THE OF BIOLCQICAL Vol. No. and U.S.A. The Rat and Human ...treated for another 24 h with serum-free medium containing dexam- ethasone (1 w) alone as a control or dexamethasone + IL-1

THE JOURNAL OF BIOLCQICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 17, Issue of April 29, pp. 12654-12661, 1994 Printed in U.S.A.

The Rat and Human Hemopexin Genes Contain an Identical Interleukin-6 Response Element That Is Not a Target of CAAT Enhancer-binding Protein Isoforms*

(Received for publication, November 22, 1993, and in revised form, January 31, 1994)

Stephan ImmenschuhS, Yasuhiro Nagael, Hiroyuki Satoh, Heinz Baumannn and Ursula Muller-Eberhardll From the Departments of Pediatrics, Biochemistry, and Pharmacology, Cornell University Medical College, New York, New York 10021 and the Wepartment of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263

Hemopexin (Hx) is an abundant acute-phase protein (APP) that binds heme with high affinity. In rat hepatic cells, the transcription rate of the Hx gene is increased by interleukin (IL)-l and IL-6. To investigate the cis- acting regulatory elements (RES) responsive to these hormones, chloramphenicol acetyltransferase constructs of rat and human H x gene sequences were tested in transiently transfected hepatoma cells. An IL-6-RE was identified in the promoter of both rat and human Hx genes, the function of which was dependent on the core sequence (CCGGGAA) common in other APP genes. The previously characterized Hx A element mediated a relatively minor cytokine response as compared with the Hx IL-6-RE. The human Hx A element, in contrast to the rat and human Hx IL-6-REs, was strongly trans-activated by cotransfected CAAT enhancer-binding proteins (C/ EBP)-P and -6. The rat gene homolog of the human Hx A element was inactive as a cytokine RE and was minimally trans-activated by CEBP isoforms. Results of electrophoretic mobility shift assays indicated that the Hx IL-6-RE is a binding site for the IL-6-inducible nuclear protein IL-6 RE-BP, which also binds to the conserved IL-6-RES of other APP genes and is distinct from CIEBPP.

The &glycoprotein hemopexin binds heme with exceptionally high affinity in an equimolar ratio (1). Heme not buried in heme-binding proteins can be detrimental, and Hx’ protects efficiently against heme-mediated oxidative damage (2-4). Such a protective function is one of the common characteristics of plasma proteins known as acute-phase proteins (APPs). In- creased expression of APPs in the liver during stress reactions is part of a series of reactions collectively referred to as acute- phase response (APR) ( 5 , 6). The circulating concentration of

26122 (to H. B.), National Institutes of Health Grant DK 30664, and a * This work was supported by National Institutes of Health Grant CA

Patterson Fund grant (to U. M-E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a postdoctoral fellowship of the Deutsche Forschun- gsgemeinschaft (Germany).

School, Tokyo 113, Japan. 6 Present address: Dept. of Internal Medicine, Nippon Medical

N-804, New York, NY 10021. Tel.: 212-746-3420; Fax: 212-746-8458. 11 To whom correspondence should be addressed: 525 E. 68th St., Rm.

tein; APR, acute-phase reaction; bp, base pair(s); CAT, chloramphenicol The abbreviations used are: Hx, hemopexin; APP, acute-phase pro-

acetyltransferase; C/EBP, CCAAT/enhancer-binding protein; EMSA, electrophoretic mobility shift assay; IL, interleukin; MUP, major urinary protein; RE, response element.

APPs varies among species; plasma levels of Hx are only slightly elevated in the human (7, 8) but increased at least 3-5-fold in the rat.2

Principle mediators of the hepatic APR are IL-1 and IL-6 type cytokines as well as glucocorticoids, all of which act largely at the level of gene transcription (9). In recent years, cis-acting cytokine RES have been identified for several APP genes, such as a,-macroglobulin (lo), a,-acid glycoprotein (11, 121, complement C3 (131, C-reactive protein (14), haptoglobin (15, 16), and Hx (17, 18). These RES contain binding sites for, and are truns- activated by, transcriptional factors of different gene families and include isoforms of C/EBP (19), NFKB (20), and the group IL-6 RE-BP (211, APRF (221, and SIF (23). The IL-6 regulation of the human Hx gene has been ascribed to an IL-6-inducible DNA-binding protein, IL-GDBP, which interacts with a sequence motif of the Hx A element (19). IL-6DBP has been sub- sequently identified to be a member of the C/EBP family and to be identical to CIEBPP (241, NF-IL6 (251, LAP (261, AGPIEBP (27), and CRP-2 (28). A similar APP gene regulatory function has been observed for the structurally related CIEBPGINF-IL6P (29, 30).

