The cellular target for the plasminogen activator, urokinase, in human fibroblasts - 66 000-dalton protein

Biochimica et Biophysica .4 cta, 720 (1982) 141 - 146

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Elsevier Biomedical Press BBA 11015

T H E CELLULAR T A R G E T FOR T H E P L A S M I N O G E N ACTIVATOR, UROKINASE, IN H U M A N F I B R O B L A S T S - 66000-DALTON P R O T E I N J O R M A KESKI-OJA and A N T T I V A H E R I

Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29 (Finland) (Received August 31st, 1981)

Key, words: Urokinase; 66 O00-Dalton protein; Human fibroblast," Cellular target," Plasminogen activator

The effects of urokinase, the plasminogen activator of human urine, on the isolated substratum-attached pericellular matrices of cultured human lung fibroblasts were studied in serum-free conditions. Pericellular matrices were prepared from dense cultures of human lung fibroblasts after labelling of the cultures with radioactive glycine. Extraction of the cultures with sodium deoxycholate and hypotonic buffer gave a reproducible pattern of polypeptides when analyzed in polyacrylamide gels. The isolated pericellular matrix was subsequently exposed to affinity chromatography-purified urokinase. Urokinase affected a 66000-dalton protein but none of the other matrix polypeptides. The appearance of a 62000-dalton protein that remained attached to the matrix was seen concomitantly suggesting that it was derived from the 66000-dalton protein. The 66000-dalton protein is the first known cellular target for urokinase.

Introduction

Plasminogen activators are specific serine proteases that are produced by malignant adherent (substratum-attached) and several types of nonmalignant cells in culture [1-3]. Urokinase is a plasminogen activator isolated from human urine. Some plasminogen activators isolated from tissues resemble urokinase in their properties while others have been classified as tissue or non-urokinase-like plasminogen activators [4-6]. The only known natural substrate for plasminogen activators is plasminogen, the zymogen precursor of plasmin. Plasmin has a broad spectrum of substrates and is considered to regulate extracellular proteolysis and functions such as cell migration and invasion [7]. N o cellular targets for plasminogen activators have been identified thus far. Different proteinases can stimulate the growth and alter the morphology of cultured cells [8-10]. The morphological changes presumably result from the effects of the proteinases on the proteins of the 0167-4889/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

plasma membrane and the pericellular matrix. However, there is no direct evidence of a causal relationship between cell surface changes in cultured malignant cells and proteolysis. Specifically, the role of plasminogen activators in malignant transformation is not understood. Using plasminogen activator-specific proteinase inhibitors, Quigley [11] found that plasminogen activator was responsible for the morphological changes observed in tumor promoter-treated fibroblasts. To study whether plasminogen activator has direct effects on cells we affinity-purified human uro_kinase and carried out experiments to analyse its effects on cultured lung fibroblasts and their cell-free pericellular matrices. The latter were prepared using sodium deoxycholate in hypotonic buffer in the presence of proteinase inhibitors-. Extraction of cultured radioactively-labelled cells with sodium deoxycholate [12,13] gave a reproducible pattern of matrix polypeptides. We exposed isolated pericellular matrices to affinitypurified urokinase and found cleavage of a 66000-

142 dalton polypeptide enriched in the matrix. The appearance of a 62000-dalton protein that remained attached to the matrix was seen concomitantly suggesting that it was derived from the 66000-dalton protein. The 66000-dalton protein is the first known cellular target for the plasminogen activator urokinase.

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Materials and Methods

Chemicals. Highly purified h u m a n thrombin (5000 N I H U / m l ) was purchased from Sigma Chemical Co, St. Louis, MO; aprotinin (Trasylol ®) from Bayer, Leverkusen, F.R.G.; plasmin (20 C U / m l ) from Kabi, Stockholm, Sweden and leupeptin from Serva, Feinbiochemica, Heidelberg, F.R.G.

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Purification of urokinase and plasminogen. tdrokinase was purchased from CalbiochemBehring Co. (La Jolla, CA) and it was purified by affinity chromatography over p-aminobenzamidine-agarose in 0.1 M sodium phosphate buffer (pH 7.0)/0.4 M NaC1. The bound urokinase was eluted by 0.1 M sodium acetate buffer (pH 4.0)/0.4 M NaC1, and dialyzed against phosphatebuffered saline [14]. The activity was quantitated in agarose gels containing plasminogen and casein [15]. No caseinolysis was observed if plasminogen was omitted. Plasminogen (15 C U / m g ; Kabi, Stockholm, Sweden) was affinity-purified over lysine-agarose (Pharmacia, Uppsala, Sweden) in 0.1 M sodium acetate b u f f e r / 0 . 4 M NaC1, p H 7.0. Bound plasminogen was eluted with 0.2 M c-aminocaproic acid and chromatographed on a Sephadex G-25 column. Purified urokinase brought about only the characteristic cleavage of purified plasminogen indicating the high purity of the preparate (Fig. 1).

