interleukin-6 responsiveness and cell-specific expression of the

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 5. Issue of February 15, pp. 2946-2952,1991 Printed in U.S.A.

Interleukin-6 Responsiveness and Cell-specific Expression of the Rat Kininogen Gene*

(Received for publication, July 24, 1990)

Huei-Mei ChenS, Karen B. ConsidineS, and Warren S . L. Liaog From the Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

The serum concentration of ra t T1 kininogen in- creases 20-30-fold in response to acute inflammation, an induced hepatic synthesis regulated primarily at the transcriptional level. To analyze the cis-regulatory elements responsible for the induced transcription, we fused a 1.6-kilobase segment of the rat T1 kininogen promoter to a reporter gene, chloramphenicol acetyltransferase (CAT). The resultant chimeric DNA was transfected into cultured cells. In transient transfection assays, this 5”flanking sequence was sufficient to confer cell-specific expression: CAT activity was read- ily detectable when the construct was transfected into liver-derived cells, but it was not detectable in nonliver cells. Furthermore, when liver cells (Hep3B) transfected with this construct were treated with conditioned medium prepared from activated mixed lymphocyte cultures or with recombinant interleukin-6 (IL-6), a 5-fold increase in CAT activity was detected. Addition of dexamethasone to the conditioned medium or to IL-6 showed synergistic effects and resulted in a 10-fold increase in CAT activity. In contrast, when IL- 1 was used with IL-6, induction of CAT activity was inhibited. Deletion analyses revealed two regions important for tissue-specific and induced regulation of T1 kininogen: sequences proximal to base pair -73 conferred enhanced expression in liver-derived cells and a distal region that conferred responsiveness to conditioned medium, recombinant IL-6, and dexamethasone. This responsive element had properties of an inducible transcriptional enhancer, and it was functional in both liver and nonliver cells when placed immediately upstream of a thymidine kinase promoter.

Acute systemic injury or infection in mammals causes a profound change in the hepatic synthesis and circulating concentration of plasma proteins (1, 2). The onset of acute phase protein synthesis in the liver following inflammation at a distant site implies the existence of humoral mediators. When released from the activated macrophages, circulating cytokines such as interleukin-1 (IL-l),’ interleukin-6 (IL-6),

* This work was supported in part by National Institutes of Health Grant AR38858 (to W. S. L. L.) and aided by National Institutes of Health Grant RR5511. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$, Supported by Training Grant HD07325.

’ The abbreviations used are: IL-1, interleukin-1; IL-6, interleukin- 6; TNF-n, tumor necrosis factor-a; RE(s), response element(s); CAT, chloramphenicol acetyltransferase; CM, conditioned medium; kb, kilobase; bp, base pair; TPA, 12-O-tetradecanoylphorbol 13-acetate; tk, thymidine kinase.

To whom correspondence should be addressed.

and tumor necrosis factor-a (TNF-a) transmit their signals to target cells, the hepatocytes, triggering the induction of acute phase protein synthesis (3, 4). IL-6 is now recognized as the hepatocyte-stimulating factor for the majority of acute phase proteins (5). Besides acting as a major regulator of acute phase response in human hepatoma cell lines and rat primary hepatocyte culture, this 26-kDa protein possesses pleiotropic functions and has been named B cell stimulatory factor-2 (6), interferon fl2 (7), and hybridoma-plasmacytoma growth factor (8). The T kininogen gene is among several acute phase genes whose expression can be modulated by IL- 6 in a dose- and time-dependent manner in vivo and in vitro (9, 10). Its protein product was known previously in the rat as al-major acute phase protein, because its level in serum could be increased 20-30-fold within 48 h of turpentine- induced inflammation and represented nearly 10% of total serum protein (11). A previous report showed that the greatly enhanced transcription activities of T kininogen led to a corresponding rise in its mRNA levels and accounted for most of the increase in the protein synthesized (In), thus demon- strating that the large increase in hepatic T kininogen synthesis during inflammation is regulated primarily at the transcriptional level.

In the rat, the kininogen gene family consists of four genes: two for highly homologous T kininogens (T1 and TZ), one for K kininogen, and finally a structurally related pseudogene (13, 14). However, in other mammals, including human, bovine, and mouse, only the gene for K kininogen is found (15- 17). Nucleotide sequence comparison has revealed extremely high sequence homology (>go%) between the two T kininogen genes and the K kininogen gene in the rat. This sequence homology extends at least 1 kb upstream of the start of transcription (13). Interestingly, in spite of the high sequence homology between the two T kininogen genes and K kininogen gene, only expression of the two T kininogen genes is induced greatly following inflammation; expression of the K kininogen gene in rat, human, and bovine is unaltered (18). Because other species lack the T kininogen genes, the inflammatory response of the kininogen gene is known only in the rat. Kininogen expression in the rat is, therefore, a unique system in which to study the mechanisms of differential gene regulation in a multigene family.

