Tissue plasminogen activator-induced secretion of type-1 plasminogen activator inhibitor in cultured human fibroblasts

THROMBOSIS RESEARCH 55; 619-625, 1989 0049-3848/89 $3.00 t .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.

TISSUE PLASMINOGEN

ACTIVATOR-INDUCED

ACTlVATORlNHlBlTORIN

SECRETION OF TYPE-l PIASMINOGEN

CULTURED HUMAN

FIBROBLABTS

T. M. Reilly. G. Greenplate and P. B. M. W. M. limmermans E. 1.du Pont de Nemours and Company, Medical Products Department, Cardiovascular Diseases Section, Experimental Station, P.O. Box 8C4C0.Wilmington, DE 19880(34oo

(Received 16.3.1989; accepted in revised form 15.6.1989 by Editor D. Galanakis)

ABSTRACT

Tiiue plasmlnogen activator (t-PA) regulation of type-l plasminogen activator inhlb itor (PAI-1) Secretion by WI-38 human fibrobla~ts wcminveStigated utiU?Inga specitlc ELIBA to measure PAI- antigen levels. Incubation of confluent fibrobla~t~wtth t-PA In serum-free medium resulted in a concentration and ttme-dependent Increase in the secretion of PAI-1, with the maximum response (a two-fold Increase) observed following a 4 hour incubation with 0.1 @I t-PA. This t-PA effect was reduced by cycloheximlde, suggesting that the involvement of specific protein Synthesis, and by inhibitors of t-PA’s enzymatic activity, suggesting a requirement for a free catalytic site. The t-PA effect could not be attributed to endotoxin contamincrtlon. Increased levels in cellassociated PAI- were abo detected following incubation of fibroblasts with t-PA. This previously undescribed effect, although Seen at relatively high concentrations of t-PA, may have important biological implications.

Tissue plasminogen activator (t-PA) is a serlne protease which catalyzes the convenlon of the Inactive precursor plasmlnogen Into plasmin. the enzyme responsible for the dlsSolutlon of flbrtn clots (19). The principal inhibitor of t-PA has been ldentlfted as type- 1 plaminogen activator lnhibltor (PAI-1). a member of the superfamily of serine protease Inhibiton or serpins (34). PAI- activity in plasma Is Increased in conditions associated with acute-phase-related responses, such as endotoxemla (!51, and may represent a riskfactor for myocardial lnfarctlon ( 6 1. In addition. PAI- ln the extracellular matrix may also be important in the regulation of a number of extravascular physiological and pathological processes (7.8). Therefore, modulatlon of PAI- levels has Important blological lmpllcatlons. PAI- has been MentRIed in many different cell tVpeS including platelets (9). endothelial cells (10.11). hepatocytes (12) and fibroblasts (7). Recent studies have identtfied a number of modulators which enhance cellular PAI- 1 activity including llpopolysaccharlde 0 (5). thrombin (13). lnterleukirr 1 (14). tumor necrosiS factor (15) and transfarmlng growth factor-C (16). The present study extends these previous flndlng by describing the stimulating effect of t-PA Itself on PAI- release by cultured WI-38 human lung flbroblasts.

