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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:39-46.)
© 1999 American Heart Association, Inc.

Original Contributions

On the Role of c-Jun in the Induction of PAI-1 Gene Expression by Phorbol Ester, Serum, and IL-1 in HepG2 Cells

Janine Arts; Jos Grimbergen; Karin Toet; Teake Kooistra

From the Gaubius Laboratory, TNO-PG, Leiden, the Netherlands.

Correspondence to Dr T. Kooistra, Gaubius Laboratory, TNO-PG, PO Box 2215, 2301 CE Leiden, the Netherlands.

	Abstract

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Abstract—We have characterized the regulation of plasminogen activator inhibitor-1 (PAI-1) gene expression by phorbol 12-myristate 13-acetate (PMA), serum, and interleukin-1 (IL-1) in the human hepatoma cell line HepG2. PMA, serum, and IL-1 induced a rapid and transient 28-fold (PMA), 9-fold (serum), and 23-fold (IL-1) increase in PAI-1 mRNA, peaking after 4 hours. These inductions of PAI-1 mRNA accumulation were reduced by pretreatment of the HepG2 cells with the protein tyrosine kinase inhibitor genistein. Conversely, stimulation of tyrosine phosphorylation by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in PAI-1 mRNA levels. The effects of PMA, serum, and IL-1 on PAI-1 mRNA expression have been compared with their ability to modulate the expression of a chloramphenicol acetyltransferase (CAT) reporter plasmid, which was under control of the -489 to +75 region of the PAI-1 promoter, and stably transfected into HepG2 cells. This region of the PAI-1 promoter was previously found to contain a tetradecanoyl phorbol acetate–response element (TRE; between -58 and -50) necessary for PMA responsiveness and with a high affinity for c-Jun homodimers. Whereas incubation of these transfected HepG2 cells with PMA and serum showed an induction profile of CAT mRNA similar to that of PAI-1 mRNA, hardly any induction of CAT mRNA was found with IL-1. In line with these findings, IL-1 poorly induced c-Jun homodimer binding to the PAI-1 TRE in gel mobility-shift assays. Pretreatment of HepG2 cells with the protein kinase C inhibitor Ro 31-8220 or the mitogen-activated protein kinase kinase (MAPKK)_1,2 activity blocker PD98059 selectively suppressed the induction of PAI-1 (and CAT) expression by PMA, but not that by IL-1. In contrast, the protein tyrosine kinase inhibitor herbimycin A blocked PAI-1 mRNA induction by IL-1 only. We propose 2 separate PAI-1 inductory pathways for PMA and IL-1 in HepG2, both involving protein tyrosine kinase activation; the serum-induced signaling pathway may (partially) overlap with the PMA-activated protein kinase C/mitogen-activated protein kinase kinase pathway, leading to c-Jun homodimer binding to the PAI-1 TRE.

Key Words: plasminogen activator inhibitor-1 • protein kinase C • interleukin-1 • gene transcription • c-Jun

	Introduction

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Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of tissue-type and urokinase-type plasminogen activators in vivo. Elevated levels of PAI-1 have been implicated in the pathogenesis of thromboembolic disease and may contribute to the risk of reinfarction in patients who have suffered a myocardial infarction. Also, increased PAI-1 gene expression has been observed in atherosclerotic human arteries. On the other hand, PAI-1 deficiencies are associated with a bleeding tendency in humans (for reviews, see References 1 through 3^{1 2 3} ). Definition of the molecular mechanisms by which PAI-1 expression is regulated in these pathological states may lead to a better understanding of the underlying pathophysiology and thereby provide new leads for the prevention of thrombosis and atherosclerosis.

Multiple factors have been identified that play a role in the regulation of PAI-1 synthesis. PAI-1 behaves as an acute-phase reactant in humans, in that plasma levels of PAI-1 are increased in patients during septicemia and after surgery or trauma.^{1 2 3} Furthermore, PAI-1 synthesis in cultured human endothelial cells and human hepatocytes has been shown to be inducible by cytokines and inflammatory mediators, such as endotoxin, interleukin-1 (IL-1), and tumor necrosis factor- (TNF-)^4–6; growth factors like insulin,^{7 8} insulin-like growth factor,⁹ transforming growth factor-ß,¹⁰ and epidermal growth factor¹¹; and the protein kinase C (PKC)–activating phorbol ester, phorbol 12-myristate 13-acetate (PMA).¹²

The human hepatoma cell line HepG2 is often used as a model of human hepatocytes. Many of the stimulators of PAI-1 synthesis in HepG2 cells, including PMA, serum, and IL-1, may be classified as agents that increase the abundance and/or activity of the transcription factor, activator protein-1 (AP-1).¹³ AP-1 is a collection of homodimeric and/or heterodimeric complexes composed of the Jun and Fos gene products. These complexes interact with a common DNA binding site, the PMA-responsive element (TRE), and activate gene transcription in response to activators of PKC, growth factors, and cytokines.^{13 14 15} Several studies have been directed at elucidating the mechanism by which PMA stimulates PAI-1 gene transcription in HepG2 cells. Transfection studies, including mutational analysis, combined with experiments with antisense c-jun and c-fos oligonucleotides and electromobility shift assays, point to an important role for c-Jun homodimer binding to the TRE at positions -58 to -50 of the PAI-1 promoter in the regulation of basal and PMA-stimulated gene transcription in HepG2 cells.^{16 17 18}

