THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 29, Issue of July 22, pp. 19095-19100, 1994 Printed in U.S.A.

Induction of Plasminogen Activator Inhibitor-1 in HepG2 Human Hepatoma Cells by Mediators of the Acute Phase Response*

(Received for publication, March 16, 1994)

Annette M. HealyS and Thomas D. Gelehrter From the Departments of Human Genetics and Internal Medicine, University of Michigan Medical School, Ann Arbot; Michigan 48109-0618

The liver plays a central role in the systemic acute phase response of an organism to injury. Plasminogen activator inhibitor-1 (PAI-l), a major regulator of fibrin- olysis, is an important component of the acute phase response in humans. The source of plasma PAL1 has been a matter of controversy, but recent in situ hybrid- ization experiments have demonstrated that human hepatocytes express the PAI-1 gene in vivo. However, little is known about regulation of human hepatic PAI-1 gene expression by mediators of the acute phase re- sponse.

We have analyzed the regulation of PAI-1 mRNA accu- mulation by interleukin (IL)-l, IL-6, and dexametha- sone, known mediators of the acute phase response, in HepG2 cells, a highly differentiated human hepatoma cell line that produces a broad spectrum of acute phase proteins including PAI-1. Incubation of HepG2 cells with IL-1 resulted in a rapid and transient 40-fold induction of the 3.2-kilobase PAI-1 mRNA and a 30-fold induction of the 2.2-kilobase PAI-1 -A. Although IL-6 alone had only a modest effect on PAL1 expression, in combination with IL-1, it caused a synergistic induction of PAI-1 -A accumulation. Dexamethasone alone did not in- crease PAI-1 mRNA accumulation but enhanced it in combination with IL-1. Using nuclear run-on experi- ments, we determined that the mechanism by which IL-1 alone, or in combination with IL-6, induced PAI-1 mRNA accumulation was to cause a 10-15-fold, transient stimu- lation of PAL1 gene transcription. We found no evidence of an effect of these cytokines on PAI-1 mRNA stability. These data demonstrate that mediators of the acute phase response induce the accumulation of PAI-1 mRNA in human hepatoma cells by rapidly and transiently in- creasing the transcription of the PAI-1 gene.

The systemic acute phase (AP)’ response to tissue injury is characterized by alterations in plasma concentrations of a large number of proteins known as acute phase reactants, which are synthesized and secreted by the liver. The physiologic role of the AP response is to reduce the systemic effects of tissue damage by enhancing clot formation, reducing proteolytic tissue dam- age, and facilitating the clearance of toxic metabolites (1-5). The major mediators of the AP response have been shown to be cy- tokines, including IL-1, IL-6, and tumor necrosis factor a. In

*This work was supported by National Cancer Institute Grant CA22729. 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. Tel.: 313-747-3159;

activator inhibitor-1; IL, interleukin; BSA, bovine serum albumin; DRB, The abbreviations used are: AF’, acute phase; PAI-1, plasminogen

5,6-dichloro-l-~-~-ribofuranosylbenzimidazole; kb, kilobase(s).

Fax: 313-763-3784.

addition, glucocorticoids can act synergistically with IL-6 and IL-1 to stimulate the synthesis of acute phase reactants (1-3,5).

Plasminogen activator inhibitor-1 (PAI-11, a serine protease inhibitor, is the primary inhibitor of tissue type and urokinase type plasminogen activators in vivo (6). PAI-1 behaves as an acute phase reactant in humans in that plasma levels of PAI-1 are increased in patients with sepsis and after surgery, trauma, or extracorporeal circulation (7-9). The magnitude and time course of the increase in plasma PAI-1 concentrations after major surgery parallels that of the AP reactant, C-reactive protein (7). Also, PAI-1 synthesis has been shown to be induc- ible by cytokines and inflammatory mediators in cultured hu- man endothelial cells and in human hepatoma cells (10, l l ) . The source of plasma PAI-1 has been a matter of controversy; both liver and endothelial cells have been suggested as the source (12-15). We have recently shown by in situ hybridization that human hepatocytes express the PAI-1 gene in vivo and that this expression is elevated in patients with sepsis,’ strongly suggesting that hepatocytes are an important site of PAI-1 synthesis during the acute phase response.

