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. 1996 Apr;16(4):1584–1594. doi: 10.1128/mcb.16.4.1584

A novel cis-acting element is essential for cytokine-mediated transcriptional induction of the serum amyloid A gene in nonhepatic cells.

A Ray 1, B K Ray 1
PMCID: PMC231144  PMID: 8657133

Abstract

Serum amyloid A (SAA) is a plasma protein which has been associated with several diseases, including amyloidosis, arthritis, and atherosclerosis, and its abnormal expression, particularly in nonhepatic cells, is implicated in the pathogenesis of these diseases. Transfection and DNA-binding studies were performed to investigate the mechanism controlling cytokine-induced, nonhepatic expression of the SAA gene. We have identified a novel promoter, located between positions -280 and 224, that confers interleukin-6 (IL-6) inducibility to an SAA-chloramphenicol acetyltransferase reporter gene in both nonhepatic and hepatic cells. DNase I protection assays revealed, within this region, three homologous highly pyrimidine rich octanucleotide sequence motifs, termed SAA-activating sequences (SAS). Specific mutations within these three SAS motifs severely reduced IL-6-mediated induction of the reporter gene in transfected nonhepatic cells but not in liver cells. A nuclear factor activated by IL-6 in both hepatic and nonhepatic cells efficiently interacts with the SAS. The induction kinetics and cycloheximide sensitivity of this SAS-binding factor (SAF) suggested that de novo synthesis of this factor itself or an activator protein is essential. Loss of DNA-binding ability as a result of in vitro dephosphorylation, induction of SAA-chloramphenicol acetyltransferase reporter gene activity in the presence of genistein, a protein kinase inhibitor, further indicate that a phosphorylation step is necessary for the activation of SAF. Our results suggest that SAF is a key regulator of cytokine-mediated SAA gene expression in some nonhepatic cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akira S., Nishio Y., Inoue M., Wang X. J., Wei S., Matsusaka T., Yoshida K., Sudo T., Naruto M., Kishimoto T. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell. 1994 Apr 8;77(1):63–71. doi: 10.1016/0092-8674(94)90235-6. [DOI] [PubMed] [Google Scholar]
  2. Aldo-Benson M. A., Benson M. D. SAA suppression of immune response in vitro: evidence for an effect on T cell-macrophage interaction. J Immunol. 1982 Jun;128(6):2390–2392. [PubMed] [Google Scholar]
  3. Banka C. L., Yuan T., de Beer M. C., Kindy M., Curtiss L. K., de Beer F. C. Serum amyloid A (SAA): influence on HDL-mediated cellular cholesterol efflux. J Lipid Res. 1995 May;36(5):1058–1065. [PubMed] [Google Scholar]
  4. Benson M. D., Cohen A. S. Serum amyloid A protein in amyloidosis, rheumatic, and enoplastic diseases. Arthritis Rheum. 1979 Jan;22(1):36–42. doi: 10.1002/art.1780220106. [DOI] [PubMed] [Google Scholar]
  5. Betts J. C., Cheshire J. K., Akira S., Kishimoto T., Woo P. The role of NF-kappa B and NF-IL6 transactivating factors in the synergistic activation of human serum amyloid A gene expression by interleukin-1 and interleukin-6. J Biol Chem. 1993 Dec 5;268(34):25624–25631. [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Brinckerhoff C. E., Mitchell T. I., Karmilowicz M. J., Kluve-Beckerman B., Benson M. D. Autocrine induction of collagenase by serum amyloid A-like and beta 2-microglobulin-like proteins. Science. 1989 Feb 3;243(4891):655–657. doi: 10.1126/science.2536953. [DOI] [PubMed] [Google Scholar]
  8. Cao Z., Umek R. M., McKnight S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991 Sep;5(9):1538–1552. doi: 10.1101/gad.5.9.1538. [DOI] [PubMed] [Google Scholar]
  9. Coetzee G. A., Strachan A. F., van der Westhuyzen D. R., Hoppe H. C., Jeenah M. S., de Beer F. C. Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition. J Biol Chem. 1986 Jul 25;261(21):9644–9651. [PubMed] [Google Scholar]
  10. De Beer F. C., Mallya R. K., Fagan E. A., Lanham J. G., Hughes G. R., Pepys M. B. Serum amyloid-A protein concentration in inflammatory diseases and its relationship to the incidence of reactive systemic amyloidosis. Lancet. 1982 Jul 31;2(8292):231–234. doi: 10.1016/s0140-6736(82)90321-x. [DOI] [PubMed] [Google Scholar]
  11. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dynan W. S., Tjian R. Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. 1985 Aug 29-Sep 4Nature. 316(6031):774–778. doi: 10.1038/316774a0. [DOI] [PubMed] [Google Scholar]