In this study, we have applied several approaches to delineate whether or not IL-1 and serum stimulate PAI-1 gene transcription through the same regulatory mechanism as found for PMA. Treatment of cells with stimuli such as PMA, serum, and IL-1 result in activation of phosphorylation cascades utilizing mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs).¹⁹ MAPKs and SAPKs comprise a family of related protein kinases that are themselves activated by phosphorylation on threonine and tyrosine residues. In addition to the differences in substrate specificities, MAPKs and SAPKs differ in their responses to extracellular stimuli. MAPKs are most efficiently stimulated by growth factors and phorbol esters, whereas SAPKs are activated in response to proinflammatory cytokines such as IL-1.¹⁹ We have investigated the role of protein tyrosine kinases in the stimulation of PAI-1 gene expression by using the protein tyrosine kinase inhibitors genistein and herbimycin A and the protein tyrosine phosphatase inhibitor sodium orthovanadate. Genistein and herbimycin A have previously been shown to suppress rather selectively the basal and IL-1–stimulated PAI-1 gene expression in cultured human endothelial cells,²⁰ and genistein has frequently been reported to inhibit the induction of c-jun.^{21 22} We have used the specific MAPK kinase 1/MAPK kinase 2 (MAPKK₁/MAPKK₂) activity blocker PD98059²³ and the specific SAPK2/p38 inhibitor SB 203580²⁴ to evaluate the role of the MAPK cascade in PAI-1 gene regulation. Furthermore, we have used stably transfected HepG2 cells, in which the expression of a reporter gene, chloramphenicol acetyltransferase (CAT), is under control of the -489 to +75 region of the PAI-1 promoter. This promoter region contains the c-Jun homodimer binding site essential for PMA induction of PAI-1 gene transcription.^{16 17 18} In addition, we have compared PMA, IL-1, and serum on their capacity to induce c-jun mRNA expression and to increase c-Jun homodimer binding to the -58 to -50 region of the PAI-1 promoter by using gel-shift analysis.

	Methods

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Materials
4ß-PMA was from Sigma Chemical Co. A stock solution of PMA (100 µmol/L) was prepared in ethanol and kept at -20°C. Human recombinant IL-1 was a gift from Dr S. Gillis (Immunex Corp, Seattle, Wash). A stock solution of IL-1 (10⁵ U/mL; specific activity, 10⁸ U/mg) was kept at -80°C. FCS was from Flow Laboratories. Genistein, herbimycin A, sodium orthovanadate, and PD98059 were from LC Laboratories. SB 203580 was from Alexis Biochemicals. Ro 31-8220 was a gift from Dr G. Lawton, Hoffmann-LaRoche (Welwyn Garden City, UK). Stock solutions of genistein (100 mg/mL), herbimycin A (5 mg/mL), PD98059 (10 mmol/L), and SB 203580 (10 mmol/L) were prepared in dimethyl sulfoxide and kept at -20°C. A stock solution of sodium orthovanadate (500 mmol/L) was freshly prepared in PBS (0.15 mol/L NaCl, 10 mmol/L Na₂HPO₄, and 1.5 mmol/L KH₂PO₄, pH 7.4) at the start of each experiment. Anti–c-Jun and anti–c-Fos polyclonal antibodies were a gift from Dr T. Oehler (Massachusetts Institute of Technology, Cambridge). Deoxycytidine 5'-[-³²P]triphosphate (3 Ci/µmol), adenosine 5'-[-³²P]triphosphate (3 Ci/µmol), [³⁵S]methionine (>1 Ci/µmol), and the Megaprime kit were from Amersham Nederland BV. Bradford protein reagent was from Bio-Rad. Human serum albumin (HSA) [20% (wt/vol), pyrogen-free] was from the Central Laboratory of the Red Cross Blood Transfusion Service (Amsterdam, the Netherlands). Other materials used in the methods described below have been specified in detail in pertinent references or were purchased from standard commercial sources.

Cell Culture
HepG2 cells were grown as monolayer cultures under a 5% CO₂–95% air atmosphere at 37°C in Dulbecco's modification of Eagle's medium supplemented with 10% (vol/vol) FCS (heat-inactivated), 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine as described previously.¹² For experiments confluent cultures were used, and cells were always refed the day before the experiment with incubation medium, viz, serum-free Dulbecco's modified Eagle's medium containing 0.1% (wt/vol) HSA, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine. Conditioned media were obtained by incubating the cells at 37°C for various times up to 24 hours with incubation medium containing the appropriate concentration of the test compound or stock solvent. After incubation, the cells were washed twice with ice-cold PBS and were used for isolation of RNA or preparation of nuclear extracts.

