NFκB Interacts with Serum Amyloid A3 Enhancer Factor to Synergistically Activate Mouse Serum Amyloid A3 Gene Transcription*

We had previously identified a distal regulatory element (DRE) in the mouse serum amyloid A3 (SAA3) promoter that functions as a cytokine-inducible transcription enhancer. Within this DRE, three functional elements interact with CCAAT/enhancer-binding protein (C/EBP) and SAA3 enhancer factor (SEF) transcription factors. In this study, we show that cotransfection of the SEF expression plasmid with an SAA3/luciferase reporter resulted in 3–5-fold activation of the SAA3 promoter. When SEF-transfected cells were further stimulated with conditioned medium or interleukin-1, SAA3 promoter activity was dramatically increased, suggesting that SEF may cooperate functionally with other interleukin-1-inducible transcription factors to synergistically up-regulate SAA3 gene transcription. Indeed, cotransfection of SEF and NFκBp65 expression DNAs resulted in synergistic activation of the SAA3 promoter. Intriguingly, no consensus NFκB-binding site was found in the SAA3 promoter region; rather a putative NFκB-binding sequence with 3-base pair mismatches was identified in the DRE. When this sequence was used in an electrophoretic mobility shift assay, it interacted with NFκBp50, albeit with binding affinities that were several hundredfold lower than that with the consensus NFκB probe. Functional cooperation between SEF and NFκB was further strengthened by the finding that SEF and NFκB formed stable cytokine-inducible protein-protein complexes. Finally, despite its weak binding, mutation of this NFκB-binding site nevertheless dramatically reduced both NFκBp65- and cytokine-mediated induction of SAA3 promoter. Therefore, the molecular basis for the functional synergy between SEF and NFκB may, in part, be the ability of SEF to recruit NFκB through physical interactions that lead to enhancement or stabilization of NFκB binding to the SAA3 promoter element.

We had previously identified a distal regulatory element (DRE) in the mouse serum amyloid A3 (SAA3) promoter that functions as a cytokine-inducible transcription enhancer. Within this DRE, three functional elements interact with CCAAT/enhancer-binding protein (C/EBP) and SAA3 enhancer factor (SEF) transcription factors. In this study, we show that cotransfection of the SEF expression plasmid with an SAA3/luciferase reporter resulted in 3-5-fold activation of the SAA3 promoter. When SEF-transfected cells were further stimulated with conditioned medium or interleukin-1, SAA3 promoter activity was dramatically increased, suggesting that SEF may cooperate functionally with other interleukin-1-inducible transcription factors to synergistically up-regulate SAA3 gene transcription. Indeed, cotransfection of SEF and NFBp65 expression DNAs resulted in synergistic activation of the SAA3 promoter. Intriguingly, no consensus NFB-binding site was found in the SAA3 promoter region; rather a putative NFBbinding sequence with 3-base pair mismatches was identified in the DRE. When this sequence was used in an electrophoretic mobility shift assay, it interacted with NFBp50, albeit with binding affinities that were several hundredfold lower than that with the consensus NFB probe. Functional cooperation between SEF and NFB was further strengthened by the finding that SEF and NFB formed stable cytokine-inducible protein-protein complexes. Finally, despite its weak binding, mutation of this NFB-binding site nevertheless dramatically reduced both NFBp65-and cytokine-mediated induction of SAA3 promoter. Therefore, the molecular basis for the functional synergy between SEF and NFB may, in part, be the ability of SEF to recruit NFB through physical interactions that lead to enhancement or stabilization of NFB binding to the SAA3 promoter element.
A prominent feature of the systemic response to acute inflammation, infection, and tissue injury is the rapid increase in the concentration of a number of plasma proteins collectively termed the acute phase proteins (1). Acute phase proteins can be divided into two groups. The type I acute phase proteins, such as serum amyloid A (SAA) 1 , C-reactive protein, and complement C3, are induced by interleukin (IL)-1-like cytokines and can be further induced by IL-6-like cytokines. The type II acute phase proteins, including fibrinogen, haptoglobin, and ␣ 2 -macroglobulin, are induced primarily by the IL-6-like cytokines (1).