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Preparation of the pericellular matrix of human fibroblasts. H u m a n embryonic lung fibroblasts

Fig. 1. The effect of affinity-purified urokinase ~n purified plasminogen; time course. The effect of the affinity-purified urokinase on plasminogen was studied in gels after the incubation of urokinase with plasminogen for different periods of time. 50 /zg plasminogen were incubated with 5 Ploug units urokinase in 0.1 M Tris-HC1 buffer (pH 7.5) at 37°C in the presence of aprotinin (200 U/ml). 2 mM benzamidine blocked the effect of urokinase on plasminogen in this assay. "l'hc conversion of plasminogen to plasmin was shown in an 8% gel (protein staining). Time (rain) is shown at the top of each track. Positions of the molecular weight markers arc shown on thc left.

(CCL-137; American Type Culture Collection) were grown to confluency on plastic tissue culture dishes. The matrices were prepared fresh for each experiment and the number of cells was about 3- 106 cells/60 m m diameter plate. The cultures were radioactively-labelled metabolically for 18 h using 5 m C i / l of [naC]glycine (113 C i / m o l ; The Radiochemical Centre, Amersham, U.K.) and the

cell-free matrices were isolated by extracting the cells three times for 5 min with sodium deoxycholate buffer (0.5% sodium deoxycholate/1 m M phenylmethane/sulfonyl fluoride in 10 mM TrisHCl-buffered saline, p H 8.0) in an ice bath on a four-way shaker followed by washing three times

143 in an ice-water bath with 2 m M Tris-HCl buffer ( p H 8.0)/1 m M phenylmethanesulfonyl fluoride [12]. The extraction results in a reproducible pattern of polypeptides [13,16] most of which have been identified as pericellular matrix proteins. About 15-20% of total cellular protein was retained in the matrix preparations. The matrices

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contained fibronectin, procollagen [12] and other polypeptides with molecular weights of 300000, 180000, 140000 [13,16,17] and 43000, evidently actin [12] (Fig. 2). This kind of matrix has been used in a number of studies to characterize pericellular structures and cell-matrix interactions [12,13,16,18]. N o major amounts of intracellular proteins have been demonstrated [12].

Analysis of the action of urokinase on the isolated pericellular matrices. Pericellular matrices of

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[14C]glycine_labelled CCL-137

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fibroblasts grown on 60 m m diameter plastic tissue culture dishes were exposed to purified human urokinase (2-50 Ploug U / m l ) in serum-free M E M (Dulbecco's modified minimal essential medium) plus 0.1% bovine serum albumin [13,16]. The incubations were carried out at 37°C from 10 to 60 min (see figure legends). After the incubation the matrices, attached on the tissue culture dishes, were washed gently with phosphate-buffered saline (0.14M NaC1 in 0.01 M phosphate buffer, p H 7.4), and extracted with gel sample buffer containing 4% sodium dodecyl sulphate/10% 2-mercaptoethanol. The matrices were scraped with a rubber policeman and more than 95% of matrix radioactivity was collected for analysis in gels. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was carried out under reducing conditions according to Laemmli [19]. Results

The effects of urokinase on the pericellular matrix of cultured human lung fibroblasts

Fig. 2. Preparation of the pericellular matrix. Human embryonic lung fibroblasts (CCL-137; American Type Culture Collection) were radioactively-labeledwith [t4C]glycine and the cell-free matrices were isolated. Autoradiogram of a 6% SDSpolyacrylamide gel is shown. (1) Total cell extract; (2) matrix extracted once with the sodium deoxycholatebuffer; (3) matrix extracted three times with the sodiuen deoxycholate buffer; (4) isolated pericellular matrix after three extractions and three washes. The isolated matrix (lane 4) represents 15-20% of total cellular protein (lane 1). Molecular weights are indicated on the right. An arrow points to the position of the 66000-dalton protein.