The human hepatoblastoma cell line HepG2 is often used as a model of human hepatocytes. These cells are highly differen- tiated and synthesize a variety of serum proteins including a broad spectrum of acute phase proteins (16,171. The expression of these AP proteins has been shown to be responsive to IL-1, IL-6, and tumor necrosis factor a (3, 18-20). HepG2 cells also synthesize PAI-1, the expression of which has been shown to be regulated by insulin (211, insulin-like growth factor (21), trans- forming growth factor p (22), epidermal growth factor (23), and phorbol esters (24).

It has recently been reported that PAI-1 protein synthesis in HepG2 cells is induced 2-3-fold by IL-1 or tumor necrosis factor a but is not induced by IL-6 (11). PAI-1 activity has also been reported to be increased by cytokines in the human hepatoma HuH-7 cell line (25). In this study, we have examined the effects of IL-1, IL-6, and dexamethasone on the accumulation of PAI-1 mRNA in HepG2 cells. We report that IL-1 causes a rapid and transient, concentration-dependent, 30-40-fold increase in PAI-1 mRNA. IL-6 has a small effect on PAI-1 expression by itself but, when combined with IL-1, causes a 50-100-fold syn- ergistic induction of PAI-1 mRNA. Dexamethasone alone did not increase PAI-1 expression but enhanced its expression 2-fold in combination with IL-1. We have used nuclear run-on assays and mRNA decay analyses to demonstrate that the mechanism of the cytokine-induced increase in PAI-1 mRNA accumulation is primarily due to a transient increase in PAI-1 gene transcription.

EXPERIMENTAL. PROCEDURES Materials-Dexamethasone, actinomycin D, and DRB were pur-

chased from Sigma. IL-lP (SA, 2 x 10’ unitdmg) was purchased from R

* A. J. Thomton and T. D. Gelehrter, submitted for publication.

19095

19096 Acute Phase Regulation of PAI-1

A B

1 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

3.2-

2.2-

- 3.2

-2.2

& D Systems IMinneapolis. MN). IL-la (SA. 3 x 10' unitsimgl was a gift from Hoffman-l,a Kochr tNutlcy, NJJ , and IL-6 (SA. 5 x 10.' unitsimg) was a gin. from Grnetics Institute tCamhridgc. MA). Eagle's minimum essential medium, glutamine. penicillinistrrptomycin. trvpsin. and fe- tal bovine serum werc purchased from Life Trchnologies. Inc. RSA rho- vine serum alhumin) was purchased from Intergen ffurchase. N Y ) . ['"PldCTP (SA, 3000 Cihrnol). [ '"PIUTP (SA, X00 Ci/mmol), and Hy- bond N wrre purchased from Amrrsham Corp. Other r ragrn ts wrre purchased from standard commercial sources.

Crll Culttrrr-HepC.2 cells were grown as monolayer cultures under 5 3 C 0 2 in Eagle's minimum essential medium supplemented with I O r ; fetal bovine serum, 2 msc glutamine, 100 unitshl penicil l in, and 100 p g h l streptomycin. At thr beginning of thr experiments. growth me- dium was removed from confluent cultures, and thr cultures were washed twice with phosphate-huffered saline and incubated in serum- free medium containing O . l r i RSA for 16 h. This medium was then replaced with fresh serum-free medium containing RSA and the indicated cytokines as drscrihcd in the figure legends.

