Alveolar Epithelial Cell Plasminogen Activator · Alveolar Epithelial Cell Plasminogen Activator CHARACTERIZATION AND REGULATION* (Received for publication, November l&1989) Bruce

THE JOVRNAL OF BIOLOGICAL CHEMISTRY % 1990 by Tbe American Society for Biochemistry and Molecular Biolo@, Inc.

Vol. 265, NO, 14, Issue of May 15, pp. 8198-82041990 Printed in U.S.A.

Alveolar Epithelial Cell Plasminogen Activator CHARACTERIZATION AND REGULATION*

(Received for publication, November l&1989)

Bruce C. Marshall~~, Daniel S. SageserS, N. V. RaoS, Mitsuru Emi% and John R. HoidalS From the DeDartments of W&man Genetics arad fMedicine, Pulmonary Division, University of Utah Medical Center,

Intra-alveolar fibrin deposition is one of the pathological hallmarks of acute lung injury. Because alveolar epithelial cells play a central role in the repair process following acute lung injury, this study was undertaken to examine their potential to produce a plasminogen activator (PA). We now report the synthesis and secretion of PA by rat alveolar epithelial cells with the catalytic properties of a urokinase-type (u-PA) rather than tissue-type plasminogen activator. Studies of regulation of epithelial cell u-PA revealed: 1) phorbol myristate acetate (P&IA) but not the inactive structural analog 4cu-PMA upregulated u-PA synthesis, putatively via the protein kinase C pathway; 2) PMA induction of U-PA activity was so~tantially inhibits by dexameth~o~e and completely inhibited by cycloheximide; 3) unstimulated alveolar epithelial cells had no detectable u-PA mRNA, whereas PMA exposure led to activation of the u-PA gene and accumulation of a 2.5-kilobase u-PA mRNA, and 4) cycloheximide did not abolish this induction of u-PA mRNA suggesting that intermediate protein synthesis was not necessary for the activation of transcription, In light of their capacity to promote fibrinolysis and their strategic anatomic location, alveolar epithelial cells are likely to play a key role in the extensive remodelling process that follows acute lung injury.

The formation of fibrin is a part of the normal tissue repair process. Fibrin not only serves a role as a hemostatic barrier and in the limitation of the exudative process, but also provides a matrix for the tissue repair that follows injury (1, 2). The plasmin/piasminogen activator system is a key compo- nent in the fibrinolysis that accompanies tissue repair. Plas- min is a potent serine proteinase that not only has enzyme activity against fibrin but also has the capacity to degrade various basement membrane components including fibronectin (3) and laminin (4). In addition, plasmin converts the latent forms of collagenase (5) and transforming growth factor-@ (6) to their biologically active forms.

Plasminogen activators (PA)’ convert the zymogen plas-

* This research was supported in part by Veteran’s Administration Career Development Award AI-859 (to B. C. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- me& in accordance with 18 USC. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Pulmonary Divi- sion. Rm. 4R240.50 North Medical Dr.. Universitv of Utah Medical Center, Salt Lake City, UT 84132. -

1 The abbreviations used are: PA, plasminogen activator: t-PA, tissue-t.ype PA; u-PA, urokinase-t.~e PA, PAI, PA inhibitor; SDS, sodiumdodeeyl sulfate; PBS, phosphate-buffered saline; PMA, phorbol mvristate acetate: TGF, transforming growth factor; PDGF, plate- Iet-derived growth factor; kb, kiloba&;TRE, lf-O-tetrade&noyI- phorbol-13-acetate-responsive elements; Hepes, 4-(Z-hydroxyethyl)- l-piperazineethanesulfonie acid.

minogen to plasmin. There are two distinct types of PA, tissue-type PA (L-PA) and urokinase-type PA (u-PA). The t- PA binds to and is stimulated by fibrin and appears to be the key form involved in intravascular thrombolysis. The u-PA has been implicated in a variety of extravascular processes including tissue remodeling and cell migration (7, 8). The proteolytic potential of the plasmin/PA system is counterbal- anced by plasmin inhibitors and also by the more recently recognized PA inhibitors. There are at least three distinct groups of PA inhibitors: the endothelial type or PAI-1, the placental type or PAI-X, and protease nexin 1 (9). The fibrinolytic balance is determined not only by the level of proteases and inhibitors but also by their localization. It is now clear that there are specific membrane receptors for u-PA that serve to localize its proteolytic action (10-13).

Acute lung injury or what has been termed adult respiratory distress syndrome is a common medical problem that contin- ues to carry a high mortality rate. One of the pathological hallmarks of the early phase of this syndrome is intra-alveolar fibrin deposition (14, 15). Recent evidence suggests that the procoa~lant/~brinol~i~ balance is altered in the early phase of adult respiratory distress syndrome such that fibrin deposition is favored (16). An extensive remodeling process occurs in survivors leading to the eventual return to near normal lung anatomy and physiologic function in the majority of individuals (17, 18). It is highly likely that the plasmin/PA system plays an important role in the solubilization of intra- alveolar fibrin deposits during this repair process.

