THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 18, Issue of June 25, pp. 10396-10401, 1989 Printed in U. S. A.

Regulation of Type 1 Plasminogen Activator Inhibitor Gene Expression in Cultured Bovine Aortic Endothelial Cells INDUCTION BY TRANSFORMING GROWTH FACTOR-@, LIPOPOLYSACCHARIDE, AND TUMOR NECROSIS FACTOR-a*

(Received for publication, September 20, 1988)

Michael Sawdey, Thomas J. Podor, and David J. LoskutoffS From the Research Institute of Scripps Clinic, La Jolla, California 92037

Cultured bovine aortic endothelial cells (BAEs) syn- thesize and secrete type 1 plasminogen activator inhib- itor (PAT-l), an M, 50,000 glycoprotein which inhibits both urokinase and tissue-type plasminogen activators. The synthesis of PAI- 1 in BAEs is positively regulated by a variety of agents. To elucidate the mechanisms which govern expression of the PAI-1 gene, total cy- toplasmic RNA was prepared from BAEs and analyzed by Northern blotting using a 1.3-kilobase (kb) human PAI- 1 cDNA probe. Hybridization under conditions of high stringency revealed two bovine PAI-1 RNA spe- cies, 3.0 and 1.6 kb in length. The ratio of the two species was approximately 4:l. The 3.0-kb mRNA was bound by oligo(dT)-cellulose, whereas the 1.6-kb form was not, suggesting that the latter form lacked a poly(A) terminus. Treatment of BAEs with transform- ing growth factor @ (TGF-@), bacterial lipopolysaccha- ride (LPS), or tumor necrosis factor a (TNF-a) mark- edly enhanced the steady-state levels of both RNA species. In each case, increases were detectable within 1 h, and maximal effects (Le. >30-fold increase) were observed between 6 and 18 h of treatment, followed by a decline to near-basal levels by 48 h. The response to each of these agents was dose-dependent, with maxi- mal induction observed at concentrations of 10 ng/ml TGF-@, 10 ng/ml LPS, and 25 ng/ml TNF-a. Induction of PAI-1 mRNA by these agents was not blocked by the protein synthesis inhibitor cycloheximide, suggest- ing that de novo protein synthesis was not required. In fact, treatment with cycloheximide (2 cLg/ml) alone also increased PAI- 1 mRNA levels. Treatment with cyclo- heximide in combination with TGF-@, LPS, or TNF-a further enhanced the accumulation of PAI-1 mRNA. Nuclear transcription run-on experiments indicated that these agents elevated the rate of PAI-1 gene tran- scription 20-30-fold and that gene template activity was temporally correlated with the accumulation of PAI-1 mRNA. These data are consistent with the con- clusion that the observed increases in PAI-1 steady- state mRNA levels result from primary effects of these agents on the rate of PAI-1 gene transcription.

* This research was supported by National Institutes of Health Grant HL-16411 (to D. J. L.). This is publication number 5553-1” from the Research Institute of Scripps Clinic. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Research Institute of Scripps Clinic/IMM-14, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-8229.

Activation of the fibrinolytic system and the generation of plasmin are subject to negative regulation by the action of specific, naturally occuring plasminogen activator inhibitors (PAIS).’ Endothelial or type 1 PA1 (PAI-1) rapidly inhibits the activity of both urokinase (uPA) and tissue-type (tPA) plasminogen activators (1-4) and appears to be the primary physiologic inhibitor of tPA (5). PAI-1 was initially purified from the conditioned medium of cultured bovine aortic endo- thelial cells (BAEs; Ref. 1) and has been detected in platelets (61, plasma (7, 8), and a variety of cell types, including fibroblasts (9-12), epithelial (11, 12), mesothelial, (11), and human endothelial cells (1, 13, 14).

