Dexamethasone Induction of an Inhibitor of Plasminogen Activator in HTC Hepatoma Cells*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY 1984 by The Americn Society of Biologicl Chemists, Inc VOl. 259, No. 11, Issue of June 1, pp , 1984 Prmted in U.S.A. Dexmethsone Induction of n Inhibitor of Plsminogen Activtor in HTC Heptom Cells* (Received for publiction, Jnury 23, 1984) Bernrd J. Cwikel, Ptrici A. Brouski-Miller, Ptrick L. Colemn, nd Thoms D. Gelehrter From the Deprtments of Internl Medicine nd Humn Genetics, University of Michign Medicl School, Ann Arbor, Michkgn Incubtion of HTC rt heptom cells with dex- inhibits both tissue-type nd urokinse-type plsminogen cmethsone cuses rpid decrese in cellulr plsmin- tivtors but not plsmin (15, 16). ogen ctivtor (PA) ctivity. Mixing experiments show The objective of this study hs been to determine whether the presence of n inhibitor of PA in dexmethsone- the presence of net PA inhibitor ctivity in hormone-treted treted cells. This study investigtes whether the de- cells represents the glucocorticoid induction of inhibitor, is crese in PA ctivity is secondry to the induction of secondry to decrese in the mount of ctivtor in the cell, n inhibitor by glucocorticoids, to decrese in the or results from combintion of both mechnisms. In order mount of PA, or to combintion of both mechnisms. to distinguish mong these lterntives, we hve utilized the PA nd its inhibitor re dissocited by sodium dodecyl observtion tht some proteses nd their inhibitors cn be sulfte-polycrylmide gel electrophoresis under nondissocited during SDS-polycrylmide gel electrophoresis (4, reducing conditions, nd both ctivities re then re- 17). Renturtion nd elution of proteins from the gel llows covered nd quntitted. HTC cells hve two mjor forms of PA with M, vlues of 11, nd 64,. seprte quntittion of both ctivtor nd inhibitor ctivi- Although PA ctivity in the unfrctionted extrcts ties. This study presents evidence tht glucocorticoid recepfrom dexmethsone-treted cells is inhibited by 9% tor-medited induction of n inhibitory protein responsible is reltive to control, there is no decrese in the totl for the inhibition of PA ctivity in HTC cells nd tht the ctivity of sodium dodecyl sulfte-dissocited PA c- mount of PA is not decresed by dexmethsone. tivity, suggesting tht dexmethsone cuses no decrese in the mount of the enzyme. PA inhibitor ctiv- EXPERIMENTAL PROCEDURES ity migrtes s single bnd of M, = 5,. The totl Cells-HTC cells were grown s monolyer cultures in Egle s ctivity of inhibitor increses in time-dependent miniml essentil medium (Gibco utoclvble powder) modified to fshion, reching mximum of >1 times control contin 5 mm Tricine, 2 mm glutmine,.5% NHC3,.2% fter 4-6-h incubtion with.1 MM dexmethsone. CC12, 5% clf serum, nd 5% fetl bovine serum. The isoltion of The induction of inhibitor requires both RNA nd pro- HTC vrint A hs been described previously (18). tein synthesis nd shows dependence en dexmeth- Confluent cultures in 35-mm dishes were rinsed twice with serumsone concentrtion identicl to tht for responses free medium nd then incubted in this medium supplemented with known to be medited by glucocorticoid receptors. We.1% BSA for 24 h prior to the ddition of fresh serum-free medium with.1% BSA nd.1% ethnol with or without.1 p~ dexmethconclude tht dexmethsone inhibits PA ctivity by sone. At the times specified, medium ws removed nd cells were inducing the synthesis of n inhibitor rther thn by scrped off the plte into cold phosphte-buffered sline (Dulbecco s decresing the mount of PA. phosphte-buffered sline without clcium or mgnesium) nd collected by centrifugtion for 5 min t 1,2 X g. Ech cell pellet ws resuspended in 1 ml of hypotonic phosphosline buffer (1:9 dilution of phosphte-buffered sline in wter), nd rpidly frozen in dry ice/ cetone. The thwed cell lyste ws