The specific cell types that contribute to the fibrinolytic balance in the alveolar space are not known. The alveolar macrophage has been recognized as a potential contributing cell in that it has the capacity to synthesize u-PA (19) and a PAI- (20). Alveolar type 2 epithelial cells proliferate and repopulate the epithelial lining layer following acute lung injury (21, 22). We hypothesized that they might also be a source of PA. To test this hypothesis we studied rat alveolar epithelial cells in vitro, and now report that these cells synthesize and secrete u-PA and that PMA upregulates u-PA synthesis via transcriptional activation of the u-PA gene.

EXPERIMENTAL PROCEDURES

~o~@r~~-Rea~ents were obtained as follows: tissue culture media, supplements, and fetal calf serum from GIBCO; tissue culture pkrsticware from Costar; the porcine pancreatic elastase for the cell &olation from worthin~o~;-~-D-Vet-Leu-Lys-~-~itroanil~de from Bachem: nlasminogen from Helena: soluble fibrin products and human t-PA from American Diagnostica; human u-PA from the Green Cross Corp (Osaka, Japan); n-casein, amiloride, biotin, bovine serum albumin, cholera toxin, dibutyryl CAMP, formaldehyde~ insulin, phorbol myristate acetate (PMA), plasmin, sodium selenite, Staphylococ- cal Protein A-Sepharose CL-4B, transferrin, trichloroacetic acid, and Triton X-100 from Sigma; cesium chloride from Boehringer-Mann- heim; acrylamide, agarose, ammonium persulfate, bis-acrylamide, Coomassie Brilliant Blue R-250, glycine, Protein Assay dye reagent,

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protein molecular weight standards, sodium dodecyl sulfate (SDS), and Tris from Bio-Rad; epidermal growth factor, platelet-derived growth factor (PDGF), and transforming growth factor-p (TGF-8) from Collaborative Research; recombinant tumor necrosis factor-e (TNF-(u), y-interferon, and recombinant interleukin-10 from Gen- zyme; 4n-PMA from LC Corp; Biotrans nylon membranes from ICN; I”“Slmethionine and la-32PldCTP from Amersham: EnHance from hu Pant; and Kodak %AR film from Eastman. Rabdit anti-mouse u- PA antisera and an SP64 plasmid containing the 660-base pair P&I, HirzdIII fragment from the coding region of the mouse u-PA gene (23) were the kind gifts of Dr. Dominique Belin (Geneva, Switzer- land).

Cell Isolation and Tissue Culture-Rat alveolar epithelial cells were isolated by enzyme dissociation followed by separation of cells based on differential adherence to IgG-coated plates (24). The type 2 cell- enriched population was plated at 5 X lo5 to 1 X lo6 cells/l6-mm well in modified Eagle’s medium-D-valine supplemented with vitamins, nonessential amino acids, penicillin (100 units/ml), streptomycin (100 fig/ml), fungizone (2.5 pg/ml), 10 mM Hepes and 10% heat-inactivated fetal calf serum. The day following isolation the adherent cell population was greater than 90% type 2 cells by standard light microscopic criteria. The contaminating cells consisted primarily of alveolar macrophages plus an occasional lymphocyte and ciliated bronchial epithelial cell. At 2 to 3 days after isolation, the confluent cells were washed with phosphate-buffered saline (PBS) and changed to a serum-free medium formulation that consisted of a high glucose modified Eagle’s medium base supplemented to a final concentration with insulin (5 rg/ml), transferrin (50 pg/ml), biotin (10 rig/ml) and sodium selenite (0.5 &ml). The supernatants were then collected at selected time points and clarified by centrifugation. Cell lysates were m-euared bv washing the cells with PBS followed bv disruntion of the monolayerwith a T&on scraper in 100-200 ~1 of O:l% TAton X-100, 50 mM Tris buffer, pH 8.0 at room temperature. The samples were stored at -70 “C until further analysis.

Alveolar macrophages were obtained from the same preparations by lavage of the lungs with PBS prior to the enzyme dissociation step. The lavage cell pellet and cell-free lavage supernatant were separated by centrifugation. The macrophages were then either lysed in 0.1% Triton, 50 mM Tris buffer, pH 8.0, or placed in culture as described for the alveolar epithelial cells.

Plasminogen Activator Assay-PA was quantitated in polyvinyl- chloride microtiter plates by measuring the enzymatic activity of the formed plasmin with the synthetic substrate, H-D-Val-Leu-Lys-p- nitroanilide (25). The assay-mixture included the following: O.Oi LM of H-D-Val-Leu-Lvs-D-nitroanilide. samule and 0.5% Triton X-100. 0.1 M Tris buffer, ifi 8.0, to bring’the final assay volume to 125 ~1: Each sample was assayed with and without plasminogen (0.03 pM) to establish PA activity. The amount of p-nitroaniline released was then measured at 410 nm with a Dynatech MR700 microplate reader. A standard curve was generated with each assay using low molecular weight human urokinase. The PA activity was expressed as IU/ milligram protein in the cell lysate. Where indicated catalytic amounts of plasmin (2 rig/ml), amiloride (1 mM), or soluble fibrin products (20 rig/ml) were included in the assay mixture.

