O-linked N-acetylglucosamine glycosylation of p65 aggravated the inflammation in both fibroblast-like synoviocytes stimulated by tumor necrosis factor-aα and mice with collagen induced arthritis

Han Byeol Kim, Sang Won Lee, Chin Hee Mun, Ji Young Yoon, Jaeyoung Pai, Injae Shin, Yong Beom Park, Soo Kon Lee, Jin Won Cho

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Abstract

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).

Original languageEnglish
Article number248
JournalArthritis Research and Therapy
Volume17
Issue number1
DOIs
Publication statusPublished - 2015 Sep 14

Bibliographical note

Funding 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.

Publisher Copyright:
© 2015 Kim et al.

All Science Journal Classification (ASJC) codes

  • Rheumatology
  • Immunology and Allergy
  • Immunology

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