Murine SAA genes belong to a small gene family consisting of four active genes (SAA1, SAA2, SAA3, and SAA5) and a pseudogene (2)(3)(4). The plasma concentrations of SAA rise from 0.5 g/ml to more than 1000 g/ml 24 h after injection of bacterial lipopolysaccharide (5). This large increase in hepatic SAA synthesis is primarily the consequence of increased transcription of SAA genes (6, 7) mediated by the proinflammatory cytokines IL-1, tumor necrosis factor, and IL-6 (1,8). This dramatic induction has therefore been used as a model system for studying differential gene expression in response to a specific stimulus.
To dissect the molecular mechanisms of SAA gene regulation, we have studied the promoters of the rat SAA1 (9 -13) and mouse SAA3 genes (8, 14 -17). Our studies of the rat SAA1 promoter have shown the functional importance and cooperative interaction between NFB and C/EBP proteins in cytokine-induced expression. Mutation of either transcription factor-binding site completely abolished SAA1 promoter activity. Studies on the mouse SAA3 promoter demonstrated that a 350-bp promoter fragment was necessary and sufficient to confer cytokine responsiveness (15). Two regulatory elements were identified in this 350-bp promoter fragment: a proximal response element that contains two adjacent C/EBP-binding sequences and enhances SAA3 gene expression in liver-derived cells (17) and a distal response element (DRE) that confers responsiveness to cytokine induction and has properties of an inducible transcription enhancer (16). The DRE consists of three functionally distinct elements: the A element, a weak binding site for C/EBP family proteins; the B element, which also interacts with C/EBP family proteins but with a much higher affinity; and the C element, which interacts with a constitutive nuclear factor termed SAA3 enhancer factor (SEF) (14,16). Functional analyses revealed that all three elements are required for maximum SAA3 promoter activity (16).
We have recently purified SEF and shown by antibody supershift and amino acid sequence analysis that it is identical to the transcription factor LBP-1c/CP2/LSF (14). LBP-1c/CP2/ LSF was initially identified as a cellular factor that binds at multiple sites in the human immunodeficiency virus type I (HIV-I) long terminal repeat (18,19), ␣-globin promoter (20), and SV40 major late promoter (21). It may function either as a transcription activator or a transcription repressor, depending on the promoter context of the gene it regulates and the transcription factors it interacts with. For example, LBP-1c/CP2/ LSF stimulates transcription from the SV40 major late promoter (21,22), whereas it cooperates with YY1 to repress HIV-1 long terminal repeat activity (23). Furthermore, inducers of cell growth can up-regulate the DNA binding activities of LBP-1c/CP2/LSF in human peripheral T lymphocytes, suggesting that it may participate in the regulation of growth-responsive genes (24). In rat pheochromocytoma PC12 cells, LBP-1c/ CP2/LSF has been shown to physically interact with neural protein Fe65 (25), but the functional significance of such interaction is yet to be determined.
Binding of IL-1 or tumor necrosis factor to their receptors leads to potent activation of the transcription factors AP-1 and NFB. Activated NFB can then rapidly translocate into the nucleus and regulate the transcription of target genes (26,27), including many effectors of the immune, inflammatory, and the acute phase responses. For example, the proinflammatory cytokines tumor necrosis factors-␣ and -␤ and IL-1 are not only potent activators of NFB but are themselves targets of NFB regulation (28,29). Other important genes regulated by NFB include IL-6, IFN-␤, the chemokines IL-8 and Gro, which summon cells to sites of inflammation (30,31), and cell surface adhesion proteins such as endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1 (32)(33)(34)(35), and the intercellular cell adhesion molecule-1 (36). Many viruses also use NFB to regulate their own expression. One example is that the expression of HIV-1 is critically dependent on the tandem NFB sites in its long terminal repeats (37). In almost all cases, NFB does not function alone. Instead, NFB often physically associates with other DNA-binding factors and functions cooperatively to regulate transcription of their target genes.
In this study, we sought the molecular mechanisms by which SEF exerts its effect on SAA3 gene transcription in response to cytokine stimulation. We provide evidence that IL-1-induced activation of SAA3 gene transcription requires cooperative interactions between SEF and NFB. The molecular basis for such functional synergy may be the ability of SEF to physically interact with NFB and thus recruit NFB to the active transcription complex.