Substrate Polyacrylamide Gel Electrophoresk-The following mod- ifications were made in the method described by Granelli-Piperno and Reich (26). The substrate gel consisted of 12.5% polyacrylamide to which plasminogen (0.03 PM) and cu-casein (2 rg/ml) were added prior to casting. Aliquots of the cell culture supernatants were di- alyzed at 4 “C against 10 mM Tris buffer, pH 8.0, lyophilized, and resuspended in SDS sample buffer. The samples were not boiled or reduced prior to the analysis; otherwise, the electrophoresis was performed by standard methods (27). Afterwards the gel was soaked for 1 h in 2.5% Triton X-100 and then overnight at 37 “C in 0.1 M glycine buffer, pH 8.0. Clear lysis zones of PA activity were localized by staining the gel with Coomassie Blue. The samples were also run on gels without plasminogen to confirm that the activity was due to a PA.

Modulation of PA A&&y-Several potential modulators of alveolar epithelial cell PA activity were examined. These experiments were all started 1 day after the cells were changed to serum-free medium. Fresh medium containing one of the following substances (final concentration) was then added to the wells: 1) PMA, 5 rig/ml and 50 rig/ml; 2) 401-PMA, 50 rig/ml; 3) dexamethasone, 1O-7 M; 4) cycloheximide, 10 *g/ml; 5) TGF-& 0.1, 1.0 or 10 rig/ml; 6) PDGF, 10 rig/ml; 7) epidermal growth factor, 5 rig/ml; 8) tumor necrosis factor-a, 200 units/ml; 9) y-interferon, 100 units/ml; 10) interleukin-l/3, 10 units/ ml; 11) dibutyryl CAMP, 0.1 mM; 12) cholera toxin, 1 pg/ml. The

supernatants and cell lysates were collected 24 h later, unless otherwise stated.

Because of variability from one primary cell preparation to the next, comparisons were made only within the same cell preparation. The potential modulator was added to three to six wells of epithelial cells and each supernatant and lysate collected and analyzed individ- ually. The PA activity resulting from each potential modulator (mean f standard error) was then compared with that of control unstimulated cells using a weighted t test. All findings were then confirmed in several other cell preparations.

Metabolic Labeling and Immunoprecipitution-Alveolar epithelial cells were incubated in medium containing either 0 or 50 rig/ml of PMA for 20 h and then labeled for 4 h in methionine-free medium supplemented with [35S]methionine (100 &i/ml, specific activity > 1100 Ci/mmol). The supernatants were clarified by centrifugation and preadsorbed with 10 rg of normal rabbit IgG and 50 ~1 of a 10% suspension of Staphylococcal Protein A-Sepharose CL-4B. Immu- noprecipitation was then performed by adding Protein A-Sepharose beads that had been preincubated with either a 50-fold dilution of rabbit anti-mouse u-PA or non-immune sera. After overnight incubation at 4 “C the immunoprecipitates were washed, solubilized in SDS sample buffer, and separated by polyacrylamide gel electrophoresis under reducing conditions. The gel was processed for autofluorography with EnHance as directed by the manufacturer.

To assess total incorporation of [“‘Slmethionine, proteins were precipitated from an aliquot of each supernatant and lysate by the addition of 100 /Lg of carrier bovine serum albumin and trichloroacetic acid to a final concentration of 10%. The precipitates were collected on fiberglass filters by vacuum and washed frie of unincorporated 135Slmethionine with ethanol. The filters and 10 ml of Outifluor were thei placed in scintillation vials and radioactivity measured in a Beckman LS-4000 scintillation counter.

Northern Blot-Total RNA was prepared by lysis of the cells in guanidine hydrochloride and ultracentrifugation of the lysate on a cesium chloride cushion (28). The RNA was quantitated by measuring absorbance at 260 nm, separated on the basis of size on a 2.2 M formaldehyde, 1% agarose gel, and transferred to a Nylon membrane by capillary action. The membranes were prehybridized overnight at 42 “C in 5 x SSC (standard sodium citrate), 50 mM sodium phosphate, 10 x Denhardt’s solution, 2.5% dextran, 0.25 mg/ml of salmon sperm DNA, 0.75% SDS, and 50% formamide. An SP64 plasmid containing the 660-base pair P&I, Hind111 fragment of the mouse u-PA gene was “P-labeled by the random primer method and hybridized at 37 “C overnight in 5 x SSC, 20 mM sodium phosphate, 1 x Denhardt’s solution, 10% dextran, 0.1 mg/ml salmon sperm DNA, 0.75% SDS, and 50% formamide. The membranes were washed in 2 X SSC, 0.1% SDS at progressively increasing temperature up to 65 “C. Autoradi- ography was performed by standard methods.