Using primary rat hepatocyte cultures, Andus et al. (19) have shown that treatment with IL-6 stimulates T kininogen synthesis 8-fold, whereas IL-1 and TNF-a showed no stimulation. Interestingly, when the cells were treated with both IL-6 and IL-1, induction of T kininogen expression was inhibited significantly. Expression was inhibited to a lesser extent when a high concentration of TNF-a, i.e. 1000 units/ ml, was used with IL-6. Similar results were obtained by Baumann et al. (20) using the rat hepatoma H35 cell line. In addition to the positive and negative effects of inflammatory

2946

Regulation of Rut Kininogen Gene Expression 2947

cytokines, the regulation of T kininogen expression is com- plicated further by the involvement of the steroid hormone glucocorticoid, because dexamethasone was required for the maximal induction of T kininogen by IL-6 (21).

To delineate further the molecular mechanisms involved in the regulation of acute phase genes by IL-6, promoter regions of human haptoglobin, hemopexin, C-reactive protein, and rat a2-macroglobulin genes (22-25) have been cloned into vectors containing appropriate reporter genes and analyzed for cis-regulatory elements important for conferring IL-6 responsiveness. Using transient transfection experiments with the deletion and site-specific mutants in combination with gel-shift and DNase I footprinting assays, cis-elements responsible for IL-6-induced transcription have been localized to the 5‘-proximal promoter region of these genes. The IL-6 response elements (RES) identified in these acute phase genes can be grouped into two consensus sequences; one is a heptanucleotide CTGG(G/A)A(A/T) and the other is a decanu- cleotide, AGTGANGNAA (5,23,26). The promoter region of human C-reactive protein was shown to contain both sequence motifs. Interestingly, distinct nuclear proteins seem to bind independently to each site but function cooperatively in IL-6-induced C-reactive protein gene expression (26). Ac- cording to the results of competition assay, it is likely that similar or at least structurally related IL-6-dependent DNA- binding proteins interact with the conserved IL-6 RES of different acute phase genes (23, 25, 26).

In this paper, we examine the regulated expression of rat T kininogen in response to inflammatory mediators by using a chimeric construct of the 1.6-kb promoter region of the T1 kininogen gene and a chloramphenicol acetyltransferase (CAT) reporter gene. Basal expression of this fusion gene, as well as its responsiveness to conditioned medium (CM) derived from activated mixed lymphocyte culture, was cell- specific. IL-6 and glucocorticoid were shown to be the primary mediators responsible for the induced expression of the rat T1 kininogen gene. An IL-6 RE was localized to a 321-bp region that confers cytokine responsiveness to a heterologous thymidine kinase promoter in both liver- and nonliver-derived cells.

EXPERIMENTAL PROCEDURES Cell Lines-Two human hepatoma cell lines, HepG2 and Hep3B

(a gift from B. Knowles, Wistar Institute), and two nonhepatoma cell lines, HeLa (human cervical carcinoma cell line) and L (mouse fibroblast cell line), were grown in basal medium consisting of modi- fied Eagle’s medium and Waymouth MAB (3:1, v/v, pH 7.0) (27) plus 10% fetal bovine serum (HyClone Laboratories, Logan, UT). All cells were maintained at 37 “C, 5% CO,, 95% air in 75-cm2 tissue culture flasks and were passaged at confluence, approximately once a week, by trypsinization in the presence of EDTA.

Conditioned Medium, Cytokirzes, and Phorbol Ester-CM derived from mixed lymphocyte cultures was prepared according to the method of Maize1 et al. (28) by incubating human peripheral blood lymphocytes (106/ml) from four healthy donors in control medium consisting of RPMI 1640 medium with 0.25% bovine serum albumin and 0.75% phytohemagglutinin (PHA M farm; GIBCO Laboratories, Grand Island, NY). Cells were cultured for 72 h at 37 “C, and the CM was separated from the cells by centrifugation and filter-sterilized. The CM prepared in this manner contains a wide variety of interleu- kins and other monokines that mediate the acute phase response (3, 19, 27, 29). CM was used as a mixture with an equal volume of basal medium.

Purified recombinant human cytokines IL-la, IL-6, TNF-a, and lymphotoxin were studied separately by adding each cytokine to 50% control medium (mixture of equal volumes of control medium and basal medium) to investigate the effect of the specific factor on the induction of acute phase gene expression. 1L-la (specific activity, 1.2 X 10’ units/mg) was generously provided by P. T. Lomedico (Hoff- man-LaRoche). IL-6 (specific activity, 2.0 X IO8 units/mg) was pur-

chased from Boehringer Mannheim; TNF-a and lymphotoxin were gifts from B. Aggarwal (M. D. Anderson Cancer Center). Dexameth- asone (Sigma) was also added at a level of 1 PM alone or in combination with the cytokine to test its effect on cytokine-induced expression. A phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) purchased from Sigma, was also added at a level of 0.1 p~ alone or in combination with IL-6 to test its effect on cytokine-induced expression.

