Role of plasmin and gelatinase matrix degradation by cultured ALVIN

P. WONG,

Department

SHIRLEY

L. CORTEZ,

in extracellular rat mesangial cells AND WILLIAM

H. BARICOS

of Biochemistry, Tulane Medical School, New Orleans, Louisiana 70112

Wong, Alvin P., Shirley L. Cortez, and William H. Baricos. Role of plasmin and gelatinasein extracellular matrix degradationby cultured rat mesangialcells.Am. J. Physiol. 263 (Renal Fluid Electrolyte PhysioL.32): F1112-F1118, 1992.-We have examined the ability of mesangialcells (MCs) to degrade extracellular matrix (ECM) using cultured rat MCs grown on thin films of radiolabeledMatrigel. ECM degradation by cultured MCs wasobservedonly in presenceof exogenouslyadded plasminogenand was a function of plasminogenconcentration (O-50 mu), cell number (O-50,000cells), and length of incubation (O-72 h). A high positive correlation (r > 0.93) was observed betweenECM degradation and plasmin activity in medium, suggesting an important role for plasmin in ECM degradationby cultured MCs. This suggestionwasconfirmed by ability of plasmin inhibitors, a,-antiplasmin (40 pg/ml) and aprotinin (216 kallikrein inhibition units/ml), to inhibit (>90%) ECM degradation. Inhibitors of cysteine proteinases [trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, 10 PM] and aspartic proteinases(pepstatin, 5.0 pg/ml) had no effect on ECM degradation.However, in presenceof plasminogen, inhibitors of matrix metalloproteinases,TIMP-1 (40 pg/ ml) and o-phenanthroline (100 PM), inhibited ECM degradation -42 t 4% and -43 * 3% (SE), respectively (n = S-10). Thus, in addition to plasmin, a matrix metalloproteinase(s)is alsoinvolved in ECM degradationby cultured rat MCs. Zymography of culture medium obtained from MCs grown on radiolabeledMatrigel films in absenceof plasminogenrevealedonly two closely migrating bands of gelatinaseactivity, relative mol wt of -7O,OOO-72,000.MCs grown in absenceof plasminogen failed to degrade ECM despite presenceof gelatinasein medium, indicating that, in absenceof plasmin, gelatinase is present in an inactive form, either as a latent proenzyme or as a gelatinase-inhibitor complex. However, in presenceof plasmin (produced from plasminogenby MC plasminogenactivators) active gelatinaseis generatedas indicated by ability of gelatinaseinhibitors to reduce ECM degradation. Thus plasmin appearsto play a key role in activation of MC gelatinase.These data indicate that ECM degradationby cultured MCs involves a proteolytic enzyme cascade.This cascadeis initiated by MC plasminogenactivator(s) which convert plasminogento plasmin, which in turn activates gelatinase(either directly or indirectly). Plasmin and gelatinasethen carry out the degradation of the ECM. type IV collagenase;metalloproteinases;proteinase cascade

(l3), and adhesion (9). In addition, both qualitative and quantitative changes in the composition of the mesangial matrix have been observed in progressive glomerular diseases (4, 24, 32), suggesting an important role for the mesangial matrix in the pathophysiology of these diseases. Despite the potential importance of the mesangial matrix in glomerular function and pathophysiology, virtually no information is available concerning the proteinases involved in degradation of the mesangial matrix by mesangial cells, the factors which regulate the synthesis and activity of these proteinases, or the potential role of such proteinases in glomerular disease. In other tissues, several lines of evidence (for review, see Ref. 26) suggest that matrix metalloproteinases, such as collagenase and gelatinase (type IV collagenase), play important roles in ECM degradation. In addition, several studies have suggested cooperative interactions between plasmin and matrix metalloproteinases in ECM degradation (10, 11, 26, 31). Mesangial cells produce several agents potentially involved (either directly or indirectly) in the degradation of the mesangial matrix including plasminogen activator(s) (12,15); plasminogen activator inhibitor (15); gelatinase (8, 19, 21); and the gelatinase inhibitor, tissue inhibitor of metalloproteinases (TIMP-1) (21). However, the role of these agents in the degradation of mesangial matrix by mesangial cells has not been reported previously. In the present study, we have examined the ability of mesangial cells to degrade ECM using a model system consisting of cultured rat mesangial cells grown on thin films of radiolabeled Matrigel, a commercially available ECM secreted by the Englebreth-Holm-Swarm (EHS) sarcoma. Although the exact composition of both the mesangial matrix and Matrigel are unknown, Matrigel contains the major constituents present in the mesangial matrix including type IV collagen, fibronectin, laminin, and heparan sulfate proteoglycan (14) and thus provides a readily available source of mesangial matrixlike material for in vitro studies of mesangial matrix ‘degradation. METHODS

