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 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. IL, interleukin base pair(s) distal response element CCAAT/enhancer-binding protein SAA3 enhancer factor human immunodeficiency virus type I electrophoretic mobility shift assays conditioned medium 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 (1Baumann H. Gauldie J. Immunol. Today. 1994; 15: 74-80Abstract Full Text PDF PubMed Scopus (458) Google Scholar). 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 α2-macroglobulin, are induced primarily by the IL-6-like cytokines (1Baumann H. Gauldie J. Immunol. Today. 1994; 15: 74-80Abstract Full Text PDF PubMed Scopus (458) Google Scholar). Murine SAA genes belong to a small gene family consisting of four active genes (SAA1, SAA2, SAA3, andSAA5) and a pseudogene (2de Beer M.C. Beach C.M. Shedlofsky S.I. de Beer F.C. Biochem. J. 1991; 280: 45-49Crossref PubMed Scopus (43) Google Scholar, 3Lowell C.A. Potter D.A. Stearman R.S. Morrow J.F. J. Biol. Chem. 1986; 261: 8442-8452Abstract Full Text PDF PubMed Google Scholar, 4Stearman R.S. Lowell C.A. Peltzman C.G. Morrow J.F. Nucleic Acids Res. 1986; 14: 797-809Crossref PubMed Scopus (35) Google Scholar). The plasma concentrations of SAA rise from 0.5 μg/ml to more than 1000 μg/ml 24 h after injection of bacterial lipopolysaccharide (5Hoffman J.S. Benditt E.P. J. Biol. Chem. 1982; 257: 10510-10517Abstract Full Text PDF PubMed Google Scholar). This large increase in hepatic SAA synthesis is primarily the consequence of increased transcription of SAA genes (6Lowell C.A. Stearman R.S. Morrow J.F. J. 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Liao W.S. Biochem. Biophys. Res. Commun. 1999; 264: 395-403Crossref PubMed Scopus (11) Google Scholar, 12Lu S.Y. Rodriguez M. Liao W.S. Mol. Cell. Biol. 1994; 14: 6253-6263Crossref PubMed Google Scholar, 13Ren Y. Reddy S.A. Liao W.S. J. Biol. Chem. 1999; 274: 37154-37160Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) and mouseSAA3 genes (8Huang J.H. Liao W.S. J. Interferon. Cytokine. Res. 1999; 19: 1403-1411Crossref PubMed Scopus (17) Google Scholar, 14Bing Z. Reddy S.A. Ren Y. Qin J. Liao W.S. J. Biol. Chem. 1999; 274: 24649-24656Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 15Huang J.H. Rienhoff Jr., H.Y. Liao W.S. Mol. Cell. Biol. 1990; 10: 3619-3625Crossref PubMed Scopus (28) Google Scholar, 16Huang J.H. Liao W.S. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar, 17Li X.X. Huang J.H. Rienhoff Jr., H.Y. Liao W.S. Mol. Cell. Biol. 1990; 10: 6624-6631Crossref PubMed Scopus (41) Google Scholar). Our studies of the rat SAA1 promoter have shown the functional importance and cooperative interaction between NFκB 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 (15Huang J.H. Rienhoff Jr., H.Y. Liao W.S. Mol. Cell. Biol. 1990; 10: 3619-3625Crossref PubMed Scopus (28) Google Scholar). 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 (17Li X.X. Huang J.H. Rienhoff Jr., H.Y. Liao W.S. Mol. Cell. Biol. 1990; 10: 6624-6631Crossref PubMed Scopus (41) Google Scholar) and a distal response element (DRE) that confers responsiveness to cytokine induction and has properties of an inducible transcription enhancer (16Huang J.H. Liao W.S. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). 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) (14Bing Z. Reddy S.A. Ren Y. Qin J. Liao W.S. J. Biol. Chem. 1999; 274: 24649-24656Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 16Huang J.H. Liao W.S. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). Functional analyses revealed that all three elements are required for maximum SAA3 promoter activity (16Huang J.H. Liao W.S. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). 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 (14Bing Z. Reddy S.A. Ren Y. Qin J. Liao W.S. J. Biol. Chem. 1999; 274: 24649-24656Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 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 (18Malim M.H. Fenrick R. Ballard D.W. Hauber J. Bohnlein E. Cullen B.R. J. Virol. 1989; 63: 3213-3219Crossref PubMed Google Scholar, 19Wu F.K. Garcia J.A. Harrich D. Gaynor R.B. EMBO J. 1988; 7: 2117-2130Crossref PubMed Scopus (109) Google Scholar), α-globin promoter (20Lim L.C. Swendeman S.L. Sheffery M. Mol. Cell. Biol. 1992; 12: 828-835Crossref PubMed Google Scholar), and SV40 major late promoter (21Huang H.C. Sundseth R. Hansen U. Genes Dev. 1990; 4: 287-298Crossref PubMed Scopus (47) Google Scholar). 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 (21Huang H.C. Sundseth R. Hansen U. Genes Dev. 1990; 4: 287-298Crossref PubMed Scopus (47) Google Scholar, 22Sundseth R. Hansen U. J. Biol. Chem. 1992; 267: 7845-7855Abstract Full Text PDF PubMed Google Scholar), whereas it cooperates with YY1 to repress HIV-1 long terminal repeat activity J. Virol. PubMed Google Scholar). of can up-regulate the binding of LBP-1c/CP2/LSF in human suggesting that it may in the of genes J. Hansen U. Genes Dev. PubMed Scopus Google Scholar). 