In the present study, the activity of the cytokine RES of the rat and human Hx genes was compared, and the presence of an identical IL-6-RE in both species was found that was structurally and functionally distinct from the previously described human Hx A element. Regulation via the Hx IL-6-RE does not involve C/EBP isoforms but does appear to involve the DNA- binding protein IL-6 RE-BP.

EXPERIMENTAL PROCEDURES Materials-All restriction endonucleases and DNA-modifying en-

zymes were purchased from New England BioLabs or Promega. Poly(d1- dC) and sonicated calf thymus DNA were obtained from Pharmacia LKB Biotechnology Inc. Radioactive nucleotides were purchased from DuPont NEN. Plasmids for chloramphenicol acetyltransferase (CAT) assays, pCAT promoter, and pCAT control vectors were products of Promega. A 7-deaza sequencing kit and Sequenase were from U. S. Biochemical Corp. IL-lp and IL-6 were provided by Immunex Corp., Seattle, WA, and Genetics Institute, Cambridge, MA, respectively.

Plasmid Constructs-All basic recombination techniques referred to were standard protocols (31). Two 5’-flanking region fragments of the rat Hx promoter region were linked in front of the bacterial CAT gene of the plasmid pSVOCAT (see Fig. 2, constructs 1 and 2), as previously described (18). A 79-bp subfragment (position -236 to -158) of the Hx promoter (see Fig. 2, construct 3 ) and multimerized copies of synthetic oligonucleotides with rat and human Hx promoter fragments were cloned into the BglII site of the plasmid pCAT promoter vector (see Fig. 2, constructs 3-9). The following oligonucleotides were used: rat Hx IL-6-RE, 5’-GATCCTGCCGGGAAGATAGTCTGAGA-3’; human Hx IL- 6-RE, 5’-GATCCTGCCGGGAAAAAGGAGTC’ITGGA-3’; human Hx A,

S. Metcalfe and U. Muller-Eberhard, unpublished results.

12654

Interleukin-6 Regulation of Hemopexin Genes 12655 5'-GATCCTA'MTGCAGTGATGTAATCAGCG-3'; rat Hx A, 5"GATC- C-GCTGTGATGTCGTCTGCG-3'; mutant Hx IL-6-RE, 5"GATC- CTGCATGGCAGATAGTCTGAGA-3'; a,-acid glycoprotein IL-6-RE, 5'- GATCTGGGC'MTTGGGAAAAACTCAAG-3'; a,-macroglobulin IL-6- RE, 5' -GATCTAATCC'ITCTGCTGGCTA-3' ; NFKB, 5'-GATC-

GAGC-3'; glucocorticoid response element, 5"GATCAGAACACAGTGT-

Oligonucleotides used for oligomerization carried a 4-nucleotide 5' extension (GATC) to allow ligation and insertion into the BglII site of the pCAT promoter plasmid. Correctness of the constructs was con- firmed by DNA sequencing.

C/EBPP and C/EBPG cDNAs inserted into a murine sarcoma virus- containing expression vector yielding pMSV-EBPP and pMSV-EBPS were provided by Dr. S. L. McKnight (24) and used for transfection into H-35 cells. High expression of the two C/EBP isoforms in human hepatoma cells was achieved by pCD-C/EBPp and pCD-CIEBPG (32).

Cell Culture Deatment and Analysis-HepG2 cells and Hep3B cells (33) were cultured in minimal essential medium with 10% fetal calf serum. H-35 cells (clone T-7-18) (34) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Confluent monolayers were treated with serum-free medium containing 1 w dexamethasone as a control or dexamethasone + IL-lP (20 ng/ml) and dexamethasone + IL-6 (100 ng/ml) as indicated.

Nuclei were prepared from individual monolayers in 10-cm-diameter culture dishes (35); transcription run-on reactions and isolation of nuclear RNA followed the procedure of Lamers et al. (36). Equal amounts of radioactivity were hybridized to nitrocellulose strips carry- ing slot-blotted denatured cDNAs encoding rat Hx and the internal reference gene mouse triose-phosphate isomerase (35). Cytoplasmic RNA was isolated from the post-nuclear cytoplasmic fractions, and the relative amount of mRNA for rat Hx was determined by dot-blot hy- bridization with 32P-labeled rat Hx cDNA.

The change in the amount of secreted Hx was measured by rocket immunoelectrophoresis in equal aliquots of the final culture medium.