centrifuged for 5 min t 1,2 X g to yield cytosol frction which contined less thn 5% of the totl cellulr PA ctivity nd ll the free PA inhibitory ctivity. The membrne pellet ws solubilized in 1 ml of.2% Triton X-1 nd clrified by centrifugtion for 5 min t 1,2 X g prior to ssy. This Plsminogen ctivtors re rginine-specific serine proteses which convert the plsm zymogen plsminogen to the ctive endopeptidse plsmin (1, 2). Proteolytic ctivity is regulted in prt by specific protese inhibitors (1, 3-5). Plsminogen ctivtors re known to be regulted by hormones nd developmentl signls (2, 6, 7); however, much less is known bout the regultion of protese inhibitors. Dexmethsone, synthetic glucocorticoid, rpidly decreses PA ctivity in HTC rt heptom cells (8-1) nd in severl other cell types (11-14). Mixing of extrcts from dexmethsone-treted nd untreted HTC cells indicted the presence of inhibitor ctivity in the hormone-treted cells (1). The inhibitor is n cid-stble cellulr product which * This work ws supported by Grnt CA from the Ntionl Institutes of Helth. The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked dvertisement in ccordnce with 18 U.S.C. Section 1734 solely to indicte this fct. The bbrevitions used re: PA, plsminogen ctivtor; SDS, sodium dodecyl sulfte; BSA, bovine serum lbumin frction from control cultures contins >95% of totl cellulr PA ctivity nd no detectble free inhibitory ctivity. The membrne frction, however, ws shown to contin inhibitor complexed with ctivtor, especilly in dexmethsone-treted cells. Preprtive Electrophoresis-To determine the totl mount of ctivtor nd inhibitor present in frctions in which both components re present, cell proteins were seprted by SDS-polycrylmide gel electrophoresis under nonreducing conditions (unless otherwise stted). The smple buffer (without -mercptoethnol), gels, nd electrode buffers were prepred ccording to Lemmli (19). Smples were incubted in smple buffer t room temperture for 1 h, nd subjected to electropboresis for 18 h t 4 V on either 3- or 1.5-mm thick slb gels contining 1% crylmide nd.27% biscrylmide. Up to 1 mg of HTC protein in mximum volume of 1 ml of smple buffer cn be pplied for every squre centimeter of gel-lne crosssectionl re without compromising resolution. After electrophoresis, gels were incubted with 2.5% Triton X-1 for 1 h t 4 C nd rinsed for 1 h in wter lso t 4 C (4). Seprte lnes of the slb gel were frctionted using rzor bed

2 6848 Dexmethsone-induced Inhibitor of HTC Cell Plsminogen Activtor nd incubted overnight t 4 C in polypropylene tubes with.1 M Tris-HCI buffer, ph 8.6, contining.1% Triton X-1 nd.1% BSA. The gel slices were then homogenized t 4 C by 5-s burst t mximum speed setting with Tekmr SDT Tissumizer (Tekmr Co., Cincinnti, OH) using n 8-mm dimeter probe. Triton X-1 ws dded to 1% finl concentrtion nd the tubes were frozen. Upon thwing, the suspensions were shken t 1 rpm in Aqutherm gyrotory shker (New Brunswick Scientific, NJ) for 1 h t room temperture with the tubes oriented horizontlly. The homogentes were centrifuged for 5 min t 1,2 X g nd portions of the superntnt frctions were ssyed for PA ctivity or urokinse inhibitory ctivity s described below. Plsminogen Actiutor Assy-Up to 1 pg of cell extrct protein or up to.25 ml of gel extrct ws ssyed for plsminogen-dependent fibrinolytic ctivity using lzsii-fibrin-coted Linbro (24-well) tissue culture dishes which were prepred using 251-lbeled humn fibrinogen (specific ctivity, 14 pci/mg, Amershm) nd unlbeled bovine fibrinogen (Clbiochem-Behring) s described previously (9, 1). Prior to use, the fibrinogen-coted dishes were incubted overnight with.5 ml/well of 5% fetl clf serum in.1 M Tris-HCI, ph 8.1, t 37 C to convert fibrinogen to fibrin, nd then wshed twice with distilled wter. Solubiliztion of 1-fibrin by cell extrcts in the presence of 2 Fg/ml of humn plsminogen ws determined s previously described (1). The plsminogen ws purified by lysinegrose ffinity chromtogrphy (2). Results re expressed s per cent of fibrinolysis (per cent of totl rdioctivity solubilized t given time corrected for bckground solubiliztion), or in terms of equivlent urokinse ctivity by comprison with urokinse stndrd (Clbiochem-Behring). Totl rdioctivity ws determined by incubting fibrin-coted wells with trypsin (25 bg/ml). Solubiliztion of rdioctivity ws liner with urokinse concentrtion up to 1.5 milliploug units/ml in 6-h ssy. Bckground relese of rdioctivity by plsminogen nd the pproprite mount of gel extrction buffer (or cell extrction buffer) ws 2-59; of totl rdioctivity, nd 1 milliploug unit of urokinse solubilized n dditionl 15-3% of the rdiolbeled fibrin under these conditions. Rdioctivity relesed in duplicte ssys vried by 1% or less. Inhibitor Assys-The mount of inhibitor ctivity ws determined by inhibition of the plsminogen-dependent fibrinolytic ctivity of urokinse. A previously described two-step procedure ws used (15) with some modifictions. In the first step, 1O-pl portions of either cytosol or gel elute were incubted with 1 milliploug unit of urokinse (totl volume, 15 pl) for 2 min t 37 C in 251-fibrin-coted wells. Residul urokinse ctivity ws determined by dding plsminogen to finl concentrtion of 2 pg/ml. The concentrtion of inhibitor in given smple is estimted from titrtion plots of residul urokinse ctivity s function of log protein concentrtion s shown in Fig. 1. We hve defined.5 milliunits of inhibitor s tht which will inhibit 5% of the ctivity of 1 milliploug unit of urokinse under these conditions. Protein Determintion-Protein ws determined by the method of Lowry et l. (21) using bovine serum lbumin s stndrd. RESULTS Effect of Dexmethsone on Activity of PA nd PA Inhibitor-The time course of inhibition of plsminogen-dependent membrne protein were ssyed for 6 h. Control ctivity corresponds fibrinolytic ctivity of the membrne frction from dexmeth- to the solubiliztion of31% of the totl rdioctivity. Bottom, PA sone-treted cells is shown in the Fig. 2, top. After lg of inhibitor specific ctivity of cytosol protein from control nd dex- 3 min, PA ctivity decresed rpidly; inhibition ws hlf- methsone-treted cells from two seprte experiments (circles nd squres) is expressed s milliunits (mu) of inhibitor/mg of protein s mximl t 5 min nd mximl (86%) fter 2 h. In 1 such defined under Experimentl Procedures. The circles represent dt experiments, hlf-mximl inhibition ws observed t 58 f 5 derived from the titrtion presented in Fig. 1 nd re from the sme min (men k SE) nd mximl PA inhibition ws 92%. experiment depicted in Fig. 2 (top). As reported previously (22), PA ctivity remins inhibited for t lest 24 h. The time course of increse of PA inhibitor ctivity in the cytosol frction from dexmethsone-treted cells is shown in Fig. 2, bottom. The circles represent dt from the sme experiment shown t the top, nd re clculted from the titrtions shown in Fig. 1. The squres represent seprte experiment depicting longer time course. PA inhibitor c- tivity, fter similr 3-min lg, incresed for 4 to 6 h, reching plteu vlue >%&fold tht of control, nd re- > c I- V 1 w ul z 5 P 3 -I 13 v) w - Control O-O Dexmethsone pg cytosol protein FIG. 1. Titrtion of intrcellulr PA inhibitor ctivity. HTC cells were incubted with.1 p~ dexmethsone () or without hormone () for the times indicted. Cytosol preprtions were nlyzed s described under Experimentl Procedures. One milliploug unit of urokinse solubilized 47% of the totl rdioctivity (3,85 cpm). Bckground relese (in the bsence of urokinse) ws 62 cpm during the 15-h incubtion period. Inhibitor ctivity in untreted cells ws unltered during the course of the experiment nd residul ctivity is presented s men? SE t,2, nd 4 h. >h I \ I Hours FIG. 2. Time