To demonstrate the biological activity of the affinity-purified urokinase we tested its effect on purified plasminogen. Uro~inase brought about only the characteristic cleavage (Fig. 1) and the cleavage could be blocked under these conditions by 2 m M benzamidine (data not shown). The isolated pericellular matrix (Fig. 2) was exposed to urokinase in serum-free medium and the changes in the matrix proteins were observed in autoradiograms of gels. Urokinase affected only the 66000-dalton protein of the matrix and the other matrix-associated polypeptides remained unaffected (Fig. 3A). The treatment resulted in the concomitant appearance of a 62000-dalton protein in the matrix. The cleavage of the 66000-dalton

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~B Fig. 3. Effect of urokinase on the pericellular matrix. Pericellular matrices of [J4C]glyeine-labeled CCL-137 fibroblasts were exposed to purified human urokinase in serum-free Dulbecco's modified minimal essential medium plus 0.1% bovine serum albumin. A. Dose dependency of the cleavage of the 66000dalton protein by urokinase. The matrices were incubated with increasing concentrations of urokinase for 30 min and the changes in the matrix polypeptides were analyzed in the autoradiogram of an 8% gel. (1-3) Matrices incubated with 2, 20 and 50 Ploug U/ml of urokinase, respectively. The untreated control matrix was comparable to lane 1, The positions of fibronectin (FN), and the 140000-dalton protein are shown on the right. Arrows point to the positions of the 66000-and the 62000-dalton polypeptides. B. Time dependence of the cleavage of the 66000-dalton protein. The matrices were exposed to urokinase (50 Ploug U/ml) for different periods of time, and the incubation was terminated by a rapid washing with the incubation buffer. The matrices were dissolved in the sample buffer and analyzed in a 6% gel. Autoradiogram of the gel is shown. (1) Control matrix 0 min. Note the abundance of actin in this matrix lost during the incubation. (2-4) Matrices incubated with urokinase for 10, 30 and 60 rain, respectively. (5) Control matrix incubated without urokinase for 60 min. The positions of fibronectin (220) and the 140000-dalton protein (140) are shown on the right. Arrows point to the 66000- and the 62000-dalton polypeptides. Both parts of the figure have been taken from the same autoradiogram of the gel. p r o t e i n b y u r o k i n a s e was dose a n d t i m e - d e p e n d e n t (Fig. 3). T h e cleavage was o b s e r v e d using u r o k i n a s e at c o n c e n t r a t i o n s b e t w e e n 5 - 5 0 Ploug U / m l . T h e release of the m a t r i x - a s s o c i a t e d p r o t e i n s (e.g., f i b r o n e c t i n a n d p r o c o l l a g e n ) into the m e d i u m was negligible u n d e r these c o n d i t i o n s ( d a t a n o t shown).

Inhibition of the action of urokinase T o exclude the p o s s i b i l i t y o f a c o n t a m i n a t i n g

protease, especially plasmin, we exposed the matrices to u r o k i n a s e in the presence of different p r o t e i n a s e inhibitors. T h e cleavage of the 66000d a l t o n p r o t e i n was i n h i b i t e d by b e n z a m i d i n e but n o t by a p r o t i n i n (Fig. 4), similar to its effects on p u r i f i e d p l a s m i n o g e n . Purified h u m a n t h r o m b i n was able to release b o t h fibronectin and procollagen from the m a t r i x (Fig. 4, l a n e 2 ) as shown previously [16]. T h r o m b i n also h a d an effect on the high m o l e c u l a r weight c o m p o n e n t s ( d o u b l e t with M r > 2 5 0 0 0 0 ) , which are p r o b a b l y glycos a m i n o g l y c a n s . W e were u n a b l e to find any reprod u c i b l e a n d clear effect of u r o k i n a s e on them. To exclude the activation a n d effects of plasm i n o g e n we e x p o s e d the matrices to p l a s m i n in the presence of different p r o t e i n a s e inhibitors. The i n h i b i t i o n p a t t e r n s of p l a s m i n a n d u r o k i n a s e were d i f f e r e n t . A p r o t i n i n was u n a b l e to i n h i b i t u rokinase, b u t b l o c k e d the effect of p l a s m i n (Fig. 4B). Plasmin, unlike urokinase, was at high conc e n t r a t i o n s ( > 1 k t g / m l ) also able to affect the o t h e r m a t r i x p o l y p e p t i d e s ( d a t a not shown). This i n d i c a t e d that the cleavage of the 6 6 0 0 0 - d a l t o n p r o t e i n was b r o u g h t a b o u t by urokinase and not b y c o n t a m i n a t i n g p l a s m i n or o t h e r proteases.

