MOLECULAR AND CELLULAR BIOLOGY, July 1994, p. 4475-4484 Vol. 14, No. 7 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology Induction of the Mouse Serum Amyloid A3 Gene by Requires both C/EBP Family Proteins and a Novel Constitutive Nuclear Factor JIANYI H. HUANG AND WARREN S.-L. LIAO* Department ofBiochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Received 1 December 1993/Returned for modification 2 February 1994/Accepted 1 April 1994

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may be induced up to 1,000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We have previously demonstrated that a 350-bp promoter fragment from the mouse SAA3 gene was necessary and sufficient to confer liver-specific and -induced expression. Deletion studies identified a distal response element that is responsible for the cytokine response and has properties of an inducible transcriptional enhancer. In this study, we further analyzed the distal response element and showed that it consists of three functionally distinct elements: the A element constitutes a weak binding site for C/EBP family proteins, the B element also interacts with C/EBP family proteins but with a much higher binding affinity, and the C element interacts with a novel constitutive nuclear factor, SEF-1. Site-specific mutation studies revealed that all three elements were required for maximum promoter activity. C/EBPoL, C/EBPP, and C/EBPB were capable of interacting with elements A and B. Under noninduced conditions, C/EBPa was the major binding factor, however, upon cytokine stimulation C/EBPID- and C/EBP8-binding activities were dramatically increased and became the predominant binding factors. Consistent with these binding studies were the cotransfection experiments in which C/EBPP and C/EBP8i were shown to be potent transactivators for the SAA3 promoter. Moreover, the transactivation required an intact B element despite the presence of other functional C/EBP-binding sites. Interestingly, although element C did not interact with C/EBP directly, it was nevertheless required for maximum transactivation by C/EBP8. Our studies thus demonstrate that both C/EBP family proteins and SEF-1 are required to transactivate the SAA3 gene.