Northern Blot Analysis
Total RNA was isolated as described by Chomczynski and Sacchi²⁵ and fractionated by electrophoresis in a 1% (wt/vol) agarose gel under denaturing conditions with 1 mol/L formaldehyde. The RNA was transferred onto Hybond-N filters by blotting, and the filters were hybridized overnight at 63°C in NaP_i hybridization mix {7% (wt/vol) SDS, 0.5 mol/L Na₂HPO₄/NaH₂PO₄ buffer (pH 7.2), and 1 mmol/L EDTA} containing 3 ng of [-³²P]dCTP–labeled probe per milliliter. The probes were labeled with a Megaprime kit, yielding an average activity of 0.2 µCi/ng DNA. After hybridization with the PAI-1, GAPDH, or CAT probe, the filters were washed twice with 2x SSC (1x SSC is 0.15 mol/L NaCl and 0.015 mol/L trisodium citrate) and 1% (wt/vol) SDS and twice with 1x SSC and 1% (wt/vol) SDS for 20-minute time periods at 63°C. In the case of hybridizations with c-jun or c-fos probe, the filters were washed with 2x SSC and 1% (wt/vol) SDS for 4 successive 20-minute periods at 63°C. The filters were then exposed to Kodak XAR-5 x-ray film with an intensifying screen at -80°C. The relative intensities of the bands present were determined on a Fujix Bas 1000 PhosphorImager.

cDNA Probes
The cDNA fragments used as probes in the hybridization experiments were as described previously¹⁸: a 2.5-kb EcoRI fragment of the human PAI-1 cDNA; a 1.2-kb PstI fragment of the rat GAPDH cDNA provided by Dr R. Offringa (Leiden University, Leiden, the Netherlands); a 1.0-kb PstI fragment of the mouse c-jun cDNA; and a 1.5-kb EcoRI fragment of the murine c-fos cDNA. A 0.6-kb NotI fragment of the pOPRSVICAT expression vector was obtained from Stratagene.

Preparation of Nuclear Extracts
Nuclear extracts for gel-shift experiments were prepared as described previously.¹⁸ HepG2 cells (25 cm²) were rinsed twice with ice-cold PBS and lysed in 2 mL of lysis buffer [10 mmol/L Tris buffer, pH 7.4; 10 mmol/L NaCl; 3 mmol/L MgCl₂; 0.5% (vol/vol) NP-40; 1 mmol/L DTT; 0.25 mmol/L sodium orthovanadate; and 1 µg/mL of the protease inhibitors leupeptin, pepstatin, and aprotinin]. The lysate was homogenized, and nuclei were collected by centrifugation (5 minutes at 1000g, 4°C) and washed with lysis buffer once more. The dry nuclear pellet was resuspended in 150 µL of 20 mmol/L HEPES, pH 7.9; 400 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L EGTA; 1 mmol/L DTT; 1 mmol/L PMSF; 0.25 mmol/L sodium orthovanadate; and 1 µg/mL of leupeptin, pepstatin, and aprotinin. This suspension was incubated for 15 minutes at 4°C while being continuously shaken and then centrifuged at 1000g, 4°C, for 5 minutes. Supernatants were stored at -80°C until use. The protein concentration in the nuclear extracts was determined using the Bradford protein assay.

Gel-Shift Experiments
For electromobility shift assays, an oligodeoxynucleotide representing the PAI-1 promoter region between -66 and -43 (5'-CTGGAACATGAGTTCATCTATTT-3') was used.¹⁸ All oligonucleotides shown and their complementary oligonucleotides were synthesized by Isogen Bioscience. The oligodeoxynucleotide was end-labeled using T4 kinase and subsequently purified by phenol/chloroform extraction and ethanol precipitation. For the electromobility shift assays, 25 fmol (10⁴ counts per minute) of radiolabeled, double-stranded oligodeoxynucleotide was mixed with nuclear extract (5 µg protein) in a total volume of 20 µL of 20 mmol/L HEPES (pH 7.9), 20 mmol/L KCl, 2 mmol/L MgCl₂, 20% (vol/vol) glycerol, 2.5 mmol/L EDTA, 2 mmol/L spermidine, 1 µg poly(dI-dC), 1 µg BSA, 1 mmol/L PMSF, and 2.5 pmol nonspecific competitor oligodeoxynucleotide (5'-CTGAGGATTCTCCACTGCA-3'). The mixture was incubated for 30 minutes at 4°C. DNA/protein complexes were separated from the nonbound oligodeoxynucleotide by electrophoresis on a 5% polyacrylamide gel in 0.25x TBE buffer (22.5 mmol/L Tris-borate, pH 8.0, and 0.5 mmol/L EDTA). Electrophoresis was carried out at room temperature at 150 V for 70 minutes using 0.25x TBE as the running buffer. After the gel had been dried on Whatman-3 MM paper, DNA/protein complexes were visualized by autoradiography.