  13. Edbrooke M. R., Burt D. W., Cheshire J. K., Woo P. Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor kappaB-like transcription factor. Mol Cell Biol. 1989 May;9(5):1908–1916. doi: 10.1128/mcb.9.5.1908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ghosh D. Status of the transcription factors database (TFD). Nucleic Acids Res. 1993 Jul 1;21(13):3117–3118. doi: 10.1093/nar/21.13.3117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ghysdael J., Gegonne A., Pognonec P., Dernis D., Leprince D., Stehelin D. Identification and preferential expression in thymic and bursal lymphocytes of a c-ets oncogene-encoded Mr 54,000 cytoplasmic protein. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1714–1718. doi: 10.1073/pnas.83.6.1714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gilmour K. C., Pine R., Reich N. C. Interleukin 2 activates STAT5 transcription factor (mammary gland factor) and specific gene expression in T lymphocytes. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10772–10776. doi: 10.1073/pnas.92.23.10772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  18. Hoffman J. S., Benditt E. P. Changes in high density lipoprotein content following endotoxin administration in the mouse. Formation of serum amyloid protein-rich subfractions. J Biol Chem. 1982 Sep 10;257(17):10510–10517. [PubMed] [Google Scholar]
  19. Husebekk A., Skogen B., Husby G., Marhaug G. Transformation of amyloid precursor SAA to protein AA and incorporation in amyloid fibrils in vivo. Scand J Immunol. 1985 Mar;21(3):283–287. doi: 10.1111/j.1365-3083.1985.tb01431.x. [DOI] [PubMed] [Google Scholar]
  20. Kinoshita S., Akira S., Kishimoto T. A member of the C/EBP family, NF-IL6 beta, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1473–1476. doi: 10.1073/pnas.89.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kisilevsky R., Subrahmanyan L. Serum amyloid A changes high density lipoprotein's cellular affinity. A clue to serum amyloid A's principal function. Lab Invest. 1992 Jun;66(6):778–785. [PubMed] [Google Scholar]
  22. Kushner I. The phenomenon of the acute phase response. Ann N Y Acad Sci. 1982;389:39–48. doi: 10.1111/j.1749-6632.1982.tb22124.x. [DOI] [PubMed] [Google Scholar]
  23. Li X., Liao W. S. Cooperative effects of C/EBP-like and NF kappa B-like binding sites on rat serum amyloid A1 gene expression in liver cells. Nucleic Acids Res. 1992 Sep 25;20(18):4765–4772. doi: 10.1093/nar/20.18.4765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liang J. S., Sipe J. D. Recombinant human serum amyloid A (apoSAAp) binds cholesterol and modulates cholesterol flux. J Lipid Res. 1995 Jan;36(1):37–46. [PubMed] [Google Scholar]
  25. Linke R. P., Bock V., Valet G., Rothe G. Inhibition of the oxidative burst response of N-formyl peptide-stimulated neutrophils by serum amyloid-A protein. Biochem Biophys Res Commun. 1991 May 15;176(3):1100–1105. doi: 10.1016/0006-291x(91)90397-p. [DOI] [PubMed] [Google Scholar]
  26. Lowell C. A., Potter D. A., Stearman R. S., Morrow J. F. Structure of the murine serum amyloid A gene family. Gene conversion. J Biol Chem. 1986 Jun 25;261(18):8442–8452. [PubMed] [Google Scholar]
  27. Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Meek R. L., Benditt E. P. Amyloid A gene family expression in different mouse tissues. J Exp Med. 1986 Dec 1;164(6):2006–2017. doi: 10.1084/jem.164.6.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meek R. L., Urieli-Shoval S., Benditt E. P. Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3186–3190. doi: 10.1073/pnas.91.8.3186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mitchell T. I., Coon C. I., Brinckerhoff C. E. Serum amyloid A (SAA3) produced by rabbit synovial fibroblasts treated with phorbol esters or interleukin 1 induces synthesis of collagenase and is neutralized with specific antiserum. J Clin Invest. 1991 Apr;87(4):1177–1185. doi: 10.1172/JCI115116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Poli V., Cortese R. Interleukin 6 induces a liver-specific nuclear protein that binds to the promoter of acute-phase genes. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8202–8206. doi: 10.1073/pnas.86.21.8202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ray A., Hannink M., Ray B. K. Concerted participation of NF-kappa B and C/EBP heteromer in lipopolysaccharide induction of serum amyloid A gene expression in liver. J Biol Chem. 1995 Mar 31;270(13):7365–7374. doi: 10.1074/jbc.270.13.7365. [DOI] [PubMed] [Google Scholar]