The biosynthesis of PAI-1 in endothelial cells is increased by a variety of physiologic or pathologic mediators, including the polypeptide growth modulators transforming growth fac- tor-@ (TGF-B; Refs. 15 and 16) and basic fibroblast growth factor (15); the toxic principle of Gram-negative bacteria, lipopolysaccharide (LPS; Refs. 17-19); and the inflammatory cytokines interleukin-1 (Il-l; 18, 20, 21) and tumor necrosis factor-a (TNF-a; Ref. 22). These molecules may potentially alter either local or systemic concentrations of PAI-1. For example, TGF-@, which promotes the formation of granula- tion tissue and the growth of new blood vessels in uiuo (23), increases the synthesis and deposition of PAI-1 into the extracellular matrix of several cell types (9, 16, 24). Localized deposition of PAI-1 into the matrix may serve to limit the generation of extracellular proteolytic activity during these processes, as recent studies have demonstrated that matrix- bound PAI-1 reacts with exogenously added uPA or tPA by forming complexes which dissociate from the matrix (24-26). Clinically, elevations in plasma PAI-1 activity have been detected in LPS-mediated Gram-negative sepsis (17) and as a component of acute phase reactions following major surgery (27,28), trauma (7,28), and myocardial infarction (29). These systemic increases in PAI-1 may derive a t least in part from the increased biosynthesis of PAI-1 in endothelium, in re- sponse to LPS, Il-l, and/or TNF-a (5).

The molecular cloning of the PAI-1 gene (30-32) has en- abled direct examination of PAI-1 gene expression through the use of cDNA probes. Two human PAI-1 mRNA species,

The abbreviations used are: PAI-1, plasminogen activator inhib- itor type 1; tPA, tissue-type plasminogen activator; uPA, urokinase plasminogen activator; BAEs, bovine aortic endothelial cells; LPS, lipopolysaccharide; TNF-a, tumor necrosis factor-a; Il-l, interleukin- 1; kb, kilobase(s); oligo(dT), oligodeoxythymidylate; poly(A), poly- adenylic acid; DMEM, Dulbecco’s modified Eagle’s medium; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid MOPS, 4-mor- pholinepropanesulfonic acid; PIPES, piperazine-N,N’-bis(2-ethane- sulfonic acid); PBS, phosphate-buffered saline; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; SDS, sodium dodecyl sulfate.

10396

Regulation of PAI-1 Gene Expression in Endothelial Cells 10397

approximately 3.2 and 2.3 kilobases (kb) in length, have been observed in all human cells known to express PAI-1, including umbilical vein endothelium (31, 32). In the current studies, we present evidence that BAEs also contain two PAI-1 RNA species, the levels of which are markedly increased in response to TGF-@, LPS, and TNF-a. We show further that these increases in steady-state PAI-1 mRNA levels result primarily from enhanced transcription of the PAI-1 gene.

EXPERIMENTAL PROCEDURES

Materials

Reagents were obtained as follows: Dulbecco's modified Eagle's medium (DMEM), HEPES buffer solution, L-glutamine, penicillin- streptomycin mixture, and 10 X concentrated Dulbecco's phosphate- buffered saline (PBS) without calcium or magnesium from M. A. Bioproducts (Bethesda, MD); Hyclone bovine calf serum from Sterile Systems (Logan, UT); fetal calf serum from GIBCO; tissue culture plasticware from Falcon (Becton, Dickinson and Co., Cockeysville, MD); Tris-HC1, EDTA, KCI, sodium acetate, NaH2P04, Na2HP04, Nonidet P-40, bovine serum albumin fraction V (BSA), PIPES, MOPS, phenylmethylsulfonyl fluoride (PMSF), antipain, a-amani- tin, cycloheximide, isoamyl alcohol, and oligo(dT)-cellulose from Sigma; glycerol, HCl, NaCl, sodium citrate, MgClz, trichloroacetic acid, and chloroform from Mallinckrodt (Paris, KY); creatine phos- phate, creatine phosphokinase, ATP, CTP, GTP, phenol (molecular biology grade), placental RNase inhibitor, yeast tRNA, and restriction enzymes from Boehringer Mannheim; dithiothreitol (DTT) and ac- tinomycin D from Calbiochem; sodium dodecyl sulfate (SDS) from Bio-Rad; vanadyl ribonucleoside complex from Bethesda Research Labs; absolute ethanol from US1 Chemicals (Tuscola, IL); BA 85 nitrocellulose from Schleicher & Schuell; Biotrans nylon membrane from ICN (Irvine, CA); [cY-~'P]~GTP (800 Ci/mmol), [a-3'P]uridine triphosphate (UTP; 3000 Ci/mmol), and ~-[~~S]methionine (800 Ci/ mmol) from Amersham, and XAR x-ray film from Eastman Kodak.