Immunoprecipitation of c-Jun and c-Fos Proteins
HepG2 cells (10 cm²) were incubated in methionine-free culture medium supplemented with 0.1% (wt/vol) HSA, 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mmol/L L-glutamine, and 0.2 mCi/mL [³⁵S]methionine. Cell extracts were prepared as described previously.¹⁸ Cells were washed twice with ice-cold PBS and harvested by scraping in 0.5 mL of lysis buffer [50 mmol/L Tris buffer, pH 8.0; 125 mmol/L NaCl; 0.5% (vol/vol) NP-40; 0.5% (wt/vol) sodium deoxycholate; 0.1% (vol/vol) SDS; and the proteinase inhibitors leupeptin (1 µg/mL), pepstatin A (1 µg/mL), aprotinin (1 µg/mL), and PMSF (0.5 mmol/L)]. The cell lysates were centrifuged in a Beckman TL-100 centrifuge at 150 000 rpm (30 minutes, 4°C). The cleared cell lysates were then incubated for 1 hour at 4°C with preimmune rabbit serum coupled to protein A–Sepharose under continuous rotation. The protein A–Sepharose was removed (30 seconds at 10 000g, 4°C), and the supernatant was subsequently used to immunoprecipitate c-Fos complexes by the same procedure, using an anti–c-Fos rabbit polyclonal antibody. Finally, the c-Fos– and c-Fos/c-Jun–depleted extract was incubated with anti–c-Jun antiserum coupled to protein A–Sepharose.²⁶ All immunocomplexes were washed 4 times with 1 mL lysis buffer and once with PBS, resuspended in Laemmli sample buffer [62.5 mmol/L Tris buffer (pH 6.8), 10% (wt/vol) glycine, 2% (vol/vol) SDS, 5% (vol/vol) ß-mercaptoethanol, and 0.02% (wt/vol) bromophenol blue],²⁷ and boiled for 5 minutes. The samples were subjected to electrophoresis (10 mA, 16 hours) on a 10% (wt/vol) SDS-polyacrylamide gel. The gel was dried on Whatman-3 MM paper, and labeled proteins were visualized with a Fujix Bas 1000 PhosphorImager.

Selection of Stable HepG2 Transfectants
HepG2 cells (10 cm²) were transfected with the calcium phosphate coprecipitation procedure with 10 µg of a -489 to +75 PAI-1-promoter CAT construct¹⁸ and 2.5 µg of the pSV2NEO plasmid, which conveys neomycin resistance under control of the SV40 promoter. Stable transfectants were obtained by selection with 0.4 mg/mL G418 sulfate (Life Technologies). Individual clones were isolated, propagated, and characterized for expression of the CAT construct.

Protein Synthesis
Overall protein synthesis was determined by measuring the incorporation of [³⁵S]methionine into the 10% (wt/vol) trichloroacetic acid–precipitable fraction of radiolabeled conditioned medium and cell extract.²⁸

	Results

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Regulation of PAI-1 mRNA Accumulation and Activity of a c-Jun–Binding Region of the PAI-1 Promoter by PMA, IL-1, and Serum
In HepG2 cells, PMA-induced PAI-1 gene expression involves c-Jun homodimer binding to the -58 to -50 region of the PAI-1 promoter.^{16 17 18} As a first approach to evaluate the role of this c-Jun–binding region in the induction of PAI-1 by serum and IL-1, we have used stably transfected HepG2 cells, in which the expression of the reporter gene CAT is under control of the -489 to +75 region of the PAI-1 promoter. This system allowed simultaneous analysis of the expression of PAI-1 mRNA and the activity of the c-Jun–binding proximal region in the PAI-1 promoter. Figure 1A shows autoradiograms of Northern blots of RNA isolated from HepG2 cells incubated for various times with PMA (100 nmol/L), serum (10%, vol/vol), or IL-1 (300 U/mL) and probed with PAI-1 and CAT. In human cells, 2 PAI-1 mRNA species of 3.2 and 2.4 kb, reflecting different polyadenylation sites, are expressed. With all 3 agents, induction of the 3.2-kb PAI-1 mRNA was evident after 2 hours, became maximal after 3 to 4 hours, and then rapidly declined, with the IL-1 response appearing markedly slower than the PMA one. Induction of the 2.4-kb mRNA started more slowly, peaked after 4 to 6 hours, and then gradually decreased. Maximal accumulation of PAI-1 mRNA (3.2- and 2.4-kb species) when normalized for the amount of GAPDH mRNA and expressed as the ratio of experimental to control at t=0 was 28-fold with PMA (at 4 hours), 9-fold with serum (at 6 hours), and 23-fold with IL-1 (at 4 hours). The induction of CAT mRNA shows a similar time profile to that of PAI-1 mRNA. However, whereas PMA and serum stimulated CAT mRNA accumulation to an extent similar to that of PAI-1 mRNA (27-fold at 4 hours for PMA and 7-fold at 6 hours for serum), IL-1 hardly induced CAT mRNA at all (maximally 2-fold at 3 hours).