EXPERIMENTAL PROCEDURES
Cell Culture and Nuclear Extracts-HepG2 cells were cultured in basal medium consisting of minimum essential medium (Life Technologies, Inc.) and Waymouth MAB (3:1 v/v) plus 10% fetal calf serum (38) and were passaged at confluence, approximately once a week.
HepG2 nuclear extracts were prepared essentially as described (39), and as modified by Singh and Aggarwal (40). Briefly, exponentially growing cells were washed twice with ice-cold 1ϫ phosphate-buffered saline and then recovered in 1 ml of phosphate-buffered saline. After centrifugation for 30 s in a microcentrifuge, the cells were resuspended in 1.2 ml of lysis buffer (10 mM KCl, 10 mM HEPES, pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 0.4 mM NaVO 4 , and protease inhibitor mixture; Roche Molecular Biochemicals) and allowed to swell on ice for 20 min. Then 37.5 l of 10% Nonidet P-40 was added to the cell suspension, and the mixture was mixed vigorously for 10 -15 s and centrifuged for 1 min in a microcentrifuge. The nuclear pellets were resuspended in 20 -30 l of ice-cold extraction buffer (0.4 M NaCl, 20 mM HEPES, pH 7.9, 1 mM EDTA, 1 mM EGTA, and a mixture of protease and phosphatase inhibitors), and the nuclear proteins were extracted by constant mixing at 4°C for 30 min. Cell debris was then removed by centrifugation, and the nuclear extracts were either used immediately or stored in small aliquots at Ϫ70°C. The protein concentrations of the nuclear extracts were determined by the Bradford method (41).
Electrophoretic Mobility Shift Assays-32 P-Labeled C element (4 ϫ 10 4 cpm) containing a single SEF-binding site was incubated with recombinant NFBp50 or purified SEF at 4°C for 30 min (16). After incubation, the reaction mixtures were loaded onto a 5% polyacrylamide gel (19:1 cross-linking ratio) in 1ϫ glycine buffer and subjected to electrophoresis at 200 V for 90 min at 4°C. The gel was dried before autoradiography. For oligonucleotide competition experiments, wildtype or mutant oligonucleotides corresponding to LBP-1c-or NFBbinding sites were used as specific competitors (see Table I).
Conditioned Medium Preparation-Conditioned medium (CM) was prepared from mixed lymphocyte cultures as described (43). Human peripheral blood mononuclear cells were isolated from multiple healthy donors by centrifugation through Ficoll-Hypaque (density, ϩ1.077 g/cm) (Life Technologies, Inc.) for 30 min at 680 ϫ g. Isolated cells were washed twice with RPMI 1640 and then cultured at 10 6 cells/ml of RPMI 1640 supplemented with 0.25% bovine serum albumin and 10 g/ml phytohemagglutinin (Life Technologies, Inc.). After incubation at 37°C for 72 h, the CM was separated from the cells by centrifugation and filtration and then stored at Ϫ20°C until use.
Transient Transfection Assay-HepG2 cells (5 ϫ 10 5 ) were seeded in 2 ml of culture medium. After overnight incubation, cells were transfected with the indicated plasmid DNAs according to the FuGENE procedure (Roche Molecular Biochemicals). All plasmids used in the transfection studies were prepared by either CsCl gradient centrifugation or the QIAfilter Plasmid Maxi kit (Qiagen). For each transfection, 0.5 g of luciferase reporter was transfected into HepG2 cells with or without one or more expression plasmids. To assess cytokine responsiveness, transfected cells were treated with 50% CM or 10 ng/ml IL-1 for 24 h before they were harvested for cell lysate preparation. Cell extracts were assayed for protein content by the Bradford method (41), and the luciferase activity was quantitated according to the manufacturer's instructions.