The systemic response to acute inflammation, infection, and scription activation of acute-phase genes (2, 14, 38, 39). All tissue injury is characterized by changes in the concentrations three C/EBP proteins have the basic leucine zipper motif of a wide variety of plasma proteins collectively called the initially found in C/EBPa. and have similar sequence specific- acute-phase reactants (23, 24, 27, 36). Expression of these ities in their DNA binding, each having the modified consensus genes is regulated primarily by the inflammatory cytokines sequence T(T/G)NNG(A/C/T)AA(T/G) (14). During acute interleukin-1 (IL-1), IL-6, and tumor necrosis factor (13, 23, inflammation, expression of C/EBPa is reduced while that of 29, 38) and by the steroid hormone (7, 13). C/EBPB and C/EBP8 is elevated, although by different mech- Elevated expression of acute-phase genes is regulated at the anisms. The increase in C/EBPfB binding and transactivation transcription level, as a result of increased interaction of activities occurs mainly through posttranslational modification transcription factors induced by inflammatory cytokines with (44, 49, 50). In contrast, elevated C/EBP8 mRNA synthesis their cognate regulatory cis-acting DNA elements (3, 14, 17, contributes to its activation (50). 38, 48, 59, 60). Analyses of many acute-phase promoters have Serum amyloid A (SAA) is one of the major acute-phase revealed three general types of regulatory elements in the proteins synthesized in hepatocytes during acute inflammation transcriptional induction by cytokines-the binding sites for (16, 23, 52). In mice, the SAA gene family consists of four members of the C/EBP transcription factors, the binding sites genes-SAAJ, SAA2, SAA3, and SAAS-and a pseudogene for the NF-KB/Rel family of proteins, and the binding sites for (19, 41, 53). In response to acute inflammation, the concentra- IL-6 response element-binding protein (IL-6 RE BP) and tion of SAA in plasma increases approximately 1,000-fold, acute-phase response factor (APRF) with the consensus bind- primarily because of an increased SAA gene transcription (42). ing sequence of CTGGGA (26) or an extended motif of Whereas expression of SAA1, SAA2, and SAA3 is dramatically (T/G)T(C/A)(C/T)(G/T)G(G/T)AA (59). induced and Several nuclear factors can interact with the C/EBP type of each contributes equally to the increased SAA cytokine response element. In addition to the liver-enriched mRNA levels in the liver (42), SAA5 expression is induced to C/EBPa, two others, C/EBPP and a much lower level and with different induction kinetics (19). C/EBP8, have been identified (1, 10, 12, 20, 35, 49) and shown In efforts to unravel the molecular mechanisms of SAA gene to be centrally involved in mediating cytokine-dependent tran- regulation, the promoters of human (22) and rat (38, 39) SAAJ and mouse SAA3 (29, 37) have been studied. A phorbol ester response element containing the NF-KB consensus binding site * Corresponding author. Mailing address: Department of Biochem- was identified in the human SAAJ promoter and shown to be istry and Molecular Biology/Box 117, University of Texas M. D. necessary for its cytokine responsiveness (22). Studies of the Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. rat SAAJ promoter demonstrated functional importance for Phone: (713) 792-2556. Fax: (713) 790-0329. and cooperative interaction between NF-KB and C/EBP pro- 4475 4476 HUANG AND LIAO MOL. CELL. BIOL. teins in cytokine-induced expression (39). Studies with the mouse SAA3 promoter demonstrated that a 350-bp promoter -169 B -126 fragment was necessary and sufficient to confer liver-specific and cytokine-induced expression (29). A proximal response DRE CGATCACATTTCTGGAAATGCCTAGATGGCGCAATCTGGGGAAAGAT element that contains two adjacent C/EBP-binding sequences DRmA enhances SAA3 in liver-derived cells (37). A distal response element (DRE) that confers responsiveness to DRmBE ------A------cytokine induction has properties of an inducible transcrip- tional enhancer (29). In this study, we demonstrate that three DRnB2 ------GAT-TC------functionally distinct elements reside within the DRE. One DRmC ------A-CA----T------element interacts with a novel transcription factor termed SAA enhancer factor 1 (SEF-1) that is detected in variety of cell -140 -128 I 1 types. The other two elements interact with C/EBP family OL-A CGATCTGGGGAAAGAT proteins. All three elements are required for maximum induc- OL-mA ----TCAA------tion by cytokines. In addition, our results show that C/EBPOL, -156 -135 C/EBPP, and C/EBP8 can transactivate the SAA3 promoter OL-B CGATGCCTAGATGGCGCAATCTGGTTAT and that the transactivation requires functional binding sites -169 -147 for both C/EBP and SEF-1. OL-C CGATCACATTTCTGGAAATGCCTAGAT OL-mC MATERIALS AND METHODS -117 -99 I I Cell lines, transient-transfection assay, and conditioned OL-117 GAAGGAAAAGTTATCTTCT medium. Hep3B cells were cultured in basal medium consist- ing of minimum essential medium and Waymouth MAB (3:1, OL-IL- 6 CGATACGTCACATTGCACAATCTTAAT vol/vol) plus 10% fetal calf serum (18) and were passaged at OL-Hpx CGATCAGTGATGTAATCA confluence by trypsinization about once a week. DNA transfections were performed by the Polybrene pro- NFXB GATCCGGGGACTTTCCG cedures (34) as described previously (29). Approximately 16 to GATCTATGCAAATG 20 h after transfection, Hep3B cells were stimulated with basal Oct-1 medium, 50% conditioned medium (CM), or 100 U of IL-lot FIG. 1. Oligonucleotide sequences. The nucleotide sequence of DRE is shown, and the regions for elements A, B, and C are indicated per ml. Cell extracts were assayed for protein content by the by the brackets. DRmA, DRmB, and DRmC are oligonucleotides in Bradford assay (9), and chloramphenicol acetyltransferase which site-specific mutations have been introduced into each of the (CAT) activity was determined by modifications (11) of pro- three elements. OL-A, OL-mA, OL-B, OL-C, and OL-mC are oligo- cedures described by Gorman et al. (25). To determine CAT nucleotides corresponding to either wild-type or mutated elements. In activities, we quantitated [14C]chloramphenicol spots corre- all cases, only the mutated nucleotides are indicated. The OL-117 sponding to acetylated and nonacetylated forms by using a sequence corresponds to bp -117 to -99 in the SAA3 promoter and Phosphor Imager (Molecular Dynamic). was used to generate a promoter fragment for the pSAA3(-117) Purified recombinant human IL-la (specific activity, 2.5 X construct. The numbers above the sequences indicate their positions in 109 U/mg) was provided by P. T. Lomedico (Hoffmann- the mouse SAA3 promoter. The sequences of OL-IL-6, OL-Hpx, NF-KB-binding site, and Oct-i-binding site are from the NF-IL6- LaRoche, Nutley, N.J.). CM was prepared from mixed human binding site in human IL-6 promoter (1), the C/EBP-binding site in the lymphocyte cultures stimulated with 0.75% phytohemaggluti- hemopexin promoter (48), the NF-KB-binding site in the immunoglob- nin as described previously (29). ulin K light-chain promoter (4), and the Oct-1-binding consensus Nuclear proteins and electrophoretic mobility shift assay. sequence (55), respectively. Nuclear extracts from Hep3B cells were prepared as described by Li et al. (37). For the induced nuclear extracts, Hep3B cells were treated with 50% CM for 16 to 20 h before harvest. For electrophoretic mobility shift assays, 20 [lI of the reaction then digested with 4 jig of freshly diluted DNase I per ml at mixture [12 mM N-2-hydroxyethylpiperazine-N'-2-ethanesul- room temperature for 30 s. The reaction was stopped by fonic acid (HEPES; pH 7.9), 60 mM KCl, 12% glycerol, 1.2 addition of 100 jil of proteinase K (60 jig/ml) in 12 mM mM dithiothreitol, 0.12 mM EDTA, 2 [Lg of poly(dI-dC), 0.5 to EDTA-0.12% sodium dodecyl sulfate-16 jig of pBR322 DNA 1 ng (1 X 104 to 2 X 104 cpm) of " P-labeled probe] was per ml at 370C for 45 min. Nucleic acids were extracted with 1 incubated with 1 to 5 ,ug of nuclear proteins. The reaction volume of phenol-chloroform (1:1), ethanol precipitated, dis- mixtures were incubated at room temperature for 30 min and solved in 99% formamide, heated at 90'C for 1 min, and then loaded onto a 4% polyacrylamide gel (19:1 cross-linking loaded onto an 8% polyacrylamide-urea gel. ratio) in 1 x glycine buffer (0.38 M glycine, 50 mM Tris, 2 mM Methylation interference analysis was performed by incuba- EDTA). In supershift experiments, nuclear extracts and DNA tion of partially methylated end-labeled DNA fragments with probes were preincubated for 10 min before addition of nuclear proteins under the gel mobility assay conditions. Free specific antisera. and bound probes were isolated after electrophoresis and DNase I footprint and methylation interference analyses. purified with Elutip-D columns. The isolated DNA probes DNase I footprint analysis was performed as described by Li et were cleaved with piperidine and loaded onto an 8% denatur- al. (37). Briefly, the reactions were performed in a final volume ing polyacrylamide gel. of 20 ,ll containing 12 mM HEPES (pH 7.9), 60 mM KCl, 12% Oligonucleotides and plasmid constructs. The wild-type and glycerol, 1.2 mM dithiothreitol, 0.12 mM EDTA, 4 jg of mutant oligonucleotides used as probes or competitors in gel poly(dI-dC), 4 ,ug of Hep3B nuclear proteins, and 3 x 104 cpm mobility shift assays are described in Fig. 1. They include the (1 to 2 ng) of end-labeled DNA fragment. The reaction DRE and mutant DREs in which element A, B, or C has been mixture was incubated for 30 min at room temperature and mutated and oligonucleotides corresponding to the individual VOL. 14, 1994 INDUCTION OF MOUSE SAA3 GENE BY CYTOKINES 4477 -169F -128 -169 C -147 -140 A -128 TTAI I II> I TGGCCACATTTCTGG&AAATGCCTAGRTGGCGCA&TCTGGGGAAAGAAGk