GLOMERULAR MESANGIUM is an intercapillary complex of mesangial cells embedded in an extracellular matrix (ECM), the mesangial matrix, which is synthesized and maintained by glomerular mesangial cells (24). Although the exact composition of the mesangial matrix is unknown, immunofluorescence and biochemical studies have documented the presence of type IV collagen, the glycoproteins fibronectin and laminin, and heparan sulfate proteoglycan as the major components of normal mesangial matrix (4, 24, 32). Recent studies suggest that, in addition to structural support, the mesangial matrix may affect a variety of mesangial cell functions including migration (29)) proliferation (6, 9)) secretion THE

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Materials. Plasminogen(from human plasma,catalog no. P 7397), plasmin (from porcine blood, 3.9 U/mg solid, catalog no. P 8644), pepstatin A (catalogno. P 4265), gelatin (from bovine skin, catalogno. G 1393),aprotinin [ 10,800kallikrein inhibition units (KIU)/mg protein, catalog no. A 62791,cu2-antiplasmin (catalogno. A 0914))fluoresceinisothiocyanate (FITC) -labeled phalloidin (catalog no. P 5282), and trarzs-epoxysuccinyl-Lleucylamido(4guanidino)butane (E-64, catalog no. E 3132) were obtained from Sigma Chemical, St. Louis, MO. RPM1 1640 cell culture medium and 0.05% trypsin-0.5 mM EDTA were obtained from GIBCO, Grand Island, NY. Methoxysuccinyl-L-Ala-L-Phe-L-Lys-7-amido-4-methylcoumarin (catalog no. GME080), was obtained from Bachem, Torrance, CA. Matrigel and Dispase,a bacterial proteinase which efficiently

0 1992 The

American

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Society

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solubilizes Matrigel, were obtained from Collaborative Research, Bedford, MA. Na1251 (451 mCi/ml) and [3H]acetic anhydride (50 mCi/mmol) were obtained from New England Nuclear, Bedford, MA. Recombinant TIMP-1 (previously referred to as TIMP) was a gift from Synergen, Boulder, CO. Antibodies against Thy-l.1 (catalog no. MAB 1406), fibronectin (catalog no. AB 1942), and factor VIII-von Willebrand factor (catalog no. MAB 037-56/3) were obtained from Chemicon, Temecula, CA. All other chemicals were reagent grade or higher and were obtained from commercial sources. Mesangial cell culture. Rat mesangial cells were cultured from isolated rat glomerular cores as described in detail by Lovett et al. (8, 19) and briefly summarized here. Glomeruli were isolated from male Sprague-Dawley rats (200-250 g) by sieving as described in our previous publications (2) except that the entire procedure was carried out under sterile conditions. Isolated glomeruli (-30,000) were incubated in 2.0 ml Hanks’ balanced salt solution (HBSS) containing 750 U/ml bacterial collagenase (Sigma type IV, containing X25 U/mg collagenase, 20-30 U/mg nonspecific proteinase, and minor amounts of clostripain) at 37°C for 30 min under sterile conditions with continuous gentle agitation. Glomerular cores (containing mainly endothelial and mesangial cells) were pelleted by centrifugation (50 g for 5 min, at 4°C) and washed three times (resuspension and centrifugation as above) with sterile HBSS. Primary cultures were established by suspending the washed cores (corresponding to 30,000 intact glomeruli) in 15 ml of RPM1 1640, supplemented with 20% heat-inactivated (56°C 60 min) fetal calf serum, 2.0 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 pg/ml), transferred to a 75-cm2 tissue culture flask, and maintained at 37°C in 5% CO, in humidified air. These primary cultures were allowed to grow to confluence (-3 wk) before passage. After this initial passage, medium was changed every 2-3 days, and cells were passaged after -21 days. Cultured cells were identified as mesangial cells by the following criteria. 1) After three to four passages, all cells were stellate or spindle-shaped with irregular cytoplasmic projections. The cells did not exhibit contact inhibition and became multilayered if allowed to grow beyond confluence without passage. 2) Immunofluorescent staining (read by Dr. Patrick Walker, Dept. of Pathology, Univ. of Arkansas for Medical Sciences) was positive for the mesangial cell markers Thy-1.1, fibronectin (double antibody technique), and actin (FITC-labeled phalloidin); and negative for the endothelial cell marker factor VIII (double antibody technique). 3) Fibroblast contamination was assessed and found to be negative by the ability of the cells to grow in medium containing D-valine in place of L-valine (19). 4) As discussed below, the cultured cells produce plasminogen activator(s) and latent gelatinase, which is also characteristic of mesangial cells (8, 12, 15, 19, 21). Mesangial cells used in these studies were between the 15th and 25th passages. However, virtually identical results were observed with cells at passage numbers 5-8. Radiolabeling of Mcztrigel. Our initial studies were carried out with [“H]Matrigel, produced by acetylation with [3H]acetic anhydride as described in detail for glomerular basement membrane (GBM) in our previous paper (1) except that the entire procedure was carried out under sterile conditions. Five microcuries of [3H]acetic anhydride in 1.0 ml of dry benzene were slowly added to 80 mg of rapidly stirring Matrigel in 40 ml of 10 mM sodium borate buffer, pH 9.5, containing 200 mM CaCl, at 0-4°C. After 1 h the [“H]Matrigel was separated from unreacted [“HIacetic anhydride and [“HIacetic acid by dialysis at 0-4°C against four changes of 6 liters of sterile distilled water. The labeled Matrigel, specific activity 1.3 x IO6 counts per minute (cpm)/mg, was stored at 4°C. Because scintillation counting consumed most of the cell-free medium available from each well (see below), in subsequent studies we utilized 12”1-labeled Matri-

