production of three plasminogen activators and an inhibitor in keratinocyte cultures

308 Biochimica et Biophysica A eta, 756 (1983) 308-318 Elsevier Biomedical Press

BBA 21420

P R O D U C T I O N OF THREE P L A S M I N O G E N ACTIVATORS AND AN INHIBITOR IN KERATINOCYTE CULTURES

H E N N I N G BIRKEDAL-HANSEN and ROBERT E D W A R D TAYLOR

Department of Oral Biology" and Institute of Dental Research, Universi(v of Alabama School of Dentisto', Birmingham, A L 35294 (U.S.A.)

(Received October 18th, 1982)

Key words: Plasminogen activator," Plasminogen inhibitor,: (Keratino~Tte)

Keratinocyte function in extraceilular proteolysis was investigated. Keratinocytes derived from rat tongue ventral epithelium were maintained and serially propagated under conditions which support continuous expansion of epithelial colonies but are restrictive to fibroblast proliferation (30-32°C and pH 6.8-7.0). These cultures, and cultures of an established, terminally differentiating keratinoeyte line, also derived from the ventral epithelium of the rat tongue, released substantial plasminogen activator activi~' as visualized by the fibrin-agar overlay technique. In addition, keratinocytes grown directly on 3H-labeled fibrin lysed this substrate in a plasmin-assisted process. The presence of serum modulated the kinetics of the reaction in a manner which suggests that a constant inhibitor tonus serves to contain the proteolytic reaction in the tissues and to prevent a chain reaction. Eiectrophoretic resolution of keratinocyte secretory, proteins and of cell lysates revealed three distinct activators migrating at molecular weights of 48000, 66000 and 95000. The keratinoeytes also manufactured inhibitor(s) of the fibrinolytic reaction mainly directed against the activation step. The inhibitory activity was present in serum-free culture harvest media in quantities sufficient to completely annihilate the endogenous activators.

Introduction

Extracellular, plasmin-dependent proteolysis is a common denominator in biologic processes which involve tissue remodeling or breakdown, and secretion of plasminogen activator, the enzyme which specifically converts plasminogen to plasmin, has emerged as a general regulatory mecha- nism in extracellular proteolysis [1-3]. Data derived largely from in vitro studies have shown that secretion of plasminogen activator is modulated by an exceptionally wide range of local and sys- temic factors [2,4,5]. The epithelial coverings of skin and mucous membranes are rich in plasmin- dependent fibrinolytic activity [6] and there is

Abbreviation: PMSF, phenylmethylsulfonyl fluoride

increasing evidence that such expressions of the epithelial phenotype as migration and terminal differentiation are governed by plasmin-dependent proteolysis. Early studies by Astrup's group [7,11] and by others [12,13] suggested that plasminogen activator production is linked to, and possibly modulated by, the state of epithelial differentiation. Recent support for this idea has emerged from the discovery that nuclear destruction in terminally differentiating keratinocytes and reduc- tion of epithelial tissue components in mammary gland involution are plasmin-assisted processes [13,14]. The precise role of plasmin-dependent proteolysis in expression of the keratinocyte phenotype, however, remains unclear. The recent identification of several distinct basement-membrane-associated proteins and proteoglycans in skin and mucous membranes such as type IV

0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

collagen, laminin and fibronectin [15-18] have generated increasing interest in basement membrane metabolism and in keratinocyte function in modulation of the extracellular environment. A correlation of migration of transformed cells and plasminogen activator secretion has been established [19] and has contributed to formulation of a pathogenic role for plasmin-dependent proteolysis in malignant invasive growth. Interestingly, an invasive physiologic process such as trophoblast im- plantation, also appears to be plasmin-dependent [20]. The search for regulatory keratinocyte functions has been greatly facilitated by the advent of improved techniques for the cultivation and maintenance of keratinocytes in vitro [21-24]. We now report evidence, derived from the use of two complementary epithelial culture systems, of keratinocyte expression of three distinct plasminogen activators and of a fibrinolytic inhibitor.