Purified human platelet-derived TGF-8 was obtained as a kind gift from Dr. Michael Sporn, National Institutes of Health, Bethesda, MD. TGF-@ was reconstituted in 4 mM HCl,O.l% BSA, to a concen- tration of 5 pg/ml and stored at -70 "C. Human recombinant TNF- a (Genzyme, Boston, MA) was supplied as a 10 pg/ml stock solution in 1 X PBS, 0.1% BSA, and stored at -70 ' C . Salmonella minnesota Re595 LPS was obtained as a gift of Drs. John Mathison and Richard Ulevitch of the Research Institute of Scripps Clinic. LPS was purified from bacteria using the phenol-chloroform-petroleum ether method (33). We have recently observed that serum alteration of LPS strongly potentiates its effect on BAE PAI-1 biosynthesis' and have thus employed serum-altered LPS in the current studies. For experimental use, serum-altered LPS was prepared by a modification of the method of Mathison et al. (34). Briefly, Re595 LPS (final concentration 0.5 mg/ml) containing biosynthetically labeled [3H]Re595 LPS added as tracer was incubated with fetal calf serum for 30 min at 37 "C. An aliquot (2.5 ml) of this solution was then diluted with an equal volume of sterile saline, mixed with 2.1 g of solid CsCl, and centrifuged for 60 h, 40,000 rpm in a SW 50.1 rotor (Beckman Instruments). This procedure results in the partitioning of the exogenously added LPS into lipoprotein-rich material, comprising the upper 20% of the gradient and in a single, visible band of lipoprotein-deficient material (i.e. protein) exhibiting intermediate density? The latter fraction was collected by aspiration and dialized against several changes of sterile saline. The final concentration of LPS in the preparation was deter- mined by scintillation counting.

Methods

Cell Culture-BAEs were isolated from adult bovine aorta by previously described methods (35). The cells employed for these studies were derived from a single cell and exhibited positive immu- nofluorescent staining for Factor VI11 (1). Cultures were serially propagated in DMEM supplemented with 100 mM HEPES buffer, 20 mM L-glutamine, 50 units/ml penicillin/50 pg/ml streptomycin, and 10% bovine calf serum. For experiments, cells were grown to conflu- ence in 100-mm tissue culture dishes, washed twice with sterile 1 X PBS, and incubated for 24 h in serum-free DMEM. The medium was

T. Podor, M. Sawdey, J. Mathison, R. Ulevitch, and D. J. Los- kutoff, manuscript in preparation.

then removed and replaced with the experimental medium as indi- cated.

RNA Isolation, Northern Blotting, and Hybridization-Total cyto- plasmic RNA was isolated from BAEs by a modification of the method of Berger and Birkenmeier (36). Cells were washed once in ice-cold PBS, removed from the culture dish by scraping with a rubber policeman, and pelleted by centrifugation at 1660 X g for 5 min at 4 "C. The pellets were resuspended vigorously in TE buffer (10 mM Tris-HC1, pH 8.0, 1 mM EDTA) containing 0.65% Nonidet P-40 and 10 mM vanadyl ribonucleoside complex, and placed on ice for 5 min. Cell nuclei were removed by centrifugation for 5 min at 10,000 X g, 4 "C. The supernatant was then mixed with 1/10 volume of a solution containing 0.58 M Tris-HC1, pH 9.0, 0.058 M EDTA, and 3% SDS, extracted twice with phenokch1oroform:isoamyl alcohol (25:25:1), once with chloroform, and precipitated with 1/10 volume 3 M sodium acetate and 2.5 volumes absolute ethanol at -20 "C for 16 h. The mixtures were then centrifuged for 20 min at 10,000 X g, 4 "C, and the RNA resuspended in distilled Hz0 for measurement of A at 260 nm. The concentration of RNA was calculated based upon an extinc- tion coefficient of 1 A unit = 42 pg/ml. Polyadenylated RNA was prepared by affinity chromatography on oligo(dT)-cellulose according to the method of Aviv and Leder (37). Fractionation of RNA on formaldehyde-containing agarose gels and blot transfer to nylon membranes were as described previously (22). Blots were prehybrid- ized for 15 min and then hybridized for 16 h at 65 "C in 50 mM PIPES buffer, pH 6.8,200 mM NaC1, 20 mM Na2P04, 30 mM NaHP04, 1 mM EDTA, and 5% SDS. The blots were then washed four times for 10 min each with prewarmed (65 "C) 1.3 X ssc (200 mM NaCl, 20 mM sodium citrate) containing 5% SDS. Where described, blots were hybridized by the procedure of Thomas (53), utilizing more stringent washing procedures (i.e. four washes for 5 min each in 2 X SSC, 0.1% SDS, at 22 "C, and two washes for 15 min each in 0.1 X SSC, 0.1% SDS at 45 "C). Blots were exposed to Kodak XAR film at -70 'C with intensifying screens. The density of autoradiographic signals was quantitated with a Zeineh soft laser scanning densitometer (Biomed Instruments, Chicago, IL), equipped with an integrator. Relative values were expressed as the number of integrator units corresponding to individual bands. Alternatively, the quantity of radioactivity corresponding to individual bands was measured directly using an Ambis Radioisotope Scanner I1 (Automated Microbiology Systems, San Diego, CA) as described (22, 38).