RNA Isolntion nr~d Norfhrm Blot Annlysis-Total cellular RNA was isolated from HrpC.2 cells hy a modification of the method o f Chom- czynski and Sacchi (261 as descrihrd by Heaton rt nl. (27) . Total RNA was isolated as previously descrihrd (2x1 from human transplant donor liver. Northern hlot analysis was carried out using 10 pg o f R N M a n e (HepG2 cells) or 25 pg of RNMane ( l iver ) as descrihed hy Heaton and Gelehrter (291. The amount of hound radioactivity in each sample was quantified using a Rrtascope 603 blot analyzer (Retagen, Waltham, MA) or a model 400 I'hosphorlmager (Molecular Dynamics, Sunnyvale, CA). The amount o f PAI-I mRNA was normalized to the amount o f glycer- aldehyde-.?-phosphate drhydrogrnase mRNA, which did not change un- der the conditions nf these experiments.

Prohcs-A 2.2-kh EwRI fragment containing a portion of the human PAI-1 cDNA was purified from a pUC13 vector containing that insert (30). A 780-hase pair XAnI-PsfI fragment containing a portion of the human glyceraldehyde-3-phosphate dehydrogenase cDNA was purified from a p13R322 vector containing a 1.2-kb human glyceraldehydc-3- phosphate dehydrogenase cDNA clone. ohtained from ATCC [Rockville, MD) (31 ). DNA fragmrnts were isolated by agarose electrophoresis and purified using the (:rneclean kit (Ria 101, La .Jolla. CA) according to the manufacturer's specifications. For Northern hlot analysis. '"P-laheled DNA probes were prepared hy the random primer method ( 3 2 ) .

7hnsrr ipt ion Assny-Nuclrar run-on assays were carried out as pre- viously descrihed (27). For rach assay. nuclei were isolated from ap- proximately 2 x 10' HrpC.2 cells. Equal total amounts o f radioactivity tlaheled RNA) from each reaction were hvhridized to separate nylon memhrane strips (Hybond N, Amersham) to which 5 pg o f rach plasmid containing the cDNAs of interest had been hound. The amount o f spe- cifically hound radioactivity was determined using a model 400 Phos- phorImagrr (Molrcular Dynamicsl.

Assny 1fI'AI-1 Artic!ity nncl Protrin-PAI-1 activity w a s measured in HepG2-conditioned medium activated with 4 \I guanidine-HCI as de- scribed ( 3 3 ) . hy titration of rrsidunl tissue type plasminogen activator activitv using the two-step esterolytic assay modified for 96-well micro- titer plates (34) . PAI-1 protein was quantified hy enzyme-linked immu- nosorhrnt assay using a human PAI-1 enzyme-linked immunosorhent assay kit from American Dingnostica. Inc. (Grrrnwich. CT).

KESI'LTS

Regulntion of PAI-1 mRNA Arcurnulntion by I I , - 1 . II,-6. a n d Dexnrnefhosonc-In human cells, two PAI-1 mRNA sprcies of 3.2 and 2.2 kh. reflecting diffrrrnt polvadenylation sitrs. arc expressed (30). Fig. L4 shows an autoradiogram of a Northrrn hlot of RNA isolated from normal human livrr from a trans- plant donor and from uninduced HepG2 crlls and prohrd with PAI-1. As reported previously (2.31. PAI-1 mRNA is detrctrd in both liver and HepG2 cells.

Incuhation of HepG2 cells with IL-I rwultrd in a rapid and transient induction of both PAI-1 transcripts. Fig. 111 sho\vs an autoradiogram of a rrpresentative Northrrn hlot. and Fin. 2A depicts pooled data from several experiments. Induction of the 3.2-kh PAI-1 mRNA accumulation was rvidrnt as early as 30 min after addition of IL-1, and the inductinn of t h r 2.2-kh mRNA was ohserved after 60 min. The accumulation of hoth PAI-1 mRNAs was maximal after 4 h of incuhation whrn nor- malized for the amount of glycrrald(.hydr-3-phosphate drhy- drogenase mRNA and rxprrssrd as thr ratio of rxprrimrntal: control; the 3.2-kh mRNA was indrrcrd 40-fold. and thr 2.2-kh mRNA was induced 30-fold. The level of hoth transcripts then fell off rapidly hut remained 2-Sfold highrr than control at 8-18 h. Similar results were ohtainrd with IL-l(t and IIA-l[3,