Experimental procedures

Materials Eagle's Minimum Essential Medium, fetal and

newborn calf sera, trypsin (2.5%), penicillin, and fungizone were from Gibco (Grand Island, NY), gentamicin was from Schering (Kenilworth, N J); culture plastics were Falcon from Becton and Dickinson (Oxnard, CA) or Nunclon manufactured by Nunc (Roskilde, Denmark) and purchased from Vanggaard (Neptune, N J); chick plasma and chick embryo extract were from Difco (Detroit, MI). Dimethylsulfoxide (silylation grade) was from Pierce (Rockford, IL). Plasminogen-free fibrogen was from Poviet Products purchased through Organon Technica (Boxtel, the Netherlands). Each lot was tested for absence of plasminogen by a standard urokinase assay (see below). Human and bovine thrombin, soybean trypsin inhibitor, c- aminocaproic acid, p-phenylguanidinobenzoate, PMSF, agarose LGT and L-lysine were from Sigma (St. Louis, MO). Urokinase was from Koch-Light (Coinbrook Bucks, U.K.) or from Leo' Pharmaceuticals (Copenhagen, Denmark). Aqua- cide II was from Calbiochem (La Jolla, CA). Elec- trophoresis grade acrylamide, N',N'-methylen- ebisacrylamide, N,N,N',N-tetramethylenedia- mine, ammonium persulfate and Triton X-100 were from Bio-Rad (Rockville Center, NY); analytical-

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grade SDS was from BDH Chemicals (Poole, U.K.); Trasylol was from Bayer (Wupperthal, F.R.G.). Gelbond film was purchased from Marine Colloids (Rockland, ME). Rabbit IgG raised against human plasminogen was from Dako (Copenhagen, Denmark) and purchased through Accurate Chemical Co. (Westbury, NY). 3H- labeled acetic anhydride (TR-381) was from Amersham International (Arlington Heights, IL). CNBr-activated Sepharose 4B and electrophoretic molecular weight standards were purchased from Pharmacia (Piscataway, N J). Bovine lenses were from Pel-Freez (Rogers, AR). Human skin fibroblasts (Ro Bel) were from the American Type Cul- ture Collection (ATCC, Rockville, MD). Human and bovine gingival fibroblasts were developed from dissected gingival tissues as previously described [25]. RTK-I rat tongue keratinocytes were from our frozen stock originally donated by Dr. Arne Jepsen (Arhus, Denmark) [21,26].

Keratinocyte cell cultures Keratinocyte cultures were established from rat

tongue explants as outlined by Jepsen et al. [21,26]. Mucosa and underlying tissue from the ventral surface of the tongue of 4-8 week old Wistar rats were cut into 1 x 1 x 1 mm pieces and affixed individually to the bottom of 35 mm plastic petri dishes by means of a chick plasma coagulum. The growth medium was Eagle's minimum essential medium with 20% fetal calf serum, 0.5% dimethylsulfoxide and antibiotics (100 ffg/ml penicillin, 50 /~g/ml gentamycin, 2.5 ffg/ml fungizone) adjusted to pH 6.8-7.0 in 5% CO 2 . Incubation was at temperatures from 29 to 32°C. The medium was changed twice weekly and the outgrowth was monitored by phase contrast microscopy. Fibrob- last contamination was monitored by microscopic surveillance of the perimeter of the advancing epithelial sheets and individual fibroblasts were removed manually by a toothpick. The explant keratinocytes were subcultured by use of a fibroblast feeder layer as originally suggested by Rheinwald and Green [24]. The feeder cells were either commercially available human skin fibroblasts, or fibroblasts derived from human gingival specimens excised during treatment for periodontal disease and grown in minimal essential medium with 10% fetal calf serum and antibiotics [25]. The

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keratinocytes were detached by 0.25% trypsin in Ca2+/Mg 2+ free phosphate-buffered saline containing 2 mM EDTA, and seeded in dishes in which the feeder cells had been grown to subconfluent density at pH 7.4 and 37°C. After seeding of the keratinocytes on the feeder cells the incubation temperature was lowered to 30-32°C and the pH to 6.8 7.0. Under these conditions the human fibroblasts do not proliferate while the rat keratinocytes are capable of clonal growth. Moreover, rat fibroblasts which may grow at the lower temperature fail to attach because of the feeder layer. Alternatively, the keratinocytes were seeded at higher density (105/cm 2) without feeder cells either in normal culture dishes or dishes coated with type IV collagen. Type IV collagen was prepared by limited pepsin digestion of bovine lens capsules, purified by chromatography on DEAE- and CM- cellulose in the native state as described by Gay and Miller [27], and subsequently used to coat the culture dishes. The bottom of each 50 mm dish was covered with 1 ml of a sterile solution of type IV collagen (0.5 mg / m l ) in 5 mM acetic acid. Excess solution was immediately removed by aspiration and the dishes were allowed to air dry at room temperature in a laminar flow hood. RTK-I , a near diploid rat tongue keratinocyte line, was grown at 37°C in minimal essential medium with 10% newborn calf serum and antibiotics as previously described [28]. The population doubling time was 16 18 h and the plating efficiency almost 100% at clonal density. The keratinocytes went through stages of colony expansion to eventually formed sheets with as many as 10-12 cell !ayers reminiscent of authentic stratified squamous epithelium. Serum-free conditioned harvest media of explants, keratinocyte subcultures and RTK-I cultures were collected after incubation with serum- free minimal essential medium for two consecutive 3-day periods. Intracellular or cell-associated plasminogen activator was prepared from lysates of whole epithelial layers detached by brief ex- posure to 1-2 mM EDTA in CaZ+/Mg2+-free phosphate-buffered saline. The cell layer was re- suspended in 0.5% Triton X-100 in the same buffer and homogenized in a Potter-Elvehjem homo- genizer. The insoluble residue was removed by low speed centrifugation and the supernatant was stored frozen at - 2 0 ° C .