Cell Dansfection and CATAssays-Monolayers of HepG2 and Hep3B cells were transfected with plasmid DNA as a calcium phosphate pre- cipitate (37) and H-35 cells as a DEAE-dextran complex (38). 1 ml of transfection solution contained 20 pg of plasmid DNA mixture com- posed of 5-15 pg of CAT gene construct, 5 pg of transcription factor expression vector or empty expression vector (where applicable), and 2 pg of PIE-MUP as internal transfection standard (11). In all experiments including hormone treatments, the cell cultures were released by trypsin after an 18-h recovery period, divided into equal portions, and placed into six-well cluster plates. 24 h later, the subcultures were treated for another 24 h with serum-free medium containing dexamethasone (1 w) alone as a control or dexamethasone + IL-1 and dexamethasone + IL-6, as indicated. CAT activity was determined in cell extracts (39) and normalized relative to the amount of major urinary protein (MUP) produced by the same cells (as a product of cotransfected p1E"UP). The normalized values are expressed in percent conversion of chloramphenicol to acetylated productslhour and grams of M U P .

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSA)-Nuclear extracts from H-35 cells were prepared ac- cording to Dignam et al. (40). The binding reaction was performed in 10 m Tris-HC1 (pH 7.5), 50 m NaCI, 0.5 m EDTA, 1 m MgCI,, 1 mM dithiothreitol, 4% glycerol, with 0.5 ng of 32P-end-labeled probe DNA and 10 pg of H-35 nuclear extracts, 500 ng of poly(dI-dC), and 100 ng of sonicated calf thymus DNA. 50- or 100-fold molar excess of the unlabeled probe was used as cold competitor in competition assays. After 30 min of incubation at room temperature, the DNA-protein complexes were resolved on 4% polyacrylamide gel in 0.5 x TBE (1 x TBE = 89 m Tris-HC1.89 m boric acid, 2 m EDTA) and 2.5% glycerol at 10 V/cm. The gels were dried and autoradiographed.

Binding reaction of recombinant protein C/EBPP was carried out as described (41). 0.5 ng of 32P-end-labeled DNA fragments was incubated in a final volume of 10 pl containing 25 m HEPES pH 7.6.60 m KCI, 7.5% glycerol, 0.1 m EDTA, 0.75 m dithiothreitol, 5 m MgCI,, and 1 pg of double-stranded poly(d1-dC) competitor DNA. C/EBPP was added last, and the binding reaction was allowed to proceed for 90 min on ice. Electrophoresis of the DNA protein complexes was carried out as described above.

GAGGGGAC'MTCCCTAGC-3'; SP-1, 5"AlTCGATCGGGGCGGGGC-

TCTCTA-3'.

RESULTS !!kanscriptional Regulation of the Hz Gene by IL-1 and IL-

6-The cytokine-specific regulation of the rat Hx gene was evaluated by determining its transcriptional rate in H-35 hepa-

3 - . - - - -- e - TPI

0 0.5 4 24 I 0.5 4 24 1L-1 I 1L-6

Treatment (hours) FIG. 1. Regulation of rat Hx gene by cytokines in H-35 cells.

Confluent monolayers of H-35 cells were treated for the indicated length of time with dexamethasone alone or a combination of dexamethasone + IL-1 or dexamethasone + IL-6. Total cellular RNA was isolated, and serially diluted cytoplasmic RNA was probed with a 32P- labeled rat Hx cDNA. Nuclei were prepared and used for run-on reactions. Labeled RNAs were hybridized to slot-blotted rat Hx cDNA (Hz) and triose-phosphate isomerase cDNA (TPI).

toma cells treated for various lengths of time with either IL-1 or IL-6 (Fig. 1). Results of run-on experiments showed maximal stimulation of the transcriptional rate of Hx as early as 30 min after exposure to IL-1. IL-6 enhanced the transcriptional rate, also within 30 min, to a maximal level that was substantially higher than that of IL-1-treated cultures. Hx mRNAlevels were increased after 4 h of exposure to IL-1 and after only 30 min of exposure to IL6. Both treatments caused continuous Hx mRNA accumulation during the subsequent 24 h. Treatment with IL-1 + IL-6 had an additive effect on the transcriptional rate, mRNA accumulation, and Hx secretion of 30-50-fold (data not shown) (see also Fig. 4A).

Regulation of Dansfected Rat Hx Gene Sequences in H-35 Cells-The regulation of the 5"flanking region and the promoter linked to theCAT gene by cis-acting cytokine RES of the rat Hx gene was evaluated in transiently transfected H-35 and HepG2 cells. Results of these experiments are summarized in Fig. 2 (see also Figs. 4-6 for representative data). A construct containing 1600 bp of the 5"flanking region of the rat Hx gene (construct 1) mediated a %fold IL-6 induction in H-35 cells. The IL-6 response was still elicited by a 5' truncated 250-bp promoter region (construct 2). The magnitude of the IL-6 regulation of constructs 1 and 2 was only a minor fraction of that of the endogenous Hx gene (Fig. 1). Contributory to this lower response was the high level of the basal expression of the Hx promoter, about 100-fold higher than that of the pCAT vector with the minimal SV40 promoter (data not shown). IL-1 alone or in combination with IL-6 was ineffective.