Others-Protein concentrations were determined by the Bradford dye-binding assay (29).

RESULTS

PA Activity in Alveolar Epithelial Cell Cultures-A repre- sentative time course of PA activity in alveolar epithelial cell supernatants is shown in Fig. 1. Virtually all of the activity was plasminogen-dependent. PA activity was not detectable until several days after the cell isolation and was maximal at day 10. Because the epithelial cells became quite attenuated and were easily dislodged from the culture plates beyond day 10, no measurements of PA activity were made beyond that point. PA activity in the cell lysates paralleled that of the supernatants (data not shown). The addition of catalytic amounts of plasmin to activate latent u-PA (30) demonstrated no significant pool of precursor u-PA in the early day culture supernatants (data not shown).

Characterization of the Alveolar Epithelial Cell PA-Late day culture supernatants demonstrate clear zones of lysis at the apparent molecular weights of 48,000 and 31,000 by substrate polyacrylamide gel electrophoresis (Fig. 2). The Mabin Darby Canine Kidney epithelial cell line with a lysis zone at the apparent molecular weight of 55,000 is shown for compar- ison. Parallel gels without plasminogen showed no areas of lysis.


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Alveolar Epithelial Cell PA

FIG. 1. PA secretion by alveolar epithelial cells: effect of time in culture. After 2 days in culture, confluent monolayers were washed with PBS and changed to serum-free medium. On days 3, 5, 7, and 10, the medium was changed and supernatants and cell lysates were collected (n = 6 for each time point) and analyzed for PA activity. The data presented are PA activity in the supernatants expressed as IU of activity/mg protein in the lysate/24 h (mean + SE., *p < 0.001 as compared to day 3). Note that PA activity increased with time in culture. This same pattern was observed in several different cell preparations; however, on occasion the rise in PA activity was detectable as early as day 4.

I 2 3 4

-45hDO -31 kDa

u-PA MDCK AEC AEC Day5 Day IC

FIG. 2. Alveolar epithelial cell PA demonstrated by substrate polyacrylamide gel electrophoresis gel. The substrate gel consisted of 12.5% polyacrylamide to which plasminogen (0.03 pm) and n-casein (2 pg/ml) were added prior to casting. The samples were not boiled or reduced prior to the analysis. After electrophoresis the gel was soaked for 1 h in Triton X-100 followed by an overnight incubation at 37 “C in 0.1 M glycine buffer, pH 8.0. Clear lysis zones of PA activity were then localized by staining the gel with Coomassie Blue. Lane I, 100 mIU of low molecular weight human u-PA; lane 2, 40 ~1 of Mabin Darby Canine Kidney supernatant; lane 3, 40 ~1 of a lo-fold concentrated day 5 alveolar epithelial cell (AEC) supernatant; lane 4, 40 ~1 of lo-fold concentrated day 10 alveolar epithelial cell supernatant. Note the clear zones of PA activity at apparent molecular weights of 48,000 and 31,000 in the alveolar epithelial cell supernatants. A parallel gel without plasminogen showed no lysis zones confirming the plasminogen dependence of this activity. The darkly stained bands at 67 kDa are likely due to serum albumin remaining from the serum-containing media.

To distinguish u-PA from t-PA, we tested the effects of amiloride and soluble fibrin products on the PA activity (Table I). Amiloride has been shown to completely inhibit u- PA and have no effect on t-PA (31). Soluble fibrin products on the other hand stimulate t-PA activity but not u-PA (32). We first tested the effects of these two agents on commercially available human u-PA and t-PA and confirmed the expected pattern of response. We found that amiloride completely inhibited PA activity in the supernatants from both late day unstimulated cells and PMA-stimulated cells. Soluble fibrin products did not significantly augment the PA activity.

Modulation of PA Activity by PMA-The epithelial cells exposed to PMA underwent subtle but distinct changes in their morphologic appearance by phase contrast microscopy. They were more flattened, had less distinct cytoplasmic bor- ders, and contained fewer lamellar inclusions. After a 24-h exposure to PMA there was a dose-dependent increase in

TABLE I

Aloeolur epitheliul cell PA uctiuity: effect of umiloride and soluble fibrin products

PA activity was measured in late day and PMA-stimulated alveolar epithelial cell supernatant (AEC sups+) and commercially available u-PA and t-PA in the standard fashion (no additives) and after the addition of amiloride or soluble fibrin products. The data are presented as percent of the value obtained with no additive. Amiloride and soluble fibrin products had the expected effect on u-PA and t- PA. Note that the response of the alveolar epithelial cell PA activity to these two agents closely paralleled that of u-PA rather than t-PA.