-156 B -135

0@ 0 NFKCB GGAA>CCC CTGGG;A IL-6 RE BP TTNNGAg consensus Oct-i TATGCA&MT G C/dEP I' APRF 3T^CG G

0 0 0 NFIL- 6 ACATTGCACAATCT

C/EBP consensus TG T A

FIG. 2. Sequence comparison between DRE and several known transcription factor-binding sites. Nucleotides in the consensus sequence that differ from those in DRE are indicated by dots. The two arrows indicate the palindromic sequences. Consensus sequences of IL-6 RE BP and APRF are from Hocke et al. (28) and Wegenka et al. (59), respectively. The NFIL-6-binding sequence and C/EBP consensus are from Akira et al. (1), and Chen and Liao (14), respectively. Consensus sequences for NF-KB and Oct-1 binding are from Baeuerle (4) and Tanaka and Herr (55), respectively. elements and their mutants. OL-117 corresponds to bp -117 binding site in the c2-macroglobulin promoter (28, 59). Ele- to -99 of the sense strand and was used as the PCR primer to ment B (-156 to -135) contained a 14-bp palindromic generate the promoter fragment for the pSAA3(-117) con- sequence that is highly homologous to C/EBP,-binding se- struct. quence in the human IL-6 promoter (1) and matches the A DNA fragment containing 306 bp of the 5'-flanking region modified C/EBP-binding consensus sequence. Element C and 45 bp of the untranslated exon 1 region of mouse SAA3 (-169 to -147) contained sequences that resemble binding promoter was inserted into the BamHI site of the pCATP' sites for NF-KB (4) and Oct-1 (55). In addition, it matches the vector to generate the pSAA3(-306) construct (29, 51). Four APRF/IL-6 RE BP binding sequences found in the promoters 5'-deletion mutants, pSAA3(-169), pSAA3(-156), pSAA3 of several acute-phase genes (17, 47, 59, 61). These compara- (-140), and pSAA3(-117), were constructed by PCR with an tive analyses suggested that DRE may be composed of multi- oligonucleotide corresponding to bp +40 to +23 of the SAA3 ple functional elements that interact with different transcrip- antisense strand (as a 3' primer) and oligonucleotide DRE, tion factors. OL-B, OL-A, or OL-117 (as a 5' primer), respectively, and Two adjacent C/EBP binding sites in DRE with different with pSAA3(-306) as a template. The site-specific mutants binding affinities. To determine whether the matched se- pSAA3(- 169mC), pSAA3(-169mB), pSAA3(- 169mA), quences described above indeed correspond to transcription pSAA3(-156mB), and pSAA3(-156mA) were constructed factor binding sites and participate in DNA-protein interac- similarly, except that the 5' primers were replaced with oligo- tions, we radioactively labeled DRE and used it as a probe in nucleotides containing the appropriate mutations. C/EBPa, gel mobility shift assays. When incubated with nuclear extracts C/EBPP, and C/EBP8 cDNAs, kindly provided by S. McKnight from Hep3B cells, three intense protein-DNA complexes and U. Schibler, were subcloned into the SmaI site of the (complexes 1, 2, and 3) were observed (Fig. 3A). When the pSVK3 expression vector (14). CM-induced Hep3B nuclear extract was used, the intensities of complexes 1 and 3 were reproducibly reduced whereas that of RESULTS a new complex, complex 4, was greatly increased. To determine which sites were involved in DNA-protein interaction, we DRE contains sequences homologous to several known performed DNase I footprint analyses with Hep3B nuclear transcription factors. We have shown previously that a 68-bp extracts. As Fig. 3B shows, only one strong DNase I-protected DNA fragment from the SAA3 promoter functions as a region (bp -152 to -134) was detected; it encompassed the transcriptional enhancer in conferring dramatic cytokine re- 14-bp palindromic sequence in element B. Identical footprints sponse onto a downstream reporter gene (29). To identify the were obtained with nuclear extracts prepared from CM-stim- regulatory DNA elements and their cognate protein factors, ulated Hep3B cells despite substantial differences in their gel the DRE sequence was compared with binding sequences of shift patterns. several known transcription factors. Three regions matched Since the footprint region contained sequences that matched either perfectly or nearly perfectly to binding sequences of the modified C/EBP-binding consensus sequence, we used several known transcription factors. As shown in Fig. 2, the competition assays to determine whether C/EBP family pro- most proximal region, element A (positions -140 to -128), teins were indeed involved in forming these complexes. As Fig. contained a sequence that not only matches the modified 3C shows, two known C/EBP-binding-site oligonucleotides, C/EBP-binding consensus sequence T(T/G)NNG(A/C/T) OL-IL-6 and OL-Hpx from IL-6 and hemopexin promoters, AA(T/G) (14) but also is homologous to the IL-6 RE BP respectively, efficiently competed with all complexes for bind- 4478 HUANG AND LIAO MOL. CELL. BIOL.

A - + B F -CM +CM C Competitor: - 0 he1. 09*2arg Fold Excess: - ho.i 00 i 100"1g1 o loo0TO1-0-0|10 1001111oi00, I00

... :. .... 1 S S . 2+ _s r +4 _ . ':"'.~.n. 3-E w oL .sl: ....

