The essential role of Oct-2 in LPS-induced expression of iNOS in RAW 264.7 macrophages and its regulation by trichostatin A.
This article reports on a study of the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) in RAW 264.7 macrophages and its underlying mechanisms. TSA pretreatment potently diminishes LPS-stimulated nitric oxide (NO) release and both mRNA and protein levels of iNOS in macrophages. The effects of TSA and LPS on transcription factors binding to two LPS-responsive elements within the iNOS promoter, one binding the NF-kappaB site and the other the octamer element, were investigated. Results show that TSA did not alter the LPS-activated NF-kappaB activity demonstrated by the nuclear translocation of p50 and p65 and by a NF-kappaB-driven reporter gene expression system. In addition, neither TSA nor LPS changed the expression of Oct-1, a ubiquitously expressed octamer binding protein. However, TSA suppressed the LPS-induced expression of Oct-2, another octamer binding protein, at both mRNA and protein levels. Chromatin immunoprecipitation assays revealed that binding of Oct-2 to the iNOS promoter was enhanced by LPS treatment; however, pretreatment with TSA resulted in loss of this binding. Moreover, forced expression of Oct-2 by transfection of pCG-Oct-2 plasmid restored the TSA-suppressed iNOS expression elevated by LPS stimulation, further indicating that Oct-2 activation is a crucial step for transcriptional activation of the iNOS gene in response to LPS stimulation in macrophages. This study demonstrates that TSA diminishes iNOS expression in LPS-treated macrophages by inhibiting Oct-2 expression and thus reducing the production of NO.
Lu
Shao-Chun
SC
Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 110, Taiwan.
Wu
Hsiao-Wen
HW
Lin
Yen-Jen
YJ
Chang
Shwu-Fen
SF
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism; D050813:Octamer Transcription Factor-2 / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D008070:Lipopolysaccharides; D050813:Octamer Transcription Factor-2; C489206:Pou2f2 protein, mouse; D009569:Nitric Oxide; C012589:trichostatin A; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse