OP0201 DYNAMIC CHANGES IN O-GLCNACYLATION REGULATE OSTEOCLAST DIFFERENTIATION AND BONE LOSS IN ARTHRITIS

Background:Bone remodeling is a constant process maintained by the balance between osteoclast-triggered bone resorption and osteoblast-mediated bone formation. In inflammatory arthritis, such as rheumatoid arthritis (RA), the pro-inflammatory environment favors osteoclast differentiation and skews the balance towards resorption, leading to progressive bone erosion and bone loss. O-GlcNAcylation is a post-translational modification, which transfers a single N-acetylglucosamine molecule to the serine or threonine of the target protein. The modification is accomplished by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Unlike other glycosylation, O-GlcNAcylation occurs in multiple cellular compartments, including the nucleus. Although O-GlcNAcylation is one of the most common modifications, its role in bone homeostasis is still poorly understood.Objectives:We aimed to investigate the role of O-GlcNAcylation in osteoclastogenesis under pro-inflammatory milieus. We also focused on dissecting the signaling pathways affected by O-GlcNAcylation during osteoclast differentiation.Methods:We examined the levels of O-GlcNAc during in vitro osteoclastogenesis by western blotting. The levels of O-GlcNAc in tissue from RA patients and experimental arthritis were detected by immunofluorescence. Pharmacological inhibition and genetic knockout were used to manipulate O-GlcNAcylaiton during osteoclastogenesis. RNA sequencing was performed to study O-GlcNAc-mediated pathways.Results:We demonstrate the dynamic changes in O-GlcNAcylation during osteoclastogenesis. The elevated O-GlcNAcylation was found in the early differentiation stages, whereas its downregulation was detected in the maturation process. TNFα elaborates the dynamic changes in O-GlcNAcylation, which further intensifies osteoclast differentiation.Targeting OGT by selective inhibitor and genetic knockout restrain O-GlcNAcylation and hinder the expression of the early differentiation marker Nfatc1. Inhibition of OGA, which forces high levels of O-GlcNAcylation throughout the differentiation, reduces the formation of multinucleated mature osteoclasts. Consistent with our in vitro data, suppressing OGT and OGA both ameliorate bone loss in experimental arthritis. We detected a reduced number of TRAP-expressing precursors and mature osteoclasts in the mice subjected to OGT inhibition. While inhibiting OGA only lowers the number of TRAP+F4/80– mature osteoclasts without affecting the number of TRAP+F4/80+ precursors.Transcriptome profiling reveals that O-GlcNAcylation regulates several biological processes. Increased O-GlcNAcylation promotes cytokine signaling and oxidative phosphorylation. The downregulation of O-GlcNAcylation is essential for cytoskeleton organization and cell fusion.Conclusion:We demonstrate that the dynamic changes of O-GlcNAcylation are essential for osteoclast differentiation. These findings reveal the therapeutic potential of targeting O-GlcNAcylation in pathologic bone resorption.Disclosure of Interests:Chih-Wei Chen: None declared, Yi-Nan Li: None declared, Thuong Trinh-Minh: None declared, ZHU Honglin: None declared, Alexandru-Emil Matei: None declared, Xiao Ding: None declared, Cuong Tran Manh: None declared, Xiaohan Xu: None declared, Christoph Liebel: None declared, Ruifang Liang: None declared, Min-Chuan Huang: None declared, Neng-Yu Lin: None declared, Andreas Ramming Speakers bureau: Boehringer Ingelheim, Roche, Janssen, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Grant/research support from: Pfizer, Novartis, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Jörg H.W. Distler Shareholder of: 4D Science, Speakers bureau: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Grant/research support from: Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB, Employee of: FibroCure

Mice Deficient for an Intestinal G Protein-Coupled Receptor Expression Have Increased Satiety During Rebound Hyperphagia

Gut-derived hormones have been successfully developed as the therapeutic targets to combat the increasing prevalence of diabetes and obesity. G protein-coupled receptors (GPCRs) in the gastrointestinal (GI) tract are involved in maintaining glucose and energy homeostasis by regulating the release of gut hormones in response to luminal dietary nutrients as well as microbial metabolites. We identified that an orphan GPCR, Gpr17, was expressed in the intestinal epithelium and found that loss of intestinal Gpr17 expression increased gut incretin hormone secretion from enteroendocrine cells (EECs). However, it is unknown how Gpr17 ablation in the intestinal epithelium affects feeding behavior and satiety regulation. To address this question, we used genetic knockout approach to generate intestinal Gpr17-deficient mice and analyzed their feeding behavior. Here we show that intestinal Gpr17-deficient mice had similar growth curve, body composition, and ad libitum food intake compared with littermate controls. Interestingly, intestinal Gpr17-deficient mice responded to fasting-refeeding challenge with reduced fasting locomotor activity and less food intake after refeeding, suggesting increased satiety during the phase of rebound hyperphagia. Moreover, we performed fasting-refeeding challenge with Gpr17-deficient mice fed on high-fat diet (HFD), and our meal pattern analysis revealed that these mice had reduced meal duration of the first meal after refeeding. In conclusion, our genetic knockout studies in rodents showed that ablating intestinal Gpr17 increased satiety during rebound hyperphagia in the fasting-refeeding experimental paradigm. Intestinal Gpr17 could be developed as a therapeutic target to treat obesity by improving energy balance through gut hormone secretion and meal pattern control.

Optogenetic manipulation of cellular communication in axolotls

Cells communicate through long cellular protrusions such as filopodia and neurites. However current approaches to study these contact-based cellular communication are largely limited to actin-depolymerizing drugs or genetic knockout of key actin modifiers which can cause severe cellular stress or semi-lethality in organisms. Here we present a versatile optogenetic toolbox of artificial myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo using light. Importantly, we discover that these long filopodial extensions are also gradually developed during axolotl limb regeneration, where we applied our toolbox to manipulate the composition and dynamics of these cellular extensions.