Deletion Mutant Constructions-A 1.6-kb fragment containing 1516 bp of proximal 5”flanking and 119 bp of exon 1 untranslated sequences of the rat T1 kininogen gene was isolated from a T1 kininogen genomic clone, pgTlK 34, obtained from a Sprague-Dawley rat liver genomic library. This fragment was cloned into the SmaI site of pSVoCAT vector (30), resulting in the hybrid gene pTlK/CAT (-1516). Cloning sites and partial restriction map of this construct are shown in Fig. 1. Three 5’ deletion mutants, pTlK/CAT (-584), pTlK/CAT (-427). and pTlK/CAT (-73), were generated subsequently by digesting pTlK/CAT (-1516) DNA with SauI (-584), Sac1 (-427), and KpnI (-73), respectively, in combination with BglII (-1516), to cut it at. the 5”distal restriction site. The DNA were blunt-ended, ethanol-precipitated, and religated with BglII linkers. The ligation products were then used to transform Escherichia coli HBIO1. The correct deletion 1nutant.s were identified by the appro- priately reduced DNA sizes as determined by agarose gel electropho- resis (31) and DNA sequencing (32).

BLCATIT1 Kininogen Constructions-Various lengths of the 5’- flanking sequence of T1 kininogen were inserted upstream of a heterologous herpes simplex virus thymidine kinase (tk) promoter, pBLCAT (33). Fragments isolated from restriction enzyme digests of pgTlK 34, i.e. SmaI-HincII (490 bp), SacI-PstI (420 bp), Sad-HincII (321 bp), and HincII-PstI (100 bp) (Fig. l), were blunt-ended, ligated to BarnHI linkers, and cloned into the BamHI site of pBLCAT vector. Plasmid DNA containing each specific insert was verified by exam- ining the size of the fragment after BamHI digestion, and the orientation was determined by dideoxy sequencing (32) using a primer derived from the tk promoter.

Transient Transfection Assay-Cells were seeded a t a density of 5- 10 X lo5 cells/100-mm culture dish and incubated a t 37 “C for 16-24 h before transfection with 20 pg of plasmid DNA by the Polybrene procedure (34). Four to six hours after transfection, cells were washed with Hanks’ balanced salt solution, subjected to a 25% glycerol shock for 2 min (35), and incubated for 16-20 h before treatment with CM or with purified recombinant cytokines in the presence or absence of 1 y M dexamethasone. To elicit acute phase response, cells were treated with 50% CM, which contained equal parts of medium from stimulated mixed lymphocytes and basal medium, or with cytokine-containing medium, which consisted of 50% control medium and one of four human recombinant cytokines. As controls, transfected cells were treated in parallel with 50% control medium. Cells were harvested 18-24 h after treatment.

CAT Assay-Transfected cells were harvested approximately 36- 40 h after transfection, washed with phosphate-buffered saline, and resuspended in 100 yI of CAT assay buffer (0.25 M Tris-hydrochloride, pH 7.8). Cell lysates were prepared by 4 cycles of freezing (-70 “C) and thawing (37 ”C) and by centrifugation at 12,000 rpm for 30 min. Supernatant fluids were collected and tested for protein content and CAT activity. Protein was quantified by the method of Bradford (36), using bovine serum albumin as a standard. The CAT assay was carried out. as described previously (30), with modifications described by Cat0 et al. (37). Our standard assay contained 5-50 yg of protein, 0.1 yci of [“C]chloramphenicol (specific activity, 50-57 mCi/mmol; Amersham Corp.), and 1 mM acetyl-coenzyme A in a final reaction volume of 120 p1. Reaction samples were incubated overnight a t 37 “C. The CAT reaction products were extracted with 1 ml of ethyl

- 1

1516 -596 -584

-427 -107 -73 AUG

I \ t CAT

Hincll Kpnl I PStI

FIG. 1. Structure of the rat TlK/CAT hybrid gene construct. A 1635-bp fragment (-1516 to +119) of the rat T1 kininogen gene sequence was ligated into the vector pSVoCAT to produce pTlK/ CAT (-1516). The horizontal arrow denotes the transcription start site (+l), and upward arrows indicate the positions of the restriction sites used for the subsequent hybrid gene constructs.

2948 Regulation of Rat Kininogen Gene Expression acetate and chromatographed on silica gel thin-layer chromatography plates using chloroform:methanol(95:5, v/v). CAT assays were quantitated in a liquid scintillation counter by measuring the radioactivity of ["Cjchloramphenicol spots corresponding to the acetylated and nonacetylated forms.

RESULTS

Differential Expression of the TlK/CAT Hybrid Gene in Cultured Cells-The rat T1 kininogen gene promoter region containing 1516 bp of the 5'-flanking sequence and 119 bp of the untranslated exon 1 sequence was cloned into the pSV,CAT vector (30). The resulting construct pTlK/CAT (-1516), was transfected into two human hepatoma cell lines (Hep3B and HepG2) and two nonliver-derived cell lines (HeLa and L) to determine whether it is expressed in a cell- specific manner. To control for transfection efficiencies, pSV2CAT DNA was transfected into duplicate cultures of the four cell lines. This control DNA contains the SV40 enhancer and early promoter in an otherwise identical plasmid and is known to be expressed in many different cell lines (30). The pSVoCAT vector was used as a negative control for the hepatoma cell lines. As shown in Fig. 2, pSV2CAT was expressed efficiently in all four cell lines, whereas no activity was detected with pSVoCAT. The pTlK/CAT (-1516) construct, however, was expressed efficiently only in hepatoma cells; no CAT activity was detected in the two nonliver-derived cell lines. This result demonstrates that regulatory sequences in the T1 kininogen promoter restrict the expression of the CAT gene to hepatocyte-derived cells.