We investigated the function of promoter sequences independent from that of the transcription initiation site by insert- ing subregions of the promoter sequence into the pCAT vector. When tested in either orientation, a 79-bp region showed low induction in response to IL-6 (construct 3 and data not shown). This 79-bp region contains an IL-6-RE at -158 to -178, previously proposed (18), with the sequence CCGGGAA (-169 to -175), which matches the core consensus sequence of the a2- macroglobulin gene (CTGGGAA) (Fig. 3) (10) in 6 out of 7 bp. To prove the functional relevance of this site, five copies of the wild type sequence from -158 to -178 in pCAT (construct 4) were tested for cytokine responsiveness. Construct 4 responded dra- matically to IL-6 and minimally to IL-1. In contrast, this 5-mer construct with three base changes in the core sequence ( C W - G&l were changed to CMGGCA) was not regulated by IL-6

12656 Interleukin-6 Regulation of Hemopexin Genes

Construct Fold Stimulation of CAT-Activity

H-35 HepG2 A

1-6 Rat Hx Gene IL-6 IL-I+IL-6 - "

IC6

3

2

2

55

1

1

22

1

1

3 2

1 2

4 2

69 10

1 1

1 1

117 12

6 1

10 1

FIG. 2. Cytokine-specific response of regulatory elements of rat and human Hx genes in transiently transfected H-35 and HepG2 cells. The indicated rat and human Hx gene sequences were cloned into pSVOCAT (constructs 1 and 2 ) or pCAT (constructs 3-9) and transiently transfected into H-35 and HepG2 cells. After a recovery period, the transfected cells were tested for their responsiveness to dexamethasone + IL-6 or dexamethasone + I L 1 + IL-6. The -fold stimulation in each experiment relative to the control with dexamethasone alone was determined. The values represent means of at least three independent experiments.

Hemopein IL-6-m

-In

475 .IS2

A Ill 1

- + - + IL-1 + + IL-6 "

B rat Hx (5xIL-6-RE) hum Hx (5xIL-6-RE)

"1

mutant CTG CATGGCA GA - - TAGTCTGAGA

consensus CTGGGAA

Hemopexin A site

-1"

. I S

eoasmsus TGNNGYAAT

FIG. 3. Sequence comparison of the &&RE and the A element

the HX IL-6-RE and the Hx A element are demonstrated with their in the rat and human Hx genes. Rat and human gene sequences of

position relative to the transcription start site. Matching sequences are indicated. The mutant Hx IL-6-RE oligonucleotide differed in 3 bp from the rat Hx IL-6-RE in the conserved CCGGGAA consensus sequence.

(Fig. 2, construct 5; see also Fig. 3). Based on these results, we henceforth denoted the 21-bp sequence from -158 to -178 the "rat Hx IL-6-RE." The extent of regulation of constructs 1-3 by cytokines was comparable in transfected H-35 and HepG2 cells. IG6 responsiveness of construct 4, however, was only 10-fold in HepG2 cells, as compared with 55-fold in H-35 cells.

- + - + - + - +IL-1 + + - - + +IL-6 "

FIG. 4. Activity of rat and human (hum) Hx &&RE in transiently transfected H-35 cells. H-35 cells were transiently transfected with either rat Hx (5xIL-6-RE) or human Hx (5xIL-6-RE) CAT plasmids. Cultures were subdivided and treated with dexamethasone alone as a control or combinations of dexamethasone and cytokines. The CAT activity was determined in equivalent amounts of cell extract, and the values for percent conversion of substrate to product are indicated at the top ofpanel B. The amount of Hx protein secreted by each culture was determined by rocket immunoelectrophoresis (panel A ) and expressed as the -fold level of control culture with dexamethasone alone.

These data indicate that the cloned rat Hx promoter does not confer the full IL-6 response level of the endogenous Hx gene in H-35 cells to CAT reporter constructs. A prominent IG6 response is, however, elicited by oligomerized copies of the sub- region between -158 and -178 containing the core consensus sequence CCGGGAA.

Rat and Human Hx Gems Contain an Identical ZL-6-RE- Acomparison of the rat Hx IL-6-RE with the homologous region of the human Hx promoter showed a high degree of sequence similarity (Fig. 3) and identity within the critical core region. To verify whether the human equivalent to the rat Hx IG6-RE is functional, we tested a pCAT construct containing five copies of the human sequence (Fig. 2, construct 7). A striking difference in regulation by cytokines between rat and human Hx ILS-RE constructs was observed (Fig. a). Whereas rat Hx

Interleukin-6 Regulation of Hemopexin Genes 12657

I 43 1 33 I 53 I 321 I 362 I 360 I L

I

n w II:

- + + - + + IL-6 - - + , , - - + IL-1 1

Vector C/EBPG

CIEBPG of the Hx A element and the Hx -6-RE in Hep3B cells. FIG. 5. Cytokine responsiveness and trans-activation by

Hep3B cells were transfected with a mixture of plasmid DNA containing the indicated CAT constructs (5 pg/ml), PIE-MUP (2 pg/ml), and either pCD-poly or an expression vector for C/EBPfi (13 pg/ml). Subcul- tures were treated with dexamethasone alone as a control or dexamethasone and cytokines as indicated. The CAT activity was determined in equivalent amounts of cell extracts and was normalized to the secreted amount of MUP for each culture. The values for percent conversion of substrate to product are shown at the top of each culture. hum, human.