Preparation of enzymes and substrates Plasminogen was prepared from outdated,

citrated human plasma by the method of Deutsch and Mertz [29]. The plasma was centrifuged at 10000 × g for 20 min and applied to a lysine-Sep- harose column prepared by the method of Cautrecasas et al. [30]. The unabsorbed fraction was tested for presence of plasminogen by radial immunodiffusion against anti-human plasminogen. Bound material was eluted with 0.3 M ,- aminocaproic acid in 0.3 M phosphate buffer. The eluted peak of plasminogen was thin desalted and the plasminogen concentration was determined spectrophotometrically assuming F l ~ = 19 [29]. ~ 2 8 0

The yield was about 100 ~g plasminogen per ml plasma. Plasminogen was removed from various serum preparations by two successive runs over a lys ine-Sepharose co lumn equi l ibrated with C a 2 + / M g 2 + - f r e e phospha t e -bu f f e r ed saline without addition of exogenous inhibitors. The eluate was restored to the original concentration by osmostically forced dialysis against aquacide and tested for presence of plasminogen by immunodiffusion and for ability to support cional growth of RTK-I cells. Acid treatment to in- activate plasma protease inhibitors (pH 2.0; 30 min; 37°C) was carried out as described by Loskutoff and Edgington [31].

[3 H]fibrinogen was prepared by acetylation with 3H-labeled acetic anhydride. Briefly, the protein was dissolved in Ca2+/Mg2+-free phosphate- buffered s~,line and dialyzed overnight against the same buffer. Labeling was performed by vigor- ously mixing 5 mCi of 3H-labeled acetic anhydride with 50 mg fibrinogen for 30 rain at 4°C in a total volume of 5 ml. The sample was then dialyzed exhaustively against phosphate buffer. The yield was 10 20 ~Ci /mg . No adverse effects on the solubility or clotting properties were observed as a result of the labeling procedure.

Enzyme assays A fibrin-agar overlay technique [32] was used to

demonstrate plasminogen activator production in keratinocyte colonies and in fibroblast-free ex- p l an t s . The cu l tu res were r insed with Ca 2+ /Mgz+- f r ee phospha te -buf fe red saline, drained and covered with a 0.4-0.6 mm layer of fibrin-agar which contained 1% agarose in minimal

311

essential medium, 2 mg/ml of fibrinogen, and 0.1 NIH * U of thrombin. Plasminogen, when used, was added at 20/~g/ml. The fibrin-agar was mixed at 37°C from preheated stock solutions, gelled by cooling to room temperature and then incubated at 37°C for 4-16 h in a humidified atmosphere. The plates were either photographed in dark field illumination or fixed with Clarke's fixative (ethanol:acetic acid, 3:1), air dried and stained w i t h 0 .25% C o o m a s s i e b l u e in an e thanol /water /acet ic acid mixture (9 : 9 : 2) and destained in the same solution.