Coimmunoprecipitation and Western Blot Analysis-HepG2 nuclear extracts (approximately 500 g) were preabsorbed with protein Aagarose-coupled beads (Santa Cruz) for 1 h at 4°C. The preabsorbed beads were then pelleted and discarded. Anti-SEF antibody (ϳ5 l) was then added to the supernatant and allowed to incubate with the nuclear extract for 2 h at 4°C. Protein A-agarose-coupled beads were then added to the reaction mixture and incubated for another hour at 4°C. The beads were pelleted and washed three times with 1ϫ phosphatebuffered saline. Immunoprecipitated proteins were then boiled in sample buffer and loaded onto a 7.5% SDS-polyacrylamide gel. After electrophoresis, the proteins were electroblotted onto polyvinylidene difluoride membranes, and specific proteins were detected in TBST solution (10 mM Tris-HCl, pH 7.5, 250 mM NaCl, and 0.05% Tween 20) containing 2% nonfat dry milk with specific anti-NFBp65 (1:5000) (Santa Cruz) or anti-SEF (1:1000) antibodies. Positions of NFBp65 and SEF were visualized with peroxidase-coupled second antibody by the ECL detection system (Amersham Pharmacia Biotech). For reprobing, membranes were stripped in 62 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM ␤-mercaptoethanol at 65°C for 45 min.

RESULTS
Identical DNA Binding Specificity between SEF and LBP-1c/CP2/LSF-Our earlier protein sequencing and antibody supershift experiments strongly suggested that SEF is identical to or highly related to the transcription factor LBP-1c/CP2/ LSF (14). To further examine whether SEF has the same DNA sequence binding specificities as those of LBP-1c/CP2/LSF, oligonucleotides corresponding to the known LBP-1c/CP2/LSFbinding sites were synthesized (Table I) and used as specific competitors in EMSA to inhibit SEF-DNA complex formation. As shown in Fig. 1A, the wild-type LBP-1c/CP2/LSF-binding sequences from the promoters of ␣-globin, SV40, and HIV specifically inhibited SEF⅐DNA complex formation (lanes 3-5), whereas a mutated binding region from the HIV promoter was ineffective as a competitor (lane 6). As expected, wild-type but not mutant SEF-binding sequences specifically competed for complex formation (lanes 7 and 8). Their identical DNA sequence binding specificities further support our conclusion that SEF is identical to LBP-1c/CP2/LSF. SEF Enhances CM-and IL-1-induced SAA3 Promoter Activity-To investigate the role of SEF in SAA3 gene transcription, the pSAA3/Luc(Ϫ165) reporter gene was transfected into HepG2 cells with increasing amounts of SEF expression plasmid. Overexpression of SEF activated SAA3 reporter gene expression in a dose-dependent manner, albeit only 3-5-fold. Intriguingly, stimulation of SEF-transfected cells with CM or IL-1 resulted in dramatic synergistic activation of reporter gene expression. In the absence of cotransfected SEF, CM, and IL-1 induced reporter gene activities by approximately 25-and 10-fold, respectively (Fig. 1B). In SEF-transfected cells, cytokine-induced reporter gene expression was even greater. At 1.0 g of SEF expression plasmid DNA, CM and IL-1 induced the pSAA3/Luc(Ϫ165) reporter gene by more than 80-and 25-fold, respectively. These results indicate that SEF plays an important role in the transcription of SAA3 promoter. More importantly, it suggests that SEF may cooperate with other cytokineinducible transcription factors to confer synergistic activation on the SAA3 gene promoter.
Overexpression of C/EBP␦ with SEF Cannot Confer Synergistic Activation of the SAA3 Gene Promoter-We had previously shown that the C/EBP family of transcription factors, C/EBP␣, ␤, and ␦, play a central role in SAA3 gene transcription (16). Although all can induce pSAA3/CAT reporter gene expression, C/EBP␦ was the most potent transactivator. Because C/EBP␦ is also induced by IL-1 (44), we tested whether C/EBP␦ could be the transcription factor that cooperates with SEF and accounts for the synergistic activation of reporter gene expression. pSAA3/Luc(Ϫ165) reporter gene was transfected into HepG2 cells with SEF and C/EBP␦ expression plasmids, individually or in combination. As shown in Fig. 1C, overexpression of C/EBP␦ alone induced the SAA3 promoter by approximately 16-fold. Expression of SEF together with C/EBP␦, however, resulted in additive rather than synergistic activation of reporter gene activity. These results therefore suggest that C/EBP␦ may not be the IL-1-induced transcription factor that cooperates with SEF to confer the observed synergistic activation of the SAA3 reporter gene.