No. Amiloride Soluble fibrin products

ImM 20 rig/ml

Late day AEC sups+ 6 0 ND PMA-stimulated AEC sups+ 6 0 111 Standard u-PA 4 1 89 Standard t-PA 4 74 1230

* ND = not determined.

8

1

PMA 5 rig/ml PMA 50 ng/m1

FIG. 3. Alveolar epithelial cell PA activity: stimulation by PMA. After 2 days in culture, confluent monolayers were washed with PBS and changed to serum-free medium. After 24 h fresh serum- free medium was placed with 0, 5, or 50 rig/ml of PMA. The cell supernatants and lysates were collected in 24 h and analyzed for PA activity. The data presented are PA activity in the supernatants expressed as IU of activity/mg protein in the cell lysate (n = 4, * p < 0.005, tp < 0.001 as compared with control). A dose-dependent increase in PA activity was observed. Note: PMA at a concentration of 200 rig/ml caused significant cell toxicity as manifested by detach- ment of many cells from the culture plate.

PA activity in the cell supernatants (Fig. 3). PA activity in the cell lysates paralleled that of the supernatants (data not shown). A structural analog of PMA that does not activate protein kinase C, FLU-PMA, had no effect on epithelial cell PA activity (data not shown). There was a significant time lag in the PMA induction of epithelial cell PA activity. No increase in PA activity was detectable at 1 or 9 h after the cells were exposed to PMA (data not shown). Preincubation of the cells in 10m7 M dexamethasone for 4 h prior to exposure to PMA inhibited the increase in PA activity in the cell supernatants by 74% (Fig. 4). A similar degree of inhibition (72%) was observed in the cell lysates.

The PMA induction of PA activity required new protein synthesis in that it was completely inhibited by cycloheximide at 10 pg/ml, a level that had no effect on cell viability as evidenced by no significant increase in the proportion of lactic dehydrogenase activity in the culture supernatants. To more specifically examine the synthesis of u-PA, we performed metabolic labeling and immunoprecipitation of control and PMA stimulated cells. We found no ongoing synthesis of u- PA in control unstimulated cells, whereas newly synthesized 48- and 31-kDa forms of u-PA were detectable in the PMA- treated cel!s (Fig. 5). There were no higher molecular weight


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FIG. 4. Alveolar epithelial cell PA activity: effect of dexamethasone. After 2 days in culture, confluent monolayers were washed with PBS and changed in serum-free medium. The following day lo-’ M dexamethasone (DEX) was added to selected wells. In 4 h fresh medium was placed containing dexamethasone lo-’ M, PMA 50 rig/ml, dexamethasone plus PMA, or no additives (Control). The cell supernatants and lysates were collected 24 h later and analyzed for PA activity. The data presented are PA activity in the supernatants expressed as IU of activity/mg protein in the cell lysate (mean f S.E., n = 3, * p < 0.001 as compared with control, t p < 0.005 as compared with PMA). Note that dexamethasone substantially (74%) inhibited the PMA stimulation of PA activity.

I 2 3 4 5 6

205-

Il6- 97’

66-

CONTROL + PMA

FIG. 5. Metabolic labeling and immunoprecipitation of u- PA. A 12.5% polyacrylamide gel electrophoresis gel was run under reducing conditions and processed for autofluorography. Lanes 1 and 4 show [S”“]methionine-labeled proteins in control and PMA-stimulated alveolar epithelial cell lysates. There were 4.6 x 10” and 6.8 x 1O’cpm incorporated/gg protein in control and PMA-stimulated cells, respectively. Lanes 2 and 5 are the immunoprecipitates of control and PMA-stimulated cell supernatants with rabbit anti-mouse urokinase. Lanes 3 and 6 are the precipitates from an equal portion of the same supernatants using non-immune sera. Note that no ongoing synthesis of u-PA was detectable in control cells, whereas newly synthesized 48- and 31-kDa u-PA was present in PMA-stimulated cells. There were no high molecular weight complexes to suggest the presence of a PA inhibitor. The high molecular weight protein (M, > 200,000) precipitated with non-immune sera is likely fibronectin.

complexes in the immunoprecipitates to suggest the presence of a PA inhibitor. Incorporation of [‘“Slmethionine into protein in the control and PMA-stimulated cell lysates was roughly equivalent (4.62 X lo4 uersu.s 6.77 X lo4 cpm/pg protein, respectively). Approximately 0.03% of the total incorporated [““Slmethionine in the PMA-stimulated cell supernatant was precipitated by antibody against u-PA.