Growth on -*H-labeled fibrin coated dishes. Twenty-four-well dishes were coated with 3H-labeled fibrinogen (10 /~g/cm 2) and dried for 48 h at 42°C. The fibrinogen was converted to fibrin by incubation for 1 h at 37°C with minimal essential medium containing 5% newborn calf serum which contains the necessary clotting factors. The plates were rinsed with distilled water and again air dried. RTK-I keratinocytes were seeded in serum-free minimal essential medium and 50 # g / m l soybean trypsin inhibitor was added to prevent premature hydrolysis of the substrate. The cells were allowed to attach overnight. The medium was then aspirated and the cultures were washed extensively to remove traces of trypsin inhibitor and supplemented with a minimal essential medium-based medium containing 10% of one of several serum fractions, all prepared from the same batch. These included control fetal calf serum, acid-treated fetal calf serum, or the same preparation depleted of plasminogen by lysine-affinity chromatography. Reconstituted sera were prepared by adding affinity-purified plasminogen at a concentration of 200 /~g/ml. This experimental design provided an opportunity to study the effect of plasminogen, of acid treatment, and of serum as a whole with its complement of protease inhibitors. Aliquots of the medium were aspirated at regular intervals and the released 3H activity was determined by liquid scintillation spectrometry and used to compute the degree 0f substrate cleavage.

Protease activity in solution. Plasminogen activator activity was measured by a modification of the assay described by Unkeless et al. [33]. The assay

* N I H , N a t i o n a l Ins t i tu tes of Hea l th .

was performed in 96-well round-bottom microtest plates, coated with 3H-labeled fibrinogen (10 /zg per well). Incubation mixtures consisted of 1-2 #g plasminogen and 5-50/~l of test sample in a total volume of "75 Izl and a final concentration of 0.05 M Tris-HC1 (pH 8.1) with 0.2% Triton X-100 and 0.5% gelatine. Incubation was at 37°C for 2 h. The enzyme activity was determined by the release of 3H activity to the supernatant during the incubation period as measured by liquid scintillation spectrometry. Controls were buffer blanks with or without plasminogen. Total available counts were measured by incubation with 5 /~g of trypsin. Fibrinolytic inhibitors were quantified by their ability to reduce the catalytic activity of standardized solutions of urokinase and of harvest fluid activators using the plasminogen activator assay described above.

Electrophoretic techniques and zymogram meth- ods. Slab gel electrophoresis in 11% acrylamide was performed as described by Neville [34] and plasminogen activator zymograms were prepared as described by Granelli-Piperno and Reich [35]. The samples were incubated for 1 h at room temperature with sample buffer [34] containing 1-5% SDS. The electrophoresis was carried out at 25 mA for 2½-3 h. Afterward, the acrylamide slab gel was washed in three changes of 2½% Triton X-100 in 50 mM Tris-HC1 (pH 8.1), over a 2 h period in order to remove the SDS, followed by a brief wash with distilled water to remove excess Triton X-100. The slab gel was then carefully overlaid with a fibrin-agar gel with or without (control) plasminogen and incubated at 37°C for 3-16 h. The 0.4-0.5 mm thick fibrin-agar gel cast on Gelbond film was composed of 1% agarose, 2 mg /ml fibrinogen, 0.1 NIH U / m l thrombin and (when present) 20 # g / m l plasminogen. The sandwiched gels were again separated when the lyric band pattern became visible in dark field illumination. The acrylamide and the fibrin-agar gels were stained with 0.2% Coomassie blue dissolved in 7% acetic acid/30% methanol, and destained in the same solvent. Molecular weight determinations were based on the migration pattern of fibrinolytic standards: plasmin (89000), human urokinase (55000, 36000), rat urine (48000) and trypsin (21 000) or on the migration of standard proteins. Moreover, the lysis of fibrin gave rise to small

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stainable peptides which diffused back into the acrylamide slab gel where their rate of migration could be compared with that of ordinary molecular weight standards.

Miscellaneous. Keratinization of epithelial colonies was demonstrated by Rhodanile blue staining after formaldehyde fixation as described by Rheinwald and Green [24].

Results

Keratinocyte explants and their early progeny were used along with an established keratinocyte line (RTK-I) capable of clonal growth in the absence of feeder cells. The tongue explants at 30 -32°C and pH 6.8-7.0 gave rise to continuously growing epithelial sheets with little or no fibroblast contaminat ion (Fig. la). The initial growth rate was slower than at 37°C but this was more than compensated for by the sustained proliferation at the lower temperature over a period of 4 - 6 weeks as previously documented [26]. Propagat ion of ' no rma l ' keratinocytes was accomplished by one of two techniques. Seeding of ( 1 -10 ) -10 2

keratinocytes per cm 2 on a fibroblast feeder layer resulted in clonal growth and a plating efficiency of about 1%. At higher densities (more than 105 cells per cm 2) the keratinocytes were amenable to serial propagat ion in the absence of feeder cells for at least 10 passages. Saturation densities were at (6 -8 ) . 105 cells per cm 2. At this stage the cells formed coherent epithelial sheets with keratinizing squames at the surface. Improvement of the plating efficiency was obtained by coating the culture

dishes with type IV collagen, but the keratinocytes remained unable to establish clonal growth in the absence of feeder cells (Fig. lb).