course of PA inhibition nd PA inhibitor induction in dexmethsone-treted HTC cells. HTC cells were incubted without (open symbols) or with.1 NM dexmethsone (closed symbols) for the times indicted. Top, the plsminogen-dependent fibrinolytic ctivity of Triton-solubilized membrne frctions is expressed reltive to urokinse stndrd curve s milliploug units (mpu)/mg of membrne protein. In ll cses, 5-pg smples of mined elevted for t lest 24 h. Bsed on 23 experiments, the ctivity of inhibitor ws 14.2 f 1.9-fold tht of the control t 4-5 h. Hlf-mximl induction of PA inhibitor ws seen by 2-h incubtion with.1 PM dexmethsone, t which time inhibition of PA ws nerly mximl. Effect of Dexmethsone on Totl PA Actiuity-The HTC cell contins both PA nd inhibitor in pprent equilibrium with n enzymticlly inctive complex. During glucocorticoid tretment, there is n increse in the mesured free inhibitor

3 _ Dexmethsone-induced Inhibitor which my result from n increse in totl inhibitor, decrese in the mount of ctivtor, or some combintion of both. To determine which lterntive is correct, we hve utilized SDS-polycrylmide gel electrophoresis under nonreducing conditions to dissocite PA from its inhibitor. Renturtion following physicl seprtion llows the mesurement of totl HTC ctivtor ctivity. To estimte the recovery of HTC PA using this technique, inhibitor-free 64,-D HTC PA (see below) ws isolted using preprtive electrophoresis. A portion of this preprtion ws then subjected to second round of preprtive electrophoresis nd renturtion. Between 7 nd 8% of I I I v v v v v Reltive mobility FIG. 3. Electrophoretic profile of SDS-dissocited HTC PA ctivity from dexmethsone-treted nd untreted cells. Membrne protein (125 pg) from control cells () nd cells treted with dexmethsone for 4 h () ws subjected to SDS dissocition, electrophoresis, renturtion, frctiontion, nd elution s described under Experimentl Procedures. The proteins in ech 4.4-mm gel slice were eluted into.7 ml of elution buffer, nd PA ctivity ws determined in both 5-p1 (not shown) nd 1-pl liquots. Plsminogen-dependent fibrinolysis (fter 15 h of incubtion) ws proportionl to gel extrct volume within this rnge. Chick myosin phosphorylse B (93,), BSA (66,), nd ovlbumin (45,) wereused s moleculr weight stndrds. 2K represents 2,, for exmple. TABLE I Effect of dexmethsone on ntive nd dissocited PA ctivity Specific PA ctivity of membrne frctions of cell cultures treted with.1 ~ LM dexmethsone for 2 nd 4 h is expressed s per cent of control (men -+ SE). Ntive PA ctivity refers to PA ctivity prior to SDS dissocition while dissocited PA ctivity refers to the totl ctivity of frctions with mobilities corresponding to M, of 6, to 9, plus 9, to 12, following SDS-polycrylmide gel electrophoresis, renturtion, nd frctiontion. The men control specific ctivities were 16 & 58 milliploug units/mg for ntive PA ctivity nd 93 k 38 milliploug units/mg for dissocited PA ctivity (n = 7 experiments). Time Ntive ctivity PA Dissocited ctivity PA h % control 2 (n = 4) 15k2 93 f 11 4 (n = 6) 1 f f 28 of HTC Cell Plsminogen Activtor 6849 HTC PA ctivity ws recovered from these preprtions. Similr recoveries were observed for PA ctivity from the conditioned medi of HTC cultures stimulted with cyclic AMP derivtives (22) in which the net effect of inhibitor is probbly insignificnt. Most of the preprtions nlyzed in this study re mixtures of both ctivtor nd inhibitor; consequently, the pprent recovery of PA ctivity my be mny fold greter thn the initil ctivity of the unfrctionted mixtures. The electrophoretic profiles of rentured plsminogen ctivtor ctivity from SDS-dissocited HTC cell membrne frctions re presented Fig. in 3. The HTC ctivtor is present in two mjor forms with M, vlues of 11, nd 64,. Dexmethsone tretment for 4 h, while inhibiting the PA ctivity in the unfrctionted membrne preprtion by 94% in