ionomycin, and this induction was further potentiated by PMA. The expression of 72-kDa gelatinase mRNA, when compared with glyceraldehyde-3-phosphate dehydrogenase mRNA levels, was not affected by ionomycin. Treatment of the cells with thapsigargin caused a dose-dependent decrease of 92-kDa gelatinase expression with notable effect at 10 nM and maximal effect at 500 nM (Fig. 3B). Collagenase mRNA levels were induced by both PMA and thapsigargin, and the induction was potentiated by simultaneous treatment of cells. Similar results on the regulation of the mRNA levels for 92- and 72-kDa gelatinases and collagenase were also obtained with ionophore A23187 (data not shown).


Figure 3: Ionomycin and thapsigargin prevent the PMA- and TNF- mediated induction of 92-kDa gelatinase mRNA. Confluent cultures of HT-1080 cells were treated with ionomycin or thapsigargin under serum-free conditions for 24 h as indicated. PMA (4 nM), concanavalin A (50 µg/ml), or TNF- (10 ng/ml) were added where indicated to induce the expression of 92-kDa gelatinase. Total RNA was then isolated and analyzed by Northern hybridization using random labeled cDNA probes for 92- and 72-kDa gelatinases. Interstitial collagenase and MT-MMP were used as controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels served as an internal standard. A, dose dependence analysis of ionomycin treatment. B, dose dependence analysis of thapsigargin treatment. C, effects of ionomycin treatment on mRNA levels in TNF- and concanavalin A-treated cells.



To compare the effects of ionomycin on the mRNA levels of 92- and 72-kDa gelatinases in PMA-stimulated cells to those obtained by TNF- or concanavalin A, the cells were treated as indicated for 12 h. Northern hybridization analysis revealed that the expression of 92-kDa gelatinase was induced by both PMA and TNF-, and the induction was prevented by ionomycin (Fig. 3C). mRNA levels of 72-kDa gelatinase were only slightly decreased by ionomycin. Concanavalin A did not have any effect on the mRNA levels of either 92- or 72-kDa gelatinases.

It has recently been reported that overexpression of the MT-MMP causes proteolytic processing of the 72-kDa gelatinase to the active 62-kDa form in COS-1 and Sf-9 cells(31) . We analyzed therefore the levels of MT-MMP expression in this system. The level of MT-MMP mRNA in serum-starved HT-1080 cells was relatively high and was enhanced 2-3-fold by PMA. Ionomycin did not cause any notable effects in the levels of MT-MMP mRNA (Fig. 3A).

Induction of 92-kDa Gelatinase by PMA Cannot Be Reversed by Subsequent Ionomycin Treatment

We analyzed next the temporal profiles of the induction and inhibition of 92-kDa gelatinase expression by PMA and ionomycin. After 6-h serum starvation, HT-1080 cells were treated with PMA and/or ionomycin as indicated for 1-24 h followed by Northern hybridization analysis. Induction of 92-kDa gelatinase in PMA-stimulated cells was detectable at 5 h and maximal at 12 h, after which the level of 92-kDa gelatinase mRNA declined slightly to 24 h. Simultaneous treatment of cells with ionomycin prevented almost completely the induction, but did not affect the temporal profile (Fig. 4A).


Figure 4: Analysis of temporal relationships between PMA induction and the inhibitory effect of ionomycin. A, ionomycin treatment does not change the temporal profile of the PMA-mediated induction of 92-kDa gelatinase. Confluent cultures of HT-1080 cells were treated with PMA and ionomycin under serum-free conditions for 1-24 h as indicated on the figure. Northern hybridization analysis of total RNA indicated that the induction profiles of 92-kDa gelatinase were similar in PMA and PMA + ionomycin-treated cells, but the amplitude of induction was lower in ionomycin-treated cells. In contrast, induction of collagenase was potentiated by ionomycin. B, induction of 92-kDa gelatinase by PMA cannot be reversed by subsequent ionomycin treatment. Confluent cultures of HT-1080 cells were first treated with PMA under serum-free conditions for 14 h to induce the expression of 92-kDa gelatinase. Subsequently, ionomycin (500 nM) was added, and the incubation was continued for 3-48 h as indicated. Total RNA was then isolated and analyzed by Northern hybridization using 92-kDa gelatinase cDNA as a probe. C, induction of 92-kDa gelatinase by PMA is inhibited by ionomycin: time dependence. Confluent cultures of HT-1080 cells were treated with PMA under serum free conditions for 12 h. Ionomycin (500 nM) was added to the cell cultures at the time points indicated on the figure. At the end of incubation, total RNA was isolated and analyzed by Northern hybridization using 92-kDa gelatinase and collagenase cDNAs as probes. After about 5 h the induction of 92-kDa gelatinase by PMA could not be prevented by ionomycin. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.