RTK-I keratinocytes when seeded at clonal density also formed stratified keratinizing colonies as evidenced by the keratin-selective Rhodanile blue staining method (Fig. lc). Colony expansion continued at a rate of 1.5 1.7 population dou- blings per day until the entire surface was covered by a multilayered epithelium.

To visualize keratinocyte secretion of plasminogen activator, fibroblast-free,explants (0.5-1.0 cm 2) and keratinocyte colonies at various stages of development were overlaid with fibrin-agar and incubated at 37°C (Fig. 2a and b). All of the explants and close to 100% of the keratinocyte colonies lysed the fibrin in the presence of plasminogen but not in its absence (Fig. 2c). Indeed, more than 48 h of incubation was required in the absence of plasminogen to detect incipient plasmin- independent substrate lysis (Fig. 2d).

A different design was employed to investigate the ability of keratinocytes to mediate extracellular proteolysis in a plasma enriched environment with

Fig. 1. Rat tongue keratinocyte phenotypes in culture. (a) Fibroblast-free explant grown at 32°C for 3 weeks. Stained with Rhodanile blue; x 1.5. (b) Third-passage rat tongue keratinocytes seeded at clonal density and grown to confluence on a bed of feeder layer fibroblasts. Stained with Rhodanile blue; x 7. (c) Clonal growth of RTK-I keratinocytes. Each colony has a stratified keratinized center. Stained with Rhodanile blue; ×7.

Fig. 2. Plasmin-dependent and plasmin-independent proteolysis in keratinocyte cultures. The cultures were incubated with a fibrin-agar film with (a and b) or without (c and d) plasminogen. (a) Explant keratinocytes, 16 h, (b) RTK-I colonies, 16 h, (c) RTK-I colonies, 16 h, (d) RTK-I colonies, 72 h, Coomassie blue; 2b and c: natural size; d: x 3.

its complement of protease inhibitors. RTK-I cells were seeded at (2 or 8). 105 cells per cm 2 on 3H-labeled fibrin coated dishes and incubated at 37°C in the presence of fetal calf serum or deriva- tives of this product. The seeding densities were chosen to give subconfluent (2.105 cells p e r cm 2) or confluent cell layers (8. 105 cells per cm2). When grown in the presence of acid-treated or untreated control fetal calf serum the cells caused a time-dependent, initially linear, release of radioactivity (Fig. 3), whereas parallel cultures, enriched with a plasminogen-depleted fetal calf serum failed to lyse the substrate. Addition of affinity- purified plasminogen (20 / tg /ml final concentration), however, restored the ability of the serum to support fibrinolysis. These results demonstrate the plasmin dependence of extracellular proteolysis mediated by keratinocyte colonies in a serum-enriched environment. It is of note that fetal calf serum with its fibrinolytic inhibitors supported extracellular proteolysis and that inactivation of endogenous serum protease inhibitors by acid treatment had little effect on the rate of substrate cleavage. In order to visualize the modulating ef-

3 0 -

25 -

20-

15-

¢n ,'7 I0-

5 -

2- I I I I I I I I

I 2 3 4 5 6 T 8 TIME, hr

Fig. 3. Plasmin-dependent fibrinolysis in RTK-I keratinocyte cultures. The cells were seeded on dishes coated with [ s H]fibrin and incubated for the time indicated in the presence of various serum preparations. C), medium containing 10% fetal calf serum; e, same, except acid treated (pH 2.0; 30 min) serum was used instead of untreated; A, medium supplemented with plasminogen-depleted, acid-treated fetal calf serum; zx, same supplemented with 20/~g/ml of plasminogen.

313

fect on extracellular proteolysis of serum protease inhibitors, a similar experiment was conducted in which parallel cultures were incubated with 20 /~g/ml of affinity-purified plasminogen in the absence or presence of plasminogen-depleted fetal calf serum (Fig. 4). The ensuing fibrinolysis revealed that plasmin-dependent degradation of fibrin occurred both in the presence and absence of serum, but in two kinetically distinct processes. In the absence of serum, a rapidly accelerating, almost exponential, reaction was observed as a result of the cumulative effect of activated plasmin molecules. In the presence of serum, the initial velocity was not much different but the release of radioactivity with time approached linearity and hence revealed a surprisingly constant rate of reaction.