View larger version (31K): [in this window] [in a new window]   Figure 1. Effect of PMA, serum, IL-1, and vanadate on PAI-1 and CAT mRNA levels in HepG2 cells incubated in the presence or absence of genistein. HepG2 cells were preincubated with serum-free medium for 16 hours, and then fresh, serum-free medium containing PMA (100 nmol/L), serum (10%, vol/vol), IL-1 (300 U/mL), or sodium orthovanadate (250 µmol/L) was added. A, At the indicated times, total RNA from PMA-, serum-, or IL-1–treated cells was isolated and analyzed by Northern blotting for PAI-1 and CAT mRNA expression. B, One hour before the addition of PMA, serum, or IL-1, serum-free medium with (+) or without (-) genistein (60 µg/mL) was added. Total RNA was isolated 3 hours after induction and analyzed by Northern blotting for PAI-1 and CAT mRNA expression. C, Total RNA was isolated 12 hours after the addition of vanadate and analyzed by Northern blotting for PAI-1 and CAT mRNA expression. The amount of RNA in each lane was equal according to a reference probe, GAPDH (shown in C only). Blots were exposed to Amersham Hyperfilm for 2 to 3 days. The experiments are representative of 2 (vanadate) or 3 independent experiments.

Effect of Genistein and Vanadate on the Induction of PAI-1 mRNA and Promoter Activity
Genistein at 60 µg/mL, a concentration that almost completely prevents the increase in PAI-1 synthesis induced by IL-1 in cultured human endothelial cells and partly reduces the basal PAI-1 production by these cells,²⁰ did not affect basal levels of PAI-1 mRNA in HepG2 cells over a 4-hour period (data not shown). Genistein (60 µg/mL), when added 1 hour before the addition of PMA, serum, or IL-1 to HepG2 cells, almost fully blocked the induction of PAI-1 and CAT mRNAs by these agents at 3 hours (Figure 1B). This inhibiting effect of genistein was concentration dependent and became detectable at concentrations as low as 10 µg/mL (data not shown). One of the possible mechanisms of genistein action on the induction of PAI-1 gene transcription may be inhibition of protein tyrosine kinase activity. To further define the role of tyrosine phosphorylation in the regulation of PAI-1 gene expression, the effect of vanadate, an inhibitor of protein tyrosine phosphatases,²⁹ was evaluated. Vanadate at 250 µmol/L slowly but continuously increased PAI-1 and CAT mRNA levels in HepG2 cells, as illustrated in Figure 1C for a 12-hour incubation period. Remarkably, only the accumulation of the 3.2-kb PAI-1 transcript was observed with vanadate.

Because the induction of PAI-1 gene expression by PMA, serum, and IL-1 is dependent on ongoing protein synthesis and because inhibition of protein synthesis by cycloheximide has been reported to result in the accumulation of PAI-1 mRNA,¹² we checked genistein and sodium orthovanadate for their effect on overall protein synthesis. Overall protein synthesis, as measured by [³⁵S]methionine incorporation, was not markedly affected by genistein or sodium orthovanadate at the concentrations used (data not shown), indicating that their effects on PAI-1 expression are directly related to their protein tyrosine kinase– and phosphatase-inhibiting activities, respectively.

Effect of PMA and IL-1 on c-jun Expression and c-Jun DNA–Binding Activity
Previous studies indicated that PMA, serum, and IL-1 all transiently induce the accumulation of c-jun mRNA in HepG2 cells.^{15 18} This would suggest that all 3 agents are able to stimulate expression of the CAT reporter gene in HepG2 cells when this reporter gene is under control of the c-Jun–binding region of the PAI-1 promoter. However, the results depicted in Figure 1A show that only PMA and serum effectively stimulated the accumulation of CAT mRNA in HepG2 cells; IL-1 proved to be a poor inducer of CAT mRNA accumulation. We therefore further evaluated the induction of c-Jun by PMA (100 nmol/L) and IL-1 (300 U/mL) in HepG2 cells. As shown in Figure 2A, the mRNA for c-jun was strongly enhanced by PMA and IL-1 after 1 hour. At this time point, the mRNA for c-fos, the heterodimeric partner of c-jun, was also substantially elevated in the PMA-treated HepG2 cells. These stimulatory effects of PMA and IL-1 on c-jun and c-fos mRNA accumulation were completely suppressed in the presence of genistein (60 µg/mL; Figure 2A). To determine whether the increase in c-jun and c-fos mRNAs was reflected at the protein level, we analyzed c-Jun and c-Fos protein induction by PMA and IL-1 by immunoprecipitation of radiolabeled cell extracts by using anti–c-Jun and anti–c-Fos polyclonal antibodies (Figure 2B). To distinguish between complexes consisting of c-Jun/c-Fos heterodimers and c-Jun/c-Jun homodimers, cell extracts were first incubated with anti–c-Fos and subsequently with anti–c-Jun polyclonal antiserum. Figure 2B shows that radiolabeled c-Fos (a broad band 55 kDa) and coprecipitated c-Jun (39 kDa) levels were strongly increased in PMA-treated HepG2 cells after 1.5 hours. Levels of radiolabeled c-Jun not complexed to c-Fos were also strongly increased after PMA treatment. These results thus confirm that the PMA-induced increases in c-jun and c-fos mRNA are reflected at the protein level. In contrast to PMA, no significant amount of radiolabeled c-Jun/c-Fos protein was observed in IL-1–treated HepG2 cells, and the induction of homodimeric c-Jun protein by IL-1 was poor (Figure 2B).