To study further the temporal relationships between PMA induction and the inhibition of 92-kDa gelatinase expression by ionomycin, HT-1080 cells were first treated with PMA for 14 h to induce 92-kDa gelatinase expression to a measurable level. Subsequently, ionomycin was added, and the incubation was continued for an additional 3-48 h. Unexpectedly, the PMA-induced mRNA level of 92-kDa gelatinase was not affected, suggesting that the induction cannot be reversed by subsequent ionomycin treatment (Fig. 4B). To determine the critical time of the inhibition by ionomycin, HT-1080 cells were incubated 12 h with PMA, and ionomycin was added 0-11 h after the onset of the incubation. At 12 h total RNA was isolated and analyzed by Northern hybridization. It was found that a time lag of 1-3 h between addition of PMA and ionomycin notably attenuated the inhibitory effect of ionomycin, and after 5-7 h ionomycin had no effect on the 92-kDa gelatinase mRNA levels (Fig. 4C). Both PMA and ionomycin induced collagenase expression, and maximal induction was seen when ionomycin was added 3 h after PMA.

Ionomycin Inhibits PMA-induced Transcription of 92-kDa Gelatinase

It is known that PMA induces the 92-kDa gelatinase mRNA levels by increasing the transcription of the gene(10) . To find out whether the inhibition of induction by ionomycin was caused by inhibition of gene transcription or by destabilization of 92-kDa gelatinase mRNA, the stability of mRNA was studied by actinomycin D treatment. HT-1080 cells were treated with PMA and ionomycin for 12 h, after which actinomycin D (5 µg/ml) was added to inhibit mRNA synthesis, and the incubation was continued for 4-8 h. Total RNA was then isolated and analyzed by Northern hybridization. The level of 92-kDa gelatinase mRNA was not affected by 8-h treatment with actinomycin D in either PMA or PMA and ionomycin-treated cells, suggesting that the half-life of 92-kDa gelatinase is quite long and is not affected by ionomycin (Fig. 5A).


Figure 5: Ionomycin inhibits PMA-induced transcription of 92-kDa gelatinase. A, effect of actinomycin D on 92-kDa gelatinase mRNA levels. Confluent cultures of HT-1080 cells were treated with PMA and ionomycin under serum-free conditions for 12 h. Actinomycin D (5 µg/ml) was then added, and incubation was continued for 2-8 h. At the end of incubation, total RNA was isolated and analyzed for the expression of the 92-kDa gelatinase. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, analysis of 92-kDa gelatinase promoter activity in stably transfected cells. HT-1080 cells were transfected with human -2.1-kb 92-kDa gelatinase promoter-luciferase construct, and stably transfected cells were selected by treatment of cells with G418 for 2 weeks (see ``Materials and Methods''). Confluent cultures were treated with PMA (10 nM) and ionomycin (500 nM) under serum-free conditions for 12 h. The cells were then lysed and analyzed for luciferase activity. The values represent the mean of three independent experiments. Standard deviation is indicated by error bars. Activity of the untreated control sample is arbitrarily set as 1.



To analyze the transcription of the 92-kDa gelatinase gene, we constructed a reporter plasmid containing a 2.2-kb fragment of the 5`-flanking region of the 92-kDa gelatinase gene (-2160 to +20) linked to luciferase gene. Transient transfection of HT-1080 cells was performed as described under ``Materials and Methods.'' After transfection, the cultures were treated with PMA and/or ionomycin for 30 h, after which cells were harvested, luciferase activity was measured and correlated to -galactosidase activity. Treatment of cells with PMA caused only a slight but reproducible increase in luciferase activity. Ionomycin (500 nM) reduced the levels of luciferase activity in both untreated and PMA-treated cells (data not shown).

