Role of the Histone H3 Lysine 27 Demethylase, JMJD3, in the Regulation of Nuclear Factor Kappa-B (NF-kB)-dependent Inflammatory Genes

Abstract

Nuclear factor kappa-light chain-enhancer of activated B cells (NF-kB) as a transcription factor can control many physiological processes, including inflammatory and immune responses. Disruption of NF-kB results in many pathological conditions including chronic inflammation and cancer. The activation of NF-kB starts from cytoplasmic site leading to its release into nucleus from its inhibitor molecules, inhibitor kappa B (IkBs). After proper activation, binding of NF-kB to particular genes also depends on other epigenetic modifiers including histone H3-lysine 4 methyltransferase, SET7/9 and coactivator proteins such as histone acetyltransferase, p300. JMJD3, a Jumonji-C domain containing 3 family histone demethylase, is induced by the transcription factor, NF-kB in response to various stimuli. JMJD3 is crucial for erasing histone-3 lysine-27 tri-methylation (H3K27me3); a histone modification associated with transcriptional repression, and is responsible for the activation of a diverse set of genes. We evaluate the involvement of NF-kB and JMJD3 for the regulation of inflammatory genes. As a modulator of inflammatory response, NF-kB along with JMJD3 plays a vital role for the lethal toxin (LeTx) intoxication resistance of RAW 264.7 cells. First, we investigated whether Jmjd3, in addition to the differential NF-kB genes, are vital for the lipopolysaccharides (LPS) induced LeTx tolerance of macrophage cells and whether these could be responsible for epigenetic regulation in intoxication-resistant cells. Bacillus anthracis LeTx is a virulence factor that causes immune suppression and toxic shock in the infected host. NF-kB is crucial for the plasticity of first level immune cells such as macrophages, monocytes and neutrophils. In macrophages, this inflammatory response, mediated by NF-kB, can regulate host defense against invading pathogens. This study showed that a 2 h exposure of macrophages to LeTx caused substantial cell death with a survival rate of around 40%. The expression of the Jmjd3 gene was induced 8-fold in intoxication-resistant cells generated by treatment with LPS of RAW 264.7 cells. Both of these intoxication-resistant cell lines, LeTx (PLx) intox and LeTx with LPS (PLxL) intox were maintained for eight passages and had a survival rate of around 100% on secondary exposure to LeTx and LPS. Analysis of NF-kB gene expression showed that the expression of p100, p50 and p65 was induced around 20, 7 and 4-fold, respectively, in both of the intoxication-resistant cell lines following a 2 h treatment with PLxL (0.1 + 0.1 + 1 􀈝 g/ml). In contrast, these NF-kB genes were not induced following treatment with PLx treatment at the same concentrations. Although LeTx influences macrophage physiology and causes defects of some key signaling pathways such as glycogen synthase kinase-3 beta (GSK3􀈕 ) which contributes to cytotoxicity, these results indicate that modulation of NF-kB by p50, p100 and Jmjd3 could be vital for the recovery of murine macrophages from exposure to the anthrax lethal toxin. Second, we investigated involvement of JMJD3 on NF-kB regulated gene and protein expression in immune cells. The objectives of this part were to identify the genes in THP-1 human monocytic cells that are significantly affected by the knockdown of JMJD3, particularly in expression levels and in downstream effects on signaling pathways. THP-1 cells were subjected to lentiviral JMJD3 shRNA (short hairpin RNA) transduction followed by analysis of gene expression by quantitative real-time RT-PCR. Global gene expression levels were detected in JMJD3 knockdown (kd) THP-1 human monocytic cells and in TNF􀄮 -stimulated JMJD3-kd THP-1 cells by a 12-plex NimbleGen human whole genome array. Datasets were analyzed using Ingenuity pathway analysis (IPA) software. IPA analysis revealed that out of 50 networks, gene expression, cell death and cellular development networks were the most affected in JMJD3-kd THP-1 cells. IPA analysis showed that the top five diseases and disorders associated with down-regulated genes in JMJD3-kd THP-1 cells were inflammatory, respiratory and hematological diseases, cancer and hypersensitivity responses. JMJD3 attenuation downregulates various key genes in NF-kB, chemokine and CD40 signaling that mostly affects inflammatory disease response molecules. Moreover, this study significantly highlights the connection of NF-kB with JMJD3, which suggests an epigenetic regulation of the inflammatory genes. Finally, we screened several novel NF-kB inhibitors where Terminalia chebula (TC) extract has enormous potential to inhibit the NF-kB activity in human lymphoma cells. TC is used in traditional medicine for the treatment of human ailments including bacterial infections, immunological malfunctions, malignant tumors and diabetes. We investigated the NF-kB inhibitory effect of TC extract and TC extract-dependent protein expression changes in human lymphoblastic T (Jurkat) cells. FRET-based real time cell-monitoring assay showed that TC extract (50 ?g/ml) inhibited NF-kB activity in Jurkat cells. Accordingly, TC extract inhibits phosphorylation and degradation of IkB􀄮 that protects NF-kB from translocation into the nucleus in Jurkat-NF-kB-bla cells. In addition, TC extract downregulated certain NF-kB regulated inflammatory genes, including IL-8 and MCP-1, in Jurkat-NF-kB-bla cells. A comparative proteomic profile was determined in TNF􀄮 - and TC extract-treated cells; the expression of beta-tubulin, beta-actin, ring finger and CHY zinc finger domain containing 1 (RCHY1) and insulin like growth factor 1 receptor kinase (IGF1R) was significantly downregulated in TC extract-treated cells. Moreover, the molecular basis for the TC extractdependent protein expression changes in Jurkat cells was determined by IPA network analysis. These results demonstrate that TC extract is able to inhibit NF-kB activity in Jurkat cells. Further studies to characterize the TC extract may uncover novel NF-kB inhibitory compounds for treating inflammatory diseases and cancer. In summary, thesis results suggest that Jmjd3 along with different NF-kBs is vital for LeTx resistance and polarization of murine macrophages. This study also predicts Jmjd3 targets in attenuated human monocytic cells focusing the connection of NF-kB with Jmjd3 in the regulation of the inflammatory genes. We also find that TC extract-dependent NF-kB inhibition in human lymphoma cells. Moreover, involvement of Jmjd3 in the subsets of NF-kB-regulated genes can be considered as an important regulator of certain inflammatory genes expression.