We next performed Northern blot analyses to test the possibility that the primary mechanism of PMA up-regulation of new u-PA synthesis occurred at the transcriptional level. We found no u-PA mRNA in control unstimulated cells and a 2.5kilobase u-PA mRNA band in the PMA-stimulated cells analogous to that found in rat kidney (Fig. 6). This induction of u-PA mRNA was readily detectable at 3 h, peaked at 9 h,

-28s

- 18s

RAT AEC AEC KID CONT PMA

FIG. 6. PMA induction of alveolar epithelial cell u-PA mRNA. After 2 days in culture, confluent monolayers were washed with PBS and changed to serum-free medium. After 24 h fresh serum- free medium was placed with 0 or 50 rig/ml of PMA. In 24 h the cells were lysed in guanidine hydrochloride and total RNA prepared (28). Total RNA (10 pg/lane) was separated on the basis of size on a 2.2 M formaldehyde, 1% agarose gel, transferred to a Nylon membrane and probed with a ‘“P-labeled SP 64 plasmid containing a 660-base pair fragment of the mouse u-PA gene. Lane I, rat kidney; lane 2, control, unstimulated alveolar epithelial cells; lane 3, PMA-stimulated alveolar epithelial cells. The position of the ribosomal RNA bands is noted to the right of the autoradiogram. Nonspecific binding to the ribosomal RNA bands at low stringency washes confirmed that there were approximately equal amounts of RNA in each lane. Note that there was no detectable u-PA mRNA in control unstimulated cells and a 2.5.kilobase u-PA mRNA band in PMA-stimulated cells analogous to that found in rat kidney,

TIME 3h 9h 24h 24h 9h

PMA t t t - t

CHX - - - - +

FIG. 7. PMA induction of u-PA mRNA: time course and effect of cycloheximide. Control and PMA-stimulated cells were prepared and Northern analysis performed as described in Fig. 6. Lanes 1-3, alveolar epithelial cells exposed to PMA for 3 h, 9 h, and 24 h, respectively; lane 4, control unstimulated cells; lnne 5, cells exposed to PMA and cycloheximide (10 pg/ml) concomitantly for 9 h. The position of the ribosomal RNA bands is marked to the right of the autoradiogram. Nonspecific binding to the ribosomal RNA bands at low stringency washes confirmed that there were approximately equal amounts of RNA in each lane. Note again that there was no detectable u-PA mRNA in control unstimulated cells. The induction of u-PA mRNA was readily detectable at 3 h, peaked at 9 h, and persisted for 24 h after exposure of the cells to PMA. Cyclo- heximide did not significantly alter the induction of u-PA mRNA by PMA.


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and persisted for 24 h after exposure of the cells to PMA (Fig. 7). While c~cloheximide completefy inhibited expression of u- PA activity, it did not alter the induction of u-PA mRNA by PMA.

Other Potential ~od~~~o~s of PA Tested--The modulation of u-PA varies according to the cell type and species. In addition to PMA and glucucorticoids, several growth factors (33-35), cytokines (36-38), and agents that increase CAMP (39, 40) have been reported to modulate u-PA. Epidermal growth factor has been reported to increase PA production by HeLa cells (411, A431 carcinoma cells (42), and mouse keratinocytes (43), but had no effect on alveolar epithelial cell production of PA. PDGF has been demonstrated to have actions similar to PMA in the 3T3/fibroblast cell line (44, 45). However, we found no change in alveolar epithelial cell PA activity in response to PDGF, consistent with the reported absence of PRGF receptors on epithelial cells (46). TGF-J?, a potent polypeptide that inhibits growth of epithelial cells (47- 49), effects matrix production (50), and modulates the balance of proteases and anti-proteases (33, 35,61) also had no effect on the alveolar epithelial cell production of PA, this same preparation of TGF-@ markedly enhanced the synthesis and release of PAI- by A549 cells (data not shown) as has been described by Keski-Oja et al. (51). Cytokines have been demonstrated to effect PA or PA inhibitor production in endothelial cells (52), macrophages (36), and synovial cells (37, 38). We found that tumor necrosis factor-a, y-interferon, and interleukin-1~ had no effect on the production of u-PA by alveolar epithelial cells. In that increased intracellular CAMP stimulates u-PA synthesis in the porcine kidney epithelial cell line, LLC-PKl (39, 40), we examined the effect of the membrane permeable dibutyryl GAMP and cholera toxin, a direct activator of adenylate cyclase. Neither agent affected PA production by the alveolar epithelial cells.

PA Activity in Rat Alveolar ~Ucro~~ffges-The major con- tami~ating cell in the alveofar epithelial cell cultures was the alveolar ma~rophage. Cultures that contained 5% or greater macrophage contamination were not used in these experiments. Using several methods we addressed the possibility that the measured PA activity might be due to contaminating macrophages. First, we assessed PA activity in freshly harvested macrophages, cell-free lavage, and macrophages placed in culture, We found a pattern analogous to that described in human alveolar macrophages (53), i.e. PA activity in freshly harvested alveolar macrophages (0.95 Z!Z 0.425 IU/mg protein, n = 5 preparations) but none in the culture supernatants or cell lysates (n = 4 preparations). We also found that PMA did not stimulate PA activity of rat alveolar macrophages in culture. Finally, alveolar epithelial cell cultures containing less than 1% alveolar macrophages were obtained by combin- ing a density gradient centrifugation step with differential adherence to IgG-coated plates (24). These nearly pure alveolar epithelial eel1 cultures secreted PA activity in amounts equivalent to those obtained by the standard cell isolation technique. We conclude that the PA activity in the epitheliaf cell cultures could not be explained by contributions from contaminating macrophages.