(5xIL-6-RE)CAT (Fig. 2, construct 4 ) was strongly stimulated by IL-6 alone, the human construct responded little to IL-6 but strongly to IL-1 + IL-6. Both rat and human Hx IL-6-RE constructs did not respond to IL-1 alone. The synergistic effect of IL-1 + IL-6 on the human Hx IL-6-RE was similar to that observed for the endogenous Hx gene (Fig. 4A). These results clearly indicate that the human sequence corresponding to the rat Hx IL-6-RE confers IL-6 responsiveness to a CAT vector, and it was therefore denoted the "human Hx IL-6-RE."

Species-specific Differences in the Function of Rat and Hu- man Hx A Elements-In a previous study on the human Hx gene, Cortese and co-workers (17, 42) reported that the Hx A element is a strong tissue-specific enhancer element conferring

hum Hx (3xA)

3OOo

h Hx (5x IL-6-RE)

.- .d

.- + 800 *

rat Hx (3% A)

- rat Hx (5% IL-6-RE)

FIG. 6. Effect of CIEBPB and CIEBPG on Hx &&RE and Hx A element in HepG2 cells. HepG2 cells were transfected with a mixture of the indicated CAT constructs ( 5 pg/ml), PIE-MUP (2 pg/ml), and either pCD-poly or expression vectors for ClEBPp and C/EBPG (13 pg ml). Subcultures were treated for 24 h with dexamethasone alone (open bars) or with dexamethasone + IL6 (striped bars). CAT activity was normalized to the secreted amount of M U P , and mean values are shown. hum, human.

IL-6 responsiveness to a reporter construct. Since the A element differs in sequence and localization from

the Hx ILS-RE (Fig. 3), we asked whether the rat equivalent to the human Hx A element functions and whether its activity is comparable with that of the Hx IL-6-RE shared by human and rat. In order to accomplish this, triplicated copies of the human and rat Hx A elements (Figs. 2 and 3) in pCAT were transfected into HepG2 and H-35 cells. Surprisingly, the human Hx A site (Fig. 2, construct 8) was not inducible by IL-6 alone but was inducible 6-fold by IL-1+ IL-6. The latter response was slightly increased in a CAT vector with five copies of the Hx A element (Fig. 2, construct 9). The rat Hx Aconstruct (Fig. 2, construct 6) did not respond to cytokine treatment. Since Hep3B cells were used in previous studies on the Hx A element (17, 421, our constructs were also transfected into this cell line. As shown for H-35 and HepG2 cells, both rat and human Hx A constructs exhibited little or no cytokine inducibility in Hep3B cells, whereas a 7-fold IL-6 induction was observed with the rat Hx (5xIL-6-RE)CAT plasmid (Fig. 5).

We conclude, therefore, that the human Hx A element medi- ates a minor cytokine response that is not specific to IL-6 and is functionally distinct from the Hx ILS-RE.

CIEBPP and CIEBPG trans-Activate the Human Hx A Ele- ment More Strongly than the Equivalent Rat Hz A Element- CiEBP isoforms have been considered to play an important role in the transcriptional regulation ofAPP genes via cytokine RES (29,32,43). One of the prime examples of this regulation is that exerted by the A element of the human Hx gene (19). Because of the differences in sequence and function of the Hx A element and the Hx IL-6-RE, we examined the trans-acting capacity of CiEBP isoforms on these elements by cotransfection with expression vectors for C/EBP isoforms. As shown in Fig. 5,


probe rat Hx IL-6-RE mutant SP-1 hum Hx A

n-n - FIG. 7. Constitutive and IC6-induc-

ible nuclear factors bind to the rat Hx IL6-RE. Synthetic oligonucleotides containing the rat Hx IL6-RE, the mutant Hx IL-6-RE (mutant), the SP-1 recognition site (SP-I ), and the human Hx A el-

induc +

ement (hum Hx A ) were incubated with cor& - nuclear extracts from control (-1 or IG6- treated (+I H-35 cells, as indicated. The DNA-protein complexes were analyzed by electrophoresis in nondenaturing polyacrylamide gel. Positions of the free probe and the constitutive DNA-protein complex (const. ) and IG6 inducible complex (induc. ) are marked. free

probe

C/EBPG elicited an 8-fold trans-activation of the human Hx A construct that was essentially unaffected by cytokine treatment. A lower (&fold) trans-activation was observed for the rat Hx A construct; C/EBPG had essentially no effect on rat and human Hx ILS-RE constructs (Fig. 5 and data not shown).