Despite ample evidence of plasminogen activator production as revealed by the overlay procedure, harvest media of explant and of RTK-I keratinocyte cultures often showed very little activity with the standard fibrin-plate assay, whereas a number of fibroblast lines which were less respon- sive with the overlay method showed ample activity in the fibrin plate method (Table I). Moreover,

60. /

I0"

o I 2 :3 4 5 6 7 8

TIME, hr

Fig. 4. Modulation of extracellular proteolysis by plasma protease inhibitors. RTK-I keratinocytes (either 2.105 (©, z~) or 8- 105 (e, A) cells per cm 2) were seeded on fibrin-coated dishes and incubated with the different serum preparations for 8 h. Fibrinolysis was measured at various time intervals during the incubation. Serum-free cultures were replenished with 20 # g / m l plasminogen. (A, zx) = + fetal calf serum; (O, C)) = - Serum.

50-

4 0 -

30" z

IIl ,'7 2O-

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TABLE I

PLASMINOGEN ACTIVATOR ACTIVITY IN KERA- TINOCYTE AND FIBROBLAST CULTURE FLUIDS

[3 H]fibrinogen was dispensed in microtest plates (10 fig/well) and converted to fibrin with 5% newborn calf serum. The wells were thoroughly washed and 2 /tg/well plasminogen, assay buffer (50 mM Tris (pH 8.1) with 0.2% Triton X-100) and 25 ,ul culture fluid as indicated below were added in a total volume of 75 ffl. Controls contained either 2 ~g plasminogen or 5 fig trypsin in assay buffer to give blank and total counts. Incuba- tion was at 35°C for 2 h. Activity released to the supernatant was measured by liquid scintillation spectrometry.

Sample Fibrin Fibrin lysis lysis (c.p.m.) (% of

total)

2 fig plasminogen, control 150 4.1 Trypsin, total count 3 700 100.0

Culture harvest medium: Bovine gingival fibroblasts 3619 97.8 Human periodontal ligament fibro-

blasts 259 7.0 Rat keratinocyte (RTK-I) 259 7.0 Rat keratinocyte (acid-treated) 245 6.6 Rat epithelial explant 155 4.2

estimates of the assay sensitivity in our hands revealed that as little as 20 pg of a standard urokinase was detectable. This suggested the possi- ble presence of a fibrinolytic inhibitor, and further studies were designed to verify its existence. Fibroblast harvest media containing an equivalent of 2. 10 2 Plough units of activator activity were incubated with increasing amounts of epithelial harvest medium and the resultant fibrinolytic activity was measured (Fig. 5). The keratinocyte harvest medium reduced the fibrinolytic activity in a dose-dependent manner consistent with the presence of an inhibitor. Inhibitory activity was also detected when urokinase rather than fibroblast or keratinocyte activators were used (Table II). In an at tempt to locate the site(s) of inhibition, urokinase-activated plasmin was incubated with fibrin-coated dishes without plasminogen under conditions (37°C, 120 min) which were chosen to mimic the standard plasminogen activator assay by liberating 20-25% of the total [3H] activity. Keratinocyte harvest media were only slightly in-

_ I 0 e- l-- z o,oo o 801 \

I \ \ \

n- 20q \

I - 5 10 15 20 25 < EPITHELIAL CULTURE FLUID, FI

Fig. 5. Inhibition of the fibrinolytic reaction by RTK-I keratinocyte harvest media. 25 #1 aliquots of a fibroblast plasminogen activator preparation (2.10 2 Plough Units) were mixed with increasing amounts of serum-free RTK-I harvest medium and resultant plasmin-dependent proteolytic activity was measured by the [3H]fibrin microtestplate assay.

TABLE II

INHIBITION OF UROKINASE AND PLASMIN BY EPI- THELIAL CULTURE FLUID

Plasminogen activator assay performed as explained in Table I except that plasminogen was omitted in the plasmin inhibition experiments. Buffer blank of 3% of total was subtracted. Incubation was at 35°C for 2 h.