NFB Participates in the Cytokine-mediated Induction of SAA3 Promoter-In addition to activating the C/EBP family of transcription factors, IL-1 is also a potent activator of AP-1 and NFB (26,27). Because NFB has been shown to play a critical role in the regulation of human and rat SAA1 genes as well as the expression of other acute phase genes, we sought to examine whether it might also participate in regulating the SAA3 promoter. To test this possibility, we transfected pSAA3/ Luc(Ϫ165) reporter DNA into HepG2 cells with the expression

5Ј-GTAagttGTCTCTCTGGTTAG-3Ј
a The underlined sequences represent the half-sites of the putative SEF-binding sequences. The lowercase characters indicate the substituted bases.
FIG. 1. DNA binding and functional properties of SEF. A, comparison of DNA binding specificities between SEF and LBP-1c/CP2/ LSF. 32 P-Labeled C element was incubated with purified SEF in standard EMSAs. The SEF⅐DNA complexes were competed with a 100-fold molar excess of oligonucleotides of known LBP-1c/CP2/LSF-binding sites (␣-globin, SV40, and HIV promoters). As controls, the SEF⅐DNA complexes were competed with mutant HIV (mHIV) and wild-type (SEF) or mutant (mSEF) C element oligonucleotides. Positions of the SEF:DNA complexes and the free probe are indicated. B, SEF enhances cytokine-dependent activation of SAA3 promoter. HepG2 cells were cotransfected with 0.5 g of pSAA3/Luc(Ϫ165) and the indicated amounts of SEF expression plasmid. Transfected cells were then treated with medium alone (Control) or with CM or IL-1 (10 ng/ml). Results were normalized to the activities of the noncotransfected control cells, to which a value of 1.0 was assigned. C, SEF and C/EBP␦ do not synergistically activate the SAA3 reporter. pSAA3/Luc(Ϫ165) reporter gene was cotransfected into HepG2 cells with SEF and C/EBP␦ expression vectors, individually or together. Results were normalized to the activities of the cells transfected with the reporter gene, to which a value of 1.0 was assigned. vectors for either the p65 or the p50 subunit of NFB. As expected, the vector control and the NFBp50 expression DNA, which lacks a functional transactivation domain, had no effect on reporter gene expression. In sharp contrast, cotransfection of NFBp65 expression DNA dramatically induced luciferase activity in a dose-dependent manner, and at 0.6 g of the expression DNA, reporter gene activity was induced by approximately 20-fold ( Fig. 2A).
To ensure that the transactivation of pSAA3/Luc(Ϫ165) reporter gene by NFBp65 was a true reflection of the participation of NFB in the regulation of SAA3 promoter activity and not merely the result of its overexpression, we examined whether cytokine-mediated induction of SAA3 promoter activities also requires NFB by expressing its inhibitor IB in the transfected cells. HepG2 cells were cotransfected with pSAA3/ Luc(Ϫ165) reporter gene and either empty vector DNA or IB␣ expression plasmid and then stimulated with CM or IL-1. As shown in Fig. 2B, in the absence of transfected IB, CM and IL-1 stimulated the SAA3 promoter by approximately 18-and 10-fold, respectively. However, overexpression of IB␣ resulted in greater than 85% inhibition of the cytokine-induced activation. This inhibition by IB was deemed specific for NFB activity because IB had no inhibitory effects on C/EBP␦-mediated transactivation but completely blocked NFBp65-mediated activation (Fig. 2B). Taken together, these results are consistent with the notion that NFB is one of the transcription factors activated by IL-1 and that it participates in the regulation of SAA3 promoter activity.
NFB Mediates the Induction of the SAA3 Promoter through DRE-We had shown previously that deletion of the DRE region from the SAA3 promoter completely abolished its cytokine responsiveness (16). To determine whether the DRE might also be necessary for NFB-mediated activation of the SAA3 promoter, two 5Ј deletion constructs (pSAA3/Luc(Ϫ93) and pSAA3/Luc(Ϫ63)) and two internal deletion constructs (pSAA3/Luc(DRE-93) and pSAA3/Luc(DRE-63)) of the SAA3 promoter were tested for their responsiveness to transactivation by cotransfected NFBp65 and to CM stimulation. As expected, the wild-type pSAA3/Luc(Ϫ165) reporter was highly responsive to CM stimulation and to transactivation by NFBp65 (Fig. 3A). However, deletions to positions bp Ϫ93 and Ϫ63 rendered the promoters completely nonresponsive to both inducing agents. Insertion of the 66-bp DRE sequences in front of these two 5Ј deletion constructs restored their responsiveness to NFB and CM, suggesting that transactivation of the SAA3 promoter by NFB is mediated through the DRE. To further examine whether DRE was responsible for NFB-mediated transactivation, we inserted one copy of the DRE se- quence in front of a minimal SV40 promoter to create the heterologous promoter construct, pSV1(DRE). When transfected into HepG2 cells, pSV1(DRE) could be induced by IL-1 and by the cotransfected NFBp65 (Fig. 3B). Consistent with the results obtained with the SAA3 promoter constructs, introduction of IB also inhibited the responsiveness of the pSV1(DRE) construct to IL-1 and NFBp65. These results therefore strongly implicate the DRE region of the SAA3 promoter as the central regulatory region that confers NFBmediated transactivation.