The harsh treatment of cells used in transient transfections could affect the ability of cells to react to PMA treatment, e.g. by increasing cell permeability to calcium. To avoid these artifacts we studied the regulation of the reporter construct in stably transfected cells and constructed a plasmid vector containing a 2.1-kb fragment of the 5`-flanking region of 92-kDa gelatinase gene (-2112 to +20) linked to luciferase gene and neomycin resistance gene. HT-1080 cells were transfected with this construct followed by selection with neomycin analogue G418 for 2 weeks. The mixed population of transfected cells was then treated with PMA and ionomycin for 12 h, and the level of luciferase in cell lysates was measured. Untreated cells showed a quite high background level of luciferase expression, which was decreased about 30% by treatment of the cells with ionomycin (Fig. 5B). Treatment of cells with PMA increased the level of luciferase expression 2-3-fold, and this increase could be inhibited by simultaneous treatment with ionomycin. Increase in the luciferase activity in cells treated with PMA was maximal as early as 6 h after the onset of PMA treatment, and at 30 h the level of luciferase expression was decreased almost to the control levels, suggesting that the induction of the transcription of the 92-kDa gelatinase gene is transient. Clones derived from single transfected cells were produced by dilution cloning and assayed for the regulation of luciferase activity by PMA and ionomycin. Basal levels of luciferase expression varied about 5-fold between different clones, but the regulation by PMA and ionomycin was similar to that seen in the mixed pool of transfected cells (data not shown).


DISCUSSION

In the present work we have characterized the effects of elevated intracellular calcium levels on the expression and proteolytic processing of 92- and 72-kDa gelatinases. To modulate intracellular calcium concentration, cultured cells were treated with two different calcium ionophores, A23187 and ionomycin, which function as mobile ion carriers and increase the permeability of plasma membrane and intracellular calcium storage compartments to divalent cations. To ensure that the effects observed were due to increased intracellular calcium levels, we also used another chemical, thapsigargin, which releases calcium from intracellular stores by inhibiting the function of endoplasmic reticulum Ca-ATPase. It was found that the conditioned medium of HT-1080 cells contained only very low levels of soluble gelatinase activity as measured by degradation of soluble radiolabeled gelatin substrate. However, when radiolabeled gelatin attached to cell culture plates was used as a substrate, we found a steady level of basal pericellular gelatinolytic activity, which was decreased by calcium ionophores and thapsigargin. Pericellular gelatinolytic activity could be induced by treatment of the cells with PMA, TNF, and concanavalin A, and their effects were abrogated by simultaneous treatment with calcium ionophores. Analysis of gelatinolytic activity by gelatin zymography revealed two distinct phenomena that could decrease the gelatinolytic potential of the cells treated with ionophores. Both the basal and PMA- or TNF-stimulated expression of 92-kDa gelatinase was decreased, and the proteolytic processing of 72-kDa gelatinase was inhibited. In contrast, treatment of rabbit synovial fibroblasts with calcium ionophores has been reported to result in endogenous activation of interstitial collagenase (40) , suggesting that the mechanisms regulating the activation processes of these two MMPs are quite different. Our data are suggestive but do not prove that the pericellular gelatinolytic activity would be totally attributable to the two gelatinases. However, these gelatinases are the only major gelatinolytic proteases present in the conditioned medium of HT-1080 cells as assayed by gelatin zymography.

Despite extensive efforts the mechanisms of 72-kDa gelatinase activation in vitro and in vivo are still unraveled. The 72-kDa gelatinase processing activity of PMA- or concanavalin A-treated cells has been localized to the plasma membrane fraction, and it is sensitive to inhibition by metal ion chelators such as EDTA and 1,10-phenanthroline as well as TIMP-2(29, 30) . Overexpression of a novel membrane-associated metalloproteinase, MT-MMP in COS-1 and Sf-9 cells, leads to the appearance of 72-kDa gelatinase processing activity in the plasma membrane fraction of the cells, and this activity could be inhibited by TIMP-2(31) . Accordingly, MT-MMP is a good candidate for the enzyme responsible for proteolytic processing and activation of 72-kDa gelatinase in phorbol ester or concanavalin A-treated HT-1080 cells.

In our study we found that MT-MMP mRNA is expressed in high levels even in serum-starved HT-1080 cells, which express only limited 72-kDa gelatinase processing activity. Furthermore, we found that treatment of the cells with PMA caused only 2-3-fold induction of MT-MMP expression, and this induction was not affected by calcium ionophores. The 72-kDa gelatinase processing activity was, however, strongly induced by PMA, and this induction was completely inhibited by ionomycin. In our studies, TNF and concanavalin A had no effect on MT-MMP mRNA levels. This is in contrast to the results of Sato et al.(31) reporting an induction of MT-MMP mRNA levels in concanavalin A-treated HT-1080 cells. Thus it appears that the level of MT-MMP mRNA expression is not directly connected with the level of 72-kDa gelatinase processing activity.