this experiment, did not significntly chnge the totl mount of ctivtor s determined by the re under the two peks. The totl dissocited PA ctivity from dexmethsonetreted cultures ws 96% of tht from control cultures. Dexmethsone ltered the reltive bundnce of the two species, however, decresing the 64,-D form nd incresing the 11, D form. The results from severl experiments re summrized in Tble I. We conclude tht dexmethsone, lthough it inhibits ntive PA ctivity by 85% or more, cuses no significnt decrese in totl PA ctivity. Locliztion nd Quntittion of Electrophoreticlly Seprted PA Inhibitor-The sme SDS-gel dissocition method ws used to determine the moleculr weight of the PA inhibitor nd to mesure the mount of ctive PA inhibitor in mixtures in which it is present with PA. The inhibitory ctivity from cell extrcts nd conditioned medi migrtes s single pek of bout 5, D. Its ctivity nd mobility re unffected by incubtion with.1 M P-mercptoethnol (which inctivtes PA) nd 1% SDS prior to electrophoresis. The estimted recovery of inhibitor ctivity from this procedure is pproximtely 3% bsed on procedure identicl to tht used to estimte the recovery of ctivtor (see bove). Gel profiles of urokinse inhibitory ctivity in cell extrcts from both dexmethsone-treted nd untreted cells re shown in Fig. 4A. In this experiment, the inhibitory ctivity ws induced 9-fold fter 4-h tretment with.1 p~ dexmethsone. The verge dexmethsone-induced increse in gel-dissocited inhibitor in nine experiments ws 1.5 f 3.1- fold over control. The time course of inhibitor induction, s mesured in SDS-dissocited cell extrcts (dt not shown), is similr to tht in unfrctionted cytosol. These results, together with those in the previous section, suggest tht the increse in free inhibitor ctivity nd decrese in net PA ctivity re due to n increse in totl inhibitor ctivity without decrese in totl ctivtor ctivity. Lck of Induction of the 5,-D Inhibitor in HTC Vr- ints-a vrint line of HTC cells selectively resistnt to dexmethsone inhibition of PA ctivity (9, 1, 18) ws nlyzed in n identicl mnner. As shown in Fig. 4B, dexmethsone filed to induce n increse in the mount of electrophoreticlly seprted PA inhibitor. Requirement for Mcromoleculr Synthesis during Inhibitor Induction-The increse in inhibitor ctivity in dexmethsone-treted cells requires both RNA nd protein synthesis (Fig. 5). Actinomycin D t.16 WM, concentrtion which inhibits [3H]uridine incorportion into RNA by >9% (23), prevented the induction of inhibitor by 85 f 1%; nd.1 mm cycloheximide, which inhibits [3H]leucine incorportion into protein by >95% (23), inhibited PA inhibitor induction by %. The inhibition of hormonl induction of PA inhibitor

4 685 Dexmethsone-induced Inhibitor of HTC Cell Plsminogen Activtor RELATIVE MOBILITY FIG. 4. Detection of dexmethsone-induced PA inhibitor in electrophoretic frctions of extrcts from wild type nd vrint HTC cells. A, 1 pg of cytosol protein from control wild type HTC cells () nd cells treted with dexmethsone for 4 h () wereincubtedwithsds-gelsmplebuffer contining.1 M - mercptoethnol nd subjected to SDS-polycrylmide gel electrophoresis. Following renturtion, proteins eluted from 2.2-mm gel slicesbetween the BSA (66,) nd ovlbumin (45,) mrkers were titrted for inhibitor ctivity, s described under Experimentl Procedures. B, 1-pg smples of cytosol protein from control (A) nd dexmethsone-treted HTC Vrint A cultures (A) were nlyzed s in A. 66K represents 66,, for exmple. mu, milliunit. I I I I DEXAMETHASONE I loglom 1 FIG. 6. Concentrtion dependence of dexmethsone induction of PA inhibitor nd inhibition of HTC PA. HTC cells were incubted with the specified concentrtion of dexmethsone for 4 h. Cytosol frctions were titrted for inhibitorctivity () nd the specific ctivity of PA () ws determined in the membrne frctions from the sme cultures. Results re