A Nonconsensus NFB-binding Site in the C Element Is Required for NFB-dependent Transactivation-Transcription factors usually exert their effects on gene transcription by binding to the promoter or enhancer regions of their target genes. As DRE conferred NFB-dependent activation of the SAA3 promoter, we searched the DRE sequence for potential NFB-binding site(s). One such site was found within the C element of the DRE; however, it contained three mismatched nucleotides when compared with the consensus NFB-binding sequence (Fig. 4A). To determine whether this putative NFBbinding sequence could function as a binding site for NFB, we incubated 32 P-labeled C element with recombinant NFBp50, and the NFBp50-C element complexes formed were analyzed by EMSA. For comparison, a consensus NFB-binding sequence was also radioactively labeled and used in EMSA with NFBp50. When the consensus NFB-binding sequence was incubated with NFBp50, as little as 5 ng of NFBp50 was sufficient to form a strong DNA-protein complex. In sharp contrast, NFBp50 binding to the C element was barely detectable even when incubated with 30 ng of the recombinant protein (Fig. 4B). Nevertheless, after longer (3 days) exposure, a weak protein-DNA complex was detected. Furthermore, formation of this protein-DNA complex could be inhibited by the wild-type NFB-binding consensus oligonucleotides but not by mutant oligonucleotides, indicating that NFBp50 can specifi-cally interact with the C element despite its low affinity. When compared with that of the consensus sequence, interaction between NFB and the C element was estimated to be several hundredfold weaker.
Such a weak NFB-binding site in the DRE was somewhat surprising because, as shown earlier in our cotransfection experiments, NFB is in fact a very potent transactivator on the SAA3 promoter. To determine whether this weak binding site is functionally important, a mutant reporter gene construct was generated in which the NFB-binding site was mutated. Because this NFB-binding site overlapped with that of SEF within the C element (Fig. 4A), we introduced a 2-bp mutation so that it affected only NFB binding but not SEF binding, as determined by EMSA (data not shown). The resulting construct, pSAA3/Luc(Ϫ165) mB , was transfected into HepG2. As shown in Fig. 4C, mutation of this weak NFB-binding site in the DRE rendered the pSAA3/Luc(Ϫ165) mB reporter nonresponsive to transactivation by the cotransfected NFBp65. Similarly, this mutant construct was also nonresponsive to CM and IL-1 stimulation (Fig. 4D). Taken together, these results strongly indicate that NFB can bind, albeit very weakly, to a nonconsensus NFB-binding site in the DRE and that this weak NFB-binding site is nevertheless functionally important for NFB-and cytokine-mediated activation of the SAA3 promoter.
Functional Cooperation and Cytokine-induced Association between NFB and SEF-We showed earlier that SEF dramatically enhanced the CM-and IL-1-mediated induction of an SAA3 reporter gene (Fig. 1B), suggesting that SEF may functionally cooperate with one or more IL-1-inducible transcription factors to synergistically activate the SAA3 promoter. Further, we provided evidence that NFB plays a key role in mediating the effects of IL-1 (Fig. 2). We therefore investigated whether SEF and NFB can function cooperatively to activate the SAA3 promoter. Wild-type pSAA3/Luc(Ϫ165) reporter gene was transfected into HepG2 cells with SEF-or NFBp65 expression plasmids, individually or together. As shown in Fig. 5, cotransfection of the reporter gene with SEF-or NFBp65 expression DNAs increased the reporter gene activity by approximately 3-and 14-fold, respectively. However, when these two expression vectors were transfected together, the luciferase activity was increased by more than 40-fold, indicating transcriptional synergy between SEF and NFB.
