Serum Amyloid A Promoter Research Articles

Characterization of G‐quadruplex Structures in Serum Amyloid A Gene as a Novel Approach for Systemic Amyloidosis Therapy

Transcriptional initiation is an important step in the regulation of transcription. When transcriptional initiation is dysfunctional, it will lead to abnormal gene expression. Overexpression of Serum Amyloid A (SAA) and self-assembly into fibrils have been associated with chronic inflammatory conditions and certain neoplasms, which lead to the deposition of AA amyloid in organs such as the liver, spleen, and kidney (systemic amyloidosis). For this reason, SAA is a highly significant molecular target. Insight into the structural biology of the promoter element that determines the combinatorial control of gene expression by different transcriptional factors is essential for understanding the dysfunction of SAA gene expression. The highly GC-rich region of the human SAA promoter is crucial for basal promoter activity and likely to assume a DNA secondary structure, such as G-quadruplex (GQ). GQs often serve as transcriptional silencer elements. Stabilization of GQ is possible with small molecule therapeutics to modulate gene expression. In the present study, G-quadruplex formation has been isolated from the GC-rich region of the 5’-untranslated region (5’UTR) of SAA2. An electrophoretic mobility shift and thioflavin T fluorescence assays were used to confirm the formation of a biologically relevant intramolecular structure. The structure of the G-quadruplex is currently under investigation by circular dichroism and DMS footprinting. These data characterizing the formation of a unique secondary structure in SAA gene are promising to develop novel anti-amyloid therapy for the treatment of systemic amyloidosis.

Salmonid Tollip and MyD88 factors can functionally replace their mammalian orthologues in TLR-mediated trout SAA promoter activation

Many functional details of the piscine Toll-like receptor (TLR) signal-mediated activation of immune defense are still elusive. We used an established reconstitution system of mammalian TLR signaling to examine if this system would allow for pathogen-dependent promoter activation of the serum amyloid A (SAA)-encoding gene from rainbow trout ( Oncorhynchus mykiss) and if the key mediators MyD88 and Tollip from trout can functionally substitute for their mammalian orthologues. Cells of the established human embryonic kidney line HEK-293 were transiently co-transfected with vectors expressing bovine TLR2 or TLR4 factors and a reporter gene driven by the promoter of the trout SAA gene. Escherichia coli stimulation increased reporter gene expression more than 3-fold. Deletion series and point mutations identified in the proximal SAA promoter a composite overlapping binding site for NF-κB and CEBP factors as crucial for promoter activation. Overexpression of NF-κB p65, but not of p50 or different members of the CEBP factor family proved this factor as an essential driver for SAA expression. Overexpression of a transdominant-negative mutant of the trout MyD88 factor reduced TLR-mediated SAA promoter activation confirming functional conservation of its TIR domain. Overexpression of the Tollip factor from trout also quenched TLR-mediated NF-κB and TLR4-mediated SAA promoter activation. The MyD88 mutant and Tollip expression studies confirm the functional homology of both piscine factors and their mammalian counterparts. We provide for the first time evidence that also the Tollip-mediated negative loop of TLR signaling may be conserved in non-mammalian organisms.

Interaction of Adenosine 3′,5′-Cyclic Monophosphate and Tumor Necrosis Factor-α on Serum Amyloid A3 Expression in Mouse Granulosa Cells: Dependence on CCAAT-Enhancing Binding Protein-β Isoform

TNFalpha is an inflammatory-related cytokine that has inhibitory effects on gonadotropin- and cAMP-stimulated steroidogenesis and folliculogenesis. Because ovulation is an inflammatory reaction and TNF specifically induces serum amyloid A3 (SAA3) in mouse granulosa cells, the effect of cAMP on TNF-induced SAA3 promoter activity, mRNA and protein was investigated. Granulosa cells from immature mice were cultured with TNF and/or cAMP. TNF increased SAA3 promoter activity, mRNA, and protein, which were further increased by cAMP. cAMP alone increased SAA3 promoter activity, but SAA3 mRNA and protein remained undetectable. Thus, there appeared to be different mechanisms by which TNF and cAMP regulated SAA3 expression. SAA3 promoters lacking a nuclear factor (NF)-kappaB-like site or containing its mutant were not responsive to TNF but were responsive to cAMP. Among four CCAAT-enhancing binding protein (C/EBP) sites in the SAA3 promoter, the C/EBP site nearest the NF-kappaB-like site was required for TNF-induced SAA3. The C/EBP site at -75/-67 was necessary for responsiveness to cAMP. Dominant-negative C/EBP and cAMP response element-binding protein or short interfering RNA of C/EBPbeta blocked TNF- or cAMP-induced SAA3 promoter activity. The combination of TNF and cAMP increased C/EBPbeta protein above that induced by TNF or cAMP alone. Thus, cAMP in combination with TNF specifically induced C/EBPbeta protein, leading to enhanced SAA3 expression but requiring NF-kappaB in mouse granulose cells. In addition, like TNF, SAA inhibited cAMP-induced estradiol accumulation and CYP19 levels. These data indicate SAA may play a role in events occurring during the ovulation process.