View larger version (42K): [in this window] [in a new window]   Figure 2. Effect of PMA and IL-1 on c-jun and c-fos expression in HepG2 cells incubated in the presence or absence of genistein. HepG2 cells were preincubated with serum-free medium for 16 hours and then incubated with serum-free medium containing PMA (100 nmol/L), IL-1 (300 U/mL), or stock solvent (ctrl). One hour before the addition of PMA or IL-1, serum-free medium with (+) or without (-) genistein (60 µg/mL) was added. A, Total RNA was isolated after 1 hour and analyzed by Northern blotting for c-jun and c-fos mRNA expression. Blots were exposed to Amersham Hyperfilm for 7 days. The experiment shown is representative of 3 independent experiments. B, [35S]Methionine was added 30 minutes after the addition of PMA, IL-1 (IL1), or stock solvent (C), and cell extracts were harvested after 1.5 hours. Immunoprecipitations were performed successively with preimmune serum (ctrl), anti–c-Fos polyclonal antiserum (-c-Fos), and anti–c-Jun polyclonal antiserum (-c-Jun). The immunoprecipitates were subjected to polyacrylamide gel electrophoresis, and radiolabeled proteins were visualized by autoradiography for 2 days on a Fujix Bas-1000 PhosphorImager screen. Brackets indicate the positions of c-Jun and c-Fos proteins on the basis of their molecular weights reported in the literature.26

To analyze the functionality of the induced c-Jun complexes, we performed electromobility shift assays. As shown in Figure 3, 2 specific DNA/protein complexes can be observed on incubation of the -66 to -43 region of the PAI-1 promoter with nuclear extracts from HepG2 cells. Competition experiments with 100-fold excess unlabeled, double-stranded, consensus TRE oligodeoxynucleotide (as present in the collagenase promoter), but not random oligodeoxynucleotide, inhibited the formation of both complexes, as illustrated previously.¹⁸ By preincubation of the nuclear extracts with anti–c-Jun or anti– c-Fos polyclonal antibodies, it was deduced that the lower complex (complex 2) predominantly consisted of DNA-bound c-Jun homodimers, whereas the upper complex (complex 1) contained c-Jun protein heterodimerized with an unidentified protein.¹⁸ We noted that PMA very strongly but IL-1 only weakly induced the formation of c-Jun homodimer–binding complexes (complex 2). No significant induction of complex formation was observed with nuclear extracts of HepG2 cells that had been stimulated by PMA or IL-1 in the presence of genistein (Figure 3).

View larger version (55K): [in this window] [in a new window]   Figure 3. HepG2 nuclear protein binding to the PAI-1 promoter AP-1–binding site as assayed by electromobility shift assay. HepG2 cells were preincubated with serum-free medium for 16 hours and then refreshed with serum-free medium with or without genistein (60 µg/mL). After 1-hour incubation, PMA (100 nmol/L), IL-1 (300 U/mL), or stock solvent (control) was added. After 2.5 hours, nuclear extracts were prepared as described in Methods. Extracts were incubated for 30 minutes with a radiolabeled oligodeoxynucleotide representing the -66 to -43 region of the PAI-1 promoter. DNA/protein complexes were separated on a 5% (wt/vol) polyacrylamide gel, and radiolabeled complexes were visualized by autoradiography for 5 days. Arrows mark specific DNA/protein complexes.