Discussion F i b r o n e c t i n is r e a d i l y r e m o v e d from the cells by different p r o t e i n a s e s [ 1,10]. T r e a t m e n t of cell layers with p l a s m i n or t r y p s i n b r i n g a b o u t a cleavage of the m o l e c u l e [20,21], whereas low c o n c e n t r a t i o n s o f t h r o m b i n can d e n u d e fibroblasts of fibronectin w i t h o u t a p p a r e n t cleavage [22]. Biologically active p h o r b o l esters affect cell s u r f a c e - a s s o c i a t e d f i b r o n e c t i n [23,24] a n d also induce p l a s m i n o g e n a c t i v a t o r activity in certain cell systems in vitro [25]. However, the roles of p l a s m i n o g e n activators in the r a p i d release of fibronectin by p h o r b o l esters [24] is not known. It has been shown that p l a s m i n o g e n activators can act catalytically on cellular s u b s t r a t e s i n d e p e n d e n t of p l a s m i n o g e n [11]. N o such substrates have been, however, identified thus far. Using fibronectin- releasing l0 000- dalton p o l y p e p t i d e s [26] derived f r o m h u m a n fibros a r c o m a cells, we were first able to observe a cleavage of the 6 6 0 0 0 - d a l t o n p r o t e i n in association with the release of intact f i b r o n e c t i n molecules

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Fig. 4. Inhibition of the action of urokinase. A. The matrices were incubated at 37°C for I h as follows: (I) untreated matrix: (2) matrix exposed to human thrombin, 5 NIH U/ml; (3) matrix exposed to urokinase (20 Ploug U/ml); (4-5) matrices exposed to urokinase (20 Ploug U/ml) in the presence of aprotinin, 400 U/ml and 200 U/ml, respectively: (6) matrix exposed to urokinase (20 Ploug U/ml) in the presence of 2 mM benzamidine. The effects of urokinase on matrix proteins were observed in audiograms of gels. Arrows point to the positions of the 66000- and the 62000-dalton polypeptides. B. Inhibition of the cleavage of the 66000-dalton polypeptide by plasmin and urokinase in the presence of different proteinase inhibitors. Isolated matrices were exposed either to urokinase (30 Ploug U/ml) or to plasmin (0.5 #g/ml) in the presence of different proteinase inhibitors at 37°C for I h. The inhibitors used were (1) none; (2) 100 U/ml aprotinin; (3) 2 mM benzamidine; (4) 0.5 mM leupeptin. An arrowhead indicates the position of the 66000-dalton protein. Fibronectin is indicated by FN.

f r o m the m a t r i c e s [13]. The t r e a t m e n t of the m a t r i c e s with collagenase r e m o v e d m a t r i x collagen [27] b u t d i d n o t affect the 6 6 0 0 0 - d a l t o n p r o t e i n [13]. Both the release of f i b r o n e c t i n a n d the cleavage of the 6 6 0 0 0 - d a l t o n p r o t e i n c o u l d b e inh i b i t e d b y p h e n y l m e t h a n e s u l f o n y l fluoride. M o r e r e c e n t l y we f o u n d that t h r o m b i n c o u l d also b r i n g a b o u t the release of f i b r o n e c t i n in a s s o c i a t i o n with the cleavage of the 6 6 0 0 0 - d a l t o n p r o t e i n [16]. M o r e o v e r , b o t h trypsin a n d p l a s m i n have b e e n o b s e r v e d to cleave the 6 6 0 0 0 - d a l t o n p r o t e i n in a s s o c i a t i o n with the a p p e a r e n c e of the 62000d a l t o n protein. H o w e v e r , the a p p e a r a n c e of the 6 2 0 0 0 - d a l t o n p r o t e i n does n o t necessarily i n d i c a t e that it is d e r i v e d f r o m the 6 6 0 0 0 - d a l t o n p o l y peptide.

In the p r e s e n t study, p u r i f i e d u r o k i n a s e cleaved the 6 6 0 0 0 - d a l t o n p r o t e i n b u t d i d not significantly affect a n y of the o t h e r m a t r i x p o l y p e p t i d e s . The cleavage of the 6 6 0 0 0 - d a l t o n p r o t e i n b y u r o k i n a s e thus a p p e a r s to be a highly specific interaction. T h e 6 6 0 0 0 - d a l t o n p r o t e i n is a s u b s t r a t e for various serine p r o t e i n a s e s b u t unlike the o t h e r s u b s t r a t e s of these enzymes, it is also cleaved b y urokinase. T h e c o n c e n t r a t i o n of u r o k i n a s e n e e d e d to b r i n g a b o u t the cleavage of the 6 6 0 0 0 - d a l t o n p r o t e i n is relatively high u n d e r these assay conditions. H o w ever, it m a y c o m p a r a b l e to the c o n c e n t r a t i o n s f o u n d locally in the cell-matrix c o n t a c t sites. T h e i n t r a c e l l u l a r c o n c e n t r a t i o n of p l a s m i n o g e n activator is k n o w n to b e high e.g., in m o n o c y t e s [28]. T h e 6 6 0 0 0 - d a l t o n p r o t e i n might thus be a mole-