-134-

.: As _. AwkM _ -152-

-169 -128

CAATTTl-s ----UA']G--- APAT'UClTAGATGCUGCAATCTGGIGAAAAGA--~------

-152 -134 FIG. 3. Determination of C/EBP binding to element B in the DRE. (A) End-labeled DRE was incubated with 1 pg of nuclear extracts from unstimulated (-) or CM-stimulated (+) Hep3B cells. Positions of specific DNA-protein complexes formed are indicated by solid arrows. The open arrow indicates the position of the free probe. (B) DNase I footprint. A 68-bp (-185 to -118) restriction fragment containing the DRE was end labeled on the noncoding strand and incubated with nuclear extracts (4 pg) prepared from control (lane -CM) or CM-treated (lane +CM) Hep3B cells. The DNA-protein mixture or the free probe (lane F) was incubated with 4 Kig of DNase I per ml for 30 s. The protected region is indicated by the bracket. Below the gel, the sequence of DRE is shown; the protected region is underlined. The palindromic sequence is indicated by arrows. (C) Competition by C/EBP consensus oligomers. End-labeled OL-B fragment was incubated with nuclear extracts from CM-treated Hep3B cells in the presence of a 10- or 100-fold molar excess of unlabeled oligonucleotides corresponding to B element (OL-B), C/EBP-binding sites in the IL-6 (OL-IL-6) and hemopexin (OL-Hpx) promoters, NF-KB-binding sequence (NFKB), and the wild-type (DRE) and mutant (DREmBl, DREmB2) DRE sequences. The open arrow denotes the position of the free probe. ing, indicating that C/EBP family proteins were the major end-labeled element C as a probe in gel mobility shift assays. proteins interacting with DRE. Binding of these protein-DNA As shown in Fig. 4A, a single protein-DNA complex was complexes was also efficiently inhibited by OL-B and the observed with nuclear extracts prepared from Hep3B cells. wild-type DRE oligonucleotides but not by two mutant DRE Nuclear extracts from CM-stimulated cells gave an identical oligonucleotides, DRmB1 and DRmB2, in which sequences in gel shift pattern. Furthermore, the intensity of the complex the footprint region had been mutated (Fig. 1). The NF-KB remained the same, indicating that CM treatment did not alter oligonucleotide served as a nonspecific negative control. These its DNA-binding activity. To determine more precisely the results therefore indicate that the protein factors interacting DNA sequence important for protein interaction, this DNA- with element B were C/EBP family proteins. protein complex was further analyzed by methylation interfer- Because element A contains sequence homologous to the ence assay. Analysis of the coding strand showed that four G C/EBP-binding consensus sequence, we examined whether it residues, when methylated, interfered with protein binding also could interact with C/EBP family proteins. An end-labeled (Fig. 4B). When the noncoding strand of the probe was fragment containing OL-A was used in a gel mobility shift analyzed, two methylated G residues, at positions -152 and assay with Hep3B nuclear extracts. The DNA-protein com- -162, interfered with protein binding. plexes formed were essentially identical to those formed with The DRE sequences surrounding these important G resi- element B; however, the intensities for these complexes were dues (indicated at the bottom of Fig. 4B) showed no obvious much weaker even when fivefold concentrations of nuclear alignment with NF-KB-, Oct-i-, or APRF-binding sites, sug- extract were used. Binding of all complexes could be specifi- gesting that the factor binding to element C is distinct from cally inhibited by several C/EBP-binding oligonucleotides these factors. To further verify whether this is indeed the case, (data not shown). These results indicate that elements A and B we used the OL-C fragment as a probe and compared the both can interact with C/EBP proteins, although element B has abilities of oligonucleotides corresponding to NF-KB-, Oct-i-, a much higher binding affinity for C/EBP than does element A. and APRF-binding sites to compete for binding. As Fig. 4C Element C interacts with a novel constitutive nuclear factor. shows, Oct-i- and NF-KB-binding-site oligonucleotides could In addition to the two adjacent C/EBP-binding sites, the more not compete for binding even at a 100-fold molar excess of distal region of DRE, designated the C element, contains competitor DNA. Oligonucleotides corresponding to the sequences that closely resemble binding sites for NF-KB, APRF-binding site also could not compete (data not shown). Oct-i, and APRF. To examine whether element C can interact In contrast, binding of the DNA-protein complex was effi- with one or more of these transcription factors, we used ciently inhibited by wild-type OL-C and DRE but not by VOL. 14, 1994 INDUCTION OF MOUSE SAA3 GENE BY CYTOKINES 4479

o Noncoding D I A -B Coding C + A9O$ +B Strand Strand Competitor: OL-C DRE DRmC Oct-i NFKB m-m1 F B F F B F Fold Excess: 10 100 10 100 10 100 10 100 10 100

.O W.R ..,,.. P; ..aXT Ai, Ar.P. AdI-. Czr: c: -b. i SEF-1 11 SEF-1 -152

.f'. -V. ,f -162

-160 -159 0

-154 0 -4*hI.>mhm

-149.

-169 -147

*- 0 0 CACATTTCTGGAAATGCCTAGAT GTGTAAAGACCTTTACGG.ATCTA

FIG. 4. Identification and characterization of the C element-binding factor, SEF-1. (A) The end-labeled fragment containing OL-C was incubated with 4 pug of Hep3B nuclear extracts without (-) or with (+) stimulation by CM. (B) Methylation interference analysis. The coding or noncoding strand of a fragment containing OL-C was end labeled, partially methylated, and used in gel shift conditions with Hep3B nuclear extracts. Protein-bound (lanes B) and free (lanes F) were eluted, cleaved, and analyzed on sequencing gels. Positions of methylated G residues that interfered with complex formation are indicated by dots and are summarized at the bottom. (C) Specificity of SEF-1 binding. The end-labeled fragment containing OL-C was incubated with 4 pug of Hep3B nuclear extracts in the presence of a 10- or 100-fold molar excess of competitors. (D) Tissue distribution of SEF-1. The end-labeled fragment containing OL-C was incubated with 20 pLg of whole-cell extracts from various cultured cell lines. Lanes: HeLa, human cervical carcinoma; T84, human colon epithelium cells; RL95, human endometrial carcinoma; K562, human chronic myelogenous ; MDCK, cocker kidney cells; JAR, human placenta choriocarcinoma; QM, quail muscle cells.