Induced Expression of TlK/CAT Gene by CM-To assess the responsiveness of the pTlK/CAT (-1516) construct to inflammatory cytokines in cultured cells, transfected cells were stimulated subsequently with CM prepared from activated mixed human lymphocyte cultures. This CM has been shown to contain polypeptide inflammatory mediators and can induce acute phase gene expression when added to hepatocyte-derived cells (29, 38). Dexamethasone was also included because it is required for maximal induction of T

- + - + - + - + - - + SVo SVt 111 SV, T l K

HepG2 - L

- + - + - + - + - + SVo SV, T1K SV, T1K

FIG. 2. Cell-specific expression and induction of TlK/CAT hybrid gene in cultured cells. Two human liver-derived cell lines (Hep3R and HepCP) and two nonliver-derived cell lines ( H e h and I,) were transfected with 20 pg of pTIK/CAT (-1516) ( T I K ) , pSV,CAT (SV,), or pSV,CAT (SV,) DNAs. Approximately 24 h after transfection, cells were treated with control medium (-) or 50% conditioned medium/dexamethasone (+). After 18 h of treatment, cells were harvested for protein and CAT assays.

kininogen expression in cultured rat liver cells (21). In the two transfected hepatoma cell lines treated with CM/dexamethasone, a 10-fold enhancement in CAT activity was observed, whereas CAT activity remained undetectable in stimulated nonliver-derived cells (Fig. 2). This induction was specific to the hepatoma cells transfected with the T1 kininogen-CAT construct, because no change in CAT activity was observed in cells transfected with the control plasmids pSV2CAT and pSVoCAT and stimulated with CM/dexamethasone (Fig. 2). These results indicate that the pTlK/CAT (-1516) construct also contains sequences responsive to dexamethasone and/or inflammatory mediators present in the CM, whereas the control plasmids do not. In addition, this response of pTlK/CAT (-1516) was specific to the hepatoma cell lines.

Induction of TlK/CAT Gene by Recombinant IL-6-As described above, pTlK/CAT (-1516) was responsive to CM from activated human mixed lymphocyte culture. Because this CM is a mixture of a number of known cytokines with hepatocyte-stimulating activities (29, 38), we sought to iden- tify the specific regulators using purified recombinant cytokines. IL-6, which has been shown to induce endogenous T kininogen gene expression in primary rat hepatocytes (19) and rat hepatoma cell lines (20), was assayed for its ability to regulate CAT expression of the pTlK/CAT (-1516) construct. Several other cytokines, i.e. IL-1, TNF, and lymphotoxin, known to be involved in the acute phase response were also tested. In addition, phorbol ester's ability to modulate IL-6- regulated expression of T kininogen was tested (39). As shown in Table I, addition of human recombinant IL-6 (200 units/ ml) to the transfected Hep3B cells produced a 5-fold induction of CAT activity, comparable to that observed with the CM treatment. Addition of dexamethasone further enhanced the IL-6-induced CAT expression, a phenomenon consistent with the CM treatment. In contrast, IL-1, TNF, and lymphotoxin had no effect on the induction of the pTlK/CAT (-1516) gene, whether with or without dexamethasone. In fact, IL-1 consistently inhibited the basal expression as well as the IL- 6-induced expression. Treatment with dexamethasone alone consistently showed approximately 2-fold induction. How- ever, when added together with CM or IL-6, it exerted a

TABLE I Induction ofpTIK/CAT hybrid gene by CM and cytokines in the

presence or absence of dexamethasone Hep3B cells were transfected with 20 pg of the pTIK/CAT (-1516)

hybrid gene and treated with dexamethasone (1 p ~ ) , conditioned medium (CM), IL-6 (200 units/ml), IL-1 (100 units/ml); TNF-CY (5 ng/ml), lymphotoxin (5 ng/ml), and TPA (0.1 p ~ ) individually or in combination for 18 h. CAT activities were quantitated by scintigra- phy. Values represent the means of three or four independent transfection experiments. In each experiment, CAT activities were expressed relative to the level of CAT activity in untreated control cells, which was assigned a value of 1.0.

Treatment Fold Induction

Control 1.0 Dexamethasone CM

2.2 f 0.2 4.6 f 0.6

CM + dexamethasone IL-6

9.5 f 1.2 5.2 f 0.8

IL-6 + dexamethasone IL-1

9.7 f 1.1 0.6 f 0.3

IL-1+ dexamethasone 1.6 f 0.5 IL-6 + IL-1 2.1 f 0.4 IL-6 + IL-1 + dexamethasone 7.5 f 0.9 TNF-CY + dexamethasone 1.5 f 0.3 Lymphotoxin + dexamethasone TPA

1.8 f 0.2 0.6 f 0.2 2.5 & 0.5 IL-6 + TPA

Regulation of Rat Kininogen Gene Expression 2949

synergistic action, resulting in a 10-fold stimulation. In our transient transfection assays, IL-1 a t a concentration of 100 units/ml effectively suppressed the induction of CAT activity mediated by IL-6 (Table I). When IL-1 was added together with IL-6 and dexamethasone, the induction by IL-6 and dexamethasone was also inhibited by IL-1, but to a lesser extent. Interestingly, phorbol ester suppressed basal expression of T kininogen to a similar extent as IL-1, and it too inhibited the IL-6-stimulated expression (Table I).