DISCUSSION The plasmin/PA system likely plays a key role in the

extensive tissue remodeling that follows acute lung injury. However, little is known about which cells pa~icipate in the ~brinol~ic process and how their responses are regulated. It has clearly been established that alveolar type 2 epithelial cells proliferate and repopulate the alveolar lining layer following acute lung injury (21,22). Our work indicates that the

alveolar epithelium must also be considered a po~ntially key contributor to the solubilization of intra-alveolar fibrin deposits that typify acute lung injury (14, 15). We have demonstrated that rat alveolar epithelial cells synthesize and secrete u-PA and that PMA stimulates u-PA synthesis via transcriptional activation of the u-PA gene, We believe this to be the first demonstration of the synthetic potential for u- PA of a parenehymal lung cell.

Several pieces of evidence confirmed that alveolar epithelial cells synthesize and secrete u-PA, as opposed to t-PA. First, we found total inhibition of PA activity by amiloride, an agent that has recently been reported to inhibit u-PA but not t-PA in all species tested (31). Second, we observed no significant stimulation of PA activity by the addition of soluble fibrin products, consistent with u-PA rather than t-PA (32). Third, we found the PA activity at n/p, 48,000 and 31,000 on substrate polyacrylamide gels, characteristic of u-PA, The higher molecular weight form was identical to the mature 48-kDa non- glycosylated mouse u-PA (28) rather than the glycosylated 55-kDa human u-PA. Finally, the immunoprecipitation and Northern blot analyses confirmed that the cells indeed synthesized u-PA.

The u-PA activity was not detectable until several days after cell isolation and was maximal at day 10. This increase in activity occurred concomitant with signi~cant mo~hologic changes in the epithelial cells. We observed, as others have reported (54, 551, that freshly isolated alveolar type 2 epithelial cells were cuboidal and contained cytoplasmic lamellar inclusions, but with time in culture they flattened and lost their lamellar bodies. These in vitro changes are analogous to the in duo transition from the cuboidal type 2 cell with lamellar bodies to the flat, elongated type 1 cell without distinctive cytoplasmic features. In addition to the morphologic changes there is loss of surfactant-rela~d markers (56, 57), a change in membrane lectin-bin~n~ profiles (58, 59) and the appearance of a type 1 cell specific antigen (60) in the in uitro culture system. There is however no consensus as to whether these changes represent squamous differentiation to a type l-like cell or dedifferentiation. Thus, until a fuller understanding of the culture system evolves, it is premature to speculate that the observed increase in PA activity is related to the squamous differentiation process. In any event, the immunolocalization of u-PA to the mouse alveolar epithelium (61) suggests that it is a physiologically relevant product of these cells.

A similar pattern of increased PA activity with time in culture has been reported in two other epithelial cell culture systems, cornea1 epithelial cells (62), and keratinocytes j63- 65). In these cells it has been postulated that the increase in PA may play a role in the process of normal maturation and terminal differentiation. Chan et at. (62) reported a substan- tial increase in detectable PA in the supernat~ts of corneal epithelial cells after confluence was reached and focal multi- layering had begun to occur. Isseroff et e1. (63) showed that normal and transformed keratinocytes secreted significantly increased levels of PA after the cells reached confluence and exhibited morphologic evidence of terminal differentiation. The highest PA activity was found in the squamous cells that were shed from the differentiating cultures.

Several potential mechanisms for the observed increase in PA activity with time in culture can be excluded. First, PA activity in the cell lysates paralleled that found in the supernatants; thus, the increase in activity in the supernatants was not due to secretion of intracellular stores. Second, measure- ment of PA activity with and without catalytic amounts of plasmin demonstrated no major pool of precursor u-PA in the


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early day samples. Third, there was no evidence for a PA inhibitor in the early day samples. The concomitant production of PA and a PA inhibitor has been reported in a variety of cell types including the A549 cell (33), a human lung adenocarcinoma cell line that has some alveolar type 2 cell like features (66,67). However, we found no evidence for a u- PA/PA inhibitor complex on either substrate gels or auto- fluorograms of newly synthesized u-PA. Thus, we believe the increased PA activity with time in culture was due to new synthesis and secretion of u-PA. The lack of ongoing synthesis of u-PA and no detectable u-PA mRNA in the early day unstimulated cells fully support this explanation.

