Background/Purpose: Osteoarthritis (OA) is a chronic degenerative joint disease caused by synovial inflammation and cartilage degradation primarily driven by interleukin-1beta (IL-1β). Studies suggest that TNF-stimulated gene 6 (TSG6) plays an important role in reducing IL-1β-induced tissue destruction in arthritis. In the present study, we evaluated the role of guanylate binding protein 5 (GBP5), an interferon gamma regulated gene, in regulating TSG6 expression, and IL-1β-induced COX-2 expression and IL-6 and MMP-1 production in human osteoarthritis synovial fibroblasts (OASFs).

Methods: Primary OASFs were isolated from synovial tissues obtained after joint replacement surgeries from OA patients under an approved IRB protocol and cultured in RPMI1640 with 10% FBS. Human OASFs were treated with IL-1β (10 ng/ml) or TNF-α (20 ng/ml) for 24 hours to study the expression of inflammatory proteins using Western immunoblotting, qRT-PCR, or ELISA method. The potential crosstalk and association of GBP5 with IL-1β stimulated signaling proteins was studied by the immunoprecipitation (IP) method. Small-interfering RNA (siRNA) mediated knockdown of GBP5 was conducted to study its effect on IL-1β-induced inflammatory mediators in human OASFs.

Results: Western blot analysis showed that the expression of GBP5 is significantly increased upon IL-1β or TNF-α stimulation in human OASFs (p< 0.05; n=3). Knockdown of GBP5 significantly increased the expression of IL-1β-induced COX-2 and the production of IL-6 and MMP-1 in human OASFs (p< 0.05; n=3). Our qRT-PCR and Western blotting results showed that the knockdown of TNF-stimulated gene 6 (TSG6) significantly upregulates IL-1β-induced MMP-1 expression in human OASFs (p< 0.05; n=3). Surprisingly, GBP5 knockdown by siRNA significantly lowered the expression of IL-1β-induced TSG6 at mRNA and protein levels (p< 0.05; n=3), which suggests GBP5 a novel regulator of TSG6 in human OASFs. Supporting evidence showed that the overexpression of GBP5 via lentiviral delivery resulted in the upregulation of TSG6 expression and a concomitant decrease in IL-1β-induced COX-2 expression and IL-6 and MMP-1 production in human OASFs (p< 0.05; n=3). Furthermore, the results from IP analysis showed that IL-1β stimulation led to enhanced association between TSG6 and GBP5 in human OASFs in vitro, thereby selectively amplifying phosphorylation of JNK/SAPK MAPK and its downstream substrate c-Jun.

Conclusion: Our study suggests that GBP5 suppresses IL-1β-induced tissue destruction primarily through TSG6, whereas it elicits anti-inflammatory actions that are independent of TSG6 in human OASFs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-interleukin-1%ce%b2-il-1%ce%b2-induced-cox-2-expression-and-il-6-and-mmp-1-production-in-human-oa-synovial-fibroblasts-by-guanylate-binding-protein-5/