Background/Purpose: O-GlcNAcylation is an important post-translational modification of nuclear and cytosolic proteins involved in the cytokine signaling networks. Studies show both pro- and anti-inflammatory roles of O-linked N-acetyl glucosamine (O-GlcNAc) depending on different cell types and diseases. However, the role of O-GlcNAcylation in chronic inflammatory diseases such as rheumatoid arthritis (RA) is yet to be explored.

Methods: Human normal SFs (NLSFs) and RASFs were isolated from healthy synovial tissues and RA synovial tissues, respectively, under the IRB-approved protocol. Cell lysates and tissue homogenates were used to determine the differences in the expression of the O-GlcNAcylation in NLSFs and RASFs by Western immunoblotting. Effects of IL-1β (10 ng/ml) on O-GlcNAcylation and two enzymes that control glycosylation (O-GlcNAc transferase, OGT and O-GlcNAcase, OGA) in human RASFs were studied. The effect of OGT inhibitor (OSMI-1; 5-50 µM) or OGA inhibitor (Thiamet-G; 1-10 µM) was evaluated on IL-1β-induced IL-6 and IL-8 production and the underlying mechanisms were studied. In rat adjuvant-induced arthritis (AIA) model, ankle, liver, and spleen homogenates from naïve and AIA rats were analyzed to determine the temporal expression pattern of O-GlcNAcylation.

Results: The expression of O-GlcNAc is upregulated in RA synovial tissues compared with healthy synovial tissues, and also showed a similar trend in SFs isolated from these tissues. We also observed IL-1β stimulation resulted in the upregulation of O-GlcNAc levels in RASFs in vitro. Interestingly, pretreatment of RASFs with OGA inhibitor (Thiamet-G) inhibited IL-1β-induced IL-6 and IL-8 production, whereas OGT inhibitor (OSMI-1) had no inhibitory effect, suggesting that OGA inhibition may be of potential therapeutic value in RA. Evaluation of the IL-1β signaling proteins (TAK1, TAB1, or NF-kBp65) that are critical in relaying downstream signaling showed a marked increase in O-GlcNAc levels upon IL-1β stimulation. Thiamet-G pretreatment showed a protective role of OGA inhibition in regulating inflammation. Similarly, in rat AIA model, we observed a higher O-GlcNAc expression pattern in rat joint homogenates, whereas no change in O-GlcNAcylation expression was observed in liver. Surprisingly, O-GlcNAcylation in spleen homogenates elicited differential expression at the onset of arthritis (day 8) and at the establishedarthritis (day 18) compared to naïve, which correlated with the clinical scores and splenomegaly in arthritic animals. Interestingly, O-GlcNAc levels of protein such as NF-kBp65 was found to be downregulated in spleen microenvironment whereas that of NF-kBp50 remained the same.

Conclusion: Our findings point to an important mediatory role of O-GlcNAc in stabilizing IL-1β signaling proteins to activate downstream inflammatory proteins. OGA inhibition may serve as a potential therapeutic target in regulating synovial inflammation in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-interleukin-1%ce%b2-signaling-by-inhibition-of-o-glc-nacase-in-rheumatoid-arthritis-synovial-fibroblasts/