Epigenetic Regulation of Lps-Induced G-Csf Expression in Macrophages

Granulocyte colony-stimulating factor (G-CSF) is an important cytokine that involves in inflammation and anti-inflammation processes due to its effect on proliferation, survival, and differentiation of hematopoietic cells and mobilization of neutrophil granulocyte precursors. G-CSF also has neuroprotective and anti-apoptotic activities for neurgenesis and cardioprotective effect. The proinflammatory role of G-CSF in the pathogenesis of inflammatory arthritis is however, one deleterious consequence of G-CSF activity. The level of G-CSF appears to associate with many inflammation processes; however, the mechanism responsible for regulating the G-CSF expression remains unclear. Our unpublished data showed that TSA, a histone deacetylase inhibitor abolished LPS-induced G-CSF expression in macrophages. In addition, our preliminary data showed that 5-aza-dC, a DNA methyltransferase inhibitor, reduced G-CSF expression in LPS-treated Raw264.7 macrophages. Since TSA and 5-aza-dC both showed inhibitory effect on LPS-regulated G-CSF expression in macrophages, we reason that there might be epigenetic alteration in G-CSF gene upon LPS-exposure in macrophages. Our emergent understanding of this process may provide a novel framework for developing treatments of G-CSF-mediated inflammation. Based on our previous results and current preliminary data, we proposed a 3-year research plan to investigate whether epigenetic mechanisms including histone modification, DNA methylation, and miRNA are involved in the regulation of LPS induced G-CSF expressions in macrophages. Specific aims in this study are: 1) evaluating whether the TSA and/or 5’-aza-dC abolished LPS-induced G-CSF expression is concentration-dependent and time-dependent; and determining the effect of other HDACi on LPS-induced G-CSF expression (the 1st year); 2) investigating if histone acetylation and DNA methylation at the promoter region of G-CSF would be changed by LPS stimulation; and validating the effect of HDACi and 5-aza-dC on this epigenetic change (the 1st year and the 2nd year); 3) clarifying transcription factors or proteins including Oct-2, NF-B and C/EBP-binding to G-CSF gene in response to LPS-triggered epigenetic modification; and validating the effect of HDACi and 5-aza-dC on LPS-recruited protein binding at the promoter of G-CSF (the 2nd year); 4) evaluating if the levels of miRNAs predicted on the 5’UTR and 3’-UTR of G-CSF transcript, especially the miR-16-2 (whose target sequence overlapped with the cis-element regulating the stability of G-CSF mRNA), would be changed by LPS stimulation (the 3rd year); 5) identifying if the specific miRNA(s), such as miR-16-2, would target G-CSF mRNAs in LPS-treated cells; and validating the roles of the miRNA(s) in LPS-induced G-CSF expression (the 3rd year). The study will further tell us alternative ways to reduce the G-CSF-triggered inflammation based on the understanding that how LPS regulates G-CSF expressions in activated macrophage