Effect of Herbimycin A, Ro 31-8220, PD98059, and SB 203580 on PMA- and IL-1–Stimulated PAI-1 Gene Expression
The above results are consistent with the notion of 2 separate PAI-inductory pathways for PMA and IL-1, both involving protein tyrosine kinase activation as indicated by our findings with genistein. To find further evidence for 2 distinct signal transduction pathways, we evaluated the effects of different kinase inhibitors on PMA- and IL-1–stimulated PAI-1 gene expression. First, we examined the effect of another protein tyrosine kinase inhibitor, herbimycin A. Herbimycin A, like genistein, was previously demonstrated to block IL-1–stimulated PAI-1 gene expression in cultured human endothelial cells,²⁰ but different target proteins have been suggested for each inhibitor.³⁰ As shown in Figure 4, herbimycin A (0.3 µg/mL) potently inhibited IL-1–induced PAI-1 mRNA expression, but unlike genistein, was ineffective toward PMA-stimulated PAI-1 and CAT mRNA induction. Also in contrast to genistein, herbimycin A had no effect on c-jun or c-fos mRNA induced by IL-1 or PMA (data not shown). Because several reports have demonstrated that PMA (via activation of PKC) and IL-1 trigger the activation of different MAPK cascades, we tested the effects of the selective PKC inhibitor Ro 31-8220, the specific MAPKK₁/MAPKK₂ activity blocker PD98059,²³ and the specific SAPK₂/p38 inhibitor SB 203580²⁴ on PMA- and IL-1–stimulated PAI-1 gene expression. As illustrated in Figure 4, Ro 31-8220 (1 µmol/L) and PD98059 (10 µmol/L) prevented the induction of PAI-1 and CAT mRNAs by PMA but did not considerably quench the IL-1–stimulated PAI-1 mRNA increase. The suppressive effect of Ro 31-8220 and PD98059 on PAI-1 and CAT mRNA induction by PMA was paralleled by comparable suppression of c-jun and c-fos mRNA levels (data not shown). With SB 203580 at concentrations up to 30 µmol/L, we found a small increase rather than a decrease in the levels of PAI-1 and CAT transcripts caused by PMA and IL-1. Taken together, the results obtained with different kinase inhibitors provide further evidence that the kinase cascade responsible for IL-1–induced PAI-1 gene activation is distinct from the PMA-triggered PKC/MAPK route.

View larger version (29K): [in this window] [in a new window]   Figure 4. Effect of herbimycin A, Ro 31-8220, PD98059, and SB 203580 on PMA- and IL-1–induced PAI-1 and CAT mRNA levels in HepG2 cells. HepG2 cells were preincubated with serum-free medium for 16 hours and then incubated with serum-free medium containing herbimycin A (0.3 µg/mL), Ro 31-8220 (1 µmol/L), PD98059 (10 µmol/L), SB 203580 (30 µmol/L), or stock solvent (vehicle). After a 4-hour (herbimycin A) or 1-hour incubation, PMA (100 nmol/L), IL-1 (300 U/mL), or stock solvent (n.a.) was added. Total RNA was isolated after 2 hours and analyzed by Northern blotting for PAI-1 and CAT mRNA expression. The amount of RNA in each lane was equal according to a reference probe, GAPDH (not shown). Blots were exposed to Amersham Hyperfilm for 2 days (PAI-1, GAPDH) or 3 days (CAT). The experiment shown is representative of 3 independent experiments.

	Discussion

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Regulation of PAI-1 gene transcription is responsive to a large variety of hormones, cytokines, and growth factors, thus reflecting the important role of the inhibitor in several physiological and pathophysiological conditions.^{2 3} Although induction of PAI-1 expression by various compounds often shows very similar characteristics, the present studies demonstrate that precisely how PAI-1 transcription is regulated is, at least in part, a function of the stimulus involved.

Early experiments showed that the PKC activating phorbol ester PMA, serum, and IL-1 induced PAI-1 expression in HepG2 cells at the transcriptional level with comparable time profiles and to a similar extent; these inductions were found to be dependent on ongoing protein synthesis.¹² Furthermore, the potent, dose-dependent inhibition of PMA-, serum-, and IL-1–induced PAI-1 expression by genistein, as demonstrated in this article, is strongly suggestive of tyrosine phosphorylation's being an intermediary step in the action of all 3 agents. Previous experiments showed that the PMA response required the PAI-1 AP-1–binding site at positions -58 to -50, and DNA-protein binding studies showed an interaction between this promoter region and c-Jun homodimers.^{16 18} Here, we demonstrate in experiments with HepG2 cells that were stably transfected with a CAT expression vector under control of the -489 to +75 region of the PAI-1 promoter that this promoter fragment is sufficient to mediate the PMA and serum responses, but that other regions of the PAI-1 promoter must mediate IL-1 induction of PAI-1 transcription. Gel-shift experiments using the PAI-1 AP-1–binding promoter region confirmed that IL-1, in contrast to PMA, hardly induced c-Jun binding activity. These results are consistent with 2 separate PAI-1 inductory pathways for PMA and IL-1, whereby the serum-mediated signal transduction pathway may (partially) overlap the PMA-activated PKC/MAPK pathway. Further evidence for 2 distinct signaling routes was obtained by using different kinase inhibitors. The protein tyrosine kinase inhibitor herbimycin A, unlike genistein, only blocked the induction of PAI-1 by IL-1. On the other hand, treatment of cells with Ro 31-8220 (a PKC inhibitor) or PD98059 (a specific MAPKK₁/MAPKK₂ activity blocker) selectively attenuated the stimulating effect of PMA on PAI-1 (and CAT) expression, but not that of IL-1. Neither of the 2 pathways involves the activation of p38/SAPK, because the selective inhibitor SB 203580²⁴ was unable to inhibit the increase in PAI-1 gene transcription caused by PMA and IL-1.