Since trans-activation levels of reporter genes may vary among different cell types (32), we tested constructs containing the rat and human Hx IL6-RES (Fig. 2, constructs 4 and 7) as well as the rat and equivalent human Hx A regions (Fig. 2, constructs 8 and 6). These constructs were cotransfected with expression vectors for C/EBPP and C/EBPG in HepG2 cells. Basal CAT expression of the human Hx(3xA)CAT plasmid was strongly increased by both C/EBP isoforms. The rat construct equivalent to the human Hx(3xA)CAT showed a significantly lower response to C/EBP than the human construct. Both C/EBP isoforms caused only minor trans-activating effects on rat and human Hx IL-6-RE constructs and did not appreciably modify the magnitude of IL-6 stimulation (see Fig. 6).

These results confirm that the human Hx A element, in contrast to the Hx ILB-RE, is a target of C/EBP isoforms.

Constitutive and ZL-6-inducible Factors Interact with the Hz ZL-6-RE-Previous results with the IL-6-RE from the rat az- macroglobulin gene showed that the consensus sequence CT- GGGAA (Fig. 3) (10, 21) or an extended motif (T/G)T(C/A)(C/ T)(G/T)G (G/T)AA (22) serve as binding sites for the nuclear factors IL-6 RE-BP (21) or APRF and SIF/GAF (22, 23). Sig- nificantly different activation kinetics of DNA binding have been shown for the IL-6 RE-BP and APRF, although the two proteins have similar molecular masses; the DNA binding activity of IL-6 RE-BP is maximally induced after 4 h (211, whereas APRF is maximally induced after as early as 15 min (22) and disappears after 1 h in IL-6-treated hepatoma cells.

EMSAs with nuclear extracts from IL-6-treated and control H-35 cell cultures (Fig. 7) identified a constitutive and an IL- 6-inducible DNA-protein complex. The IL-6-inducible complex appeared after 2 h of IL-6 treatment in H-35 cells and was detectable up to 20 h but was not noticed within the 1st h of IL-6 induction (data not shown). Thus, the time course of IL- 6-dependent DNA binding to Hx IL-6-RE was in agreement with that of IL-6 RE-BP rather than with that of APRF. A detailed study on this issue has been performed in our labora-

The mutant Hx IL-6-RE sequence (Fig. 3) did not form any

S. Lai, S. Immenschuh, D. Gearing, S. F. Ziegler, and H. Baumann,

tory.3

manuscript in preparation.

- + - + - + - + DNA-protein complexes (Fig. 7) with the same nuclear extracts. There was no significant difference in binding of nuclear proteins to the SP-1 consensus sequence (44). The complexes of the human Hx A element formed with nuclear extracts from IL-6- treated cells was stronger than that with control extracts indicating a higher binding activity (Fig. 7).

Sequence specificity of DNA-protein complexes was evaluated by competition experiments (Fig. 8). Formation of DNA- protein complexes was inhibited by a 50-fold molar excess of the rat or human Hx IL-6-RE oligonucleotides (Fig. 8 A , lanes 2 4 ) but was only weakly affected by a 100-fold molar excess of the human and rat Hx A oligonucleotides (Fig. 8 A , lanes 6 and 7). The complex formation with the rat Hx IL-6-RE was unaffected by the mutant Hx IG6-RE oligonucleotide, the recognition sequence of NFKB (45) (Fig. 8B, lane 5), or the glucocorticoid response element (46) (Fig. 8B, lane 6). By contrast, sequences containing the IL-6-RES of the rat a,-acid glycoprotein (12) and a,-macroglobulin genes (10) (Fig. 8B, lanes 3 and 4 ) prevented DNA-protein complex formation.

These data demonstrate that the binding properties of an IL-6-inducible nuclear factor to the Hx IL-6-RE coincide with that of IL-6 RE-BP and critically depend on the intact CCGG- GAA consensus motif.

Recombinant CIEBPP Binds Human and Rat Hx A Elements with Different Affinity-To determine to what extent identical proteins might interact with both Hx IL-6-RES and Hx A elements, the human Hx A was used as a probe for EMSAs. Nuclear extracts of IL-6-induced H-35 cells produced two major complexes with the Hx A oligonucleotide (Fig. 9, lane 1). This interaction was shown to be specific, since a 25-fold excess of unlabeled human Hx A oligonucleotide prevented complex formation (Fig. 9, lane 2). By contrast, a 50-fold excess of rat Hx A oligonucleotide did not completely extinguish the bands (Fig. 9, lane 51, suggesting that the rat element had less affinity for the A-binding protein than the human sequence. A 100-fold excess of rat or human Hx IL-6-RE failed to alter the observed band pattern (Fig. 9, lanes 6 and 7).