To characterize further the expression of T kininogen induced by IL-6, Hep3B cells transfected with pTlK/CAT (-1516) were treated with increasing concentrations of purified recombinant IL-6. In a parallel experiment, dexamethasone (1 PM) was added in combination with IL-6 to examine whether the observed synergistic action depended on IL-6 concentration. The results indicated that IL-6-induced expression of TlK/CAT was do&-dependent, with maximal induction observed a t 100-200 units/ml (Fig. 3). In addition, the glucocorticoid analogue clearly showed synergistic effects and enhanced TlK/CAT gene expression a t all IL-6 concentrations examined. Although the maximal inductions for these two treatments were different, the amounts of IL-6 required to attain half-maximal induction were essentially identical (approximately 40 units/ml) (Fig. 3).

Taken together, these results indicate that IL-6 acts in a dose-dependent manner on the transcriptional induction of the CAT gene in hepatoma cells under the control of the rat T1 kininogen promoter. I t demonstrates further that IL-6 is the primary cytokine in the CM that participates in the regulation of T kininogen gene in the liver and that dexamethasone was required for the maximal induction by either CM or IL-6. In contrast, IL-1 and TPA effectively inhibited the induction by IL-6.

Localization of 5"Flanking Sequences Responsive to IL-6- To localize the DNA sequences in the T kininogen gene promoter necessary to confer IL-6 responsiveness, portions of the promoter region were deleted from the pTlK/CAT (-1516) hybrid gene by restriction enzymes. Constructs resulting from these deletions, pTlK/CAT (-584), pTlK/CAT

S O L .DEX I

I I t 1

100 200 300 400

IL-6 (U/ml) FIG. 3. Dose-dependent induction of TlK/CAT gene by IL-

6 in the presence and absence of dexamethasone (DEX). Hep3B cells were transfected with 20 pg of pTlK/CAT (-1516) DNA and treated with increasing amounts of IL-6 in the presence (+) or absence (-) of dexamethasone (1 p ~ ) . Cells were harvested after 18 h of treatment. Protein concentration and the percent of ['.'C]chloramphenicol converted were quantitated as described under "Experimen- tal Procedures." Values represent the means of three independent transfection experiments.

(-427), and pTlK/CAT (-73), with the 5' deletion end points a t -584, -427, and -73, respectively, from the transcription start site (Fig. 1) were transfected into Hep3B cells, which in turn were stimulated with either IL-6 or dexamethasone individually or with both agents in combination. The results were compared with those of pTlK/CAT (-1516). We sur- mised that this experiment would indicate whether the glucocorticoid RE is co-localized in the same region with the IL- 6 RE and whether both cis-acting regulatory elements are required for the observed synergistic effects. Differential CAT expression of these deletion mutants with or without the inducers are presented in Fig. 4A. CAT activities were then quantitated and expressed relative to the CAT activity ob-

1L-m - - + + - - + + - - + + - - + + - ~ ~ x , ~ ~ - + - + ~ ~ ~ ~ - + ~ ~ - + -

B . H.Lo -1510 -684 -427 -73 SV,

""

l o o t - \ k -J w a

25 ' - ""-\ - - 1500 - 1000 -500 . l

DISTANCE FROM TRANSCRIPTION START SITE

FIG. 4. Deletion analysis of TlK/CAT fusion gene in Hep3B and HeLa cells. Cells were transfected with 20 pg of pTlK/CAT (-1516) and three 5' deletion mutants: pTlK/CAT (-584). pTIK/ CAT (-427), and pTlK/CAT (-73). pSVpCAT was used as control. Approximately 24 h after transfection, cells were treated with control medium, dexamethasone ( D E X 1 p ~ ) , IL-6 (200 units/ml), or IL-6/ dexamethasone. Protein concentration and CAT activity were determined after 18 h of stimulation. A, results of the CAT activity of each construct transfected into Hep3B cells; R, results of the CAT activity for each construct transfected into HeLa cells; C, quantitation of the CAT activity for each mutant in Hep3B cells. Results were calculated relative to the CAT activity in pTlK/CAT (-1516)-transfected cells and treated with IL-6/dexamethasone, to which a value of 100 was assigned. Values represent the means of three independent transfection experiments.