  33. Ray A., Ray B. K. Analysis of the promoter element of the serum amyloid A gene and its interaction with constitutive and inducible nuclear factors from rabbit liver. Gene Expr. 1993;3(2):151–162. [PMC free article] [PubMed] [Google Scholar]
  34. Ray A., Ray B. K. 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. Mol Cell Biol. 1994 Jun;14(6):4324–4332. doi: 10.1128/mcb.14.6.4324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ray B. K., Gao X., Ray A. Regulation of rabbit alpha 1-acid glycoprotein gene expression in acute-phase liver. Identification of inducible and constitutive proteins like CCAAT-enhancer binding protein that interact with the 5'-proximal promoter elements. Eur J Biochem. 1993 Aug 15;216(1):127–136. doi: 10.1111/j.1432-1033.1993.tb18124.x. [DOI] [PubMed] [Google Scholar]
  36. Ray B. K., Ray A. Expression of the gene encoding alpha 1-acid glycoprotein in rabbit liver under acute-phase conditions involves induction and activation of beta and delta CCAAT-enhancer-binding proteins. Eur J Biochem. 1994 Jun 15;222(3):891–900. doi: 10.1111/j.1432-1033.1994.tb18937.x. [DOI] [PubMed] [Google Scholar]
  37. Ray B. K., Ray A. Functional NF-kappa B element in rabbit serum amyloid A gene and its role in acute phase induction. Biochem Biophys Res Commun. 1993 Jun 30;193(3):1159–1167. doi: 10.1006/bbrc.1993.1747. [DOI] [PubMed] [Google Scholar]
  38. Ray B. K., Ray A. Sp1-mediated transactivation of the rabbit alpha 1 acid glycoprotein-encoding gene involves a cis-acting element in the 5'-proximal promoter region. Gene. 1994 Sep 30;147(2):253–258. doi: 10.1016/0378-1119(94)90076-0. [DOI] [PubMed] [Google Scholar]
  39. Rokita H., Shirahama T., Cohen A. S., Meek R. L., Benditt E. P., Sipe J. D. Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes. J Immunol. 1987 Dec 1;139(11):3849–3853. [PubMed] [Google Scholar]
  40. Ron D., Habener J. F. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 1992 Mar;6(3):439–453. doi: 10.1101/gad.6.3.439. [DOI] [PubMed] [Google Scholar]
  41. Saffer J. D., Jackson S. P., Annarella M. B. Developmental expression of Sp1 in the mouse. Mol Cell Biol. 1991 Apr;11(4):2189–2199. doi: 10.1128/mcb.11.4.2189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schindler C., Darnell J. E., Jr Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem. 1995;64:621–651. doi: 10.1146/annurev.bi.64.070195.003201. [DOI] [PubMed] [Google Scholar]
  43. Sen R., Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell. 1986 Aug 29;46(5):705–716. doi: 10.1016/0092-8674(86)90346-6. [DOI] [PubMed] [Google Scholar]
  44. Shuai K., Schindler C., Prezioso V. R., Darnell J. E., Jr Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein. Science. 1992 Dec 11;258(5089):1808–1812. doi: 10.1126/science.1281555. [DOI] [PubMed] [Google Scholar]
  45. Trautwein C., Caelles C., van der Geer P., Hunter T., Karin M., Chojkier M. Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain. Nature. 1993 Aug 5;364(6437):544–547. doi: 10.1038/364544a0. [DOI] [PubMed] [Google Scholar]
  46. Urieli-Shoval S., Meek R. L., Hanson R. H., Eriksen N., Benditt E. P. Human serum amyloid A genes are expressed in monocyte/macrophage cell lines. Am J Pathol. 1994 Sep;145(3):650–660. [PMC free article] [PubMed] [Google Scholar]
  47. Wakao H., Gouilleux F., Groner B. Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J. 1994 May 1;13(9):2182–2191. doi: 10.1002/j.1460-2075.1994.tb06495.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wen Z., Zhong Z., Darnell J. E., Jr Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell. 1995 Jul 28;82(2):241–250. doi: 10.1016/0092-8674(95)90311-9. [DOI] [PubMed] [Google Scholar]
  49. Williams S. C., Cantwell C. A., Johnson P. F. A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Dev. 1991 Sep;5(9):1553–1567. doi: 10.1101/gad.5.9.1553. [DOI] [PubMed] [Google Scholar]
  50. Wilson D. R., Juan T. S., Wilde M. D., Fey G. H., Darlington G. J. A 58-base-pair region of the human C3 gene confers synergistic inducibility by interleukin-1 and interleukin-6. Mol Cell Biol. 1990 Dec;10(12):6181–6191. doi: 10.1128/mcb.10.12.6181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhong Z., Wen Z., Darnell J. E., Jr Stat3 and Stat4: members of the family of signal transducers and activators of transcription. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4806–4810. doi: 10.1073/pnas.91.11.4806. [DOI] [PMC free article] [PubMed] [Google Scholar]

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