Since the Hx A element was trans-activated by C/EBP isoforms (Figs. 5 and 6), we used the recombinant protein CEBPP, synonymous with LAP (261, for EMSAs with the Hx A oligonucleotide. Indeed, a C/EBP-mediated complex with the human Hx A oligonucleotide (Fig. 10, lanes 1 and 2 ) was detected that was sequence-specific (Fig. 10, lane 3) and co-migrated closely to the lower band seen with total nuclear extracts (Fig. 9 and data not shown). The complex produced with the rat Hx A

Interleukin-6 Regulation of Hemopexin Genes 12659

A probe rat Hx IL-6-RE -

indue. "C

const. - free

1 2 3 4 5 6 7

B probe rat Hx IL-6-RE -

indue. 4 const. 4

probe human Hx A k

free

1 2 3 4 5 6 7

FIG. 9. Competition EMSA of the hum Hx A element with NE of H-35 cells. 0.5 ng of radiolabeled human Hx A oligonucleotide was used as probe for EMSA with nuclear extracts from IL6-treated H-35 cells. The following non-labeled competitor oligonucleotides were added. Lune 1, no competitor; lunes 2 and 3, human Hx A, 25- and 50-fold molar excess; lanes 4 and 5, rat Hx A, 25- and 50-fold molar excess; lanes 6 and 7, rat and human Hx IL-6-RE, 100-fold molar excess.

H x IL-6-RE human HxA rat H x A human rat

" " - free probe - m m

1 2 3 4 5 6

FIG. 8. Competition EMSA analysis. Panel A, sequence specificity of constitutive and IL6-inducible complexes. Nuclear extracts of IL-6- treated H-35 cells were incubated using the radiolabeled rat Hx IL 6-RE oligonucleotide as a probe. Either no competitor (lane 1 ) or the following non-labeled competitor oligonucleotides were present during the binding reaction. Lanes 2 and 3, rat Hx IL-6-RE, 100- and 50-fold molar excess; lunes 4 and 5, human Hx IL-6-RE, 100- and 50-fold molar excess; lunes 6 and 7, human and rat Hx A element, 100-fold molar excess. Panel €3, competition EMSA with oligonucleotides of other APP genes. Nuclear extracts from IL-6-treated H-35 cells were incubated with the rat Hx IL-6-RE oligonucleotide as radiolabeled probe. A50-fold molar excess of the following non-labeled competitor oligonucleotides was added. Lune 1, no competitor; lane 2 , mutant Hx IL-6-RE; lune 3, a,-acid-glycoprotein IL-6-RE; lune 4, a,-macroglobulin IL-6-RE; lane 5 , NFKB; lune 6, glucocorticoid response element.

oligonucleotide yielded a significantly weaker band than that produced with the human Hx A element (Fig. 10, lanes 4 and 5) when tested under identical conditions. No binding of CIEBPP to rat or human Hx IL6-RE oligonucleotides (Fig. 10, lanes 6-91 was detected.

From these results, we conclude that the human Hx Aelement serves as a stronger binding site for C/EBPP than the rat Hx A element and that neither rat nor human Hx IL-6-RE is a binding site for C/EBPP. The EMSAdata agree with the results obtained from functional analyses in transfected cells (Figs. 5 and 6).

DISCUSSION The major findings of this study are as follows: 1) the Hx

gene of rat and human contains an identical IL6-RE with the

free r .A"" ""

probe +

1 2 3 4 5 6 7 8 9

FIG. 10. Human and rat Hx A elements bind C/EBPB with different affinities. 0.5 ng of radiolabeled oligonucleotides containing the human Hx A element (lunes 1 3 ) , rat Hx A element (lunes 4 and 51, human Hx IL6-RE (lanes 6 and 7), or rat Hx IL-6-RE (lanes 8 and 9) were incubated with 15 ng (lanes 1,4,6, and 8 ) or 30 ng (lunes 2 , 3 , 5 , 7, and 9) of purified recombinant CIEBPP. The incubation mixture in lane 3 contained a 100-fold molar excess of human Hx A oligonucleotide as specific competitor.

sequence CCGGGAA common to other AF'P genes, 2) this I L 6-RE is not trans-activated by C/EBP isoforms, and 3) it binds the IL6-inducible nuclear factor IL-6 RE-BP.

The plasma levels of Hx increase during an AF'R (47) and in various forms of cancer and metabolic disorders (1,48). To ex- amine the molecular basis of cytokine induction of the Hx gene, we performed transient transfection experiments of rat Hx promoter CAT constructs in hepatoma cell lines of the rat and hu-


man. These cell lines show a prominent transcriptional regulation of APP genes by inflammatory mediators (34, 49, 50).