Incubation of HepG2 cells with IL-6 resultrd in a small. time-dependent induction of PAI-1 mRNA (Fig. 2 n 1. The indrrc- tion of hoth transcripts was maximal aftrr a 2 4 - h incuhation. The 3.2-kb mRNA was induced 3.5-fold,and the 2.2-kh mRNA was induced 2.5-fold. The levrl of hoth transcripts then p a d u - ally decreased.

Although IL-6 alone had only a modest effrct on PAI-1 rx- pression, in comhination with IL-1, it rrsultrd in a rapid syn- ergistic induction of PAI-1 mRNA (Fig. 2C 1. As w a s the casr with IL-1, induction of the 3.2-kh PAI-1 mRNA was rvidrnt 30 min after addition of IL-1 + IL-6,and induction of t h r 2.2-kh mRNA was apparent after 60 min. >laximal induction of hoth transcripts occurred 2 h after addition of IL-l + IL-6 and was much greater than that ohserved for IL-I or IL-6 alone. The 3.2-kh transcript was induced >100-fnld, and the 2.2-kh tran- script was induced >5O-fold. Aftrr 2 h. the PAI-1 mRNA Irvrl decreased rapidly but remained .3-:',-fold ahovr control Irvrls.

Ry itself, the synthetic glucocorticoid drxamcthasonr did not affect the accumulation of PAI-1 mRNA accumulation in HepG2 cells (data not shown). Howelvr. in comhination with IL-1, it enhanced accumulation of the 3.2-kh PAI-I mRNA ap- proximately 2-fold; exprrssion of the 2.2-kh transcript was rn- hanced approximately 1.3-fold ( n = 4. p < 0.01 pairrti t t rs t 1. In addition, drxamethasone had little effrct on PAI-1 rxprrssion

Acute Phase Regulation of PAI-1 19097

100

80

60

40

2 0

0

0.1 1 10 100 1000

6. [IL- 11 (unitslml)

FIG. 3. IL-1 concentration dependence for the induction of PAI-1 mRNA. The experiment was carried out as described in Fig. 2. HepG2 cells were incubated with the indicated concentrations of IL-la for 4 h. Each data point represents the average of two to four cultures.

mRNA by IL-1, IL-6, and IL-1 + IL-6 (data not shown). These results indicate that RNA synthesis is required for induction of PAI-1 mRNA by IL-1 and IL-6 and are consistent with a tran- scriptional mechanism of action for IL-1 and IL-6 in HepG2

lation of PAI-1 mRNA in HepG2 cells involves stimulation of gene transcription, nuclear run-on assays were carried out (Fig. 4). Transcription of the glyceraldehyde-3-phosphate dehy- drogenase gene, which was not affected by these cytokines, was used as an internal control. In the presence of IL-1 or IL-1 + IL-6, an increase in PAI-1 transcription is evident after 30 min; the transcription rate peaks at 2 h and returns to basal levels by 4 h (Fig. 4, B and C). IL-1 increases the PAI-1 transcription rate 10-fold at 2 h, and IL-1 + IL-6 causes a 15-fold increase a t 2 h. We could not detect a change in the rate of PAI-1 tran- scription in the presence of IL-6 alone (Fig. 4A).

Effect of Cytokines on PAI-1 mRNA Stability-Because nuclear run-on experiments indicated that the rate of PAI-1 mRNA synthesis was essentially basal after 4 h of incubation with cytokines, we were able to measure the rate of PAI-1 mRNA decay in the absence of inhibitors of RNA synthesis by

Tima fh\ continued presence of IL-1 or IL-1 + IL-6. In the continued

2.

