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RECEPTORS AND SIGNAL TRANSDUCTION

The essential role of Oct-2 in LPS-induced expression of iNOS in RAW 264.7 macrophages and its regulation by trichostatin A

¹Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei; and ²Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan

Submitted 16 January 2009 ; accepted in final form 24 February 2009

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-B site and the other the octamer element, were investigated. Results show that TSA did not alter the LPS-activated NF-B activity demonstrated by the nuclear translocation of p50 and p65 and by a NF-B-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.

inducible nitric oxide synthase; lipopolysaccharide; macrophage