Background/Purpose: Interleukin-1 (IL-1) is a crucial mediator of inflammatory cartilage and bone destruction in rheumatoid arthritis (RA). IL-1β signaling relies on the activation of TGF-beta activated kinase 1 (TAK1), a serine/threonine kinase that centrally regulates the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways, which makes it an attractive therapeutic target. Takinib is a small molecule inhibitor proposed to inhibit TNF-α-induced inflammation by targeting TAK1, however, its efficacy against IL-1β-driven inflammation and the underlying mechanism of its action remains to be tested. In the present study, we evaluated the effect of takinib on IL-1β-activated RA synovial fibroblasts (RASFs).

Methods: Human RASFs isolated from RA synovial tissue were serum starved overnight followed by two-hour pretreatment with takinib at various concentrations (0.1-10 µM) before treatment with IL-1β (10 ng/mL) for 24 hours to determine the cell viability and the production of soluble proteins by ELISA, or for 30 minutes to evaluate the effect on IL-1β-activated signaling pathways. Irreversible type I (5z-7-oxozeaenol; 5Z-7o; 1 µM) and reversible type II (NG-25; 1 µM) inhibitors of TAK1 served as experimental controls. Effect of takinib on the kinome was determined using a human proteome profiler phospho-kinase array. Conditioned media was used for ELISA, whole cell lysates and nuclear/cytoplasmic extract were used for Western blot analysis. Effect on the cell viability was determined using an MTT-based assay. Molecular docking of Takinib as a ligand was performed on human STAT3 protein.

Results: Human phospho-kinase array results from RASFs identified phosphorylation of STAT3 at Ser⁷²⁷ and Tyr⁷⁰⁵ as a primary target of takinib (10 µM) in response to IL-1β stimulation. Pretreatment of RASFs with takinib (0.1-10 µM) showed a dose-dependent reduction in ENA-78/CXCL5, IL-6, IL-8 and MCP-1/CCL2 production, which were completely abrogated by 5Z-7o and NG-25 at 1 µM (n=3; p< 0.05). We also observed a significant reduction in cell viability by takinib at 10 µM (n=4; p< 0.05). Evaluation of the signaling pathways in RASFs further confirmed that takinib abrogates the non-canonical activation of STAT3 in response to IL-1β stimulation by suppressing Ser⁷²⁷ and Tyr⁷⁰⁵ activation. Surprisingly, takinib showed a dose-dependent increase in IL-1β-induced phospho-TAK1 at its kinase domain (Thr^184/187) in human RASFs. In corroboration of the kinome results, Western blot analysis showed that takinib inhibited the nuclear translocation of IL-1β-induced STAT3, but not of NF-κBp65, in human RASFs (n=3; p< 0.05). Molecular docking of takinib with STAT3 protein suggests binding of takinib at Gln⁶⁴⁴ and Tyr⁶⁵⁷ residues in the DNA binding domain. These may be essential for STAT3 activation and translocation to the nuclear compartment.

Conclusion: Our findings suggest STAT3 is an important target of takinib in the IL-1β signaling pathway, which may further be utilized for therapeutic purposes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/takinib-inhibits-il-1%ce%b2-induced-activation-of-signal-transducer-and-activator-of-transcription-3-stat3-in-human-rheumatoid-arthritis-synovial-fibroblasts/