FRI011 Exploring The Contribution Of PARP1-mediated Site-specific ADP-Ribosylation Of C/EBPb In Macrophages To Diet-induced Obesity

Abstract

Abstract Disclosure: A.L. Whitaker: None. R. Gupte: None. T.S. Nandu: None. Inflammation is an important component of the pathological effects of obesity, which are mediated in part by adipose-associated macrophages (TAMs). Signal-regulated post-translational modifications in the TAMs help modulate the transcriptional programs that mediate biological outcomes. ADP-ribosylation is a posttranslational modification that regulates various cellular processes, including macrophage activation. ADP-ribosylation is the covalent attachment of ADP-ribose units on proteins by poly(ADP-ribose) polymerase (PARP) enzymes, mainly PARP1 in the nucleus. We have shown that ADP-ribosylation of key transcription factors (e.g., C/EBPβ and STAT1α) by PARP1 regulates adipogenesis, as well as pro-inflammatory responses in macrophages. Here we aim to investigate the role of PARP1-mediated ADP-ribosylation of C/EBPβ in inflammation and obesity using monocyte-specific mouse models. We have found that knockout (KO) of PARP1 in the monocyte/macrophage lineage (i.e., using a LysM-Cre driver) results in dramatic weight gain in mice with a nearly 2-fold increase in fat body mass in animals fed a high fat diet. Further analysis revealed decreases in adipose-associated macrophages and adipose hypertrophy, as well as increased hepatic lipid accumulation. Our findings exemplify the critical role that PARP1 in macrophages plays in maintaining homeostasis in key metabolic tissues. Moreover, our results indicate that regulation by PARP1 in macrophages contributes to resistance of diet-induced obesity through tissue-collaborative mechanisms. Our ongoing experiments suggest that PARP1 regulates LPS-induced inflammatory gene expression in macrophages. PARP1 controls LPS-mediated gene expression, in part, by ADP-ribosylating two sites (Glu 135 and Glu 139) in the regulatory domain of C/EBPβ; mutation of these sites inhibits PARP1-mediated PARylation of C/EBPβ and LPS-mediated gene expression through C/EBPβ. Additionally, treatment with a PARP inhibitor decreases LPS-mediated gene expression associated with upstream C/EBPβ enhancers. Moreover, treatment with a PARP inhibitor also decreases histone H3 lysine 27 acetylation (H3K27ac) at these C/EBPβ enhancers. To investigate the physiological impact of loss of C/EBPβ ADP-ribosylation in macrophages, we have generated a knock-in mouse model for inducible monocyte-specific expression of a C/EBPβ ADP-ribosylation site mutant (CebpbE135A/E139A). This is the first physiological model for the study of site-specific ADP-ribosylation in vivo. Using this model, we are investigating the impact of site-specific ADP-ribosylation of C/EBPβ in mice fed a high fat diet. With these studies, we hope to reveal the specific roles of PARP1-mediated ADP-ribosylation in diet-induced obesity. Presentation: Friday, June 16, 2023