Key words: type 1 plasminogen activator inhibitor. fibroblasts 619

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Materi& Human one-chain t-PA. provided in a solution of 1 M NHqHC03, and the low molecular weight form of urokinase (u-PA) were from American Diagnostica. Plasminogen activator inhibitor-l, also from American Diagnostica, was activated using treatment with SDS as previous&’described (17). Cycloheximide. Polymyxin B and lipopolysaccharide were from Sigma. Cell The human lung fibroblast cell line, WI-38, was obtained from the American Type Culture Collectlon. Cells were maintained in Eagle’s minimal essential medlum (MEM) containing 10% fetal Calf serum. Confluent monolayer cultures in 24-well tissue culture plates (7.5 x lo5 cells per well) were washed two times with MEM, and incubated at 37°C for various times in 200 fl of MEM containing either t-PA or an appropriate dilution of 1 M NHqHC03. Other components were added as indicated in the text. FollowinQ incubations, cell supematants were collected while IySateS were prepared by washing cells three times quickly with MEM, adding 5QOflof 0.5% Triton X-100 to each well, agitating the plates on a shaker for 5 min at room temperature, collecting the material and centrifuging for 5 min at 4“ at 1UZlDxg to pellet insoluble material. Supematants and lysates were stored frozen ot -20” C until tested in the PAI1 EUSA. PAI_ Cell-secreted PAl-1 from supematant fractions and cell-associated PAI- from lysates was measured using a PAI- specific ELISA (American Diagnostica). Data is expressed In terms of nQ/ml. Dther k&~ratorv methads. Protein content of cell supematant fractions was determined by means of the BIO-RAD protein assay utilizing bovine serum albumin as the standard. Endotoxin content of t-PA preparations was determined with a pyrogen testing kit from Whittaker Bloproducts. Enzymatic activity of t-PA was determined by means of a amidolytlc assay employing the chromogenic substrate S-2251 according to the manufactureh instructions (Kabi Viirum).

Prior to measuring PAI- antigen from fibroblasts, we characterized the ELISA for its ability to detect PAI- complexed with t-PA. SDS-activated PAI- was incubated for 1 h at 37°C in the presence or absence of an equivalent amount of t-PA, and both PAI-1 antigen and t-PA enzymatic activity were determined in each sample. As shown in FIG. 1, the two curves generated in the PAI- EUSA by measuring antigen content in dilutions of each sample were nearly superimposable. FOlIOWIng lncubatlon of the t-PA with PAI-1, a 68% loss in the serine protease’s enzymatic actlvtty, as measured In an amidolytic assay, was noted. These results indicate that the ELISA is capable of detecting PAIunder conditions where its compiexing to t-PA can be verified through loss of t-PA’s enzymatic activity. Therefore, the EUSA was suitable for measuring PAI- from cells incubated with large concentrations of t-PA, where complexes may be expected to form.

o.o! . 0.0

I

0.4

.

I

0.8

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1.2

.

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PAI- (nglml) FIG 1 P ISA q&&~n of PAl-1 In the m of t-PA, PAI- (40 nQ/mDwas incubated in the absence (0) or presence @) of an equivalent amount of t-PA for 1 h at 37°C. Standard curves were generated by measuring the PAl-1 content In a series of dilutions made from each sample.

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To determine whether t-PA affects the secretion of PAi- by WI-38 fibrobiasts. conditioned medium was collected at various times from ceiis incubated continuously for 24 h in the presence or absence of 0.1 p&l t-PA, and assayed for PAi- content. The t-PA treatment did not affect ceil viability, as assessed by trypan blue exclusion. nor did it otter the integrity of the monolayer. Rbrobiasts produce measurable amounts of PAi-1 in their supematant medium, and this level increased siightiy during the course of a 24 h incubation (FIG. 2). inclusion of t-PA in the medium resulted in an approximate doubling of the secreted PAI-1, evident by 4 h and lasting for the entire 24 h duration. Elevated PAI- levels were aiS0 observed in extracts prepared from t-PA-treated ceils (FIG. 2). These results indicate that both secreted and cell-associated PAi-1 are aitered by incubation with t-PA. 20-

Cell

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Cell

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and as&&@d PAi-1, Confluent monoiayers were incubated for the indicated times in the absence (01 or presence @> of 0.1 PM t-PA. Supematant and iysate fractions were then prepared and measured for PAI- 1. Each point represents the mean f SEM of three representative cuitures. FIG.2

Additional studies to characterize the t-PA effect on ceil-secreted PAi- were performed by assaying medium conditioned by an 18 h incubation with fibrobiasts. Serum. at concentrations ranging from 0.1 to 10%. increased the basal levels of PAi- without increasing the stimulatory effect of t-PA (FiG. 3). 40 -

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PG. 3 Fff[ three representative cuitures.