TGF-beta down-regulates IL-1alpha-induced TLR2 expression in murine hepatocytes.

We have previously reported that the proinflammatory cytokine interleukin (IL)-1alpha can up-regulate functional Toll-like receptor 2 (TLR2) expression in primary-cultured murine hepatocytes, and bacterial lipopeptide (BLP) is capable of signaling through TLR2 to induce serum amyloid A (SAA) expression in hepatocytes. In the present study, we investigated the effect of the anti-inflammatory cytokine transforming growth factor-beta (TGF-beta) on TLR2 expression in primary-cultured murine hepatocytes. At the mRNA and protein levels, TGF-beta up-regulated TLR2 expression but inhibited TLR2 expression induced by IL-1alpha at 24 h. BLP-induced SAA promoter activity could be augmented by pretreatment with IL-1alpha but not TGF-beta or the combination of TGF-beta and IL-1alpha. TLR2 promoter activity and nuclear factor (NF)-kappaB activation by IL-1alpha were inhibited by TGF-beta treatment. Pretreatment with TGF-beta strongly suppressed IL-1alpha-induced TLR2 promoter activity and NF-kappaB activation, which was consistent with the down-regulation of type I IL-1 receptor (IL-1RI) mRNA expression. IL-1alpha up-regulated IL-1RI mRNA, but it was inhibited by the treatment with TGF-beta. These results suggest that TGF-beta suppresses the induction of TLR2 expression by IL-1alpha through down-regulation of IL-1RI expression. These results also demonstrate the disparity between IL-1alpha and TGF-beta in regulating TLR2-mediated SAA production in hepatocytes.

Tumor necrosis factor-alpha induces serum amyloid A3 in mouse granulosa cells.

TNF-alpha has significant inhibitory effects on steroidogenesis and folliculogenesis and is associated with several inflammatory responses. Because ovulation is an inflammatory reaction, the effects of TNF on the family of acute-phase proteins in granulosa cells were investigated. Granulosa cells from immature mice at 28 d of age were cultured in the presence of 10 ng TNF/ml for 24 h. Serum amyloid A3 (SAA3), a main acute-phase protein, was induced by TNF in granulosa cells. The other isoforms of serum amyloid proteins SAA1, SAA2, and SAA4 were neither expressed in granulosa cells nor induced by TNF. TNF did not induce SAA3 mRNA in granulosa cells from TNF receptor type 1 (TNFR1) knockout mice, although SAA3 mRNA was induced within 3 h after TNF treatment in wild-type cells. Two SAA3 promoters, -617/+73 and -198/+73, were responsive to TNF and to p65, a component of the TNF signaling molecule nuclear factor (NF)-kappaB. The -106/+73 promoter of SAA3 lacking a NF-kappaB-like site was not responsive to TNF or p65. In granulosa cells from TNFR1 knockout mice, the SAA3 promoter (-198/+73) was responsive to transfection with the p65 component of NF-kappaB, but neither TNF treatment nor overexpression of the p50 component of NF-kappaB increased promoter activity. Similar results were observed in the murine ovarian granulosa tumor cell line (OV3121-1). Overexpression of the inhibitor of NF-kappaB (called IkappaB) blocked SAA3 promoter activity induced by TNF and by p65 in OV3121-1 cells. Closer analysis of deletion mutants of the SAA3 promoter revealed the necessity of a NF-kappaB like site for responsiveness to TNF in the OV3121-1 cells. TNF rapidly increased p65 in OV3121-1 nuclei when compared with controls not treated with TNF. TNF also increased phospho-IkB and SAA3 in whole-cell homogenates as determined by Western blots. Thus, TNF likely increased SAA3 promoter activity and protein by activating NF-kappaB signaling via TNFR1 in mouse granulosa cells. SAA3 is a novel gene in granulosa cells with yet unknown functions in the ovary.