0- 0

. H-H . 2 4 6 8 1 2 1 8 cells. To determine directly if the mechanism of cytokine regu-

0 2 4 6 6 12 18 measuring the decline in PAI-1 mRNA between 4 and 7 h in the

FIG. 2. Time course of the effects of cytokines on PAI-1 mRNA accumulation in HepG2 cells. After monolayer cultures of HepG2 cells were incubated in serum-free medium containing 0.1% BSA for 16 h, 500 unitdml IL-1 (A), 40 ng/ml IL-6 ( B ) , or IL-1+ IL-6 (C) was added. Cells were harvested at the times indicated, cellular RNA was isolated,

mental Procedures." The amount of PAI-1 mRNA was normalized to the and Northern blot analysis was performed as described under "Experi-

amount of glyceraldehyde-3-phosphate dehydrogenase mRNA and is presented as the ratio of experimenta1:control. Each data point repre- sents the average ? S.E. of four to eight cultures (two to four separate experiments). Tkiangles and circles represent the 3.2- and 2.2-kb mRNA

been included. Note that a different scale was used on they axis for the species, respectively. Data from experiments using IL-la or IL-1p have

IL-6 data (panel B ) .

in combination with IL-6 or IL-1 + IL-6 (data not shown). Concentration Dependence of PAI-1 mRNA Induction-Fig. 3

shows the IL-1 concentration dependence for the induction of PAI-1 mRNA. Both PAI-1 transcripts responded similarly to various concentrations of IL-la. Half-maximal responses oc- curred at 20-25 unitdm1 and maximal responses at 100-500 units/ml after 4 h of incubation. Similar results were obtained with IL-1p (data not shown).

Cytokine Regulation of PAI-1 Gene lkanscription- Actinomycin D, a t 5 pg/ml, a concentration that inhibits RNA synthesis by >98% in HepG2 cells, blocks the induction of PAI-1

presence of IL-1, the 3.2-kb PAI-1 transcript had a half-life of 0.8 h, and the 2.2-kb transcript had a half-life of 1.2 h (Fig. 5A). Removing IL-1 after 2 or 4 h of incubation by washing the cells and incubating in medium without IL-1 (35) did not alter the half-life of either transcript (data not shown). In the continued presence of IL-1 + IL-6, the half-lives of the two PAI-1 tran- scripts were similar to those observed in the presence of IL-1 alone; the 3.2-kb transcript had a half-life of 1.0 h, and the 2.2-kb transcript had a half-life of 1.4 h (Fig. 5B) . Similar PAI-1 mRNA decay rates were also observed in the presence of IL-1+ dexamethasone (data not shown). Determination of the rate of PAI-1 mRNA decay in the absence of cytokines was not possible because of the low levels of PAI-1 mRNA in uninduced HepG2 cells (Fig. 1) (24) and the stabilizing effects of RNA synthesis inhibitors (see below).

It has been previously reported that both actinomycin D (5 pg/ml) and DRB (200 p ~ ) block the decay of the 3.2-kb PAI-1 mRNA in HepG2 cells (24). We have observed that addition of as little as 0.5 or 1 pg/ml actinomycin D (concentrations that inhibit RNA synthesis in HepG2 cells by 90 and 97%, respec- tively), 2 h after addition of IL-1, significantly blocked decay of both PAT-1 transcripts (data not shown). 50 and 200 PM DRB also completely blocked decay of both PAI-1 transcripts (data not shown). These results suggest that decay of PAI-1 mRNA

19098 Acute Phase Regulation of PAI-1

A Cont IL- 1 IL-6 IL- 1 +IL-6 mediators of the acute phase response. IL-1 strongly induced the accumulation of PAI-1 mRNA, and