Nuclear Transcription Run-om-Nuclear transcription run-on as- says were performed essentially according to the procedure of Green- berg and Ziff (39). Briefly, cell pellets containing 3 X 10' BAEs were vortexed for 10 s in 10 ml of prechilled (4 "C) Nonidet P-40 lysis buffer (10 mM Tris-HC1, pH 7.4; 10 mM NaC1; 3 mM MgC12; 1 mM EDTA; 0.1 mM PMSF 1 mM DTT; M antipain; 0.5% Nonidet P-40), and placed on ice for 5 min. The nuclei were collected by centrifugation at 1660 X g for 5 min at 4 "C, and the lysis procedure

buffer (50 mM Tris-HC1, pH 8.3; 0.1 mM EDTA; 1 mM DTT; 40% repeated once. The nuclei were then resuspended in glycerol storage

glycerol) at lo7 nuclei/100 pl, and snap-frozen in liquid nitrogen. For run-on reactions, the nuclei were mixed with an equal volume of 2 X reaction buffer (10 mM Tris-HC1, pH 8.0; 5 mM MgC12; 0.3 M KC1; 1 mM each ATP, CTP, GTP; 5 mM DTT; 25 units/ml placental RNAse inhibitor; 10 mM creatine phosphate; 20 unitslml creatine phospho- kinase; 0.1 mM PMSF) containing 100 pCi [c~-~'P]UTP, and incubated for 30 min at 26 "C. As a control, replicate run-on reactions were performed in the presence of the RNA polymerase inhibitor 01-

amanitin (final concentration 10 pg/ml). Isolation of nuclear RNA, and prehybridization, hybridization, and washing of filters were ex- actly as described (40). Autoradiography and densitometric scanning procedures were as detailed for Northern blots,

Preparation of DNA Probes-A human PAI-1 cDNA fragment, corresponding to bases 1-1249 of the human PAI-1 cDNA (30), was purified by preparative agarose gel electrophoresis, employing an Elutrap filter (Schleicher & Schluell) according to the manufacturers instructions. The purified fragment was nick-translated in the pres- ence of [cI-~'P]~GTP to a specific activity 510' dpm/pg for use in Northern blot hybridizations. For nuclear run-on experiments, 1.0 pg of the PAI-1 cDNA fragment, or 1.0 pg plasmid DNA linearized with the appropriate restriction enzyme, was denatured and slot-blotted on nitrocellulose filters essentially as described (39). Plasmids con- taining cDNA inserts encoding human von Willebrand factor (vWF; Ref. 41) or the Chinese hamster gene choB (42) were obtained as gifts from Dr. Dennis Lynch of Harvard Medical School and Dr. Michael Wilson of the Research Institute of Scripps Clinic, respec- tively. The choB gene encodes an mRNA of presumed "housekeeping"

10398 Regulation of PAI-1 Gene Expression in Endothelial Cells

function (54,55). The choB plasmid was thus employed as a negative control probe in the Northern blot experiments in Figs. 2-4 and in the nuclear run-on experiments in Figs. 5 and 6.

