title: Use of the acute phase serum amyloid A2 (SAA2) gene promoter in the analysis of pro- and anti-inflammatory mediators: differential kinetics of SAA2 promoter induction by IL-1 beta and TNF-alpha compared to IL-6.
abstract: A cytokine responsive construct, pGL2-SAA2pt, was generated by cloning the acute phase promoter of human serum amyloid A2 (SAA2) upstream of a luciferase reporter gene. The construct responds to the inflammatory mediators MoCM, IL-1 beta, TNF-alpha, and IL-6 in a manner that closely mimics the response of the endogenous SAA2 gene to such stimuli: i.e. single treatments induce transcriptional activation by IL-1 beta and TNF-alpha to a greater extent than by IL-6 at 12-24 h. However, timecourse experiments show that the kinetics of induction generated by IL-1 beta and TNF-alpha are quite distinct from IL-6, IL-6 having a much greater effect at 3-6 h. IL-1 beta and TNF-alpha synergize with IL-6 to give a 10-fold increase in transcriptional readout over single cytokine treatments. The kinetics of this synergistic response resembles that generated by IL-6 alone. The IL-1 receptor antagonist, hIL-1ra, can specifically block the IL-1 beta driven transcriptional activation of pGL2-SAA2pt, but not that driven by TNF-alpha or IL-6. Furthermore, in synergistic cytokine combinations, it blocks only the IL-1 beta driven component indicating that the effect is biological and not attributable to toxicity. Consequently assays utilizing pGL2-SAA2pt will be useful both for the investigation of the kinetics of inflammatory signalling in a cytokine specific manner, and for the evaluation of the pro- and anti-inflammatory properties of novel natural and synthetic molecules.
authors.affiliation: Department of Genetics and Biotechnology Institute, Trinity College, University of Dublin, Ireland.
authors.forename: C M
authors.initials: CM
authors.lastname: Uhlar
authors.forename: S
authors.initials: S
authors.lastname: Grehan
authors.forename: D M
authors.initials: DM
authors.lastname: Steel
authors.forename: A
authors.initials: A
authors.lastname: Steinkasserer
authors.forename: A S
authors.initials: AS
authors.lastname: Whitehead
mesh_terms: D000209:Acute-Phase Proteins / Q000187:drug effects / Q000235:genetics*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001483:Base Sequence; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D018836:Inflammation Mediators / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000819:agonists / Q000494:pharmacology*; D015850:Interleukin-6 / Q000819:agonists / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000276:immunology*; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors; D000685:Serum Amyloid A Protein / Q000187:drug effects / Q000235:genetics*; D012795:Sialoglycoproteins / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000819:agonists / Q000494:pharmacology*
chemical_list: D000209:Acute-Phase Proteins; D000894:Anti-Inflammatory Agents, Non-Steroidal; C507763:IL1RN protein, human; D018836:Inflammation Mediators; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D015850:Interleukin-6; D017472:Receptors, Interleukin-1; D000685:Serum Amyloid A Protein; D012795:Sialoglycoproteins; D014409:Tumor Necrosis Factor-alpha