expressed s the per cent of mximl response (men f SE). Dt were derived from three experiments, ech using t lest two replicte cultures t ech dexmethsone concentrtion. of PA Inhibitor-The dexmethsone concentrtion-dependence for the induction of cytosolic inhibitor is shown in Fig. 6. Hlf-mximl induction of inhibitor ws observed t 5.5 k 1.1 nm (n = 3) which is the sme concentrtion s tht which cuses hlf-mximl induction of tyrosine minotrnsferse nd inhibition of mino cid trnsport in these cells (24). Thus the induction of PA inhibitor ppers to be medited by the glucocorticoid receptor. In contrst, hlf-mximl inhibition of PA ctivity in prticulte frctions of the sme cultures ws observed t 1.2 &.2 nm dexmethsone. Thus, 1% of mximl inhibitor induction is pprently sufficient to cuse 25% inhibition of PA ctivity. Control Dexmethsone Control Dexmethscne FIG. 5. Effect of ctinomycin D nd cycloheximide on the glucocorticoid induction of PA inhibitor. Actinomycin D (.16 $M; htched brs) or cycloheximide (.1 mm; stippled burs) ws dded to cultures 2 min prior to the ddition of dexmethsone. After 4 h of incubtion, PA inhibitor ctivity ws mesured in ntive cytosol preprtions (A) nd in elutes of slices from the M, = 4,-7, region of SDS-polycrylmide gels run under reducing conditions (B). In order to pool the results from severl experiments, PA inhibitor ctivity is expressed s the -fold control ctivity (men & SE). The verge bsl specific ctivity of PA inhibitor in these experiments ws 5.3 milliunits/mg of protein (n= 4) for ntive cytosol nd 67 milliunits/mg of protein loded on the gel (n = 4) for gel elutes. ws demonstrble in both ntive cytosol (Fig. 5A) nd in gel elutes (Fig. 5B). Concentrtion Dependence of the Dexmethsone Induction DISCUSSION Previous studies from this lbortory hve shown tht dexmethsone-treted HTC cells contin specific inhibitor of PA (9,1, 15). The hormone-dependent expression of this inhibitor did not occur in either nuclete HTC cells (25) or in vrint clones of HTC cells which re selectively resistnt to dexmethsone inhibition of PA (1). A decrese in PA ctivity coupled with n increse in free PA inhibitor following dexmethsone tretment is comptible with two very different models. Dexmethsone might directly decrese the totl mount of cellulr PA nd thereby mke constitutively produced mounts of inhibitor pper elevted. Alterntively, the decrese in HTC PA ctivity could be secondry to the induction of inhibitor without decrese in the mount of PA. A combintion of these two models is lso comptible with these observtions. Becuse the interction between inhibitor nd ctivtor from HTC cells is reversed by SDS-polycrylmide gel electrophoresis, we hve been ble to mesure the ctivity of both proteins from dexmethsone-treted cells. After the first 4 h of hormone tretment, by which time PA ctivity is mximlly inhibited, dexmethsone hs no effect on the totl mount of PA ctivity recovered fter dissocition nd renturtion (Fig. 3 nd Tble I). This observtion suggests tht the rpid decrese in HTC PA ctivity is not due to decrese in the mount of the enzyme in these cells. Dexmethsone does,

5 Dexmethsone-induced Inhibitor of HTC Celt Plsminogen Activtor 6851 however, cuse shift in the distribution of PA ctivity from The glucocorticoid-induced inhibitor ppers to inhibit the M, = 64, form to the M, = 11, form. The reltion HTC PA ctivity without irreversibly inctivting the enof the two forms of HTC PA to ech other nd the cuse of zyme. In contrst, the sme inhibitor irreversibly inhibits the hormone-induced shift re unknown nd re currently under investigtion. The Mr = 11, form could represent precursor of the M, = 64, form, or could be covlent complex of the M, = 64, form with the PA inhibitor which is enzymticlly ctive fter SDS-polycrylmide gel electrophoresis nd renturtion. urokinse nd forms covlent complex with '251-urokinse