2950 Regulation of Rat Kininogen Gene Expression

tained from the cells transfected with pTlK/CAT (-1516) and stimulated with IL-6/dexamethasone (Fig. 4C). Basal CAT expression gradually declined as the 5'-flanking region of the T kininogen promoter was deleted progressively, with the shortest construct, pTlK/CAT (-73), retaining only 40% of the basal CAT activity. However, this lower basal activity was still specific to liver cells; no CAT activity was detected in HeLa cells with this construct (Fig. 4B). In contrast, loss of IL-6 responsiveness was much more dramatic. Although there was no effect on the magnitude of induction by IL-6/ dexamethasone when the T kininogen promoter was deleted from bp -1516 to -427, further deletion to -73 abolished the induction. These observations were identical for the transfected cells whether treated with IL-6 alone or IL-6/dexamethasone. The loss in glucocorticoid response, however, seemed to be more pronounced with deletion from bp -584

These results demonstrate that the 5' promoter region of the rat T1 kininogen gene contains a t least one regulatory region around -427 to -73 that confers responsiveness to both IL-6 and dexamethasone. Both the IL-6 RE and the glucocorticoid RE are likely to reside in this region and are responsible for the synergism observed with the pTlK/CAT (-427) construct. These results further suggest that sequences proximal to bp -73 are sufficient to confer T1 kininogen gene expression in a liver-specific manner.

A 321-bp Fragment from the TI Kininogen Gene Promoter Confers IL-6 Responsiveness to a Heterologous Promoter-To examine whether the identified IL-6 RE spanning from bp -427 to -73 could function on a heterologous promoter, a larger DNA fragment (-596 to -8) encompassing this region was isolated and subjected to further restriction digests. The resulting DNA fragments subsequently were inserted 5' to the thymidine kinase (tk) promoter in the pBLCAT vector and the orientation determined by DNA sequencing. Con- structs ptk/TlK (-107/-596), ptk/TlK (-8/-427), and ptk/ T1K (-107/-427), contained 490, 420, and 321 bp, respectively, of the T1K promoter region inserted in the opposite orientation, and constructs ptk/TlK (-427/-107) and ptk/ T1K (-107/-8) contained 321 and 100 bp, respectively, of the T1K promoter inserted in the same orientation as the tk promoter (Fig. 5A). These constructs were then transfected into Hep3B, and the responsiveness to IL-6 and dexamethasone, individually or in combination, was determined. The pBLCAT vector was included as a control. The orientation of the insert in each construct and quantitation of the transfection results are summarized in Fig. 5A. For comparison, fold induction was expressed as CAT activity relative to that of the untreated controls, and a value of 1.0 was assigned to the control of each construct.

Under noninduced conditions, the pBLCAT vector and the chimeric ptk/TlK constructs showed similar conversions of ['4C]chloramphenicol, within a range of 5 7 % . With the ex- ception of the ptk/TlK (-107/-8) hybrid gene, all other ptk/ T1K constructs displayed a similar magnitude of induction in response to IL-6 and IL-6/dexamethasone. A 2-fold stimulation was observed with IL-6 alone and approximately 5-fold when dexamethasone was added together with IL-6. The lower fold of induction observed in cells transfected with the ptk/ T1K constructs, as compared with those obtained from the pTlK/CAT constructs, was perhaps due to the higher basal expression of the t k promoter constructs, which in turn reduced the magnitude of induction. Indeed, under identical conditions, the tk promoter constructs gave a 7-%fold higher basal activity than that with the T1K promoter (data not shown). In contrast to other ptk/TlK constructs, the ptk/

to -427.

A Fold Induction

Dex IL-6 IL-WDex e 1.1 0.9 0.8

4 T & 1.4 1.8 4.5

-107 -596 e 0.9 1.9 4.7

-8 -427

1.1 2.1 4.8

-10 -427

6 1.2 2.0 5.4 -427 -107

0.8 1.0 0.8 107 -8

B Hop3B

9

FIG. 5. Effect of 321-bp fragment from the T1 kininogen promoter on a heterologous tk promoter. Hep3B and HeLa cells were transfected with 20 pg of pBLCAT vector or ptk/TlK constructs and treated with control medium, dexamethasone (DEX; 1 pM), conditioned medium (CM), CM/dexamethasone, IL-6 (200 units/ml), or IL-6/dexamethasone. Cells were harvested after 18 h of treatment, and protein concentration and CAT activity were analyzed. A, quantitation of the CAT assay for each ptk/TlK construct transfected into Hep3B cells. Fold induction was calculated relative to the CAT activity for each construct in control cells, to which a value of 1.0 was assigned. The thin arrow denotes the direction of transcription, and the thick arrow indicates the orientation of the T1K promoter fragment with respect to the direction of transcription. B, results of the CAT activity of pBLCAT vector and ptk/TlK (-427/-107) construct transfected into Hep3B and HeLa cells under various treatments.

T1K (-107/-8) construct did not show any detectable induction under any condition. These results, in accord with 5' deletion studies, indicated that the elements containing the IL-6 responsiveness, as well as the synergistic effects between IL-6 and dexamethasone, are localized within a 321-bp region spanning from bp -427 to -107. Moreover, these results demonstrated that the IL-6 RE from the rat T1 kininogen promoter functions equally well on a heterologous promoter.

