Lu S, Wu H, Lin Y, Chang S. The essential role of Oct-2 in LPSinduced expression of iNOS in Raw 264.7 macrophages and its regulation by trichostatin A. Am J Physiol Cell Physiol 296: C1133-C1139, 2009. First published March 11, 2009 doi:10.1152/ajpcell.00031.2009.-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 HISTONE MODIFICATIONS, such as acetylation, methylation, and phosphorylation, play important roles in gene expression. Acetylation is the most frequent posttranslational modification of histone. The level of histone acetylation is controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) (11). Higher levels of histone acetylation lead to relaxation of the local structure of chromatin and thus increase the accessibility of transcription factors to the DNA, subsequently enhancing gene transcription. In contrast, silent genes are usually located at nucleosomes with hypoacetylated histone proteins (35).Trichostatin A (TSA) is a potent and nonselective inhibitor of HDACs and is widely used to study the role of histone acetylation in genes. TSA treatment induces histone acetylation in mammalian cells (25,38). However, several studies have shown that TSA treatment changes the expression of only about 2% of genes (24, 39). Aung et al. (1) reported that TSA enhanced LPS-induced expressions of Cox-2, Cxcl2, and Lfit2, whereas LPS-...