Miscellaneous-radiolabeling of BAEs with methionine and trichloroacetic acid-precipitation of cell lysates to assess incorpora- tion was by the procedure of Mimuro et a/. (16).

RESULTS

Total cytoplasmic RNA prepared from BAEs was analyzed by Northern blotting as described under “Methods,” employ- ing a 1.3-kb human PAI-1 cDNA probe corresponding to the PAI-1 coding region (30). Two bovine PAI-1 RNA species, approximately 3.0 and 1.6 kb in length, were detected (Fig. 1). Similar results were observed in replicate Northern blot experiments employing more stringent conditions for washing of the blots (53; data not shown). In previous studies employ- ing sucrose gradient centrifugation to characterize oligo(dT)- selected BAE PAI-1 mRNA (43), we detected only one mRNA species, of estimated length 3.1 kb. To determine if this apparent discrepancy was due to oligo(dT) selection of the RNA, total cytoplasmic BAE RNA was again subjected to chromatography on oligo(dT)-cellulose and the results ana- lyzed by Northern blotting. Fig. l shows the starting material (lane I ) , the column eluate (lane 2), and the column flow- through (lane 3) . The 3.0-kb species was greatly enriched in the polyadenylated RNA (Fig. 1, lane 2) relative to the 1.6-kb form. Multiple cycles of oligo(dT) selection resulted in the depletion of the 3.0-kb PAI-1 mRNA species from the total RNA (Fig. 1, compare lunes 1 and 3) , whereas the level of the 1.6-kb species did not appear to diminish under these condi- tions. Thus, the two PAI-1 RNA species appear to differ in their affinity for oligo(dT)-cellulose, suggesting that the 1.6- kb form lacks a poly(A) terminus.

The effects of agents known to alter production of PAI-1 protein on steady-state PAI-1 mRNA levels in BAEs were assessed. Confluent cultures of BAEs were incubated for various times in the presence of TGF-@ (1 ng/ml), LPS (10 ng/ml), or TNF-a (2 ng/ml) and total RNA extracted and analyzed by Northern blotting (Fig. 2). Each agent induced rapid elevations in the levels of both PAI-1 transcripts, with

1 2 3

3.OKb-

1.6Kb-

FIG. 1. Northern blot hybridization analysis of total cyto- plasmic and oligo(dT)-selected BAE RNA. Total cytoplasmic RNA was prepared from BAEs treated for 5 h with 10 ng/ml LPS. Polyadenylated BAE RNA was then selected by three repetitive cycles of affinity chromatography on an oligo(dT)-cellulose column (37). The results were analyzed by Northern blotting with a 1.3-kb human PAI-1 cDNA probe, as described under “Experimental Procedures.” Lune I,,total cytoplasmic BAE RNA, 10 pg; lane 2, column eluate, 2 pg; lane 3, column flow-through, 10 pg. Autoradiographic exposure times were varied for optimal visualization of the result. The length in kilobases ( K b ) of the PAI-1 RNA species was estimated by co- electrophoresis of BAE RNA with RNA markers (not shown).

B

- C

D

I n u

200.

175

150.

125. .! .,

.. ’..

FIG. 2. Time course of PAI-1 RNA induction by TGF-8, LPS, and TNF-a. Confluent cultures of BAEs were preincubated 24 h in serum-free DMEM, and fresh media containing 1 ng/ml TGF- p ( A ) , 10 ng/ml LPS ( B ) , or 2 ng/ml TNF-a (C) was added. Total cytoplasmic RNA (10 pg/lane) was then extracted at the indicated times and analyzed by Northern blotting for PAI-1 mRNA. Identical time course experiments were performed with serum-free DMEM (autoradiograms not shown). The results corresponding to the 3.0-kb PAI-1 mRNA species were quantitated by use of a &scanning device. To control for variability in gel loading, the blots were rehybridized with a cDNA probe for the Chinese hamster ovary gene, choB, and the level of choB mRNA was determined by densitometric scanning of the autoradiograms (see “Methods”). The data for the 3.0-kb PAI- 1 mRNA were normalized to the level of choB mRNA by dividing the PAI-1 mRNA counts/min value for each time point by the respective choB relative values. The normalized data are plotted in D. W, LPS; 0, TGF-8; A, TNF-CY; 0, control.