DRmC, in which element C had been mutated. These results wild-type pSAA3(-169) construct, which contained an intact demonstrate that although element C contained sequences DRE, showed approximately 15- and 10-fold increases in CAT that are homologous to NF-KB-, Oct-i-, and APRF-binding activity when stimulated by CM and IL-1, respectively. In sharp sequences, the detectable protein interacting with this element contrast, the 5' deletion construct pSAA3(-156), which had was distinct from these known transcription factors. It was the C element deleted but still retained intact B and A subsequently designated SAA enhancer factor-1 (SEF-1). elements, showed approximately 20 and 12% of the basal and To examine the tissue distribution of SEF-1-binding activity, induced CAT activity, respectively. Further deletion of B and we prepared whole-cell extracts from a variety of cell lines and A elements to positions -140 and -117, respectively, com- performed gel mobility assays. Except for the MDCK cells, pletely abolished promoter activity (Fig. 5A). SEF-1-binding activity was readily detectable in all cells exam- In addition to the 5' deletion constructs, we analyzed a series ined, albeit to significantly different levels (Fig. 4D). Interest- of constructs in which each of the three elements had been ingly, like hepatoma Hep3B cells, levels of SEF-1-binding mutated (Fig. SB). Consistent with the pSAA3(-156) con- activity were very high in HeLa cells and in the leukemia cell struct, the pSAA3(- 169mC) construct, in which the C element line K562, indicating that SEF-1 is not restricted to liver cells had been mutated, showed five- and eightfold reduction in but has a rather broad tissue distribution. basal and cytokine-induced CAT activity, respectively. These All three elements are required for maximum induction. results clearly demonstrate that element C played an important Our studies demonstrated that DRE contained three distinct role in conferring maximum cytokine response on the SAA3 elements, each capable of interacting with distinct transcrip- promoter. To examine the functional role of the two adjacent tion factors. To assess the functional importance of these C/EBP-binding sites, we individually mutated elements B and elements in conferring cytokine responsiveness, we generated A to generate pSAA3(-169mB) and pSAA3(-169mA), re- two series of SAA3 promoter-CAT constructs in which one or spectively. Results of the functional analysis in Hep3B cells of more of these elements had been either deleted or mutated these two mutants were compared with results for the wild- (Fig. 5). Resulting constructs were assayed for promoter type construct pSAA3(-169). Whereas CAT expression of activities in Hep3B cells treated with control medium or pSAA3(- 169) induced by cytokines was 10 to 15 times as high medium containing 50% CM or 100 U of IL-1 per ml. The as that of the control, CAT activity was not detectable with 4480 HUANG AND LIAO MOL. CELL. BIOL.

A. Relative CAT Activity -CM +CM r .. C B A 0 4 8 12 16 - CL 3 8 C E-!. Antibody: -169 =h~ Fold Dilution: - 20 100 20 100 10 50 - 20 100 20 100 10 50 -156 AG-CT

-140 E CCAT 4 - 2 -117 1 A Control 3 U [ .+CM

B. Relative CAT Activity 0 4 8 12 16

-169 C +40

-169 mC[ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 FIG. 6. Identification of C/EBP proteins interacting with element -169mBA [ B. An end-labeled OL-B fragment was incubated with unstimulated (-CM) and CM-stimulated (+CM) Hep3B nuclear extracts (1 jig) in the presence of various dilutions of specific antisera raised against C/EBPco, C/EBPI3, and C/EBP8. Positions of specific complexes formed and free probe are indicated by solid and open arrows, respectively. -156 mB F"' [

_ Control -156 mA | CA [ E= +CM I-I +IL-1 antibody (Fig. 6, lanes 2 and 3). Anti-C/EBPP partially super- shifted complex 2, whereas anti-C/EBP8 had no effect on these FIG. 5. Functional analysis of DRE by 5' deletion and site-specific complexes (lanes 4 to 7). However, with nuclear extracts mutations. Constructs with 5' deletions (A) or site-specific mutations prepared from CM-stimulated Hep3B cells, no complex was (B) of the SAA3 promoter were transfected into Hep3B cells. After supershifted by anti-C/EBPa (lanes 9 and 10), whereas anti-C/ approximately 16 to 20 h, the cells were treated with control medium EBPP completely supershifted complex 2 and partially super- or medium containing 50% CM or IL-1 (100 U/ml). CAT activities shifted complex 4 (lanes 11 and 12). Anti-C/EBP8 abolished were quantitated by using a Phosphor Imager. Results were calculated the formation of complex 4, although no supershifted complex relative to CAT activity for pSAA3(- 169)-transfected cells, to which a was value of 1.0 was assigned. The left panels show schematic diagrams of observed (lanes 13 and 14). These results demonstrate that various promoter constructs used for transfection. C/EBPa is the major C/EBP protein in the noninduced nuclear extracts, accounting for the majority of the binding activity. After induction, its binding activity was drastically reduced whereas that of C/EBPP was increased. C/EBP8-binding activ- pSAA3(- 169mB) even after stimulation. Mutation of the ity was detectable only with the induced extracts. Thus, com- weak C/EBP-binding site (A element), however, reduced CAT plexes 1 and 3 were formed from C/EBPoa, complex 2 was activity to approximately 50% of control levels. These results formed from C/EBPr3, and the inducible complex 4 was formed demonstrate that mutation of either element B or C essentially primarily from C/EBPB. Identical supershift patterns were abolished the basal and inducible promoter activity, while observed when the OL-A fragment was used as the probe (data mutation of element A significantly lowered its activity. Taken not shown). together, these results indicate that all three elements were C/EBP8 is the most potent transactivator. To determine required for the full cytokine-induced expression of the mouse whether different binding activities of these C/EBP family SAA3 gene and suggest that the induced expression of the proteins could be correlated with increased SAA3 expression mouse SAA3 gene required cooperative interaction between following cytokine stimulation, we cotransfected various SEF-1 and C/EBP family proteins. amounts of C/EBPoL, C/EBPP, or C/EBP6 expression plasmids Interaction of C/EBP family proteins with DRE. Members with pSAA3(- 169) into Hep3B cells. Overexpression of of C/EBP family proteins can form homodimers or het- C/EBPoa resulted in 12-fold-higher CAT activity; however, the erodimers of essentially identical binding specificities (10, 35). magnitude of induction was reduced at higher levels of However, their expression is differentially regulated in re- C/EBPa (Fig. 7A). In contrast, C/EBPP and C/EBP8 transac- sponse to acute inflammation. While the expression of C/EBPot tivated the reporter gene in a dose-dependent manner, and 15 decreases following lipopolysaccharide-induced inflammation, ,ug of expression plasmid resulted in 12- and 26-fold induction C/EBPI3 and C/EBPB expression increases (2, 30). Therefore, for C/EBPB and C/EBP8, respectively (Fig. 7B and C). In all although our gel mobility data demonstrate that C/EBP family cases, treatment with CM further increased CAT activity, proteins could bind to elements A and B in the DRE, it was not although to different degrees. These results demonstrated that clear which members of the C/EBP family proteins actually all three C/EBP proteins can effectively transactivate mouse contributed to the observed DNA-protein complexes. To ad- SAA3 promoter, C/EBP8 being the most potent transactivator. dress this question, we used antisera against each of these The C element is required for transactivation by C/EBP8. proteins in supershift analyses. With Hep3B cell nuclear To determine whether transactivation by C/EBP5 occurs extracts, complexes 1 and 3 were supershifted by anti-C/EBPot through its binding site at the A or B element, we cotransfected VOL. 14, 1994 INDUCTION OF MOUSE SAA3 GENE BY CYTOKINES 4481