or orme

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the t-PA_ of PAI_1 bv raJ of t-PA, Each point represents the mean f SEM of

The addition of increasing concentrations of t-PA to WI-38 ceils resulted in a dose-dependent increase in secreted PAi- levels (FIG. 4). No effects were observed with 0.001 PM t-PA while near maximum ievets of PAC1 (a two-fold increase) were observed with 0.1 &I t-PA. Total proteln

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concentration was similar in the conditioned medium from control cells (60 f 3 Pg/ml) and from cells treated with 0.1 pM t-PA ( 59 ? 9 Pg/mB, suggesting that t-PA-stimulated release was specitlc for PAI-1,

0

0.001

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FIG. 4 Fffeedpf increg&a t-PA wn PACT secrmn represents the mean f SEM of three representative cultures.

bv WI-38 fibrc&jg$& Each point

The enhanced release of PAI- by fibroblasts cultured with t-PA was further characterized in additional experlments where various test agents were included in the incubation medium. Cycloheximide. which by itself had little effect on PAi- release, attenuated the t-PA effect on cellular PAIsecretlon (Table 1) suggesting involvement of new protein synthesis in this process, This concentration of cycloheximide (25 Pg/ml> has been shown to be effective in blocking cellular protein synthesis (18) in other cell lines. Endotoxin at low levels is known to increase cellular secretlon of PAI- in endothelial cells (19) . Although it seems unlikely that endotoxin concentration of t-PA (less than 3 EU/mg according to in house assays 1 would have been responsible for the PAI- increase in fibroblasts, we investigated this possibility in experiments with Polymyxin B and lipopolysaccharide (LPSI. Poiymyxln B has been shown to prevent the induction of PAI- by LPS in human endotheliil cells (2021). In fibroblasts. 10 Pg/ml of LPS induced an approximate twofold increase in secreted PAI-1. Polymyxin B, at 30 pg/ml, reduced this effect of LPS without modifying the PAI- increase induced by 0.1 P.Mt-PA CTable1). To investigate whether t-PA enzymatic activity was required for the enhanced PAI- secretion by fibroblasts, we utilized two different agents: the modifled tripeptide. D-Phe-L-Pro-L-Argchloromethylketone, which inhibits the enzymatic activity of t-PA by irreversibly alkylatlng an active site histldine (22). and a specific t-PA monoclonal antibody. CD2, which abolishes the catalytic activity of the serine protease, probably by binding to an epitope at or near its active site (23). 0.1 @l t-PA was incubated with 100 $vl of the peptMe for 1h at 37” C. a treatment which completely neutralized the enzymatic activity of t-PA, the mixture dialyzed overnight in MEM to remove free peptide. and the resulting complex incubated for 18 h with fibroblasts. As shown in Table 1, PAI- levels in the conditioned medium from such cells were considerably lower in comparison with those observed In medium from t-PA treated cells. The tripeptide by itself had a small stimulatory effect on PAIsecretion by fibroblasts (data not shown). Similar results were observed following incubation of cells with t-PA that had been complexed to CD2 by a 1h, 37% incubation wtth a lOO-foldmolar excess of the antibody. t-PA complexed to monoclonal antibody AE5. which blnds to the serine protease wtth a similar affinity as CD2 without impairing its catalytic activity (23). differed very little from free t-PA In Its ability to induce a PAI- secretion from flbroblasts. Neither antibody by themselves induced a significant change in PAI- secretlon (data not shown). These results suggest that this t-PA-mediated effect upon fibroblast PAI- is dependent upan a free acttve site. We also evaluated the ability of U-PA, a plasminogen activator with similar eruymatlc actMty as t-PA. to induce a PAI- effect in flbroblasts. As with t-PA, incubation of fibroblasts with 0.1 t&l u-PA induced an approximate doubling in the secretion of PAi- (Table 1).

