Abstract
Chronic inflammation is a major contributing factor in the pathogenesis of many age-associated diseases. One central protein that regulates inflammation is NF-κB, the activity of which is modulated by post-translational modifications as well as by association with co-activator and co-repressor proteins. SIRT1, an NAD+-dependent protein deacetylase, has been shown to suppress NF-κB signaling through deacetylation of the p65 subunit of NF-κB resulting in the reduction of the inflammatory responses mediated by this transcription factor. The role of SIRT1 in the regulation of NF-κB provides the necessary validation for the development of pharmacological strategies for activating SIRT1 as an approach for the development of a new class of anti-inflammatory therapeutics. We report herein the development of a quantitative assay to assess compound effects on acetylated p65 protein in the cell. We demonstrate that small molecule activators of SIRT1 (STACs) enhance deacetylation of cellular p65 protein, which results in the suppression of TNFα-induced NF-κB transcriptional activation and reduction of LPS-stimulated TNFα secretion in a SIRT1-dependent manner. In an acute mouse model of LPS-induced inflammation, the STAC SRTCX1003 decreased the production of the proinflammatory cytokines TNFα and IL-12. Our studies indicate that increasing SIRT1-mediated NF-κB deacetylation using small molecule activating compounds is a novel approach to the development of a new class of therapeutic anti-inflammatory agents.
Highlights
Inflammation is a physiological response to remove injurious stimuli and initiate the healing process
Unresolved or sustained low-grade inflammation leads to development of chronic diseases including chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, type 2 diabetes (T2D), cancer, Alzheimer’s disease, cardiovascular, and renal diseases, many of which are associated with aging
We developed a high throughput cellular assay to examine the ability of compounds to promote SIRT1-mediated deacetylation of p65 protein in cells
Summary
Inflammation is a physiological response to remove injurious stimuli and initiate the healing process. Upregulation of inflammatory biomarkers is a characteristic of the aging process [1]. One key protein that regulates inflammatory responses is the transcription factor NF-kB which is held quiescent in the cytoplasm when in complex with IkBa. In response to a proinflammatory stimulus (e.g. lipopolysaccharide (LPS), tumor necrosis factor (TNFa), or interleukin-1 (IL-1)) via Toll-like receptors or cytokine receptors, IkBa is phosphorylated by IKK and subject to ubiquitin-dependent proteasomal degradation, thereby allowing NF-kB to translocate to the nucleus and activate the transcription of a cascade of proinflammatory cytokines and chemokines to induce inflammatory responses [2,3]. Of particular interest is the acetylation of p65/RelA, a subunit of the heterodimeric NF-kB protein, which can either potentiate or diminish NF-kB signaling depending on the particular acetylated lysine residue [7,8]. Among the seven lysines (lysine 122, 123, 218, 221, 310, 314, 315) that are acetylated by p300/CBP and PCAF [7,8,9,10,11,12,13,14], acetylation of lysine 310 is critical for full activation of NF-kB transcription potential [7], which can be deacetylated by SIRT1 [15]
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