Sample Fibrin lysis Inhibi- tion

(c.p.m.) (%) (%)

2 fig plasminogen, control 43 0.9 Trypsin, total count 4578 100.0

0.10 Plough Units urokinase 1007 22.0 0 Urokinase + keratinocyte

(RTK-I) culture fluid 127 2.8 87.4 Urokinase + explant culture

fluid 45 1.0 95.5

Fibroblast activator 1 093 23.8 0 Fibroblast activator + kera-

tinocyte culture fluid 47 1.0 95.7

20 ng plasmin 1 153 25.2 20 ng plasmin +

keratinocyte culture fluid 855 18.6 26.2

40 ng plasmin I 808 39.5 40 ng plasmin + keratinocyte

culture fluid 1 431 31.3 20.8

hibitory (20-30%) toward plasmin but completely abolished the overall fibrinolytic reaction which suggests that the inhibitor is more effective at the activator than at the effector (plasmin) level.

Electrophoretic resolution of keratinocyte harvest media proteins followed by fibrin-agar gel overlay revealed three distinct activators migrating with apparent molecular weights of 95 000, 66 000 and 48000 (Fig. 6). Parallel fibrin-agar gels prepared without plasminogen showed no lysis in the same incubation period (3-16 h). Each of the activators occasionally resolved into two closely spaced bands. Among different preparations, there was a considerable variatoin in the relative inten- sity of the three major activator bands. Occasion- ally a weak fourth band (29000) believed to be a breakdown product of the 48000 activator was observed. Survey of a large numbre of different keratinocyte harvest media did not show a sys-

125K

90K

75K

60 K

50K

40K

I 2 3 4 5 6 7 8 9 1 0 1 1 Fig. 6. Fibrin-agar overlay of electrophoretogram. Keratinocyte harvest media proteins and Triton X-100 extracts were resolved by 11% acrylamide slab gel electrophoresis and overlaid with a fibrin-agar gel supplemented with plasminogen. During the incubation the activator diffused into the fibrin-agar gel and activated the plasminogen which in turn cleaved the fibrin and gave rise to visible lysis zones. Lane 1: Human plasmin (89000) activated by human urokinase (55000); lane 2: Rat urine (48000); Lane 3: Rat plasma; Lane 4: Explant harvest medium; lane 5: Third-passage keratinocyte harvest medium; Lane 6: Third-passage keratinocyte lysate; Lanes 7-9: RTK-I harvest media collected under identical conditions; Lanes 10 and 11: RTK-I lysates.

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tematic change from one form to another. Triton X-100 extracts of keratinocyte lysates contained the same three activator bands and again in vary- ing proportions (Fig. 6). The continuous keratinocyte line, RTK-I , had retained the ability to secrete all three activators and no apparent and systematic change in the relative activities of these distinct activators was evident when compared to harvest media and cell lysates prepared from explant keratinocytes.

Discussion

This study has shown that rat tongue keratinocytes express three electrophoretically distinct plasminogen activators in primary culture and continue to do so even after several years of continued cultivation. A variety of mammalian cells produce activators which can be classified in two main groups on the basis of immunologic reactiv- ity and molecular weight [36-39]. The urokinase- type activator from rodents migrates with an apparent molecular weight of 48 000 (55 000 in humans, [38]) and is inhibited by antibodies raised against urokinase, the plasminogen activator present in urine. It is often associated with its major breakdown product (29000 in rodents; 35000 in humans) which has retained catalytic activity [36]. The other major group of activators (vascular activators) fails to react with anti-urokinase IgG. Its predominant component, a 70000-75000 enzyme [35,36,39], is produced in large quantities by endothelial cells and is believed to mediate throm- bolytic control in the vascular system [40,41]. De- spite the difference in the molecular weight estimates we assume that the enzyme which migrates in our system as a closely spaced double band at 66000 and 62000 is identical to the 'vascular ' activator. Previous investigations have provided strong immunologic evidence that the urokinase and vascular activators are separate gene products [38,42,43]. A third activator (95 000-105 000) which is immunologically closely related to urokinase has recently been identified in several mesenchymal and epithelial cells [39,44-46]. It has variously been interpreted as a distinct activator [39], a precursor of urokinase [45], prekallikrein a n d / o r factor XlIa [45] and plasmin [35]. Based on the m o l e c u l a r weights we a s sume that our

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95000-100000 bands correspond to this high molecular weight human activator. The signifi- cance of secretion of a multitude of activators by keratinocytes (and other cells) remains unknown, since specific functions have not yet been assigned to the different activators with any degree of cer- tainty. The secretion of plasminogen activator(s) in culture, however, suggests that keratinocytes utilize plasmin-dependent proteolysis in the modulation of the extracellular compartment, either in connec- tion with cell movement/cell migration, basement membrane metabolism or terminal differentiation.