IL-6 had a modest effect. However. IL-6 in combination with IL-1 resulted in a synergistic induction of PAI-1 mRNA. By

pKS-

PAI- 1 - 0 blocking RNA synthesis with actinomycin D, wr determinrd

0 tha t dr norw RNA synthesis is required for induction of PAI-I GAP 0 0 - 2 0

16

1 2

8

4

I B c I C T c I

0 ' I I I I

0 2 4 0 2 4

mRNA by IL-1 and IL-6; these results, together with thr rapid accumulation of PAI-1 mRNA. are consistrnt with a transcrip- tional mechanism of action for IL-1 and IL-6 in HepG2 cells. Using nuclear run-on assays to measure the rate of PAI-I genr transcription directly, we have shown that thr mechanism of the cytokine-induced increase in PAI-1 mRNA is primarily an increased rate of PAI-1 gene transcription.

Because the magnitude of the increase in PAI-I gene tran- scription was apparently less than that of mRNA accumulation. we also asked whether post-transcriptional mechanisms might play a role in the cytokine regulation of PAI-I mRNA accumu- lation. We were able to measure PAI-I mRN.4 decay in the absence of inhibitors of RNA synthesis by taking Advantagr of the fact that the cytokine-induced rate of PAI-I grne transcrip- tion had declined to essentially basal Irvrls hy 4 h of incuha- tion. Therefore. by measuring the decay in PAI-I mRNA lrvels between 4 and 7 h, we could directly assrss the rate of drcay of the mRNA. In the continued presence of IL-I, the half-life ofthr 3.2-kb PAI-1 mRNA was approximately O.X h, and that of th r 2.2-kb species was 1.2 h. These half-lives were not nlterrd by

ase mRNA and is presented as the ratio of experimental:control. Each data point represents the average 2 S.E. of three to six cultures. G A f , Rlvceraldehyde-3-phosphate dehydrogenase.

requires ongoing RNA synthesis; therefore, inhihitors of RNA synthesis cannot be used when measuring the half-lives of the two PAI-1 transcripts in HepG2 cells.

Cvtokinr Rrgrrlntion of PAI-I Activitv and Protein-To deter- mine whether IL-I and IL-6 induce the accumulation of PAI-1 protein, PAI-1 antigen and activity were measured in HepG2- conditioned medium. Incubation of HepG2 cells with IL-1 re- sulted in a 6-fold increase in PAI-1 protein after 8 h and a 4-fold increase after 24 h (Fig. 6A ). PAI-1 activity was increased about 3-fold a t both 8 and 24 h (Fig. 6 R ) . IL-6 did not cause any changes in the level of PAI-1 protein (data not shown). Incuha- tion of HepG2 cells with IL-1 + IL-6 resulted in a n %fold in- crease in PAI-1 protein after 8 h and a 6-fold increase after 24 h. PAI-1 activity was induced 6-7-fold at 8 and 24 h.

DlSCUSSION

Human PAI-1 behaves like an acute phase reactant in uiuo; plasma PAI-1 concentration is significantly increased in vari- ous disease states and after acute stress (7-9,. The source of plasma PAI-I is controversial. We (Fig. lA), and others (23), have demonstrated the presence of PAI-1 transcripts in RNA isolated from human liver. In addition, we have shown directly by in situ hybridization that human hepatocytes express the PAI-1 gene in ~j iuo and that this expression is elevated in pa- tients with sepsis.2 Thus, hepatocytes may be an important site of PAI-1 synthesis during the acute phase response. In this study, we demonstrate that PAI-1 mRNA accumulation in the human hepatoma cell line, HepG2, is dramatically increased by

expression. Although i t has been reported that in uninduced HrpG2 crlls

treated with high concentrations of actinomycin D (10-30 pg/ ml), the 3.2-kb PAI-1 transcript has a half-life of 0.9-1.2 h. and the 2.2-kh transcript has a half-life of 2.8-3.5 h (36.37,. wr. and others (24). have found that the decay of PAI-I mRNA is blocked by inhibition of RNA synthesis with actinomycin D or DRR, suggesting that RNA synthesis is necessary for PAI-1 mRNA decay. We were able to avoid this potential prohlrm by determining the half-livrs of PAI-I transcripts in the ahsencr of these inhibitors.