DRE PRE A. mPORA-A-Ml ., 4'41 o +CM m 0 - CAT U 31 * Control CBA 0 21 Z:X 11 0 _ 0 5 10 15 jg C/EBPa - exp B. 40. o +CM 0 .a:a) 30- * Control a)

20 - - '

a) 10 0 0 -169 -169 mC -169 mB 169 mA 0 5 ~~~~~~10 15 jig C/EBPO - exp FIG. 8. Effects of site-specific mutations on C/EBP8 transactiva- tion. Hep3B cells were cotransfected with 5 [ig of pSAA3(-169), C. pSAA3(-169mC), pSAA3(-169mB), and pSAA3(-169mA) target 40 DNAs and 15 [ig of C/EBP8 expression plasmid. As control, 15 jig of pSVK3 vector DNA was also cotransfected with these target DNAs, 30 - and the transfected cells were not stimulated (-CM) or were stimu- lated with CM (+CM). Results were calculated relative to the activity 0 20 - of pSAA3(-169) cotransfected with pSVK3, to which a value of 1.0 was assigned. 0 10 cc 01 required for C/EBP8 transactivation. This indicates that bind- 0 5 10 ing of SEF-1 to the C element was necessary for C/EBP8 gg C/EBP8 - exp function and suggests that these two transcription factors are FIG. 7. Differential transactivation of C/EBP family proteins. both required to transactivate the SAA3 promoter. Hep3B cells were cotransfected with 5 jig of pSAA3(- 169) and 0, 5, 10, or 15 [ig of C/EBPa, C/EBPr3, or C/EBP& expression plasmids. DISCUSSION Transfected cells were treated with medium alone (control) or with 50% CM (+CM). CAT activities were quantitated a by using Phosphor We sought a molecular explanation for cytokine-induced Imager. Results were normalized to the activity of the control and mouse SAA3 gene acute noncotransfected cells, to which a value of 1.0 was assigned. expression following injury by analyz- ing the sequences in the promoter that are responsible for this phenotype. Our previous studies had shown that a DRE could confer cytokine responsiveness and has properties of an induc- the C/EBP6 expression plasmid with SAA3 promoter con- ible transcriptional enhancer. In this study, we identified three structs in which element A, B, or C had been mutated. pSVK3 functionally distinct sequences, referred to as A, B, and C vector DNA was included as a control. While cotransfection of elements, that were required for maximum cytokine response. C/EBPB with the wild-type pSAA3(-169) construct increased The A element, a weak C/EBP-binding site, appeared to affect CAT activity approximately 26-fold, C/EBPB transactivated the magnitude of SAA3 expression but not its responsiveness to pSAA3(-169mB) only about 5-fold despite the presence of cytokines. The B element, identified as a strong C/EBP-binding several other C/EBP-binding sites (element A and two addi- site, was crucial for both basal and cytokine-induced expres- tional sites in the proximal response element [37]) in the SAA3 sion. The C element, which interacts with a novel constitutive promoter (Fig. 8). When the A element corresponding to the nuclear protein, was likewise necessary for both basal and weak C/EBP-binding site was mutated, transactivation by induced expression of SAA3 promoter. Moreover, a functional C/EBP5 was reduced to about 50% of that of the wild-type C element was required for the transactivation by C/EBP construct. Interestingly, the construct with a mutated C ele- family proteins. ment, pSAA3(- 169mC), also drastically reduced the ability of Multiple response elements are required for full promoter C/EBPS to transactivate. These results demonstrate that the activity. One main issue concerning the complex organization high-affinity C/EBP-binding site (B element) is specifically of the cytokine response region in the SAA3 promoter is recognized by C/EBP5 and is necessary for maximum transac- whether interaction of distinct elements is required for full tivation. The lower-affinity C/EBP-binding site (A element) promoter activity. Two lines of evidence suggest that maximum had a similar but smaller effect. The effect of mutating the C promoter activity requires all three elements. First, when element is most intriguing. Although there was no direct compared with a promoter construct that contained an intact interaction between element C and the C/EBP transcription DRE, constructs with mutated B or C elements showed factor and mutation of this element did not alter C/EBP- drastically reduced basal expression and were nonresponsive to binding to elements A and B, element C was nevertheless cytokine stimulation. Mutation of element A resulted in a 4482 HUANG AND LIAO MOL. CELL. BIOL. twofold reduction in SAA3 promoter activity. Second, con- the acute-phase gene regulation through increased de novo structs with mutations in the strong and weak C/EBP-binding protein synthesis in the induced cells (2, 50). In contrast, sites-pSAA3(- 169mB) and pSAA3(- 169mA), respective- elevated C/EBPf-binding activity is mediated primarily ly-showed significantly reduced activity when cotransfected through protein phosphorylation of preexisting protein (2, 49, with C/EBP8 expression plasmid. The magnitude of the effect 50). However, increased C/EBPI mRNA has also been re- correlated with their binding affinities to C/EBP; that is, ported (2). mutation of the stronger C/EBP-binding site, element B, We have also attempted to correlate the binding activity of reduced activity more than mutation of the weaker binding site C/EBP proteins with their transactivating function by perform- did. It is important to note that although the C element does ing cotransfection experiments with C/EBPa, C/EBPr3, and not interact with C/EBP family proteins directly, mutation of C/EBP8 expression vectors and the SAA3 promoter. All three this element nevertheless drastically reduced the transactiva- C/EBP proteins were able to transactivate the reporter gene, tion ability by C/EBP8. We conclude that concerted action of albeit to different degrees. At low levels, C/EBPot transacti- all three elements is required for maximum induction by vated the SAA3 promoter; however, at higher levels it was cytokines and for maximum transactivation by C/EBP family inhibitory. On the other hand, both C/EBPP and C/EBP5 proteins. transactivated the target gene in a dose-dependent manner, Cooperative interaction of multiple cis-regulatory elements