Because of the overlapping nature of their binding sites and their functional cooperation in SAA3 promoter activation, we investigated whether SEF and NFB in fact physically interact with each other and thus provide an underlying molecular basis for their synergistic activation. Exponentially growing HepG2 cells were serum-starved for 16 h before they were stimulated with either IL-1 or CM for various time periods. After stimulation, cells were harvested and nuclear extracts were prepared and incubated with anti-SEF antibody to immunoprecipitate SEF and its associated proteins. The presence of NFB in the immune complexes was then determined by Western blotting with anti-p65 antibodies. As shown in Fig. 6, in untreated HepG2 cells, a low level of NFBp65 was detected in the anti-SEF immunoprecipitates. However, within 5 min of IL-1 or CM stimulation, the amount of NFBp65 in the immunoprecipitates was increased substantially. The levels of NFBp65 in the immunoprecipitates were maintained at elevated levels even at 60 min after stimulation (Fig. 6). When these samples were probed with anti-SEF antibodies, the amount of SEF detected in each lane was approximately the same, indicating that differences in the levels of NFBp65 were not due to unequal loading or uneven immunoprecipitation with anti-SEF. To further demonstrate the specificity of the coimmunoprecipitation procedure, we used preimmune serum in the immunoprecipitation reactions and were not able to detect any NFBp65 (data not shown). These data therefore indicate that SEF can form protein-protein complexes, directly or indirectly, with NFBp65. Further, CM and IL-1 stimulation increases their association with a rapid kinetics. Thus, the transcriptional synergy between SEF and NFBp65 may be facilitated through their physical interactions.
SEF-binding Site Is Critical for Cytokine-mediated Induction of SAA3 Promoter-To assess its functional importance in conferring the cytokine response, we constructed a reporter gene in which the SEF-binding site was specifically mutated. The resulting construct pSAA3/Luc(Ϫ165) mSEF was then assayed for its responsiveness to cytokine stimulation. As shown in Fig.  7, the wild-type pSAA3/Luc(Ϫ165) construct showed an approximately 25-and 10-fold increase in luciferase activities when stimulated by CM and IL-1, respectively. In sharp contrast, the pSAA3/Luc(Ϫ165) mSEF construct was nonresponsive to cytokine stimulation. This result clearly demonstrates that SEF and its binding site play an important role in conferring maximum cytokine response on the SAA3 promoter. DISCUSSION We sought a molecular mechanism for cytokine-induced mouse SAA3 gene expression following acute inflammation or tissue damage by analyzing its regulatory elements in the 5Ј promoter regions and their interacting transcription factors. Our earlier studies identified a 66-bp DRE region that could confer cytokine responsiveness and had properties of an inducible transcriptional enhancer. Within the DRE, three functionally distinct regions, referred to as the A, B, and C elements, proved important for SAA3 promoter function. The A element, a weak C/EBP-binding site, appeared to affect the magnitude of SAA3 expression but not its responsiveness to cytokine stimulation. On the other hand, the B element, identified as a strong C/EBP-binding site, was crucial for the basal and cytokineinduced activities of the SAA3 promoter. The C element, which interacts with the constitutive transcription factor SEF, was important for both basal and cytokine-induced activation of SAA3 promoter (16).
In the present study, we analyzed further the function of SEF in SAA3 gene regulation. Surprisingly, whereas SEF by itself can only moderately activate the SAA3/Luc reporter, stimulation of SEF-transfected cells with IL-1 or CM resulted in dramatic synergistic activation of the reporter gene. We interpreted this result as a strong suggestion that SEF cooperates functionally with one or more cytokine-activated transcription factors to up-regulate SAA3 gene transcription. We had previously shown that C/EBP␦, an IL-1-inducible transcription factor, could transactivate the SAA3 promoter through the DRE. We therefore tested for functional coopera-  6. Cytokine-inducible association between SEF and NFBp65. HepG2 cells were serum starved for 16 h before they were stimulated for the indicated time periods with IL-1 or CM and harvested for nuclear extract preparation. The nuclear extracts were first precleared with protein A-agarose before immunoprecipitated (IP) with anti-SEF antibodies. The immunoprecipitated proteins were then resolved on a 7.5% SDS-polyacrylamide gel, transferred onto polyvinylidene difluoride membrane, and blotted sequentially with anti-p65 and anti-SEF antibodies. WB, Western blot. tion between SEF and C/EBP␦. However, no synergistic induction was observed.