increases detectable within 1 h of incubation. Maximal induc- tion was observed at 6-18 h. The levels of mRNA began to decrease by 18-24 h and returned to near-basal levels by 48 h. An apparent biphasic response of PAI-1 mRNA to TGF-@ was observed, with peaks at 6 and 18 h (Fig. 2A). However, only a single peak a t 6 h was observed in three independent repetitions of this experiment (data not shown). To determine if this discrepancy was due to inconsistencies in quantitation,

Regulation of PAI-1 Gene Expression in Endothelial Cells

loading, or transfer of total RNA, the Northern blots shown in Fig. 2 were rehybridized with a negative control probe, choB (42). This probe detects a 1.1-kb mRNA of presumed housekeeping function (54, 55) expressed a t constant levels under the conditions employed for these experiments (see Fig. 4). The level of the 3.0-kb PAI-1 mRNA in Fig. 2, A-C was quantitated by use of a @-scanning device and corrected for variation in gel loading by normalization to the choB signal. The results are shown in Fig. 2 D. An essentially monophasic response was observed for each agent. The maximal level of induction corresponded to greater than 30-fold increases in the 3.0-kb PAI-1 mRNA, relative to control incubations with serum-free media (Fig. 2 0 ) . The 1.6-kb species displayed a similar pattern of induction, as is apparent from the autora- diograms (Fig. 2, A-C). In general, the two species exhibited coordinate regulation in these experiments, with the ratio of the larger to smaller form remaining constant a t approxi- mately 4:l.

The dose dependence of the response of PAI-1 mRNA to TGF-P, LPS, and TNF-a was investigated. BAEs were incu- bated for 10 h with increasing concentrations of each agent, and total RNA was prepared for Northern blot analysis. Changes in PAI-1 mRNA levels were quantitated by densi- tometric scanning of the autoradiogram (Fig. 3). Increases in the level of the 3.0-kb PAI-1 mRNA species could be detected with doses as low as 0.1 ng/ml for each agonist. LPS induction was maximal at 10 ng/ml, with a decline in mRNA levels a t higher doses, most likely reflecting cytotoxic effects. TGF-P and TNF-a each exhibited a similar dose dependence, with maximal responses observed a t 10 and 25 ng/ml, respectively.

To determine whether de novo protein synthesis was re- quired for induction of PAI-1 mRNA, total RNA was isolated from BAEs incubated for 4 h with TGF-P (5 ng/ml), LPS (10 ng/ml), TNF-a (5 ng/ml), or serum-free DMEM, in the presence or absence of cycloheximide (2 pg/ml). This concen- tration of cycloheximide inhibited ~-[~‘S]methionine incor- poration in BAEs by 93% (data not shown). Northern blot analysis revealed that the effects of TGF-@, LPS, and TNF- a were not blocked by cycloheximide at these concentrations (Fig. 4). Instead, PAI-1 mRNA accumulation was further induced (ie. superinduced) by cycloheximide. Incubation of BAEs with cycloheximide alone also significantly increased

Relative Value

2o : 15

LP s- TGF-B TNF-a Concentration, ng/ml

FIG. 3. Dose response of PAI-1 mRNA induction by LPS, TGF-B, and TNF-a. BAEs were preincubated 24 h in serum-free DMEM and then incubated in fresh media containing the indicated concentrations of each agent. Total cytoplasmic RNA was extracted after 10 h and analyzed by Northern blotting. The results show the levels of the 3.0-kb PAI-1 mRNA as quantitated by densitometric scanning of the autoradiogram. Rehybridization of the blot with the control probe, choB, gave an essentially constant signal intensity for the choB mRNA in each lane (data not shown).

Control +TGF-I) +LPS - + - + - +

I, ” --

A B C

10399

+TNF-a - +

” ] PAL1

(I, * -CHO B

D FIG. 4. The effect of cycloheximide on PAI-1 mRNA accu-

mulation. BAEs were preincubated 24 h in serum-free DMEM and refed with serum-free DMEM ( A ) or serum-free DMEM containing 5 ng/ml TGF-P ( B ) , 10 ng/ml LPS ( C ) , or 5 ng/ml TNF-n (D) in the presence (+) or absence (-) of cycloheximide (2 pglml). Total cyto- plasmic RNA (10 pg/lane) was extracted after 4 h and analyzed by Northern blotting with probes for PAI-1 and choB as indicated.