Like other matrix metalloproteinases, MT-MMP has an NH-terminal latency-associated domain, which is presumably removed upon activation. Activation of 72-kDa gelatinase by PMA and concanavalin A could thus be mediated by induction of proteolytic processing and activation of latent MT-MMP, which could then activate the 72-kDa gelatinase. We have reported earlier that treatment of isolated extracellular matrices of human embryonal lung fibroblasts with urokinase-type plasminogen activator, thrombin, plasmin, and two mast cell-derived enzymes, tryptase and chymase, causes processing of matrix-bound 72-kDa gelatinase to 64- and 62-kDa forms(43, 50) . On the other hand, in experimental settings using purified 72-kDa gelatinase as a substrate, urokinase-type plasminogen activator, plasmin, stromelysin, and even trypsin have lacked the ability to activate 72-kDa gelatinase(30, 51) . These contradictory results could be explained by a model of a cell surface-associated proteolytic cascade, which would involve activation of MT-MMP by some of the above-mentioned serine proteases, perhaps receptor-bound urokinase-type plasminogen activator, as also proposed by Strongin et al.(35) . Active cell surface-bound MT-MMP would then bind and activate 72-kDa gelatinase to achieve cell surface-targeted gelatinolytic activity. Constitutive production of 72-kDa gelatinase by stromal fibroblasts and its deposition in the pericellular matrix would provide a potent, rapidly and targetedly activable store of latent gelatinolytic enzyme. Still another possibility is that some other membrane-associated metalloproteinase like a homologue of MT-MMP is involved. In addition, there are also membrane metalloproteinases that are not homologous to matrix metalloproteinases like endothelin-converting enzyme-1 (ECE-1), but their possible effects in the activation of matrix metalloproteinases are yet to be characterized(52) .

Recent evidence indicates that the presence of some TIMP-2 is mandatory for the binding of 72-kDa gelatinase to the MT-MMP at the cell surface and for the subsequent membrane activation of 72-kDa gelatinase(35) . However, the optimal concentration of TIMP-2 for 72-kDa gelatinase activation is quite low and narrow, since an excess of TIMP-2 leads to the inhibition of the activation reaction. In our experiments we could not see remarkable differences in the mRNA or protein levels of TIMP-2 between cells treated with PMA alone or PMA together with ionomycin (data not shown). Accordingly, regulation of TIMP-2 levels seems not to be the cause of ionomycin-mediated inhibition of 72-kDa gelatinase activation.

In the assays for pericellular gelatinolytic activity, PMA and TNF were more potent inducers of gelatinolysis than concanavalin A (Fig. 2A). However, in gelatin zymography the processing of 72-kDa gelatinase to lower molecular weight forms was more complete in the conditioned medium of concanavalin A-treated cells, whereas PMA- or TNF-treated cells secreted high levels of 92-kDa gelatinase. Although 92-kDa gelatinase was found to be in the 92-kDa progelatinase form and not in the 84-kDa proteolytically processed form, it may still be active in the pericellular environment.

To analyze the mechanisms of the effects of calcium ionophores on the expression of 92-kDa gelatinase, total RNAs were extracted and analyzed by Northern hybridization. The basal level of 92-kDa gelatinase expression in serum-starved HT-1080 cells was quite low and was strongly induced by PMA and TNF. Maximal levels of 92-kDa gelatinase expression were achieved at 12 h. Because the half-life of the mRNA is rather long, the lacking of further accumulation suggests that the induction of transcription is only transient. Addition of ionomycin at the same time with PMA almost completely blocked the induction, but if ionomycin was added only about 1 h after PMA, its inhibitory effect on 92-kDa gelatinase expression was significantly attenuated. If ionomycin was added 12 h after the onset of PMA stimulation, no effect on the 92-kDa gelatinase expression could be observed, suggesting that the effect of ionomycin is mediated by inhibition of gene transcription rather than destabilization of 92-kDa gelatinase mRNA. This was also confirmed by our observation that the half-life of 92-kDa gelatinase is rather long and not affected by ionomycin.