The employment of secretory proteases in the modulation of the extracellular compartment natu- rally raises the question of how control is maintained over secretory products outside the cells. The idea that plasminogen activator is associated with the plasma membrane has gained some experimental support [47-49]. Although the data is difficult to interpret, this hypothesis has several attractive aspects in explaining the ability of neu- tral secretory proteases to effect extracellular proteolysis in a plasma-enriched environment with its high concentrations of protease inhibitors, c~2-mac- roglobulin, al-antitrypsin and c~l-antiplasmin. A primary or secondary binding of plasminogen activator to the plasma membrane, however, is not necessary to explain the sustained extra-cellular proteolysis revealed by Fig. 3. The constant inhibitor tonus provided by plasma dampens the accel- eration of the reaction by continuously capturing newly activated plasmin molecules but does not prevent it from occurring. Each plasminogen molecule is protected from elimination in the zymogen state but once activated it binds either to a substrate or to an inhibitor molecule. In the latter case it becomes eliminated. Molecules bound to the substrate later dissociate from the cleavage products and once more face the possibilities of encountering either an inhibitor or a substrate molecule. The stochastic probability of a given activated enzyme molecule encountering and cleaving the substrate depends on the relative concentration of competing inhibitor and substrate. In a non-replenished experimental system all plasmin molecules are eventually captured and the overall (gross) reaction stops. On the other hand, continuous secretion of low levels of the activator results in continuous activation of plasmin molecules and

in a steady-state overall reaction fueled by the balanced generation and capture of plasmin molecules. Ideally, the reaction rate approaches linearity until plasminogen or substrate concentration become rate limiting. This is indeed the case in serum supplemented cultures (Fig. 4) before the reaction eventually levels off, most likely because of exhaustion of plasminogen. These findings per- mit some general considerations regarding the spa- tial relations between effector cell and substrate matrix. The probability of an activated plasmin molecule becoming captured increases linearly with distance. The presence of excess plasmin inhibitors, therefore, limits the proteolytic effect to the immediate vicinity of the cell surface whether the activator is membrane-associated or not. The requirement for proximity is met through compartmentalization of the extracellular space. Com- ponents targeted for breakdown are brought in close contact with the cell membrane. One may assume that the breakdown of fibrillar structures such as fibrin and collagen which, by nature of their infinite size, cannot be phagocytized before they are cleaved, requires compartmentalization of the extracellular space and possibly membrane contact. In avascular tissues such as cartilage [50] endogenous tissue inhibitors maintain a tonus of inhibitory activity which serves a dampening function similar to that of serum anti-plasmins and there is increasing evidence that these specific inhibitors contribute to the regulation of extracellular proteolysis [51-53]. This study provides evidence for the existence of hitherto unrecognized keratinocyte inhibitors capable of blocking the fibrinolytic reaction. An inhibitor with properties similar to the one detected in keratinocyte cultures was recently described also in cultures of endothelial cells derived from aorta [54]. Smokovitis and Astrup [55] investigated the distribution of fibrinolytic activity and of inhibitors of the fibrinolytic reaction in a number of tissues by a histochemical screening technique but found little or no inhibitory activity in squamous epithelium. J~rvinen et al. [56] identified and purified a truly epidermal protease inhibitor, but its activity was limited to thiol-proteases. The precise role of endogenous epithelial inhibitors is not clear, but it is reasonable to assume that they participate in the regulation of extracellular proteolysis either within

t h e e p i t h e l i u m or a t t he e p i t h e l i o m e s e n c h y m a l in-

t e r p h a s e .

Acknowledgements

T h i s p r o j e c t w&s s u p p o r t e d b y g r a n t s f r o m t he

N a t i o n a l I n s t i t u t e o f D e n t a l R e s e a r c h ( D E 02670

a n d D E 05817) a n d b y a gif t f r o m P r o c t e r a n d

G a m b l e . W e are g r a t e f u l to Dr . K e l d D a n o fo r h is

c o n t r i b u t i o n to t h e m e t h o d o l o g y u s e d in th i s s tudy .

T h e ski l l fu l t e c h n i c a l a s s i s t a n c e of Ms . R o s a l y n

P ie rce a n d Mr . R o d n e y M. R o b i n s o n is a c k n o w l -

edged .

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production of three plasminogen activators and an inhibitor in keratinocyte cultures

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