C TGF-P LPS TNF-a CHX

PAI-1

CHO B

vWF

FIG. 5. The effect of TGF-B, LPS, TNF-a, and cyclohexi- mide on PAI-1 gene transcription. Nuclei were isolated from BAEs treated for 8 h with either serum-free DMEM, 1 ng/ml TGF- 0, 10 ng/ml LPS, 2 ng/ml TNF-n, or 2 pg/ml cycloheximide ( C H X ) . Run-on reactions and hybridization of the purified run-on RNA to the indicated cDNA probes were performed as described under “Ex- perimental Procedures.”

the level of PAI-1 mRNA (Fig. 4A). Rehybridization of the blot with the control probe, choB, gave an essentially constant signal intensity for the choB mRNA in each lane. Additional experiments were performed employing more stringent crite- ria for the inhibition of protein synthesis. BAEs were prein- cubated for 1 h in either serum-free DMEM or serum-free DMEM containing 100 pg/ml cycloheximide, which inhibited L-[””Slmethionine incorporation in these cells by >99.5%. TNF-a was then added to a final concentration of 25 ng/ml, and RNA extracted 1 h later for analysis by Northern blotting. The level of PAI-1 mRNA was again enhanced in cyclohexi- mide-treated cells receiving TNF-a, relative to TNF-a con- trols (data not shown), consistent with the results shown in Fig. 4 0 .

To determine the effects of these agents on the rate of PAI- 1 gene transcription, nuclear transcription run-on experi- ments were performed with nuclei isolated from BAEs. Nas- cent nuclear transcripts were elongated in the presence of [a- “PIUTP and hybridized to various cDNA probes immobilized on nitrocellulose filters. The results are shown in Fig. 5. A pronounced increase of radiolabeled “run-on” RNA hybridiz- ing to PAI-1 cDNA was found in nuclei prepared from BAEs treated with TGF-P, LPS, and TNF-a. A smaller but signifi- cant increase was detected in cells treated with cycloheximide.

10400 Regulation of PAI-1 Gene Expression in Endothelial Cells

......I..... ................................. ...... h

0 3 8 24 Time (h)

FIG. 6. Time course of PAI-1 gene transcription. Nuclei treated as in Fig. 5 were isolated at 3, 8, and 24 h and assayed as above for PAI-1 gene transcription. The results were quantitated by scanning densitometry of the autoradiograms. Input counts/min were equalized to 2 X 106/hybridization. A, TNF-a, W, LPS; 0, TGF-8; +, cycloheximide; 0, control.

Control probes for the Chinese hamster gene, choB (42), or the human von Willebrand factor gene (41) were included in the hybridization reactions. Relatively little change in the transcription of these genes was observed under the condi- tions employed. The addition of a-amanitin (10 pg/ml) to replicate run-on reactions resulted in a complete loss of hy- bridization signal (data not shown), indicating that the PAI- 1 gene is transcribed by RNA polymerase 11.

Time course experiments were then performed in order to compare the changes in PAI-1 gene transcription relative to changes in steady-state RNA levels. Nuclei were isolated from BAEs incubated for 3,8, or 24 h with each agonist. The results of the run-on hybridizations were quantitated by densitomet- ric scanning of the autoradiograms and are presented in Fig. 6. TGF-P, LPS, and TNF-a each elevated PAI-1 gene tran- scription within the first 3 h. The peak responses were ob- served at 8 h and represented 20-30-fold increases over con- trols. Cycloheximide also induced a modest increase in PAI- 1 gene transcription, exhibiting a peak at 3 h, and declining slowly thereafter.