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gel, because the same sample of cell-free medium used for gamma counting could be used for the measurement of additional parameters (e.g., measurement of enzyme activity) following gamma counting. 1251-Matrigel was produced by the chloramine-T method as described by McConahey and Dixon (23). Five hundred microcuries of Na1251 (451 mCi/ml) were added to 5.0 mg of rapidly stirring Matrigel at 0-4°C followed by dropwise addition of 50 pg of chloramine-T dissolved in 100 ~1 of 100 mM phosphate buffer, pH 7.0. After 10 min, unreacted chloramine-T was neutralized by the addition of 50 pg of sodium metabisulfite. Labeled Matrigel was separated from unreacted 125I by extensive dialysis at 0-4°C against at least three changes of 6 liters of sterile distilled water. The 1251-labeled Matrigel (specific radioactivity of 2 x lo6 cpm/mg protein) was stored at 4°C. Production of Mutrigel films. Thin films of radiolabeled Matrigel were produced in 24-well Costar multi-well culture plates essentially as described by Collaborative Research. Immediately prior to use, an aliquot of the radiolabeled Matrigel was diluted with unlabeled Matrigel to a final concentration of 100 pg/ml; 250 ~1 of this suspension (25 I,cgof Matrigel protein, 25,000-35,000 cpm) were added to each well, gently swirled to evenly coat the bottom of the well, and allowed to air dry for 48 h by keeping the plates at room temperature with the cover slightly ajar in a sterile laminar flow hood. After drying, plates can be stored under sterile conditions for several weeks. Measurement of ECM degradation by cultured rat mesangial cells. For studies of ECM degradation, wells containing dried Matrigel films (25 pg of protein, 25,000-35,000 cpm) were washed three times with 1.0 ml of serum-free RPM1 1640 medium immediately before addition of mesangial cells (100,000 cells/well, unless specified otherwise) in RPM1 1640 medium containing 10% fetal calf serum. Plates were then incubated for 48 h (37°C 5.0% CO,) to allow the mesangial cells to attach to the Matrigel films. Each well was then washed three times with 1.0 ml of serum-free RPM1 1640 medium (to remove proteolytic enzyme inhibitors potentially present in the serum) and then incubated (O-72 h) in 500 ~1 of serum-free RPM1 1640 containing 0.2% lactalbumin hydrolysate (time 0) as previously described by Lovett and co-workers (8, 19, 21) for the culture of mesangial cells in serum-free medium. Cell viability in RPM1 1640 containing 0.2% lactalbumin hydrolysate was greater than 90% for at least 72 h as measured by Trypan blue exclusion. Exogenously added agents (e.g., plasminogen, proteinase inhibitors) were dissolved at the concentration indicated in RPM1 1640 containing 0.2% lactalbumin hydrolysate. ECM degradation by cultured mesangial cells was measured by the release of radioactivity into the cell-free culture medium as follows. At the end of the incubation period (usually 72 h) the medium from each well (solution A) was carefully removed for the measurement of radioactivity and other parameters as indicated. Undigested Matrigel remaining in each well was solubilized by overnight treatment at 37°C with 500 ~1 of Dispase (1 mg/ml) and subsequently counted (solution B). Preliminary studies documented complete solubilization of Matrigel films under these conditions. Unless otherwise indicated, results are means t, SE as percent degradation calculated from the radioactivity released into the medium (solution A) divided by the total radioactivity present in each well (radioactivity present in solution A + radioactivity present in solution B). Differences in total radioactivity among wells were usually less than 10%. Plasmin assay. Plasmin activity of medium obtained from mesangial cells cultured on Matrigel films, appropriate controls, and plasmin standards was determined by a modification of the method of Castellino and Powell (5) using the synthetic fluorometric plasmin substrate methoxysuccinyl-L-Ala-L-Phe-L-Lys7-amido-4-methylcoumarin. In a 12 x 75-mm glass culture tube, up to 100 ~1 of sample (cell-free medium or plasmin standards)