When analyzing the promoter activity of 92-kDa gelatinase using transient transfection of cells with promoter/luciferase constructs, we observed only very limited induction of promoter activity in PMA-treated cells. However, promoter activity was significantly decreased by ionomycin. The low levels of luciferase expression could be caused by leakage of the plasma membranes during the transfection process, which would allow influx of calcium. To circumvent the harsh treatment of transient transfections, we produced cells that were stably transfected with the 92-kDa gelatinase-luciferase reporter construct. The basal level of luciferase expression in these cells was relatively high, which could be explained by the absence of a silencer that would exist in the vicinity of the 92-kDa gelatinase gene but which would be lacking in the transfected cells. In stably transfected cells promoter activity was induced by PMA and TNF-, and these inductions were prevented by ionophores, which confirms that 92-kDa gelatinase is regulated at the transcriptional level.

Treatment of cells with calcium ionophores or thapsigargin causes a transient increase in the intracellular calcium levels as calcium flows into the cytoplasm from extracellular space and intracellular calcium stores (see (37, 38, 39) ). After the initial surge of calcium the intracellular calcium concentrations reaches a stable level that is higher than before stimulation. However, in our study the actual calcium levels were not measured. Absence of calcium waves and oscillations typical of in vivo calcium signaling may also modulate the effects of calcium ionophores. Another phenomenon that is associated with prolonged treatment of cells with calcium ionophores or thapsigargin is depletion of intracellular calcium stores, which might have diverse and unappreciated effects on cellular signal transduction and even on protein folding in the Golgi apparatus(38) . The in vivo counterpart to the effects of calcium ionophores remains also unraveled. Possible candidates are growth factors and peptides, which signal through tyrosine kinase or G-protein-linked receptors to increase intracellular calcium levels. Cancer cells often have abnormal calcium signaling mechanisms; they need much less external calcium for proliferation, and they no longer obey calcium signals to differentiate or die by apoptosis(36) . This aberrance of the calcium signaling network might also include absence of calcium-mediated inhibition of 72-kDa gelatinase activation and 92-kDa gelatinase expression. In any case, our data suggest the presence of a calcium-dependent signaling mechanism leading to two separate phenomena capable of decreasing the pericellular gelatinolytic activity. Specific inhibition of 72-kDa processing activity by calcium ionophores may also serve as a valuable tool in the further analysis of the 72-kDa gelatinase activation cascade.


FOOTNOTES

*
This work was supported by the Academy of Finland, the Finnish Cancer Foundation, Oskar flund Foundation, the University of Helsinki, and Sigrid Juselius Foundation. Presented in part at the Fifth International Conference on the Molecular Biology and Pathology of Matrix, Philadelphia, PA (53). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Research associate of the Academy of Finland.

To whom correspondence should be addressed: Dept. of Virology, University of Helsinki, Haartmaninkatu 3, FIN-00290 Helsinki, Finland. Tel.: 358-0-434-6672; Fax: 358-0-434-6491.

The abbreviations used are: MMP, matrix metalloproteinases; TNF, tumor necrosis factor-; PMA, phorbol 12-myristate 13-acetate; TIMP-2, tissue inhibitor of metalloproteinases, type 2; MT-MMP, membrane-type matrix metalloproteinase; MEM, Eagle's minimal essential medium; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s).

J. Lohi, J. Westermarck, V.-M. Kähäri, and J. Keski-Oja, submitted for publication.


ACKNOWLEDGEMENTS

We thank Drs. Tapio Vartio, Ulpu Saarialho-Kere, and Jussi Taipale for critical comments and Sami Starast for fine technical assistance.