IL-I increases PAI-1 mRNA accumulation in HepG2 cells in a concentration-dependent manner. Half-maximal responsrs occurred at approximately 20 unitslml (60 pp/ml) 11,-1, a con- centration similar to that detected in plasma of healthy indi- viduals (38 ) . Maximal responses occurrrd at approximatrly 100 unitslml (300 pg/ml) IL-1, a concentration comparable with IL-Ip levels measured in plasma of some septic patirnts (3x1. The concentration dependence on IL-1 of the induction of PAl-1 mRNA is similar to that of other AP reactants in hrpatic crlls; 100-1000 unitslml IL-1 has been found to be maximally rffrc- tive (39). The time course. magnitude, and concentration dr- pendence of the induction of PAI-I mRNA was th r s amr for IL-la and IL-1p. The concentration deprndrncr of IL-6 could not be readily determined hrcause of its small rffrct on PAI-I mRNA accumulation.

No single factor can elicit the synthesis of the entire sprc- t rum of acute phase reactants. In addition. each AP rractant requires a specific comhination of mediators for maximal r x - pression (1-3, 5 ) . Two classes of AP reactants havr bern dr- scribed in rat H-35 hepatoma cells as follows: typr 1 A J ' pra-

Acute Phase Regulation of PAI-1 19099

FIG. 5. Effect of IL-1 and IL-6 on PAI-1 mRNA decay. The experiment was carried out as described in Fig. 2.

unitdml IL-la (A) or IL-la + 40 ng/ml HepG2 cells were incubated with 500

IL-6 ( B ) . Each data point represents the average S.E. of four cultures.

100

2 7 5 . C 0

C ai .- * 5 0 g w

26

r

0 8 h 2 4 h

a

v- I

Time (h)

.-. FIG. 6. Effects of IL-1 and IL-6 on

8 h

teins were induced by IL-1 and required IL-1 in combination with IL-6 for maximal expression, and type 2 AP proteins were stimulated maximally by IL-6 alone and were not induced by IL-1(1). Studies in human hepatoma cells indicate that human AP reactants can also be categorized in this way; for example, a,-antichymotrypsin and qantitrypsin are stimulated by IL-6, and C3 and a,-acid glycoprotein are stimulated by IL-1 in com- bination with IL-6 (39, 40). The regulation of human PAI-1 in HepG2 cells is characteristic of type 1 AP proteins, requiring IL-1 + IL-6 for maximal expression.

Unlike the induction of several other AP reactants, which are maximal in 6-12 h, the induction of PAI-1 mRNA by IL-1 and IL-6 in HepG2 cells occurs rapidly, peaking at 2-4 h, and is very short-lived. Nuclear run-on assays reveal that transcription of the PAI-1 gene peaks at 2 h and returns to basal levels by 4 h; thus, the transient nature of the PAI-1 mRNA induction is due to a rapid reduction in PAI-1 gene transcription. This reduction in transcription could be due to depletion of cytokines in the medium, reduction in the number or affinity of IL-1 receptors, or a post-receptor event. We determined that IL-1 is not being rapidly depleted from the medium and remains biologically active; conditioned medium from HepG2 cells treated with 500 unitdm1 IL-1 for 4 h that was transferred to fresh cells elicited a full response. Also, readdition of 500 unitdm1 IL-1 to cells that had been incubated with IL-1 for 4 h did not prolong the induction of PAI-1 mRNA (data not shown). Down-regulation of IL-1 receptors by IL-1 has been reported in a human large granular lymphocyte cell line and in a murine T-helper cell line (41, 42). In both systems, maximal down-regulation reached >90% after 16-24 h. However, virtually complete down-regula- tion of IL-1 receptor by 2-4 h would be necessary to account for