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were mixed with 450 ~1 of 0.2 M tris(hydroxymethyl)amiTable 1. Plasminogen dependence of ECM degradation nomethane hydrochloride (Tris . HCl), pH 7.4, containing 0.2 M by cultured rat mesangial cells NaCl and 0.05% NaN,, and 125 ~1 of water. Each reaction was started at 15-s intervals by the addition of 225 ~1 of methIncubation Conditions n Degradation, % oxysuccinyl-L-Ala-L-Phe-L-Lys-7-amido-4-methylcoumarin in water (final substrate concentration is 5.0 PM). Immediately Matrigel + after the addition of substrate, each tube was mixed well and RPM1 1640 + 0.2% LH only 5 7tO.l Mesangial cells only transferred to a 37°C water bath. Each tube was capped to avoid 12 12t0.5 Plasminogen (15 mu) only 7 12tl.O evaporation during the incubation period. After 40 min, each Mesangial cells and plasminogen (15 mu) 29 43k2.0 reaction was stopped at 15-s intervals by the addition of 100 ~1 Values are means & SE for n determinations. Cultured rat mesangial of soya bean trypsin inhibitor (0.25 mg/ml) followed by vigorous mixing. The fluorescence of each tube was then measured in a cells (100,000 cells/well) were grown for 72 h on thin films of [3H]Matrigel(35,OOO cpm/well), and degradation was measured as release of Sequoia-Turner fluorometer (model 450) equipped with approradioactivity into the cell-free medium. See METHODS for further depriate filters for aminomethylcoumarin fluorescence (excitation tails. ECM, extracellular matrix; LH, lactalbumin hydrolysate. at 360 nm; emission at 450 nm). Before each series of readings, the fluorometer was set to zero with water and to 1,000 relative the absence of mesangial cells) under conditions identical fluorescence units with 0.5 PM 7-amino-4-methylcoumarin to those described in Table 1, the conversion of plasmistandard. Tubes containing plasmin standards (0.25 and 0.50 mu) were included in each set of assays, and the plasmin con- nogen to plasmin was less than 10% of that observed in the presence of mesangial cells. Thus plasminogen actitent of each sample was determined by comparison to a standard curve generated by the purified plasmin standards. In vator activity of the Matrigel does not significantly contribute to plasminogen activation in this system. preliminary studies we demonstrated that hydrolysis of methoxysuccinyl-L-Ala-L-Phe-L-Lys-7-amido-4-methylcoumarin by Further studies using identical conditions documented medium obtained from cultured rat mesangial cells incubated in that Matrigel degradation by cultured rat mesangial cells the presence of plasminogen was >90% inhibitable by cu,-antiwas a function of plasminogen concentration (O-50 mu, plasmin, a specific inhibitor of plasmin, establishing that plas- Fig. l), cell number (O-50,000 cells, Fig. 2), and length of min accounts for essentially all of the hydrolysis of this sub- incubation (O-72 h, Fig. 3). These results document that strate under the conditions of this assay. Results are means t cultured rat mesangial cells degrade ECM only in the SE (in mU of plasmin/well), corrected for appropriate blanks. Zyrnography. Sodium dodecyl sulfate (SDS) -substrate gel presence of plasminogen. Role of plasmin in ECM degradation by cultured rat electrophoresis(zymography) wascarried out by incorporating gelatin (500 pg/ml) into standard 10% polyacrylamide gel elec- mesangial cells. Plasminogen is an inactive zymogen with trophoresis(PAGE) gelsessentiallyasdescribedin our previous little if any proteolytic activity until it is converted to an paper (16). After electrophoresisat 0-4°C (using the Bio-Rad active proteinase, plasmin, by the action of plasminogen Mini Protean II System), eachgel waswashedtwo times for 15 activators. In addition to fibrin, plasmin has been shown min each time with 2.5% Triton X-100 (to remove SDS) and to degrade a variety of ECM components including type then incubated overnight in 50 mM Tris HCl, pH 7.4 contain- IV collagen, laminin, fibronectin, and proteoglycan (17, ing 10mM CaC12.Each gelwasstained with Coomassieblue for 26, 31). These observations, coupled with the ability of 30 min and then destainedin 30% methanol-lo% acetic acid. Clearbandson a blue background,due to the degradationof the cultured rat mesangial cells to produce plasminogen actientrapped gelatin, indicate the presenceof gelatin-degrading vators (12, l5), prompted us to examine the role of plasproteinaseswhosemolecularweightscan be estimatedfrom the min in ECM degradation by cultured rat mesangial cells. position of molecular weight standardssimultaneouslyrun on We first examined the ability of purified plasmin to deWhen purified plasmin was incubated the samegel. Inclusion of proteinaseinhibitors in the overnight grade Matrigel. incubation buffer allows further classification of the proteinases.BecauseSDS activates latent matrix metalloproteinases, this technique allowsthe detection and characterization of both latent and active forms of theseproteinases. RESULTS