REFERENCES
  1. Woessner, J. F., Jr. (1991)FASEB J. 5, 2145-2154 [Abstract/Free Full Text]
  2. Birkedal-Hansen, H., Moore, W. G. I., Bodden, M. K., Windsor, L. J., Birkedal-Hansen, B., DeCarlo, A., and Engler, J. A.(1993)Crit. Rev. Oral Biol. Med. 4, 197-250 [Abstract]
  3. Kleiner, D. E., Jr., and Stetler-Stevenson, W. G.(1993)Curr. Opin. Cell Biol. 5, 891-897 [Medline] [Order article via Infotrieve]
  4. Brinckerhoff, C. E., Gross, R. H., Nagase, H., Sheldon, L., Jackson, R. C., and Harris, E. D., Jr.(1982)Biochemistry 21, 2674-2679 [Medline] [Order article via Infotrieve]
  5. Postlethwaite, A. E., Lachman, L. B., Mainardi, C. L., and Kang, A. H.(1983) J. Exp. Med. 157, 801-806 [Abstract]
  6. Conca, W., Kaplan, P. B., Krane, S. M.(1989)J. Clin. Invest. 83, 1753-1757 [Medline] [Order article via Infotrieve]
  7. MacNaul, K. L., Chartrain, N., Lark, M., Tocci, M. J., Hutchinson, N. I.(1990) J. Biol. Chem. 265, 17238-17245 [Abstract/Free Full Text]
  8. Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A., and Goldberg, G. I. (1989)J. Biol. Chem. 264, 17213-17221 [Abstract/Free Full Text]
  9. Gaire, M., Magbanua, Z., McDonnell, S., McNeil, L., Lovett, D. H., and Matrisian, L. M. (1994)J. Biol. Chem. 269, 2032-2040 [Abstract/Free Full Text]
  10. Sato, H., and Seiki, M. (1993)Oncogene 8, 395-405 [Medline] [Order article via Infotrieve]
  11. Brinckerhoff, C. E., Harris, E. D.(1981)Biochim. Biophys. Acta 677, 424-432 [Medline] [Order article via Infotrieve]
  12. Matrisian, L. M., Leroy, P., Ruhlmann, C., Gesnel, M.-C., and Breatnach, R.(1986) Mol. Cell. Biol. 5, 1679-1686
  13. Edwards, D. R., Murphy, G., Reynolds, J. J., Whitham, S. E., Docherty, A. J. P., Angel, P., and Heath, J. K.(1987)EMBO J. 6, 1899-1904 [Abstract]
  14. Huhtala, P., Tuuttila, A., Chow, L., Lohi, J., Keski-Oja, J., Tryggvason, K.(1991) J. Biol. Chem. 266, 16485-16490 [Abstract/Free Full Text]
  15. Mainardi, C. L., Hibbs, M. S., Hasty, K. A., and Seyer, J. M.(1984)Collagen Relat. Res. 4, 479-492
  16. Hibbs, M. S., Hasty, K. A., Seyer, J. M., Kang, A. H., and Mainardi, C. L.(1985) J. Biol. Chem. 260, 2493-2500 [Abstract]
  17. Saarialho-Kere, U. K., Welgus, H. G., and Parks, W. C.(1993)J. Biol. Chem. 268, 17354-17361 [Abstract/Free Full Text]
  18. Salo, T., Lyons, J. G., Rahemtulla, F., Birkedal-Hansen, H., and Larjava, H.(1991) J. Biol. Chem. 266, 11436-11441 [Abstract/Free Full Text]
  19. Reponen, P., Sahlberg, C., Munaut, C., Thesleff, I., and Tryggvason, K.(1994) J. Cell Biol. 124, 1091-1102 [Abstract]
  20. Reponen, P., Leivo, I., Sahlberg, C., Apte, S. S., Olsen, B. R., Thesleff, I., and Tryggvason, K.(1995)Dev. Dynamics 202, 388-396 [Medline] [Order article via Infotrieve]
  21. Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Selzer, J. L., Kronberger, A., He, C., Bauer, E. A., and Goldberg, G. I.(1988) J. Biol. Chem. 263, 6579-6587 [Abstract/Free Full Text]
  22. Tryggvason, K., Huhtala, P., Tuuttila, A., Chow, L., Keski-Oja J., and Lohi, J.(1990) Cell Differ. Dev. 32, 307-312 [Medline] [Order article via Infotrieve]
  23. Huhtala, P., Chow, L. T., and Tryggvason, K.(1990)J. Biol. Chem. 265, 11077-11082 [Abstract/Free Full Text]
  24. Stricklin, G. P., Jeffrey, J. J., Roswit, W. T., and Eisen, A. Z.(1983) Biochemistry 22, 61-68 [Medline] [Order article via Infotrieve]
  25. Grant, G. A., Eisen, A. Z., Marmer, B. L., Roswit, W. T., and Goldberg, G. I.(1987) J. Biol. Chem. 262, 5886-6889 [Abstract/Free Full Text]
