Introduction: We investigated the inflammatory potential of O-linked N-acetylglucosamine glycosylation (O-GlcNAcylation) of p65 in rheumatoid arthritis (RA). Methods: Fibroblast-like synoviocytes (FLS) and MH7A cells were treated with synthetic ThiaMet-G (200 μM), an O-GlcNAcase (OGA) inhibitor, followed by tumor necrosis factor (TNF)-aα (10 μg/mL). Proliferation of synovial cells was measured by MTT assay, and the levels of mRNAs encoding pro-inflammatory molecules were quantitated by RT-PCR. The nuclear localization of O-GlcNAcylated of p65 and its DNA binding affinity and transcriptional activity were assessed. The severity assessment of arthritis and a histopathological examination were done in mice with collagen induced arthritis (CIA). ThiaMet-G (20 mg/kg) intraperitoneal injection was done every other day for 26 days. Fluorescence-activated cell sorting (FACS) analysis of T cells was performed. Results: Hyper-O-GlcNAcylation increased the proliferation and mRNA expression of pro-inflammatory genes in synoviocytes stimulated by TNF-aα. Moreover, O-GlcNAcylation of p65 enhanced its proportion of nuclear localization, DNA binding affinity and transcriptional activity. In CIA mice, ThiaMet-G significantly aggravated the severity of arthritis clinically and histologically, and it also increased CD4 + IFN-γ + T cells and CD4 + IL-17+ T cells. Conclusions: O-GlcNAcylation of p65 increased the effects of TNF-aα-mediated inflammation both in vitro (in synovial cells) and in vivo (in mice with CIA).
Bibliographical noteFunding Information:
We thank Prof. Yoon HG (Yonsei University, South Korea) for providing MH7A cells, Prof. Vocadlo DJ (Simon Fraser University, Canada) for providing a synthetic method for ThiaMet-G, and Prof. Suh PG (Pohang University, Republic of Korea) for providing the Lentivirus-based OGA shRNA construct. Also the authors are grateful to Dong-Su Jang, (Medical Illustrator, Medical Research Support Section, Yonsei University College of Medicine, Seoul, Korea) for his help with the figures. This study was supported by a faculty research grant of Yonsei University College of Medicine (6-2012-0186) and research fund from Handok (2011–36) to Sang-Won Lee. This study was also supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A2A1A01008067) to Jin Won Cho. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/ certificate/THjDDG.
© 2015 Kim et al.
All Science Journal Classification (ASJC) codes
- Immunology and Allergy