Because the tk promoter does not function in a cell-specific manner, it was of interest to examine whether IL-6 RE from the T1 kininogen promoter could function in nonliver cells. HeLa cells were transfected with ptk/TlK (-427/-107) DNA

Regulation of Rat Kininogen Gene Expression 2951

and were stimulated subsequently with cytokines and dexamethasone. As shown in Fig. 5B, IL-6 RE was equally effective in conferring cytokine responsiveness to nonliver- and liver- derived cells. This result is in sharp contrast to that obtained from pTlK/CAT deletion mutants, where CAT activity was not detectable in transfected HeLa cells induced with IL-6/ dexamethasone (Fig. 4B) or CM/dexamethasone (data not shown). The pBLCAT vector consistently displayed a low level of induction by CM (1.5-fold) and CM/dexamethasone (2.3-fold). Similar levels of induction have been reported for HeLa cells transfected with pBLCAT vector and treated with interferon-a (40). Accordingly, it is the interferon-a compo- nent, which is known to be present in our CM (28), that may have contributed to the observed background induction of pBLCAT in our study. When treated with IL-6 alone or with IL-6/dexamethasone, pBLCAT gave no detectable induction (Fig. 5). Therefore, this result clearly demonstrated that the IL-6 RE in the T1 kininogen can function in nonliver cells, such as HeLa cells, when linked to a noncell-specific promoter such as the tk promoter.

DISCUSSION

Inuolvernent of IL-6, IL-I, and Glucocorticoid in Acute Phase Response-An acute inflammation induced by subcutaneous injection of turpentine in the rat causes a 20-30-fold increase in T kininogen mRNA levels in 24 h (12, 13). However, T kininogen mRNA reaches the maximal level in 4 h after a single dose injection of IL-6 into the rats, implying the direct action of this cytokine on hepatocytes (10). Thus, the delayed response in rats treated with turpentine is probably due to the acute phase cascade of events, including activation of monocytes and macrophages and the formation and secretion of inflammatory mediators and their transport to the target cells that must proceed before the mediators can exert their effects (10). This investigation was therefore carried out primarily to localize the ck-regulatory elements in the T1 kininogen gene and to characterize its responsiveness to the several acute phase mediators, particularly IL-6.

Using a hybrid gene constructed by fusing the 1516-bp 5’- flanking region and the 119-bp untranslated exon 1 sequence of the rat T1 kininogen gene to a CAT reporter gene, we demonstrated that this chimeric gene, when transfected into Hep3B and HepG2 cells, was responsive to stimulation by the CM derived from activated mixed human lymphocyte culture. This hybrid gene also responded to dexamethasone stimulation; however, the responsiveness was magnified when both CM and dexamethasone were added together. With the use of purified recombinant human cytokines, we identified IL-6 as the main inflammatory mediator in the CM responsible for inducing CAT expression and showed that this cytokine can work cooperatively with dexamethasone to elicit a synergistic induction of the TlK/CAT hybrid gene.

It is interesting to note that IL-1, a known inflammatory mediator (29), instead of stimulating TlK/CAT expression, actually down-regulated its basal activities and to a similar extent effectively inhibited the induction by IL-6 when IL-1 and IL-6 were added together. The down-regulation of the endogenous T kininogen gene by IL-1 was also observed by Andus et al. (19) using rat primary hepatocyte cultures. IL-1 also effectively reduced the induction of the endogenous T kininogen by IL-6. Thus, T kininogen may be classified as a member of genes such as albumin and fibrinogen whose expression is regulated negatively by IL-1 (27, 41). As for the lower CAT activities observed in the combined treatments of IL-1 and IL-6, as compared with those observed with IL-6 alone, it is unclear whether the lowering of basal expression

as the result of IL-1 treatment may contribute to the reduced level of induction. The details of the mechanism by which IL- 1 suppresses IL-6-induced expression are not known. The inhibitory effect of IL-1 is unlikely to be associated with IL- 6 receptor, because these two cytokines have their own specific membrane receptors. Furthermore, IL-1 and IL-6 act addi- tively or synergistically in the regulation of other acute phase genes such as al-acid glycoprotein and serum amyloid A (42).* In fact, inhibition of IL-6 action by IL-1 has been demonstrated only for fibrinogen, a,-macroglobulin, and T kininogen genes (42). Therefore, specific DNA sequences are likely to play an important role in this type of inhibition. The antagonistic action was also examined when dexamethasone was included in the treatment. As shown in Table I, IL-1 exerted some inhibition even in the presence of dexamethasone. This observation is in agreement with previous results in rat primary hepatocyte cultures as well as rat hepatoma cell line H35 (19, 21). In addition to IL-1, a protein kinase C- inducing agent, TPA, was included to assess its ability to modulate basal expression as well as IL-6-regulated expression of T kininogen. Phorbol ester exerted some inhibitory effects on both basal and IL-6-stimulated CAT activities. The analogy between IL-1 and TPA in inhibiting basal activity and IL-6-induced T kininogen expression raises the possibility that they mediate their effects by a similar mechanism.