2 4 h

PAI-1 protein and activity in HepG2 cells. Confluent monolayers of HepG2 cells were incubated for 16 h in serum- free medium containing 0.1% BSA. Then, 500 unitdml IL-la (cross-hatched bars) or IL-la + 40 ng/ml IL-6 (black bars) was added for the times indicated. Condi- tioned medium was harvested, and PAI-1 antigen and activity levels were deter- mined as described under “Experimental Procedures.” Data represent the average of two experiments and varied by less than 20%. 1 unit of PAI-1 activity was defined as the amount that inhibited 50% of the activity of 2 international units of tissue type plasminogen activator. White bars, control.

the total loss of response to the high concentrations of IL-1 that we observed. These results suggest that a post-receptor event is responsible for the transient nature of PAI-1 mRNA induction.

IL-1 is known to bind to the monomeric type 1 IL-1 receptor on the cell surface. The mechanism of IL-1 signal transduction, however, is unknown and controversial. I t is likely that protein kinases are involved in the signaling mechanism that eventu- ally results in the activation of transcription factors, including NF-KB and AP-1 (43). The molecular basis for the synergistic interaction of IL-1 and IL-6 is only beginning to be understood but apparently involves the interaction of NF-KB and CIEBPP in the promoter region of genes such as serum amyloid A, which show synergistic induction (44).

IL-1 has been reported to induce transcription of the IL-6 gene in HepG2 cells (45). However, because IL-6 has little effect on PAI-1 mRNA accumulation, it is unlikely that IL-1 induces PAI-1 expression by this mechanism.

Although IL-1 alone and in combination with IL-6 dramati- cally induces the accumulation of PAI-1 mRNA, the increases in PAI-1 protein and activity levels in conditioned medium are more modest. However, the transient nature of the PAI-1 mRNA induction may be a factor in this discrepancy, because the effect of a transient increase in PAI-1 mRNA would be diluted over time. In addition, the appearance of a protein in the medium often lags behind the synthesis of the correspond- ing mRNA. Induction of plasminogen activators could also ac- count for the modest induction of PAI-1 activity. I t has been reported that HepG2 cells do not express tissue type plasmin- ogen activator (231, but urokinase type plasminogen activator activity has been detected (46). We could not detect urokinase type plasminogen activator mRNA in untreated HepG2 cells,

19100 Acute Phase Reg

but urokinase type plasminogen activator mRNA accumulation was induced by IL-1 (data not shown). IL-6 did not induce urokinase type plasminogen activator expression, nor did it enhance urokinase type plasminogen activator expression in combination with IL-1.

Glucocorticoids can act synergistically with IL-1 and IL-6 in inducing the acute phase response. We, and others (471, have shown that the PAI-1 gene in HepG2 cells is not induced by dexamethasone alone; however dexamethasone does enhance the effects of IL-1 approximately 2-fold. This enhancement does not seem to occur at the level of PAI-1 mRNA stability. HepG2 cells express low levels of glucocorticoid receptors (18), but it is not clear whether these receptors are capable of acting inde- pendently as transcriptional activators. Dexamethasone syner- gistically enhances the cytokine induction of other acute phase reactants such as q a c i d glycoprotein in HepG2 cells but, by itself, is unable to stimulate a,-acid glycoprotein expression (18).

In this study, we have demonstrated that the human PAI-1 gene in HepG2 hepatoma cells is regulated by IL-1 and IL-6, known mediators of the acute phase response. PAI-1 gene ex- pression is dramatically induced by IL-1 and requires IL-1 in combination with IL-6 for maximal expression. The mechanism of this induction is primarily increased PAI-1 gene transcrip- tion; we found no evidence for an effect on PAI-1 mRNA stability.

Roche for cytokines and R. Hart at American Diagnostica for the PAI-1 Acknowledgments-We thank Genetics Institute and Hoffman-La-

enzyme-linked immunosorbent assay kit. We also thank Joanne Heaton for helpful advice, June Nagy for secretarial assistance, and the Howard Hughes Medical Institute for use of the PhosphorImager.

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