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J. 1994; PubMed Scopus Google Scholar). also NFκB to expression. is that the expression of HIV-1 is on the NFκB sites in its long terminal D. PubMed Scopus Google Scholar). In all NFκB function NFκB with other factors and functions to transcription of In this study, we the molecular mechanisms by which SEF its on SAA3 gene transcription in response to cytokine We that activation of SAA3 gene transcription cooperative interactions between SEF and The molecular basis for such functional synergy may be the ability of SEF to interact with NFκB and recruit NFκB to the active transcription cells were in medium consisting of medium and serum J. Biol. 1986; PubMed Scopus Google Scholar) and were at a nuclear were as E. Nucleic Acids Res. 1989; PubMed Scopus Google Scholar), and as by and S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with and in of for in a the cells were in of and and to on for of was to the and the was for and for in a The nuclear were in of and a of and and the nuclear proteins were by at for was by and the nuclear were either used or in small at The protein concentrations of the nuclear were by the Biochem. PubMed Scopus Google Scholar). C element a site was with or purified SEF at for (16Huang J.H. Liao W.S. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). the were a in and to at for at The was For or to or NFκB-binding sites were used as specific sequences from gene promoters or used as in sequences the of the putative The the The sequences the of the putative The the in a A fragment of the and of the of mouse SAA3 promoter was into site of the to the Two and with in the and NFκB-binding were by with that mismatches to specific The with the shown in are as and We also two and and two and (15Huang J.H. Rienhoff Jr., H.Y. Liao W.S. Mol. Cell. Biol. 1990; 10: 3619-3625Crossref PubMed Scopus (28) Google Scholar). The human SEF was by as The SEF was into the site of a J.A. J. Biol. 1991; PubMed Scopus Google Scholar), which contains of the in at the of SEF were by medium was from as S. D. J. Immunol. 1981; Google Scholar). cells were from multiple by through for at cells were with and at of with serum and μg/ml at for the was from the cells by and and at cells were in of cells were with the plasmid DNAs to the used in the studies were by either or the For 0.5 of reporter was into cells with or or more expression To cytokine cells were with or for 24 h were for were for protein by the Biochem. PubMed Scopus Google Scholar), and the activity was to the nuclear were with protein for h at The were and antibody was to the and to with the nuclear for h at were to the and for at The were and three with proteins were in and a the proteins were and specific proteins were in and with specific or of NFκBp65 and SEF were with antibody by the system For were in and at for Our protein and antibody supershift that SEF is identical to or to the transcription factor LBP-1c/CP2/LSF (14Bing Z. Reddy S.A. Ren Y. Qin J. Liao W.S. J. Biol. Chem. 1999; 274: 24649-24656Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). To further SEF has the sequence binding as of to the sites were and used as specific in to shown in the sequences from the promoters of and whereas a binding from the promoter was as a but sequences for identical sequence binding further that SEF is identical to To the of SEF in SAA3 gene the reporter gene was into cells with of SEF expression of SEF SAA3 reporter gene expression in a albeit Intriguingly, of SEF-transfected cells with or resulted in dramatic synergistic activation of reporter gene expression. In the of and induced reporter gene by and In SEF-transfected cytokine-induced reporter gene expression was of SEF expression plasmid and induced the reporter gene by more than and that SEF an in the transcription of SAA3 promoter. it that SEF may cooperate with other cytokine-inducible transcription factors to confer synergistic activation on the SAA3 gene promoter. We had previously shown that the C/EBP family of transcription and a in SAA3 gene transcription (16Huang J.H. Liao W.S. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). all can reporter gene was the is also induced by S. Immunol. PubMed Google Scholar), we be the transcription factor that cooperates with SEF and for the synergistic activation of reporter gene expression. reporter gene was into cells with SEF and expression or in shown in of induced the SAA3 promoter by of SEF with resulted in rather than synergistic activation of reporter gene activity. therefore that may be the transcription factor that cooperates with SEF to confer the synergistic activation of the SAA3 reporter In to the C/EBP family of transcription is also a activator of and NFκB C.A. PubMed Google Scholar, S. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). NFκB has been shown to a in the of human and rat SAA1 genes as as the expression of other acute phase we to it also in the SAA3 promoter. To this we reporter into cells with the expression for either the or the of the and the expression which a functional had no on reporter gene expression. In cotransfection of NFκBp65 expression dramatically induced activity in a and at of the expression reporter gene activity was induced by To that the of reporter gene by NFκBp65 was a of the of NFκB in the of SAA3 promoter activity and the of its we cytokine-mediated induction of SAA3 promoter also NFκB by its in the cells were with reporter gene and either or expression plasmid and stimulated with or shown in in the of and stimulated the SAA3 promoter by and of resulted in than of the cytokine-induced This by was specific for NFκB activity had no on but completely activation are with the that NFκB is of the transcription factors by and that it in the of SAA3 promoter activity. We had shown previously that of the DRE from the SAA3 promoter completely abolished its cytokine responsiveness (16Huang J.H. Liao W.S. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). To the DRE also be necessary for activation of the SAA3 two and and two and of the SAA3 promoter were for responsiveness to by NFκBp65 and to the reporter was to and to by NFκBp65 to and the promoters completely to both of the DRE sequences in of two responsiveness to NFκB and suggesting that of the SAA3 promoter by NFκB is mediated through the DRE. To further DRE was for we of the DRE sequence in of a SV40 promoter to the promoter When into be induced by and by the NFκBp65 with the with the SAA3 promoter of also the responsiveness of the to and therefore the DRE of the SAA3 promoter as the regulatory that confers factors on gene transcription by binding to the promoter or enhancer of DRE activation of the SAA3 we the DRE sequence for NFκB-binding such site was found the C element of the it three with the consensus NFκB-binding sequence To this putative NFκB-binding sequence function as a binding site for we C element with NFκBp50, and the element formed were by For a consensus NFκB-binding sequence was also and used in with When the consensus NFκB-binding sequence was with NFκBp50, as as of was sufficient to a In binding to the C element was with of the protein after a weak was of this be by the NFκB-binding consensus but by that can interact with the C element despite its When with that of the consensus interaction between NFκB and the C element was to be several hundredfold a weak NFκB-binding site in the DRE was as shown in cotransfection NFκB is in a on the SAA3 promoter. To this weak binding site is functionally a reporter gene was in which the NFκB-binding site was this NFκB-binding site with that of SEF the C element we a mutation that it NFκB binding but SEF binding, as by The was into shown in mutation of this weak NFκB-binding site in the DRE the reporter to by the this was also to and that NFκB can albeit to a NFκB-binding site in the DRE and that this weak NFκB-binding site is nevertheless functionally for and cytokine-mediated activation of the SAA3 promoter. We that SEF dramatically the and induction of an SAA3 reporter gene suggesting that SEF may functionally cooperate with or more transcription factors to synergistically the SAA3 promoter. we that NFκB a in the of We therefore SEF and NFκB can function to the SAA3 promoter. reporter gene was into cells with or NFκBp65 expression or shown in cotransfection of the reporter gene with or NFκBp65 expression DNAs increased the reporter gene activity by and two expression were the activity was increased by more than synergy between SEF and of the of binding sites and functional cooperation in SAA3 promoter we SEF and NFκB in interact with other and an molecular basis for synergistic cells were for h were stimulated with either or for cells were and nuclear were and with antibody to SEF and its The of NFκB in the was by with shown in in a of NFκBp65 was in the of or the of NFκBp65 in the was increased The of NFκBp65 in the were at at after When were with the of SEF in was the that in the of NFκBp65 were to or with To further the of the we used serum in the and were to NFκBp65 therefore that SEF can protein-protein or with and with a rapid the synergy between SEF and NFκBp65 may be through physical To its functional importance in the cytokine we a reporter gene in which the site was The was for its responsiveness to cytokine shown in the an and increase in stimulated by and IL-1, In the was to cytokine This that SEF and its binding site an in maximum cytokine response on the SAA3 promoter. We a molecular for cytokine-induced mouse SAA3 gene expression acute or tissue by its regulatory elements in the promoter and transcription factors. Our studies identified a DRE that confer cytokine responsiveness and had properties of an inducible enhancer. Within the DRE, three functionally distinct to as the and C for SAA3 promoter The A element, a weak C/EBP-binding to the of SAA3 expression but its responsiveness to cytokine the other the B element, identified as a C/EBP-binding was for the and cytokine-induced of the SAA3 promoter. The C element, which interacts with the constitutive transcription factor was for both and cytokine-induced activation of SAA3 promoter (16Huang J.H. Liao W.S. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). In the study, we further the function of SEF in SAA3 gene whereas SEF by can the of SEF-transfected cells with or resulted in dramatic synergistic activation of the reporter We this as a that SEF cooperates functionally with or more transcription factors to up-regulate SAA3 gene transcription. We had previously shown that an transcription factor, the SAA3 promoter through the DRE. We therefore for functional cooperation between SEF and no synergistic induction was NFκB is of the transcription factors that the we NFκB by the SAA3 promoter. we found that it The that NFκB the SAA3 promoter was sequence analysis of the SAA3 promoter an NFκB-binding and functional for an functional of NFκB in the of SAA3 in response to cytokine cotransfection of NFκBp65 with a reporter gene dramatically induced reporter gene expression in a and activation of SAA3 promoter were completely by are show that NFκB can SAA3 promoter in an more that NFκB in such as the cytokine-mediated transcription to SAA3 promoter. Finally, a NFκB-binding sequence was identified the C element. 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