As NFB is one of the key transcription factors that mediates the IL-1 effects, we then examined whether NFB could, by itself, activate the SAA3 promoter. Surprisingly, we found that it did. The fact that NFB potently activated the SAA3 promoter was unexpected because sequence analysis of the SAA3 promoter did not reveal an obvious NFB-binding site. However, our biochemical and functional results presented here argue for an important functional role of NFB in conferring the transcriptional up-regulation of SAA3 in response to cytokine stimulation. First, cotransfection of NFBp65 with a SAA3/Luc reporter gene dramatically induced reporter gene expression (20-fold) in a dose-dependent manner. Second, CMand IL-1-mediated activation of SAA3 promoter were completely inhibited by IB␣. These results are particularly significant because they show that NFB not only can transactivate SAA3 promoter in an overexpression system, but, more important, they demonstrate that NFB participates in normal physiological conditions, such as the cytokine-mediated transcription activation, to regulate SAA3 promoter. Finally, a nonconsensus NFB-binding sequence was identified within the C element. Intriguingly, this putative NFB-binding sequence overlapped with that of the SEF-binding site. This binding sequence showed very weak NFB binding, several hundredfold lower than that of a consensus NFB-binding sequence. It is noteworthy that despite its weak binding, mutation of this site nevertheless completely abolished NFB-dependent and cytokine-induced activation of the SAA3 promoter. Thus, our results clearly demonstrate that NFB plays a critical role in SAA3 gene regulation.
As a potent transcription activator, NFB often does not act alone to regulate its target gene promoters. Instead, NFB functions cooperatively with other DNA-binding factors to induce gene transcription. This functional cooperation usually involves physical interactions between these transcription factors. Some of the transcription factors that have been shown to physically interact with NFB include AP-1 (45), Sp-1 (46, 47), C/EBP (48 -50), SRF (51), Stat6 (52), and components of the basal transcription factors TBP and TFIIB (53). Other factors, such as HMGI/Y, have also been shown to interact directly with NFB to induce transcription activation through the PDR-II region of the IFN-␤ gene promoter (54 -56). Physical interactions have also been shown to result in antagonistic effects on the expression of the target genes. For example, interactions between the glucocorticoid receptor and NFB resulted in inhibition of gene activation (57)(58)(59)(60).
In the case of the SAA3 promoter, we also observed strong synergy when NFB and SEF were coexpressed in HepG2 cells with a SAA3/Luc reporter. The notion of functional cooperation between NFB and SEF is further strengthened by the demonstration that these two factors can interact with each other. Moreover, their interaction is dependent entirely or is greatly enhanced upon cytokine stimulation. At present, we do not know whether the interaction between NFB and SEF is through direct protein-protein interaction or whether it involves other protein mediators. Because NFB is normally localized in the cytoplasm and translocated to the nucleus only after cytokine-dependent activation, this cytokine-dependent interaction may merely reflect their nuclear colocalization and may not require post-translational modification induced by the cytokines.
The underlying mechanism for their functional cooperation therefore appears to be facilitated by the ability of SEF to physically associate with NFB. Studies have shown that protein-protein contacts between heterologous factors can stabilize DNA binding or alter sequence specificity of binding. For example, sequence-specific binding of the homeodomain proteins Ubx, Hox, and Ftz-F1 depends on their stable interactions with other sequence-specific transcription factors (61)(62)(63). Because the NFB site in the C element is estimated to be several hundredfold weaker than that of the consensus NFB-binding sequence, it raises an intriguing possibility that SEF may participate to stabilize or enhance NFB binding to this site. Thus, the striking functional synergy between SEF and NFB may be facilitated by their ability to physically interact with each other and perhaps the ability of SEF to enhance or stabilize NFB binding to its weak binding site in the DRE. It is interesting to note that in the regulatory regions of both SV40 and HIV-I, binding sites for SEF and NFB have been described. Although it is not known whether these two factors also function cooperatively to regulate the expression of viral genes, it would be tempting to speculate that SEF may enhance the function of NFB and thus contribute to the expression of viral genes.