DISCUSSION

Previous observations in our laboratory have demonstrated that the synthesis of PAI-1 is increased in BAEs exposed to TGF-P (16), LPS (44), and in human endothelial cells exposed to TNF-a (22). The current studies establish that the stimu- latory effects of these agents on PAI-1 biosynthesis in BAEs are a consequence of large-scale increases in PAI-1 mRNA (Fig. 2). Elevations in steady-state PAI-1 mRNA levels have been reported in cultured human fibrosarcoma cells in re- sponse to dexamethasone (40,45) and in cultured human lung fibroblasts in response to TGF-P (10, 46). These elevations were shown to result at least in part from increased PAI-1 gene transcription in these systems (40, 47). In addition, the human PAI-1 promoter region has recently been isolated and shown to confer glucocorticoid inducibility on heterologous reporter genes (47, 48). The magnitude and kinetics of TGF- p, LPS-, and TNF-a-induced alterations in the rate of BAE PAI-1 gene transcription (Fig. 6) essentially parallel the con- comitant alterations in BAE PAI-1 mRNA levels (Fig. 2). These data are consistent with the conclusion that the ob- served increases in steady-state PAI-1 mRNA in BAEs result primarily from events at the level of transcription. The rapid onset of induction (Fig. 2) and the lack of inhibition by cycloheximide (Fig. 4) suggest further that these events are primary in nature, reflecting signal transduction mechanisms

independent of de nouo protein synthesis. The 3”untranslated region of human PAI-1 mRNA con-

tains AU-rich sequences homologous to functionally charac- terized 3‘ AU sequences implicated in mRNA turnover (49), raising the possibility that increases in PAI-1 mRNA half- life could also account for its increased accumulation. As our data do not fully exclude this possibility, we performed prelim- inary experiments employing the transcriptional inhibitor actinomycin D to examine the rate of PAI-1 mRNA decay in BAEs. The half-life of the 3.0-kb mRNA in the basal state (t1h = 2.2 h) was not increased in BAEs preincubated for 3 h with TGF-P, LPS, or TNF-a, but was increased by cyclohex- imide (tlh = 3.8 h).3 Similar results were observed for the 1.6- kg ~pecies.~ Thus TGF-P, LPS, and TNF-(Y do not appear to stabilize PAI-1 mRNA in these cells. However, it should be noted that actinomycin D has been shown to both decrease (50) and prolong (51) mRNA half-life and thus may conceiv- ably perturb cellular signaling mechanisms which influence mRNA decay.

Two human PAI-1 transcripts, 3.2 and 2.3 kb in length, have been demonstrated in several human cell lines (10, 30, 40,45, 46), including cultured human umbilical vein endothe- lial cells (22, 31, 32). Sequencing of human PAI-1 cDNA clones and Northern blotting of polyadenylated RNA isolated from human cells (31, 45, 46) have indicated the presence of poly(A) termini on both human PAI-1 mRNAs. Available evidence suggests these mRNAs arise from the use of alter- native polyadenylation signals, and differ only in the length of their 3”untranslated regions (30-32, 52). The Northern blot hybridization experiments of total cytoplasmic BAE RNA presented here reveal the existence of two bovine endo- thelial PAI-1 transcripts, 3.0 and 1.6 kb in length. These two transcripts appear to differ in their affinity for oligo(dT)- cellulose (Fig. l) , suggesting that the smaller 1.6-kb form lacks a poly(A) tail. These findings are in agreement with earlier studies in our laboratory on the characterization of BAE PAI-1 mRNA, where the existence of a single 3.1-kb bovine mRNA was postulated based on the in vitro translation of oligo(dT) selected, size-fractionated BAE RNA (43). In preliminary experiments performed to further analyze the two bovine transcripts, a 3‘ cDNA probe, corresponding to nucle- otides 1998-2478 of the human PAI-1 cDNA (30) hybridized to the 3.0-kb bovine PAI-1 mRNA but not to the 1.6-kb specie^.^ Thus, the two bovine PAI-1 RNAs are analogous to the two human mRNAs in that they also differ in the length of their 3’-untranslated regions. However, the apparent lack of polyadenylation of the 1.6-kb RNA species is suggestive of novel processing mechanisms distinct from the use of alter- native polyadenylation sites (e.g. cleavage by endonucleases involved in mRNA degradation). This possibility is currently under investigation.

Acknowledgments-We thank Robert Medcalf, Wolf-Dieter Schleuning, and Fedor Bachmann for assistance with nuclear run-on studies and Peggy Tayman for manuscript word processing.

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