which is stble to SDS- nd sulfhydryl-reducing gents (29). This represents nother difference between tissue ctivtors nd urokinse-type ctivtors (1). PA-specific inhibitors hve been described in humn skin fibroblsts (3), humn plcent (3), nd bovine endothelil In contrst to the lck of glucocorticoid effect on totl PA cells (31), the ltter two of which hve moleculr weights ctivity, dexmethsone cuses 1-fold increse in the similr to tht of the HTC inhibitor. Bovine endothelil mount of PA inhibitor ctivity recovered from the M, = inhibitor ctivity cn lso be recovered following sulfhydryl 5, region of SDS-polycrylmide gels. The dexmeth- reduction nd SDS-polycrylmide gel electrophoresis (31). sone-medited induction of SDS-dissocible PA inhibitor pr- However, only the HTC nd fibroblst inhibitors hve been lleled its ppernce in the unfrctionted "ntive" cytosol. Thus, the glucocorticoid inhibition of PA ctivity in HTC cells ppers to be medited primrily, if not solely, by the induction of specific inhibitor of this protese without ny chnge in the mount of the enzyme. We suggest model in shown to be under hormonl control. The PA inhibitor in HTC cells is clerly different from the protese nexins, one species of which hs moleculr weight similr to the HTC inhibitor (32). The HTC PA inhibitor, unlike protese nexin, is cid stble nd does not inhibit plsmin (15) or thrombin which newly synthesized molecules of inhibitor combine with PA to form n enzymticlly inctive complex. This hs the (9). The present studies show tht the net plsminogen-deeffect of diminishing PA ctivity but initilly preventing the pendent fibrinolytic ctivity of HTC cells depends on the detection of incresed mounts of inhibitor. Proof of this ctivities of both plsminogen ctivtor nd specific inhibmodel will require direct mesurement of the mount of itor. Glucocorticoid hormones rpidly inhibit PA ctivity by enzyme nd inhibitor by immunologic techniques. The glucocorticoid induction of PA inhibitor ctivity in inducing PA inhibitor without mount of plsminogen ctivtor. directly ltering the totl HTC cells, like tht of tyrosine minotrnsferse ctivity (s), requires concomitnt RNA nd protein synthesis. The Acknowledgments-We thnk J. Worley for secretril ssistnce nd M. Mrshll for preprtion of the figures. concentrtion dependence for both responses is lso the sme suggesting tht the induction of the PA inhibitor is lso medited by the glucocorticoid receptor. The role of the inducible inhibitor in mediting the dexmethsone effect on PA ctivity is lso supported by studies using vrint HTC cells selectively resistnt to dexmeth- sone inhibition of PA. No inhibitory ctivity could be detected in the cytosol frction of these cells (1) nd very little in electrophoretic frctions from these cells either before or fter dexmethsone tretment. Thus, PA inhibitor ppers to be mde constitutively in vrint cells s it is in wild type cells, but its production is not inducible by dexmethsone. The lesion in the vrints, therefore, is presumbly not loss of the structurl gene for the PA inhibitor, but rther defect in its inducibility by glucocorticoids. Two relted observtions re lso consistent with the interprettion tht dexmethsone inhibition of PA ctivity is secondry to the induction of the inhibitor. First, 5% inhibition of HTC PA ctivity occurs within 1 h of dexmethsone tretment, t time when there is only prtil induction of inhibitor ctivity. Second, inhibition of PA ctivity is redily detected t subnnomolr concentrtions of dexmethsone when no increse cn be detected in inhibitory ctivity. Anlysis of the reltionship between the concentrtion-dependence of PA inhibition nd tht of inhibitor induction in the sme cultures suggests tht 1% of mximl induction of PA inhibitor is sufficient to cuse >5% of mximl inhibition of HTC PA. 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