ECM degradation by cultured rat mesangial cells. In our first series of experiments, we examined the ability of cultured rat mesangial cells to degrade the model ECM, Matrigel. When radiolabeled Matrigel films were incubated for 72 h in RPM1 1640 medium containing 0.2% lactalbumin hydrolysate (RPMI-LH) only, with mesangial cells in RPMI-LH (no plasminogen), or in RPMI-LH + 15 mU of plasminogen (no cells), only small amounts of radioactivity (7- 12%) were released into the medium (Table 1). However, when radiolabeled Matrigel films were incubated under identical conditions in the presence of mesangial cells and 15 mU of plasminogen, ~43% of the ECM was degraded (Table 1). These results document that both mesangial cells and plasminogen are required for significant ECM degradation to occur. When 15 mU of plasminogen were incubated with Matrigel films (in

10

20 Plasminogen

30 40 (mUnits/well)

50

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Fig. 1. Effect of increasing amounts of plasminogen on extracellular matrix (ECM) degradation by cultured rat mesangial cells. Cultured rat mesangial cells (100,000/we11) were grown on thin films of 1251-labeled Matrigel (-25,000 cpm/well) in presence of varying amounts of plasminogen. After 72 h radioactivity released into the cell-free medium was determined. Results are means t SE (in % degradation) of triplicate determinations corrected for triplicate blanks (cells grown on labeled Matrigel in absence of plasminogen) included on the same 24-well plate. Further details are given in METHODS.

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Effects of proteinase inhibitors on ECM degradation by cultured mesangial cells. To further substantiate the role

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Fig. 2. Effect of cell number on ECM degradation by cultured rat mesangial cells. Varying numbers of cultured rat mesangial cells were grown on thin films of 1251-labeled Matrigel (-25,000 cpm/well) in presence of 15 mU of plasminogen. After 72 h, radioactivity in the cell-free medium was determined. Results are means & SE (in % degradation) of triplicate determinations corrected for triplicate blanks (identical number of cells grown on labeled Matrigel in absence of plasminogen) included on the same 24-well plate. Further details are given in METHODS.