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_ ofv_anPAI-1 seq&jpnbvWl38f&fgj&& ----___-_____-__-___-------------~-~~~~~~~~~~~~~~~~~~~~~-~ n Tredment ------------------------____-_---------------------------None t-PA (0.1 PM) Cyclcheximide (25 Pg/ml) t-PA + Cycloheximide Lps (10 erg/ml) Polyrnyxin I3 (30 Pg/ml> LPS + Polymyxin B t-PA + Polymyxin 6 t-PA - (Phe-Pro-Arg-CMKI t-PA - CD2 t-PA - AE5 u-PA (0.1 PM)

12 12 9 9 3 : 3 6 !

623

Pfil

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11.2f 0.9 22.0+ 1.2 10.5f 2.9 12.5* 2.8 24.Oi 2.3 14.1+ 2.5 13.5+ 2.2 22.0+ 3.0 15.5+ 1.0 13.6& 1.9 21.7* 0.7 213* 1.1

Values are expressed as mean f 3EM. ‘n’is the number of independent measurements for each treatment.

To examine the effects of t-PA incubation on PAI- production and secretlon by WI-38 human flbroblasts, a PAI- immunoassay rather than an activity assay, was utlllzed to prevent possible misinterpretations due to the presence of large concentrations of t-PA or to other fibrobk%t inhibitors. Fibroblast incubation with t-PA resulted in a concentration and time-dependent increase in the secretion of PAI-1, with a two-fold increase in the PAl-1 level observed following a 4 h incubation with 0.1 @I t-PA. Celkissocltied PAl-1 was also found to increase following incubation of flbroblasts wttht-PA. The specificity of the t-PA effect on fibroblast PAI- secretion was addressed In a number of different experiments. This effect did not require the presence of serum, but was blocked by cyclohexlmkte suggesting the involvement of new protein synthesis, The PAI- increase could not be attrtbuted to endotoxln contamination of the t-PA. Inhiblton of t-PA’s enzymatic acthrlty blunted the sertne protease’s effect in fibroblasts suggesting that catalytic activity is required for enhanced PACl production. This concept is supported by our observation that u-PA. which has a similar enzymatic activity as t-PA, also enhanced PAL1 secretion by flbroblasts. A number of reports have shown that cellular PAI- levels are subject to regulation by different modulators including thrombin (13). H-1 (14 1. TNF(15 1, and LPS (19 1. while many of these studies were performed with human endothelial cells, WI-38 ftbroblasts also represent a useful model far studying PAI-1 regulatlon. For example, these cells, which produce both t-PA and PAl-1 (161, respond to transforming growth factor- B with elevated mRNA leveb for PAl-1 (24) as well as wtth enhanced PAt-1 secrehon and deposit&n into the growth substratum (16). Abroblast PAl-1 has been imptlcated as important. nat onjy in madujattng tlbrtnolvtlc actjvity. but also in protecttng the extra&l&r matrix from proteotysis by plaJminagen activators (162425). Regulation of fibroblast PAI- production and secretion apparently has important biologIcal consequences. Recently. additlon of PAS to the extracellular matrix of human endotheltal cells or flbrcbtasts has been shown to release PA hhlbltar into the supematant (26). Our stud& with cycloheximlde suggest that the much of the PAC1 ln medium from t-PA-treated WI-38 ftbrobk&s represents newly synthesized Protein and not matertal released from the matrix. In other expertments. we were unable to detect a signtflcant increase in PAt-1 antigen, as measured by the ELlSA. folbwiig incubation of Wl-35 mat&es with t-PA.

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The effects of t-PA on WI-38 PAI- levels are consistent wtth an earlier report where induction of secreted PAI- by human hepatoma cells (Hep 62) was observed in response to t-PA incubation (27). However unlike the present study with flbroblasts, it was concluded that t-PA in Hep G2 cells is acting on a post translational step of PAI- processing since PAI-1 mRNA and protein synthesis were not affected by t-PA incubation. Despite possible mechanistic differences. both studies suggest that t-PA at high concentrations, such as those administered as thrombolytlc therapy to patients with myocardial infarction, has the capacity to increase cellular secretion of PAI-1.

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