Functional analysis of a minimal mouse serum amyloid A3 promoter in transgenic mice

We report the generation of transgenic mice harboring the SAA3/LacZ transgene and analysis of its expression patterns in vivo following LPS-induced inflammation. Our results show that a 210-bp fragment of the mouse SAA3 promoter when placed in front of the LacZ gene was sufficient to confer basal and inflammation-induced reporter gene expression. Consistent with endogenous SAA3 expression, the basal level of LacZ expression was high in the lung and liver of newborn and 1-week-old transgenic mice. Its expression however decreased with increasing age and at 3-weeks of age, LacZ expression was very low in the lung and was essentially undetectable in the liver. When SAA 3/LacZ transgenic mice were injected with lipopolisaccharide to induce inflammation, β-gal activities were increased approximately 6- and 16-fold in the lung and liver, respectively. Histological examination of lung and liver tissues stained with X-gal revealed that the LacZ transgene was expressed primarily in the macrophages. Thus, this minimal SAA3 promoter fragment contains the necessary regulatory sequences for its expression and cytokine responsiveness in macrophages albeit is insufficient to confer expression in hepatocytes.

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 NFkappaBp65 expression DNAs resulted in synergistic activation of the SAA3 promoter. Intriguingly, no consensus NFkappaB-binding site was found in the SAA3 promoter region; rather a putative NFkappaB-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 NFkappaBp50, albeit with binding affinities that were several hundredfold lower than that with the consensus NFkappaB probe. Functional cooperation between SEF and NFkappaB was further strengthened by the finding that SEF and NFkappaB formed stable cytokine-inducible protein-protein complexes. Finally, despite its weak binding, mutation of this NFkappaB-binding site nevertheless dramatically reduced both NFkappaBp65- and cytokine-mediated induction of SAA3 promoter. Therefore, the molecular basis for the functional synergy between SEF and NFkappaB may, in part, be the ability of SEF to recruit NFkappaB through physical interactions that lead to enhancement or stabilization of NFkappaB binding to the SAA3 promoter element.

Synergistic induction of mouse serum amyloid A3 promoter by the inflammatory mediators IL-1 and IL-6.

Serum amyloid A (SAA), one of the major acute-phase proteins, increases several hundredfold in concentration in plasma following acute inflammation, primarily as a result of a 200-fold increase in its transcription rate. We have previously demonstrated that a 350-bp promoter fragment from the mouse SAA3 gene could confer conditioned medium-induced expression in cultured cells. The induction is mediated through a 42-bp distal response element (DRE) consisting of three functional regulatory elements. In this study, we show that interleukin-1 (IL-1) is the major cytokine in the conditioned medium responsible for SAA3 induction, and the induction by IL-1 can be effectively blocked by H-7, a protein kinase C inhibitor. Although IL-6 alone had no effect on SAA3 promoter activity, the addition of IL-6 and IL-1 resulted in dramatic synergistic activation of the reporter gene. We further show that the DRE is both necessary and sufficient to confer synergistic induction by IL-1 and IL-6. Moreover, individual mutation of the three regulatory elements within DRE either abolished or drastically reduced the synergistic induction. Our results indicate that synergistic activation of SAA3 promoter by IL-1 and IL-6 is achieved through integration of signals triggered by these two cytokines onto the DRE and that all three functionally distinct regulatory elements in the DRE are required to effectively and fully activate SAA3 gene transcription.

Activation of Sp1 and Its Functional Co-operation with Serum Amyloid A-activating Sequence Binding Factor in Synoviocyte Cells Trigger Synergistic Action of Interleukin-1 and Interleukin-6 in Serum Amyloid A Gene Expression

The serum amyloid A (SAA) protein has been implicated in the progression and pathogenesis of rheumatoid arthritis through induction of collagenase activity in synovial fibroblast cells that line the joint tissues. We demonstrate that SAA is synergistically induced in synovial cells by interleukin (IL)-1 and IL-6 that are present at significantly high level in the synovial fluid of arthritis patients. These cytokines induced phenotypic changes in synovial cells, promoting protrusion and increased cellular contact. Induction of SAA under this condition is mediated by promoter elements located between -254 and -226, which contains binding sites for transcription factors Sp1 and SAA activating sequence binding factor (SAF). Mutation of these sequences abolishes SAA promoter response to IL-1 and IL-6. The role of Sp1 in SAA induction was demonstrated by increased DNA binding activity, phosphorylation, and increased protein content of Sp1 during cytokine treatment. Sp1 interacts with the SAA promoter in association with SAF as an SAF. Sp1 heteromeric complex. Furthermore, using a phosphatase inhibitor, we demonstrated increased transactivation potential of both Sp1 and SAF as a consequence of a phosphorylation event. These results provide first evidence for cytokine-mediated activation of Sp1 in synovial fibroblast cells and its participation in regulating SAA expression by acting in conjunction with SAF.