146

cule that is somehow involved in the association of fibronectin with the matrix. The present studies do not distinguish whether the 66000-dalton protein is located on the cell surface or whether it is on the cytoplasmic side and has affinity to the components of the pericellular matrix. Because of a similar type of localization it will be of interest to determine its possible relationship to fimbrin, a 68000-dalton protein [29]. Studies on the interactions of the 66000-dalton protein may also elucidate its possible role in malignant transformation. Acknowledgements We thank Ms. Satu Cankar and Ms. Terttu Jelve for competent technical assistance. This work was supported by grants from the Finnish Cancer Foundation, the Finnish Medical Research Council, and National Cancer Institute D H E W (Grant No. 24605). References 1 Reich, E. (1975) in Proteases and Biological Control (Reich, E., Rifkin, D.B. and Shaw, E, eds.), Cold Spring Harbor Conf. Cell Prolif., Vol. 2, pp. 333-341 2 Christman, J.K., Silverstein, S.C. and Acs, G. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A.J., ed.), pp. 91-149, Elsevier/North-Holland, Amsterdam 3 W,ilson, E.L. and Doudle, E. (1978) Int. J. Cancer 22, 390-399 4 Aoki, N. (1974) J. Biochem. (Tokyo) 75, 731-741 5 Binder, B.R., Spragg, J. and Austen, K.F. (1979) J. Biol. Chem. 254, 1998-2003 6 Tucker, W.S., Kirsch, W.M., Martinez-Hernandez, A. and Find, L.M. (1978) Cancer Res. 38, 279-302

7 Reich, E., Rifkin, D.B. and Shaw, E. (cds.l (1975) Protcases and Biological Control, Cold Spring Harbor Conf. ('ell Prolif., Vol. 2 8 Burger, M.M. (1970) Nature 227, 170-171 9 Shefton, B.M. and Rubin, H. (1970) Nature 227, 843-845 l0 Blumberg, P.M. and ~.obbins, P.W. (1975) Cell 6, 137 147 I1 Quigley, J.P. (1979) Cell 17, 131-141 12 Hedman, K., Kurkinen, M., Alitalo, K., Vaheri, A., Johansson, S. and H66k, M. (1979) J. Cell Biol. 81, 83-91 13 Keski-Oja, J. and Todaro, G.J. (1980) Cancer Res. 40, 4722-4727 14 Holmberg, L., Bladh, B. and Astedt, B. (1976) Biochim Biophys. Acta 445,215-222 15 Saksela, O. (1981) Anal. Biochem. 11 I, 276-282 16 Keski-Oja, J., Todaro, G.J. and Vaheri, A. (1981) Biochim. Biophys. Acta 673, 323-33 I 17 Lehto, V.-P., Vartio, T. and Virtanen, I. (1980) Biochem. Biophys. Res. Commun. 95, 909-9t6 18 Hedman, K. (1980) J. Histochem. Cy¢ochem. 28, 1233-1241 19 Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685 20 Vaheri, A., Mosher, D.F., Saksela, O., Keski-Oja, J., Kurkinen, M. and Hedman, K. (1978) Proc. Fed. Eur. Biochem. Soc. 1977 47, 137 145 21 Vaheri, A., Ruoslahti, E. and Mosher, D.F. (eds.) (1978) Ann. N.Y. Acad. Sci. 312 22 Mosher, D.F. and Vaheri, A. (1978) Exp. Cell Res. 112, 323 334 23 Blumberg, P.M., Driedger, P.E. and Rossow, P.W. (1976) Nature (London) 264, 446-447 24 Keski-Oja, J., Shoyab, M., De Larco, J.E. and Todaro, (i.J. (1979) Int. J. Cancer 24, 218--224 25 Wigler, M. and Weinstein, I.B. (1976) Nature (London) 259, 232-233 26 Keski-Oja, J., Marquardt, H., De Larco, J.E. and Todaro, G.J. (1979) J. Supramol. Struct. 11,217-225 27 Vaheri, A., Kurkinen, M., Lehto, V.-P., Linder, E. and Timpl, R. (1978) Proc. Natl. Acad. Sci. USA 75, 4944-4948 I 28 Hovi, T., Saksela, O. and Vaheri, A. (1981) FEBS Lett. 129, 233-236 29 Bretscher, A. and Weber, K. (1980) J. Cell Biol. 86, 335-340