Background/Purpose: Histone deacetylase-6 (HDAC-6) functions as a non-epigenetic deacetylase for non-histone substrates and regulates microtubule-mediates processes such as cell migration, cell cycle arrest, and angiogenesis. We have previously demonstrated that Tubastatin A suppresses synovial inflammation and joint destruction in a collagen antibody-induced arthritis mouse model. However, the exact mechanism through which Tubastatin A exerts its anti-arthritic effect is not fully understood. The aim of the study is to investigate whether ubiquitin-proteasome pathway is involved in the anti-arthritic mechanism of Tubastatin A.

Methods: Fibroblast-like synoviocytes (FLS) were treated with TNF-α in a time-dependent manner (control, 5, 15, 30 minutes) and the phosphorylation activity of NF-κB and IκB was measured using Western blot analysis. FLS were stimulated with TNF-α after treatment with different doses of proteasome inhibitor (MG-132) or Tubastatin A, and the expression of IL-6 were measured using ELISA. FLS were stimulated with TNF-α after treatment with different doses of Tubastatin A and phosphorylation of nuclear NF-κB and of cytosolic IκB were measured using Western blot analysis. FLS were stimulated with TNF-α in a time dependent manner (15, 30, 45, 60 min) after pretreatment with Tubastatin A or control and expression of phosphorylated IκB was measured using Western blot analysis. FLS were treated with TNF-α after treatment with different doses of Tubastatin A, and cytosolic proteasome activity was measured using 20S Proteasome Activity Assay Kit.

Results: The phosphorylation of NF-κB and IκB were increased after stimulation of TNF-α at 5 minutes. After stimulation with TNF-α, the expression of IL-6 was attenuated in FLS treated with Tubastatin A or MG-132 compared with controls. At 3µM of Tubastatin A, the phosphorylation of NF-κB induced by TNF-α was suppressed, and phosphorylated IκB in cytosol was increased. Furthermore, the total cytosolic IκB remained the same. Phosphorylated IκB appeared in the cytosol 30 min after incubation with TNF-α after treatment with Tubastatin A, but no effect was seen when treated with TNF-α alone. After stimulation with TNF-α, the cytosolic proteasome activity was significantly decreased after treatment of Tubastatin A or MG-132 compared with controls.

Conclusion: Our study demonstrates that Tubastatin A exerts its anti-arthritic effect, at least in part, through preventing degradation of IκB by attenuating ubiquitin-proteasome pathway, and thus inactivating nuclear translocation of NF-κB.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-arthritic-effect-of-tubastatin-a-a-novel-histone-deacetylases-6-inhibitor-is-mediated-by-stabilization-of-ikb-via-suppression-of-ubiquitin-proteasome-pathway/