Because the response of T kininogen to inflammatory cytokines and hormones observed in vivo or in rat primary hepatocyte culture and hepatoma cell lines (10, 19, 21) was faithfully reproduced in our transfection system, our results together with others clearly demonstrate that the acute phase response of T kininogen is likely to be composed of a rather complex mechanism involving several cytokines, like IL-6, IL-1, possibly hepatocyte-stimulating factor I11 (21), and steroid hormones such as glucocorticoid and estrogen (43). The ultimate level of T kininogen expression thus depends on the absolute and relative concentrations of each cytokine and the steroid hormones and how they interact with each other to impose the final action. Clearly, IL-6 and dexamethasone play a major role as positive regulators of T kininogen. Two different mechanisms may explain the synergism between IL- 6 and dexamethasone. First, glucocorticoid RES are co-localized with IL-6 RES such that the strength of the enhancer elements that serve as transcription activators is enhanced through protein-protein interactions of nuclear factors binding with them (44, 45). Another mechanism involves stimulation of IL-6 receptor synthesis in hepatoma cells by dexamethasone, resulting in an increased number of IL-6 receptors at the cell surface. Results from two independent studies support this mechanism. An increase in IL-6 receptor mRNA levels was observed after dexamethasone treatment of HepG2 cells, and this elevation of hepatic IL-6 receptors possibly promoted an efficient signal amplification event (5). In a separate experiment, hepatocytes were shown to respond to dexamethasone stimulation by increasing IL-6 receptor synthesis (46). The most likely possibility, in fact, is that a combination of both mechanisms contributes to the synergy between IL-6 and dexamethasone.

Regulatory Element(s) for Tissue-specific Expression-Since the pTlK/CAT (-1516) construct conferred hepatoma-specific transcription, we sought to localize the cis-regulatory element(s) for tissue-specific expression. A series of 5’ deletion mutants were transfected into nonliver cells (HeLa), and CAT activities were quantitated under control or stimulated (by IL-6/dexamethasone) conditions. No CAT activities were detectable under either condition, indicating progressive dele-

J. Huang and W. S. L. Liao, unpublished data.

2952 Regulation of Rat Kininogen Gene Expression

tion from -1516 to -73 did not lead to a loss in the tissue specificity of the fusion gene. Moreover, when transfected into Hep3B cells the shortest construct, pTlK/CAT (-73), still retained 40% of the basal activity. These results demonstrate that sequences within the 73 bp immediately upstream from the CAP site are necessary and sufficient for hepatocyte- specific transcription. This conclusion was supported further by two additional observations. First, when transfected into HeLa cells, the ptk/CAT (-427/-107) hybrid gene displayed a similar magnitude of induction by IL-6/dexamethasone as that observed in the hepatoma-derived cells. This result im- plied that this 321-bp DNA fragment did not contain an additional cis-regulatory element for tissue-specific expression. Second, the ptk/CAT (-107/-8) construct, when transfected into Hep3B cells, showed a 50% higher basal expression than that of the pBLCAT vector. However, the gradual loss of basal activity in Hep3B cells that followed the progressive deletion from -1516 to -73 underlines the presence of mul- tiple regulatory elements in the upstream regions that contribute to the enhanced expression of TI kininogen in liver cells. IL-6 Regulatory Element Responsible for Induced Expres-

sion-Deletion analyses of the T1 kininogen promoter located a region between bp -427 and -107 relative to the transcription start site that contains IL-6 response activities. When transfected into Hep3B cells, this 321-bp fragment conferred IL-6 responsiveness as well as synergy of IL-6 and dexamethasone to either its own promoter or a heterologous tk promoter. IL-6 RES derived from other acute phase genes such as human haptoglobin and hemopexin are also equally functional when inserted upstream of a heterologous promoter (25, 47). In comparing the 1516-bp proximal 5"flanking and 119-bp exon 1 untranslated sequences of the Sprague-Dawley rat TI kininogens (48) with the consensus sequences for IL- 6 RE (5, 23, 26), only one perfectly matched heptanucleotide, CTGGAAA, was found; it resides at 228 bp upstream from the transcription start site. This sequence is located within the 321-bp region containing the IL-6 response activity. How- ever, no good match was found with the other less conserved IL-6 RE consensus sequence (AGTGANGNAA) (26) within this region. Interestingly, a near-match, partially palindromic glucocorticoid RE sequence (TGTACATTGTCCT) (49) is located at bp -296, which is also within the 321-bp fragment. Whether these sequences represent functional IL-6 and glucocorticoid RES remains to be determined. When linked to the tk promoter the 321-bp fragment still retained the inhibitory effect of IL-1 on IL-6 stimulation (data not shown). Therefore, in addition to containing positive regulatory sequences for IL-6 and dexamethasone responsiveness, this 321- bp region may also contain recognition sequences for negative regulation in response to IL-1 treatment.

The identification of a proximal element that confers liver specificity and a distal element that confers responsiveness to cytokines permits the analysis of trans-acting factors that confer the appropriate patterns of regulation to this gene. Ongoing studies are aimed at defining the nature of such factors and exploring their regulation and mechanisms of action. Additionally, mechanisms underlying the differential regulation between the T and K kininogen genes during inflammation will be examined.

Acknowledgments-We would like to thank Jianyi Huang and

Xiaoxia Li for their advice and discussion throughout this study and Alisha Tizenor for the preparation of the manuscript.

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