trichostatin-a and 5--methylthioadenosine

Group IVA cytosolic phospholipase A2 (cPLA2 or PLA2G4A) is a key enzyme that contributes to inflammation via the generation of arachidonic acid and eicosanoids. While much is known about regulation of cPLA2 by posttranslational modification such as phosphorylation, little is known about its epigenetic regulation. In this study, treatment with histone deacetylase (HDAC) inhibitors, trichostatin A (TSA), valproic acid, tubacin and the class I HDAC inhibitor, MS-275, were found to increase cPLA2α messenger RNA (mRNA) expression in SH-SY5Y human neuroblastoma cells. Co-treatment of the histone acetyltransferase (HAT) inhibitor, anacardic acid, modulated upregulation of cPLA2α induced by TSA. Specific involvement of class I HDACs and HAT in cPLA2α regulation was further shown, and a Tip60-specific HAT inhibitor, NU9056, modulated the upregulation of cPLA2α induced by MS-275. In addition, co-treatment of with histone methyltransferase (HMT) inhibitor, 5'-deoxy-5'-methylthioadenosine (MTA) suppressed TSA-induced cPLA2α upregulation. The above changes in cPLA2 mRNA expression were reflected at the protein level by Western blots and immunocytochemistry. Chromatin immunoprecipitation (ChIP) showed TSA increased binding of trimethylated H3K4 to the proximal promoter region of the cPLA2α gene. Cell injury after TSA treatment as indicated by lactate dehydrogenase (LDH) release was modulated by anacardic acid, and a role of cPLA2 in mediating TSA-induced injury shown, after co-incubation with the cPLA2 selective inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3). Together, results indicate epigenetic regulation of cPLA2 and the potential of such regulation for treatment of chronic inflammation.

Topics: Anacardic Acids; Anilides; Benzamides; Cell Line, Tumor; Chromatin Immunoprecipitation; Deoxyadenosines; Epigenesis, Genetic; Fluorescent Antibody Technique; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Group IV Phospholipases A2; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; L-Lactate Dehydrogenase; Lysine; Neuroblastoma; Pyridines; Real-Time Polymerase Chain Reaction; RNA, Messenger; Thiazoles; Thionucleosides; Valproic Acid

The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression.

Topics: Blotting, Western; Cell Line, Tumor; Chromatin; Deoxyadenosines; Enzyme Inhibitors; Gene Expression Regulation; Histones; Hormones; Humans; Hydroxamic Acids; Immunoprecipitation; Interleukin-1beta; Molecular Sequence Data; Peptide Nucleic Acids; Promoter Regions, Genetic; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Double-Stranded; Thionucleosides; Up-Regulation