  26. He, C., Wilhelm, S. M., Pentland, A. P., Marmer, B. L., Grant, G. A., Eisen, A. Z., and Goldberg, G. I.(1989)Proc. Natl. Acad. Sci. U. S. A. 86, 2632-2636 [Abstract]
  27. Mignatti, P., and Rifkin, D. B.(1993)Physiol. Rev. 73, 161-195 [Free Full Text]
  28. Saarinen, J., Kalkkinen, N., Welgus, H. G., and Kovanen, P. T.(1994)J. Biol. Chem. 269, 18134-18140 [Abstract/Free Full Text]
  29. Brown, P. D., Kleiner, D. E., Unsworth, E. J., and Stetler-Stevenson, W. G.(1993) Kidney Int. 43, 163-171 [Medline] [Order article via Infotrieve]
  30. Strongin, A. Y., Marmer, B. L., Grant, G. A., and Goldberg, G. I.(1993)J. Biol. Chem. 268, 14033-14039 [Abstract/Free Full Text]
  31. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994)Nature 370, 61-65 [CrossRef][Medline] [Order article via Infotrieve]
  32. Cao, J., Sato, H., Takino, T., and Seiki, M.(1995)J. Biol. Chem. 270, 801-805 [Abstract/Free Full Text]
  33. Brown, P. D., Levy, A. T., Marguiles, I. M. K., Liotta, L. A., and Stetler-Stevenson, W. G. (1990)Cancer Res. 50, 6184-6191 [Abstract]
  34. Overall, C. M., and Sodek, J.(1990)J. Biol. Chem. 265, 21141-21151 [Abstract/Free Full Text]
  35. Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grant, G. A., and Goldberg, G. I.(1995)J. Biol. Chem. 270, 5331-5338 [Abstract/Free Full Text]
  36. Whitfield, J. F. (1992)Crit. Rev. Oncog. 3, 55-90 [Medline] [Order article via Infotrieve]
  37. Berridge, M. J. (1993)Nature 361, 315-325 [CrossRef][Medline] [Order article via Infotrieve]
  38. Pozzan, T., Rizzuto, R., Volpe, P., and Meldolesi, J.(1994)Physiol. Rev. 74, 595-636 [Free Full Text]
  39. Clapham, D. E. (1995)Cell 80, 259-268 [Medline] [Order article via Infotrieve]
  40. Unemori, E. N., and Werb, Z.(1988)J. Biol. Chem. 263, 16252-16259 [Abstract/Free Full Text]
  41. Rodland, K. D., Lenormand, P., Muldoon, L. L., and Magun, B.(1992)J. Invest. Dermatol.98,12S-16S [Abstract]
  42. Kohn, E. C., Jacobs, W., Kim, Y. S., Alessandro, R., Stetler-Stevenson, W. G., and Liotta, L. A. (1994)J. Biol. Chem. 269, 21505-21511 [Abstract/Free Full Text]
  43. Lohi, J., Harvima, I., and Keski-Oja, J.(1992)J. Cell. Biochem. 50, 337-349 [Medline] [Order article via Infotrieve]
  44. Laemmli, U. K. (1970)Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  45. Taipale, J., Koli, K., and Keski-Oja, J.(1992)J. Biol. Chem. 267, 25378-25384 [Abstract/Free Full Text]
  46. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J.(1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  47. Goldberg, G. I., Wilhelm, S. M., Kronberger, A., Bauer, E. A., Grant, G. A., and Eisen, A. Z.(1986)J. Biol. Chem. 261, 6600-6605 [Abstract/Free Full Text]
  48. Forth, P., Marty, L., Piechaczyk, M., El Sabrouty, S., Dani, C., Jeanteur, P., and Blanchard, J. M.(1985)Nucleic Acids Res. 13, 1431-1442 [Abstract]
  49. Sambrook, J., Fritsch, E. F., and Maniatis, T. E. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 16.66-16.67, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  50. Keski-Oja, J., Lohi, J., Tuuttila, A., Tryggvason, K., and Vartio, T.(1992) Exp. Cell. Res. 202, 471-476 [Medline] [Order article via Infotrieve]
  51. Okada, Y., Morodomi, T., Enghild, J. J., Suzuki, K., Yasui, A., Nakanishi, I., Salvesen, G., and Nagase, H.(1990)Eur. J. Biochem. 194, 721-730 [Abstract]
  52. Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWit, D., and Yangisawa, M. (1994)Cell 78, 473-485 [Medline] [Order article via Infotrieve]
  53. Lohi, J., and Keski-Oja, J.(1994)Matrix Biol.14,421

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