The extent of the cytokine-dependent transcriptional regulation of the endogenous Hx gene (Fig. 1) is not reflected by the level of cytokine-induced stimulation elicited when two Hx promoter constructs (Fig. 2, constructs 1 and 2) are transfected into H-35 cells. A major reason for this fact is the exceptionally high basal activity of these constructs caused by a strong enhancer element proximal to the rat Hx ILS-RE (18, 51). This high basal activity may preclude that the CAT activity substantially increases in response to cytokine treatments. In addition, it has to be considered that the Hx promoter in the episomal plasmid context has a structurally distinct conformation and may be recognized by a variety of transcription factors or that the cloned gene promoter sequences are epigenetically modified as compared with the chromosomal Hx gene (52).

A relatively low IL-6 response level, as that found for the rat Hx IL-6-RE, has also been reported for the IL-6-RES of other APP genes (12, 14, 21, 35) that contain the core sequence CT- GGGAA (Fig. 3). Our data (Fig. 2, construct 4 ) with oligomerized copies of the Hx IL-6-RE support the conclusion that we identified a specific IL-6 element in the rat Hx gene. Somewhat unexpected was the observation of a synergistic effect of IL - l+ IL-6 on the human Hx IL-6-RE but not on the rat Hx IL-6-RE (Fig. 4B). This finding is most likely due to the sequence difference between the two elements (Fig. 3). Similarly, two homologous APP cytokine RES of the rat and human haptoglobin genes are regulated differentially. A single base substitution in the rat sequence caused a significant loss of cytokine responsiveness (16, 35). A genuine IL-1-RE of the Hx gene still re- mains to be defined, because the 1600-bp construct transfected into H-35 cells shows only a minimal response to IL-1 (Fig. 2). Such an IL-1-RE might consist of a site for C/EBP and/or NFKB, as reported for angiotensinogen (20, 531, complement C3 (13), C-reactive protein (14), and serum amyloid A genes (54).

Two different signaling pathways appear to be involved in the induction of the Hx gene by IL-6, as suggested when com- paring the Hx A element and the Hx IL-6-RE in cotransfection experiments (Figs. 5 and 6) and EMSAs (Figs. 7-10). The first pathway involves CBBP isoforms and appears to be mediated via the human Hx A element. The activity of CBBP isoforms is enhanced by cytokine treatment of hepatic cells, indicating that C/EBP-sensitive gene elements act as IL-6-RES (29, 32, 43). However, IL-6 effects on trans-activation capacities of C/EBP isoforms are inconsistent. Our results agree with previous data obtained for cytokine RES of the thiostatin and haptoglobin genes (32) and. those published by Ramji et al. (29). These studies indicate that the cotransactivation potential of C/EBP isoforms is not enhanced by cytokine treatment. Contradictory observations have been made for the ILB-RE of C-reactive protein (19). It has been proposed that a protein kinase C activation may be involved in the enhanced trans-activation efficacy of C/EBPP by IL-6 (55).

The second signaling pathway contributing to IL-6 induction of the Hx gene appears to be directly mediated via the Hx IL-6-RE (Fig. 3) independently of C/EBP isoforms (Figs. 5 and 6). This control mechanism is not well defined, and different transcription factors (IL-6 RE-BP, APRF, or SIFIGAF) have been proposed to interact with the core consensus motif found in the Hx ILS-RE. Results of our EMSA experiments (Figs. 7 and 8) together with the DNA binding kinetics of an IL-6- inducible factor (data not shown) point to its identity with IL-6 RE-BP (21) rather than with APRF or SIF/GAF (22, 23). Pre- cedents that APP genes are regulated via CBBP recognition sequences (type I IL-6-RES) and IL-6 RE-BP recognition sequences (type I1 IL-6-RES) have been found for human p-fi- brinogen (56,57) and T-kininogen genes (58). However, further

experiments will be necessary to clarify to what extent the demonstrated mechanisms are cooperative in APP gene regulation.

A sequence comparison of the promoter elements of rat and human Hx reveal that functional differences have evolved in both genes despite extensive sequence conservation. One of these differences is that the A element in the rat Hx gene is unresponsive to cytokines. This unresponsiveness implies that the rat Hx gene is less or not at all sensitive to C/EBP trans- activation. However, the functional analysis of the Hx gene sequences is still incomplete and does not allow such a conclusion.

Acknowledgments-We thank Drs. S. Dower and G. Wong for provid- ing recombinant cytokines, Dr. M. Chojkier for recombinant CIEBPP, and Karen K. Morella for technical assistance.

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THE OF BIOLCQICAL Vol. No. and U.S.A. The Rat and Human ...treated for another 24 h with serum-free medium containing dexam- ethasone (1 w) alone as a control or dexamethasone + IL-1