with radiolabeled Matrigel films under conditions identical to those above (Figs. l-3), a time-dependent (O-24 h) and dose-dependent (O-25 mu) degradation of Matrigel occurred with up to 60% degradation observed with 25 mU of plasmin over a 24-h period. This degradation was completely blocked by aprotinin (an inhibitor of several serine proteinases including plasmin) and az-antiplasmin (a specific plasmin inhibitor), establishing that plasmin, and not a contaminant proteinase, was responsible for the ECM degradation. These results are in keeping with previously published studies documenting the ability of plasmin to degrade ECM and/or ECM components (17, 26, 31). We then examined the relationship between plasmin activity in the medium and ECM degradation by cultured rat mesangial cells. Using medium obtained from the studies presented in Figs. 2 and 3, we observed an excellent correlation (r > 0.93) between ECM degradation and plasmin activity in the medium (Fig. 4). These data sug-

of plasmin and examine the potential role of other proteinases in ECM degradation by cultured rat mesangial cells, we examined the effects of selected proteinase inhibitors. As shown in Table 2, both aprotinin, an inhibitor of several trypsin-like proteinases including plasmin, and aZ-antiplasmin, a specific inhibitor of plasmin, nearly completely blocked Matrigel degradation by cultured mesangial cells. In addition, both o-phenanthroline, a me tal chelator that inhibits metalloproteinases, and TIMP- 1 (tissue inhibitor of metalloproteinases), a specific inhibitor of matrix degrading metalloproteinases, including gelatinase (26)) produced a significant reduction in ECM degradation by cultured rat mesangial cells (Table 2). These inhibitors had no effect on the conversion of plasminogen to plasmin by cultured mesangial cells, on plasmin activity in the in vitro plasmin assay, or on mesangial cell growth or viability. In contrast to inhibitors of plasmin and gelatinase, inhibitors of cysteine proteinases (E-64, 10 PM) and aspartic proteinases (pepstatin, 5 hg/ml) had no effect on ECM degradation, establishing that these classes of proteolytic enzymes did not contribute significantly to Matrigel degradation by cultured rat mesangial cells. These data provide direct evidence for the role of plasmin in ECM degradation by cultured rat mesangial cells and, in addition, establish that a TIMPinhibitable metalloproteinase is also involved in this process. Evidence for role of gelatinase in ECM degradation by cultured rat mesangial cells. Lovett and co-workers (8,19,

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gest an important role for plasmin in ECM degradation by cultured mesangial cells and confirm that mesangial cells also produce plasminogen activators as previously reported by other laboratories (12, 15). We further documented the production of plasminogen activator by mesangial cells using plasmin .ogen-contain ing substrate gels as previously described by Glass et al. (12) for cultu red rat mesangial ccl 1s (not shown). Taken togeth .er, these results document an important role for the plasminogen activator-plasminogen-plasmin system in ECM degradation by cultured rat mesangial cells.

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21) have reported that cultured rat and human mesangial I

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1I

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rat Fig. 3. Effect of incubation time on ECM degradation by cultured mesangial cells. Cultured rat mesangial cells (100,000/we11) were grown on thin films of 1251-labeled Matrigel (~25,000 cpm/well) in presence of 15 mU of plasminogen. At time indicated, radioactivity released into the cell-free medium was determined. Results are means & SE (in % degradation) of triplicate determinations corrected for triplicate blanks (cells grown on labeled Matrigel in absence of plasminogen for each time period) included on the same 24-well plate. Further details are given in

Fig. 4. Correlation between plasmin activity and by cultured rat mesangial cells. Plasmin activity was free medium obtained from studies presented in plotted against percent ECM degradation. Further

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ECM degradation determined in cellFigs. 2 and 3 and details are given in

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Table 2. Effect of proteinase inhibitors on plusminogen-dependent ECM degradation by cultured rat mesangial cells. Inhibitor

n

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%

6 91+2 a,-Antiplasmin (20 pg/well) 8 963tr3 Aprotinin (108 KIU/well) TIMP-1 (20 pg/well) 10 42+4 8 43+2 o-Phenanthroline (100 wM) 3 2+1 E-64 (10 PM) 3 3+1 Pepstatin A (2.5 rg/well) Values are means rt SE for n determinations based on ECM degradation under identical conditions in the absence of any inhibitors. Cultured rat mesangial cells (100,000 cell/well) were grown for 72 h on thin films of [aH]Matrigel (35,000 cpm/well) in presence of 15 mU plasminogen and the proteinase inhibitor indicated, and degradation was measured as release of radioactivity into cell-free medium. See METHODS for further details. KIU, kallikrein inhibition units.