with C/EBP6 being the more potent transactivator. In all cases, has been described in the induction of several acute-phase stimulation with CM further increased the expression of the genes by inflammatory cytokines (3, 17, 26, 39, 43, 47, 48, 56, reporter gene. These cotransfection results are consistent with 60, 62). In most cases, full transcriptional activation requires those of the gel shift experiments, indicating that C/EBPB and the combined action of a constitutive factor-binding site and an C/EBP8 are the major proteins involved in cytokine-induced inducible transcription factor-binding site. For example, induc- SAA3 expression. It is intriguing that different C/EBP proteins tion of P-fibrinogen by IL-6 requires the cooperative interac- have different transactivation capabilities. Thus, they may serve tion of three regulatory elements: binding sites for the consti- distinct functions by acting through similar but distinct C/EBP- tutively expressed transcription factor hepatocyte nuclear binding sites. In the alcohol dehydrogenase I, II, and III factor 1 (HNF-1), an IL-6-inducible C/EBP protein, and an promoters, the diverged C/EBP sites respond differentially to unidentified IL-6 responsive factor (17). In the C-reactive cotransfected C/EBP homodimers and heterodimers (58). Sim- protein promoter, two HNF-1-binding sites and one C/EBP- ilarly, cotransfected C/EBP proteins show different transacti- binding sequence are required for full promoter activity fol- vating activities for acute-phase gene promoters containing lowing cytokine induction (43, 56). In our study of the mouse distinct C/EBP-binding sequences (6, 14). Further support for SAA3 promoter, we also found that both the SEF-1-binding the differential role of C/EBP proteins is provided by the fact site and C/EBP-binding sites were necessary for its cytokine- that site-specific mutation of the B element in the SAA3 induced expression. It is noteworthy that in all these cases, promoter completely abolished promoter activity despite the binding sites for the constitutively expressed transcription presence of additional C/EBP-binding sites downstream. Thus, factors alone were not sufficient to elicit a cytokine response; additional sequence specificity must exist within or in the however, mutation of these sites dramatically reduced the flanking regions of the consensus site to account for these cytokine-induced expression of the target gene, indicating that differential effects. these elements are necessary to confer the cytokine response. Binding of SEF-1 is necessary for SAA3 promoter activity. An underlying mechanism for the link between basal and Two general classes of transcription factors have been reported induced expression is protein-protein interaction between con- to be involved in the regulation of acute-phase genes: consti- stitutive and inducible transcription factors (31). Alternatively, tutive and inducible factors. Several constitutive factors, in- binding of constitutive factors facilitates the formation of a cluding HNF-1, HNF-3, and HNF-4, are highly enriched in the more stably associated preinitiation complex (45). Thus, it liver. They not only confer liver cell enhanced expression to the remains to be elucidated how factors binding to elements A, B, downstream gene but also are required for their induced and C may interact with each other or with the general expression during the acute-phase response, presumably by transcription machinery to elicit the full cytokine response. cooperating with inflammation-induced transcription factors Differential binding and transactivation activities of C/EBP (17, 43, 57). The C/EBP family proteins C/EBP, and C/EBP8, family proteins. In noninduced Hep3B cells, C/EBPot is the NF-KB, and, more recently, APRF are well-characterized major C/EBP protein interacting with elements A and B. inflammation-induced transcription factors (2, 4, 10, 35, 59). However, its binding activity is reduced to undetectable levels Whereas the binding activities of C/EBPB and C/EBP8 can be following cytokine treatment. Reduced C/EBPao-binding activ- induced by both IL-1 and IL-6, NF-KB-binding activities are ity is consistent with the down-regulation of its mRNA levels in regulated by IL-1 and tumor necrosis factor and those of mice during the acute-phase response (2, 30, 40). Concomitant APRF are induced by IL-6 only (4, 14, 33, 59). In this report, with the reduction in C/EBPot-binding activity is the dramatic we demonstrate that the distal cytokine response element in increase in C/EBPB- and C/EBP8-binding activities, which the SAA3 promoter contains two adjacent C/EBP-binding sites contribute to the major inducible complexes observed with capable of interacting with C/EBPP and C/EBP8. In addition, elements A and B. This C/EBPo-to-C/EBPI or C/EBPoa-to-C/ this response element contains a region homologous to the EBP5 transition of binding activities was also reported for the NF-KB- and APRF-binding sites. However, its binding factor, human C3 promoter and the rat kininogen promoter in SEF-1, is clearly distinct from these two transcription factors. response to IL-1 and IL-6 stimulation, respectively (14, 33). In Both NF-KB- and APRF-binding activities are very low in addition, C/EBPI and C/EBP5 have been shown to be the control cells but can be induced to high levels by cytokines. major components of the cytokine-induced complex interact- SEF-1-binding activity, on the other hand, is readily detectable ing with the acute-phase response elements from hemopexin, in noninduced cells. Moreover, its activity is not further haptoglobin, C-reactive protein, and aol-acid glycoprotein pro- increased when stimulated by cytokines. While no binding moters (50). C/EBP8 mRNA is normally expressed at low activities were detected for NF-KB and APRF, their potential levels; however, during the acute-phase response its level role in SAA3 regulation cannot be ruled out completely. C increases dramatically (50). Therefore, C/EBP8 participates in element also contains sequences similar to the binding site for VOL. 14, 1994 INDUCTION OF MOUSE SAA3 GENE BY CYTOKINES 4483