Isolation and functional characterization of cDNA of serum amyloid A-activating factor that binds to the serum amyloid A promoter.

Serum amyloid A (SAA), a plasma protein inducible in response to many inflammatory conditions, is associated with the pathogenesis of several diseases including reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. We have previously reported an element of the SAA promoter, designated SAA-activating sequence (SAS), that is involved in the inflammation-induced SAA expression, and a nuclear factor, SAS-binding factor (SAF), that interacts with the SAS element has been identified previously (A. Ray and B. K. Ray, Mol. Cell. Biol. 16:1584-1594, 1996). To evaluate how SAF is involved in SAA promoter activation, we have investigated structural features and functional characteristics of this transcription factor. Our studies indicate that SAF belongs to a family of transcription factors characterized by the presence of multiple zinc finger motifs of the Cys2-His2 type at the carboxyl end. Of the three cloned SAF cDNAs (SAF-1, SAF-5, and SAF-8), SAF-1 isoform showed a high degree of homology to MAZ/ZF87/Pur-1 protein while SAF-5 and SAF-8 isoforms are unique and are related to SAF-1/MAZ/ZF87/Pur-1 at the zinc finger domains but different elsewhere. Although structurally distinct, all members are capable of activating SAS element-mediated expression and display virtually identical sequence specificities. However, varying levels of expression of members of this gene family were observed in different tissues. Functional activity of SAF is regulated by a posttranslational event as SAF DNA-binding and transactivation abilities are increased by a protein phosphatase inhibitor, okadaic acid, and inhibited by a protein kinase inhibitor, H7. Consistent with this observation, increased DNA binding of the cloned SAF and its hyperphosphorylation, in response to okadaic acid treatment of the transfected cells, were observed. Taken together, our results suggest that, in addition to tissue-specific expression, SAFs, a family of zinc finger transcription factors, undergo a modification by a posttranslational event that confers their SAA promoter-binding activity and transactivation potential.

Involvement of an SAF-like transcription factor in the activation of serum amyloid A gene in monocyte/macrophage cells by lipopolysaccharide.

Serum amyloid A (SAA) has been linked to atherosclerosis because of its ability to remodel high-density lipoprotein by the depletion of apolipoprotein A1, its ability to bind cholesterol, and its presence in the atherosclerotic plaques of coronary and carotid arteries. In the present study, we investigated the induction mechanism of SAA gene in THP-1 monocyte/macrophage cells which play a critical role in the development of atherosclerotic fatty streak and plaque formation. We and others have shown that SAA gene is induced in monocyte/macrophage cells by lipopolysaccharide (LPS). By promoter function analysis, we show that the SAA promoter sequence between -280 and -226 can confer LPS responsiveness. Gel electrophoretic mobility shift assay detected an induced DNA-binding activity in these cells in response to LPS. Characterization of the DNA-binding protein by UV cross-linking, Southwestern blot, and antibody ablation/supershift assays revealed that it is similar to a recently reported nuclear factor designated SAF. These results demonstrated that LPS-mediated SAA gene induction in monocyte/macrophage cells is primarily due to the induction of SAF activity.

Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.

We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal luciferase expression observed prior to inflammatory challenge, markedly increased expression from the C3 promoter was detected in liver in response to both lipopolysaccharide (LPS) and turpentine, and lower-level inducible expression was also found in lung. In contrast, expression from the SAA3 promoter was found only in liver and was much more responsive to LPS than to turpentine. After LPS challenge, hepatic luciferase expression increased rapidly and in proportion to the LPS dose. Use of cytokine-inducible promoters in gene transfer vectors may make it possible to produce antiinflammatory proteins in vivo in direct relationship to the intensity and duration of an individual's inflammatory response. By providing endogenously controlled production of recombinant antiinflammatory proteins, this approach might limit the severity of the inflammatory response without interfering with the beneficial components of host defense and immunity.

