Fig. 1

Effects of O-linked N-acetylglucosamine glycosylation on proliferation and gene expression in synovial cells. Fibroblast-like synoviocytes (FLS) and MH7A cells were treated with synthetic ThiaMet-G (200 μM) for 6 h, an O-GlcNAcase (OGA) inhibitor, followed by TNF-α (10 μg/mL) for 12 h. Proliferation of synovial cells was measured by MTT assay. a TNF-α significantly increased proliferation of FLS, compared to controls and proliferation was further enhanced following treatment with ThiaMet-G. b The human OGA gene was cloned into a pRK5-FLAG vector and transiently transfected into MH7A cells. In contrast to treatment of ThiaMet-G, overexpression of OGA in FLS significantly attenuated the effects of TNF-α on proliferation, compared to controls. c The levels of mRNAs encoding pro-inflammatory molecules including matrix metalloproteinase-1 (MMP-1), chemokine ligand 5 (CCL5) and IL-8, were quantitated by RT-PCR. ThiaMet-G did not affect the expression levels of pro-inflammatory molecules. But, ThiaMet-G significantly increased expression levels in TNF-α-stimulated FLS, compared to untreated controls. d The pLKO.1 vector and packaging plasmid pMD.2G-VSVG were used to generate a lentivirus-based OGA shRNA construct. OGA shRNA significantly decreased the expression of OGA by about 75 %, compared to sh-luciferase (sh-Luc) control. Similar effects were seen following shRNA-mediated knockdown of OGA, resulting in substantial increase in IL-6 expression, compared to those of sh-Luc controls in TNF-α-stimulated MH7A cells (*P <0.05). GAPDH glyceraldehyde-3-phosphate dehydrogenase