Oct-1; however, Oct-i-binding-site oligonucleotides could not regulation by distinct protein subunits. Biochim. Biophys. Acta inhibit SEF-1 binding. Thus, SEF-1 may represent an addi- 1072:63-80. tional constitutively expressed transcription factor that inter- 5. Baeuerle, P. A., and D. Baltimore. 1988. lKB: a specific inhibitor of the NFKB transcription factor. Science 242:540-546. acts with cytokine-inducible factors, such as C/EBPP and 6. Baumann, H., K. K. Morella, S. P. Campos, Z. Cao, and G. P. C/EBP8, in the up-regulation of a subclass of acute-phase Jahreis. 1992. Role of CAAT-enhancer binding protein isoforms genes. in the cytokine regulation of acute-phase plasma protein genes. J. In our transfection studies, mutation of element B or C Biol. Chem. 267:19744-19751. drastically reduced promoter activity. Furthermore, mutation 7. Baumann, H., C. Richards, and J. Gauldie. 1987. Interaction of the C element rendered the promoter nonresponsive to among hepatocyte-stimulating factors, interleukin-1 and glucocor- transactivation by C/EBP8. These results strongly suggest that ticoids for the regulation of acute phase plasma proteins in human induction of the SAA3/reporter gene results from the cooper- hepatoma (HepG2) cells. J. Immunol. 139:4122-4128. ative effects of the constitutively expressed SEF-1 and induc- 8. Bengal, E., L. Ransone, R. Scharfmann, V. J. Dwarki, S. J. Tapscott, H. Weintraub, and I. M. Verma. 1992. Functional ible C/EBP family proteins. We do not know, however, antagonism between c-Jun and MyoD proteins: a direct physical whether this cooperative effect is through direct interaction association. Cell 68:507-519. between SEF-1 and C/EBP or through their independent 9. Bradford, M. M. 1976. A rapid and sensitive method for the interaction with the general transcription machinery. It is quantitation of microgram quantities of protein utilizing the evident that regulation of gene expression is not mediated principle of protein-dye binding. Anal. Biochem. 72:248-254. solely by the presence or absence of one particular set of 10. Cao, Z., R. M. Umek, and S. L. McKnight. 1991. Regulated transcription factors. Physical and functional interactions of expression of three C/EBP isoforms during adipose conversion of factors within a particular family have been well documented 3T3-L1 cell. Genes Dev. 5:1538-1552. (5, 10, 35); however, recent reports have shown that cross- 11. Cato, A. C., R Miksicek, G. Schutz, J. Arnemann, and M. Beato. 1986. The hormone regulatory element of mouse mammary tumor occur in family interactions of specific transcription factors also virus mediates progesterone induction. EMBO J. 5:2237-2240. modulating gene expression. For example, interaction between 12. Chang, C. J., T. T. Chen, H. Y. Lei, D. S. Chen, and S. C. Lee. 1990. the receptor and the AP-1 proteins Jun and Fos Molecular cloning of a transcription factor, AGP/EBP, that be- (21, 32, 63) and interaction between the leucine zipper protein longs to members of the C/EBP family. Mol. Cell. Biol. 10:6642- Jun and the helix-loop-helix protein MyoD (8) have been 6653. reported to occur in the regulation of collagenase and MyoD 13. Chen, H.-M., K. B. Considine, and W. S.-L. Liao. 1991. Interleu- gene expression, respectively. Direct interaction between kin-6 responsiveness and cell-specific expression of the rat kinino- C/EBP and several other transcription factors has also been gen gene. J. Biol. Chem. 266:2946-2952. observed. Nishio et al. (46) reported that direct protein-protein 14. Chen, H.-M., and W. S. L. Liao. 1993. Differential acute-phase response of rat kininogen genes involves type I and type II interaction between human C/EBPP and glucocorticoid recep- interleukin-6 response elements. J. Biol. Chem. 268:25311-25319. tor contributes to the synergistic activation of rat aol-acid 15. Chen-Kiang, S., W. Hsu, Y. Natkunam, and X. Zhang. 1993. glycoprotein gene. The physical and functional interactions Nuclear signaling by interleukin-6. Curr. Opin. Immunol. 5:124- observed between C/EBPP and NF-KB may explain the func- 128. tional synergy observed between IL-1 and IL-6 in several 16. Cohen, A. S., and L. A. Jones. 1993. Advances in amyloidosis. Curr. physiological responses, including acute-phase gene regulation Opin. Rheumatol. 5:62-76. (39, 54). Lastly, a selective and direct association between 17. Dalmon, J., M. Laurent, and G. Courtois. 1993. The human 1B human C/EBPB and AP-1 family proteins has been reported fibrinogen promoter contains a hepatocyte nuclear factor 1-depen- (15). In the case of the mouse SAA3 gene, we currently have no dent interleukin-6-responsive element. Mol. Cell. Biol. 13:1183- evidence to support direct protein-protein interaction between 1193. 18. 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