Concerted Participation of NF-κB and C/EBP Heteromer in Lipopolysaccharide Induction of Serum Amyloid A Gene Expression in Liver

The promoter region of the rabbit serum amyloid A (SAA) gene contains two adjacent C/EBP and one NF-kappa B binding element. Involvement of these elements in SAA gene induction, following lipopolysaccharide (LPS) stimulation of the liver, has been studied by investigating LPS-activated transcription factors and their interaction with the promoter elements of the SAA gene. Appearance of complexes in the electrophoretic mobility shift assay has indicated that DNA-binding proteins that interact with the NF-kappa B element of the SAA promoter are induced in the LPS-treated rabbit liver. Presence of RelA (p65 subunit of NF-kappa B) in these complexes was demonstrated by the ability of RelA-specific antisera to supershift the DNA-protein complexes. LPS also induced several members of the C/EBP family of transcription factors, which interacted with the C/EBP motifs of the SAA promoter. Activated C/EBP and RelA form a RelA-C/EBP heteromeric complex that associates with varying affinity to NF-kappa B and C/EBP elements of the SAA gene. Transfection assays using both transcription factor genes have demonstrated that the heteromeric complex of NF-kappa B and C/EBP is a much more potent transactivator of SAA expression than each transcription factor alone. The heteromeric complex efficiently promotes transcription from both NF-kappa B and C/EBP sites.

Serum amyloid A gene expression under acute-phase conditions involves participation of inducible C/EBP-beta and C/EBP-delta and their activation by phosphorylation.

Serum amyloid A (SAA) is a plasma protein whose synthesis is markedly increased in the liver during the inflammatory process. Previous analysis of SAA promoter function implicated the involvement of the CCAAT/enhancer-binding protein (C/EBP) in controlling this process. In this study, using antibodies against three C/EBP isoforms in DNA-binding and Western blot (immunoblot) assays, we found that in response to inflammatory signals, both C/EBP-delta and C/EBP-beta are induced and that their interactions with the SAA promoter element are necessary for the increased SAA gene expression. Cotransfections of liver cells with an SAA promoter-linked reporter chloramphenicol acetyltransferase gene and murine sarcoma virus-expressed C/EBP-delta or C/EBP-beta confirm such phenomena. The increased transactivating ability in the presence of the cellular phosphatase inhibitors okadaic acid and sodium orthovanadate, coupled with the observation that dephosphorylation severely inhibits the DNA-binding ability in vitro, implicates a role of phosphorylation in the regulation of the activities of the C/EBP-delta isoform. Consistent with these findings, we have detected higher levels of DNA-binding activity of C/EBP-delta prepared from cells treated with phosphatase inhibitors. We also present evidence that C/EBP-delta is a phosphoprotein. These results suggest that C/EBP-delta is regulated by phosphorylation and, in conjunction with C/EBP-beta, is one of the major proteins responsible for the increased transcription of the SAA gene in response to inflammatory stimuli.

Serum amyloid A gene expression under acute-phase conditions involves participation of inducible C/EBP-beta and C/EBP-delta and their activation by phosphorylation.

Serum amyloid A (SAA) is a plasma protein whose synthesis is markedly increased in the liver during the inflammatory process. Previous analysis of SAA promoter function implicated the involvement of the CCAAT/enhancer-binding protein (C/EBP) in controlling this process. In this study, using antibodies against three C/EBP isoforms in DNA-binding and Western blot (immunoblot) assays, we found that in response to inflammatory signals, both C/EBP-delta and C/EBP-beta are induced and that their interactions with the SAA promoter element are necessary for the increased SAA gene expression. Cotransfections of liver cells with an SAA promoter-linked reporter chloramphenicol acetyltransferase gene and murine sarcoma virus-expressed C/EBP-delta or C/EBP-beta confirm such phenomena. The increased transactivating ability in the presence of the cellular phosphatase inhibitors okadaic acid and sodium orthovanadate, coupled with the observation that dephosphorylation severely inhibits the DNA-binding ability in vitro, implicates a role of phosphorylation in the regulation of the activities of the C/EBP-delta isoform. Consistent with these findings, we have detected higher levels of DNA-binding activity of C/EBP-delta prepared from cells treated with phosphatase inhibitors. We also present evidence that C/EBP-delta is a phosphoprotein. These results suggest that C/EBP-delta is regulated by phosphorylation and, in conjunction with C/EBP-beta, is one of the major proteins responsible for the increased transcription of the SAA gene in response to inflammatory stimuli.

Identification of a Transcriptional Enhancer in a Mouse Amyloid Gene

We have employed a mouse liver-derived cell line (BNL) to identify DNA sequences in the promoter of the serum amyloid A3 (SAA3) gene essential for expression. In both transient DNA transfection assays and BNL cells stably transformed with SAA3 fusion genes, deletion analysis of the SAA3 promoter indicates that sequences between -185 and -138 base pairs 5' to the transcription initiation site are essential for expression of heterologous genes. A 69-base pair sequence spanning this region markedly augmented expression of the bacterial chloramphenicol transacetylase gene regardless of orientation or position. The homology of this sequence with sequences in the promotors of other genes expressed by liver during inflammation suggests a common mechanism of regulation.