cells produce the ECM-degrading, matrix metalloproteinase, gelatinase (type IV collagenase). This observation, coupled with the reduction of ECM degradation by ophenanthroline and TIMP-1 (Table 2), inhibitors of gelatinase, suggested that the ECM-degrading metalloproteinase in our model system was gelatinase. Because of the small number of cells used in our studies (100,000 cells/well) we were not able to utilize quantitative gelatinase assays for the measurement of gelatinase activity in medium obtained from cells cultured in either the absence or presence of plasminogen. However, SDS-substrate PAGE (zymography), is a sensitive and commonly used technique for identifying and characterizing proteolytic enzymes (8, 16, 21, 27). We utilized this technique to examine the gelatin-degrading proteinases produced by cultured mesangial cells. As shown in Fig. 5, zymography of cell-free medium obtained from rat mesangial cells grown on Matrigel films in the absence of plasminogen revealed only two closely migrating bands of proteinase activity [relative molecular mass (M,) of -70-72 kDa]. The activity of these bands in the zymogram was completely inhibited by the addition of 10 mM EDTA to the overnight incubation buffer (see METHODS) but unaffected by the addition of aprotinin or cY2-antiplasmin, documenting the metalloproteinase nature of these pro-

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teinases and indicating that these bands represent truncated forms of gelatinase. No bands of gelatinase activity could be detected in the medium obtained from the following: wells containing only Matrigel films, from wells containing Matrigel + plasminogen (no cells), or when 25 pg of Matrigel itself was examined in this same system. In keeping with the previous studies of Lovett et al. (19), we observed little if any gelatinase activity in homogenates of cultured mesangial cells. Zymography of medium obtained from rat mesangial cells grown in the presence of plasminogen revealed an additional band of gelatinase activity (M, - 68 kDa; Fig. 5). The activity of this band was completely inhibited by EDTA and unaffected by aprotinin or cu2-antiplasmin (when present in the overnight incubation buffer, see METHODS), documenting that this band was a metalloproteinase and not plasmin. This band was absent (or very faint) in medium obtained from mesangial cells grown in the absence of plasminogen or in the presence of plasminogen and the plasmin inhibitors, aprotinin or a2-antiplasmin (Fig. 5). Thus these data document the ability of cultured rat mesangial cells to produce gelatinase and, combined with the effects of TIMP-1 and o-phenanthroline on ECM degradation, suggest that gelatinase also contributes to ECM degradation by cultured mesangial cells. In addition, these data indicate an important role for plasmin in the activation of gelatinase (see below). DISCUSSION

The ability of cultured mesangial cells to produce latent gelatinase and, by the action of plasminogen activator(s), to convert plasminogen to plasmin is well documented (8, 12, 15, 19, 21). However, the role of these proteinases in ECM degradation by mesangial cells has not previously been reported. Our data document important roles for the plasminogen activator-plasmin system and gelatinase in ECM degradation by cultured mesangial cells. Mesangial cells grown in the absence of plasminogen fail to degrade ECM (Table 2) despite the presence of gelatinase in the medium (Fig. 5). This observation indicates that, in the absence of plasmin, gelatinase is present in an inactive form, either as a latent proenzyme or as a

Fig. 5. Zymogram of cell-free medium obtained from cultured rat mesangial cells grown on thin films of Matrigel. Cultured rat mesangial cells (lOO,OOO/well) were grown on thin films of ‘s”I-labeled Matrigel (-25,000 cpm/well) under the following conditions: in absence (lane 1) or presence of 15 mU of plasminogen (lane 3); in presence of 15 mU of plasminogen + 20 gg as-antiplasmin (lane 5); or in presence of 15 mU of plasminogen + aprotinin (216 KIU/ml, lane 7). After 72 h, 5 ~1 of cell-free medium were subjected to SDS substrate-gel electrophoresis (zymography) as described in METHODS. Molecular weight standards are present in lane 8. Arrow indicates position of albumin, molecular mass of 68 kDa. Lanes 2,4, and 6 are empty. Further details are given in METHODS.