Studies of regulation revealed that PMA but not the inactive structural analog 4cu-PMA induced epithelial cell u-PA activity, putatively via the protein kinase C pathway. In the early day unstimulated cell cultures we found no PA activity, no ongoing u-PA synthesis and no detectable u-PA mRNA. By Northern analyses we discovered that the primary mechanism of PMA up-regulation of u-PA synthesis occurred at the transcriptional level. The 2.5-kilobase u-PA mRNA was readily detectable at 3 h, peaked at 9 h, and persisted for 24 h after exposure of the cells to PMA. In addition, the induction of u-PA mRNA by PMA was not abolished by cycloheximide, suggesting that no intermediate protein synthesis was necessary. Our data are consistent with transcriptional activation of the u-PA gene as part of the early cellular response to PMA as has been described in several other cell types (34, 68-70). The complexity of the effect of protein synthesis inhibition on PMA modulation of u-PA gene expression has been clearly demonstrated; cycloheximide both stimulates u- PA gene transcription and increases u-PA mRNA stability (68). Thus, the finding of PMA induction of u-PA mRNA in the presence of cycloheximide leads us to conclude only that activation of the u-PA gene appears to be a primary effect of PMA. These observations suggest that the mechanism of regulation of the alveolar epithelial cell u-PA gene by PMA is likely a post-translational modification, perhaps phos- phorylation, of a gene activator or repressor.

Several other PMA responsive genes including collagenase have been found to have a common g-base pair sequence in the 5’-flanking region termed the 12-O-tetradecanoylphorbol- 13-acetate (PMA)-responsive element or TRE (71). The protein products of two nuclear proto-oncogenes, c-jun and c-fos, have been implicated in the regulation of these PMA-responsive genes via binding to the TRE and transcriptional activation of coupled genes (71-74). The mouse and human u-PA genes do not contain any TRE-like sequences in the 5’- flanking region. Therefore, further investigation will be necessary to fully elucidate the mechanisms of u-PA gene regulation by PMA.

The glucocorticoids are another class of potent modulators of the fibrinolytic system. They have been demonstrated to decrease u-PA gene transcription, likely via a cis-acting glucocorticoid-responsive negative regulatory element (75, 76), and stimulate production of PA inhibitors (75, 77). In the current investigation we found that dexamethasone substantially inhibited the PMA induction of u-PA activity. Dexa- methasone alone had no effect on PA activity and did not induce the production of a PA inhibitor.2 Its effect on PA activity was thus likely due to decreased transcription of the u-PA gene. This finding may have relevance to the clinical setting of acute lung injury where glucocorticoids have been a commonly used treatment modality. Recent controlled trials have clearly demonstrated no benefit and perhaps harm in

‘B. C. Marshall, D. S. Sageser, N. V. Rao, M. Emi, J. R. Hoidal, unpublished observation.

the administration of steroids in this setting (78). Animal studies have also shown that steroids can exacerbate acute lung injury and lead to significantly poorer outcome (79, 80). We speculate that in part this may be due to glucocorticoid inhibition of the remodeling process via its inhibition of u- PA production.

The extensive remodeling that follows acute lung injury likely involves the coordinated actions of several cell types. The role of the keratinocyte in skin wound healing provides a precedent for the epithelial cell to play a key role in this process. Keratinocytes not only migrate and proliferate to re- epithelialize denuded surfaces (81, 82), but also express proteinases such as u-PA (63-65) and collagenase (83) that may be important in migration and solubilization of provisional matrix. In addition they have the synthetic capacity to recon- stitute a basement membrane (84). Much less is known about the role of the alveolar epithelial cell in repair of injury. It has been clearly shown that type 2 epithelial cells proliferate and repopulate the alveolar lining layer following lung injury (21,22). Several investigators have recently demonstrated the ability of these cells to synthesize a variety of basement membrane components (85, 86). However, the proteolytic capacity of the alveolar epithelial cell has received little atten- tion. Rannels et al. (87) demonstrated that rat alveolar epithelial cells have the capacity to actively degrade matrix proteins in uitro. The mechanism of this degradation was not characterized but almost certainly involved proteinases. We have demonstrated that rat alveolar epithelial cells synthesize and secrete u-PA and that PMA up-regulates u-PA synthesis via transcriptional activation of the u-PA gene. Further investigation of modulation of the alveolar epithelial cell u-PA may provide new insight into the alveolar response to acute lung injury and potential ways to favorably manipulate this response.

Acknowledgments-We gratefully acknowledge Jerri Duncan-Goff and Hollie Kounalis for their secretarial assistance, and Dr. Domi- nique Belin and Dr. Edward J. Campbell for their careful review of the manuscript.

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B C Marshall, D S Sageser, N V Rao, M Emi and J R HoidalAlveolar epithelial cell plasminogen activator. Characterization and regulation.

1990, 265:8198-8204.J. Biol. Chem.

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Alveolar Epithelial Cell Plasminogen Activator · Alveolar Epithelial Cell Plasminogen Activator CHARACTERIZATION AND REGULATION* (Received for publication, November l&1989) Bruce