In support of our finding that the IL-1 response mechanism in HepG2 cells differs from that of the PKC activator PMA, Fandrey et al³¹ found no translocation of PKC isoenzymes with IL-1 (or TNF-) in HepG2 cells, strongly suggesting that these cytokines do not activate PKC in these cells. Furthermore, Daffada et al³² reported relatively poor IL-1 induction of an AP-1–responsive reporter construct in HepG2 cells. Also, IL-1 has been reported to be a weak inducer of the AP-1–responsive region of the collagenase promoter in fibroblasts.³³ Finally, Bird et al³⁴ showed that, in HepG2 cells, IL-1 does induce the p54/SAPK, which mediates c-jun transcriptional induction, but in contrast to PMA, is unable to activate the p42/44 MAPKs, which induce c-Jun DNA–binding activity.³⁵ These latter findings may also explain the lack of c-Jun activation by IL-1 in our studies, even though c-jun mRNA levels were effectively induced.

Several studies implicate a role for nuclear factor-B (NF-B) in IL-1 signal transduction in HepG2 cells. For example, Daffada et al³² showed that IL-1 strongly increased NF-B activity in HepG2 cells by using a CAT expression vector under control of an NF-B region. Whether NF-B plays a role in IL-1–induced PAI-1 gene transcription in HepG2 cells or through which site the IL-1 response is mediated is not clear at present. One candidate region is the IL-1–inducible site between -675 and -669 reported by Dawson et al.³⁶ This site has similarities to an NF-B binding site, and NF-B has also been implicated in the PAI-1 transcriptional induction by TNF- in human endothelial cells.³⁷ In this context it is of interest that both genistein and herbimycin A have been reported to inhibit NF-B activation by IL-1.^{38 39}

We demonstrated that genistein effectively suppressed the induction of PAI-1 gene transcription by PMA, serum, and IL-1. Genistein probably acts by inhibiting a protein tyrosine kinase, because the structural genistein analogue daidzein, which has low protein tyrosine kinase activity,⁴⁰ did not inhibit stimulated PAI-1 synthesis. Consistent with an involvement of tyrosine kinases in PAI-1 gene transcription are the data obtained with sodium orthovanadate, a potent inhibitor of protein tyrosine phosphatases.²⁹ We found that incubation of stably transfected HepG2 cells with sodium orthovanadate resulted in the accumulation of (the 3.2-kb form of) PAI-1 mRNA and CAT mRNA. Similarly, we found that genistein effectively suppressed the induction of c-jun mRNA by PMA, serum, and IL-1. Interestingly, another protein tyrosine kinase inhibitor, herbimycin A, only blocked the induction of PAI-1 by IL-1 but not that by PMA. Also, herbimycin A did not suppress IL-1–increased c-jun mRNA levels. It is unlikely that the inhibition of c-jun mRNA induction by genistein is a direct effect on a PKC-dependent pathway, because genistein is a poor inhibitor of PKC activity, with an apparent IC₅₀ >100 µg/mL.⁴⁰ Discrepant findings with different protein tyrosine kinase inhibitors as shown here for genistein and herbimycin A were also recently described for the regulation of inducible nitric oxide synthase mRNA in primary rat hepatocytes,³⁰ suggesting different target proteins for each inhibitor. However, exactly where genistein and herbimycin A interfere with the 2 separate PAI-1 inductory pathways for PMA and IL- is not clear at present and requires further research.

An interesting observation during our studies on the role of protein tyrosine phosphorylation in PAI-1 transcriptional regulation was the fact that in the presence of sodium orthovanadate, only the upper band of PAI-1 mRNA (the 3.2-kb form) accumulated in HepG2 cells, whereas in experiments with genistein, predominantly the lower band (the 2.4-kb form) was detectable. How such a shift in the ratio between the 2 PAI-1 mRNAs is brought about cannot be deduced from our work. One possibility is a shift in the use of the 2 alternative polyadenylation sites, as has been observed for the mouse dihydrofolate reductase gene during cell growth and for the rat GsN1 signal transduction protein gene after dexamethasone treatment.^{41 42} The other possibility is a change in posttranscriptional regulation of PAI-1 gene expression depending on protein tyrosine phosphorylation. In this respect, it might be significant that genistein has been found to decrease c-myc mRNA in NIH-3T3 cells at a similar concentration that inhibits PAI-1 expression in HepG2 cells⁴³; c-myc protein has been suggested to affect PAI-1 gene expression at the level of RNA processing, nuclear RNA turnover, and RNA export.^{44 45}

In conclusion, stimuli as different as PMA, serum, and IL-1 appear to have similar effects on PAI-1 gene transcription regulation in HepG2, but their mechanism of action is fundamentally different. These findings might be of relevance in understanding elevated PAI-1 gene expression in various disease states.

	Acknowledgments

 
This study was supported by a grant from the Netherlands Heart Foundation (90.267 to J.A. and J.G.).

Received September 29, 1997; accepted May 6, 1998.

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