Date: Saturday, November 6, 2021
Session Title: RA – Etiology & Pathogenesis Poster (0011–0045)
Session Type: Poster Session A
Session Time: 8:30AM-10:30AM
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 (Thr184/187) 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.
To cite this abstract in AMA style:singh A, Panipinto P, Siegel R, Shaikh F, Chourasia M, Ahmed S. Takinib Inhibits IL-1β-Induced Activation of Signal Transducer and Activator of Transcription 3 (STAT3) in Human Rheumatoid Arthritis Synovial Fibroblasts [abstract]. Arthritis Rheumatol. 2021; 73 (suppl 9). 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/. Accessed June 1, 2023.
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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/