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ECM

DEGRADATION

gelatinase-inhibitor complex. However, in the presence of plasmin (produced from plasminogen by mesangial cell plasminogen activators) active gelatinase is present as indicated by the ability of gelatinase inhibitors to reduce ECM degradation (Table 2). Thus plasmin appears to play a key role in the activation of mesangial cell gelatinase. Previously published data suggest the following three mechanisms by which plasmin potentially could increase gelatinase activity: direct activation of a latent gelatinase (7, 10, 11); activation of a second latent proteinase, which then directly activates latent gelatinase (25); and inactivation of TIMP (28), the gelatinase inhibitor produced by mesangial cells (21). Our data do not allow us to distinguish among these mechanisms, although Okada et al. (27) failed to observe activation of purified latent synovial fibroblast gelatinase in the presence of plasmin. Although the precise role of plasmin in the activation of gelatinase remains to be determined, our results indicate that ECM degradation by cultured mesangial cells involves a proteolytic enzyme cascade. This cascade is initiated by mesangial cell plasminogen activator(s) , which converts plasminogen to plasmin, which in turn results in the activation of gelatinase (directly or indirectly). Plasmin and active gelatinase then carry out the degradation of the mesangial matrix. It is important to point out that, although Matrigel may contain transforming growth factor (TGF)-P and other cytokines which might alter the relative contributions of plasmin and gelatinase to ECM degradation (see below), such changes would be quantitative in nature and would not alter the major conclusion of our study, which is that ECM degradation by cultured mesangial cells is mediated by a proteinase cascade involving both plasmin and gelatinase. An important aspect of this proteinase cascade is its potential for regulation by multiple mechanisms operating at several levels of metabolic control. For example, at the transcriptional level, a variety of cytokines (e.g., TGF-P, interleukin-1, tumor necrosis factor-a) could alter the expression of these matrix degrading proteinases and/or their inhibitors as has been shown in other tissues (20,26,30) and mesangial cells (l&22). Regulation at the posttranslational level could be effected by specific proteinase inhibitors such as plasminogen activator inhibitors and TIMPs, both produced by the mesangial cells (12,15,21), and acz-antiplasmin, derived from the plasma. Additional points of posttranslational control could include regulation of the activation of latent proteinases and inactivation of proteinase inhibitors such as TIMP, and plasminogen activator inhibitor. arz-antiplasmin, Taken together, these interactions among proteinases, their inhibitors, and various modulators (e.g., cytokines, reactive oxygen metabolites) would establish the net proteinase-proteinase inhibitor balance within the mesangium and determine the overall direction of matrix metabolism, i.e., accumulation or degradation. Agents that decrease the amounts of proteinase inhibitors and/or increase the amounts of active proteinases would result in net degradation of the mesangial matrix. In contrast, agents that increase the amounts of proteinase inhibitors and/or decrease the amounts of active proteinases would

BY MESANGIAL

F1117

CELLS

result in net accumulation of the mesangial matrix. Several independent lines of evidence provide support for the proteinase-proteinase inhibitor balance as an important determinant of mesangial matrix metabolism. Such evidence includes the following: the ability of TGF-p to decrease the production of ECM-degrading proteinases and increase the production of their inhibitors in other tissues (26, 30); in vivo studies by Border and co-workers (3) showing that administration of antibodies against TGF-0 blocked the accumulation of mesangial matrix which occurs in the anti-Thy- 1.1 model of glomerulonephritis; and recent preliminary studies of Lovett et al. (18) demonstrating that, in cultured human mesangial cells, low concentrations (0.1-1.0 rig/ml) of TGF-P increase the production of TIMP (although high concentrations of TGF-P increase gelatinase activity). Although the significance in vivo of the plasminogen activator-plasmin-gelatinase cascade pathway remains to be determined, we feel that it provides a viable working hypothesis for the degradation of ECM by mesangial cells, which should serve as a useful conceptual framework for the design and interpretation of further studies in this area. In addition, similarities in the composition of the mesangial matrix and the GBM, the other major ECM of the glomerulus, suggest that the same proteinase cascade may also be involved in the degradation of GBM as well. We thank Dr. Gillian Murphy (Strangeways Research Laboratories, Cambridge, UK) for providing help and encouragement in the early phases of this work and for critical review of the manuscript, Sue Atkinson (Strangeways Research Laboratories) for help with the early cell culture work, Dr. S. V. Shah (Little Rock, AR) for critical review of the manuscript, and Dr. David Lovett (San Francisco, CA) for helpful comments concerning interpretation of our data. This work was supported by a grant from the National Center of the American Heart Association and a Student Summer Research Fellowship from the American Heart Association, Louisiana Affiliate, to A. P. Wong. Portions of this work have been presented in abstract form at the Annual Meeting of the American Society of Nephrology in Baltimore, MD, November 17-20, 1991, and at the International Symposium on Cell and Molecular Biology of Basement Membranes in Health and Disease, in Airlie, VA, September 20-22, 1991. Address for reprint requests: W. H. Baricos, Dept. of Biochemistry, Tulane Medical School, 1430 Tulane Ave., New Orleans, LA 70112. Received

17 March

1992; accepted

in final

form

29 June

1992.

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