Suppression of monosodium urate crystal-induced inflammation by inhibiting TGF-β-activated kinase 1-dependent signaling: role of the ubiquitin proteasome system

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Abstract

Monosodium urate (MSU) crystals activate inflammatory pathways that overlap with interleukin-1β (IL-1β) signaling. However, the post-translational mechanisms involved and the role of signaling proteins in this activation are unknown. In the present study, we investigated the intracellular signaling mechanisms involved in MSU-induced activation of THP-1 macrophages and human nondiseased synovial fibroblasts (NLSFs) and the in vivo efficacy of an inhibitor of tumor growth factor-β (TGF-β)-activated kinase 1 (TAK1), 5Z-7-oxozeaenol, in MSU-induced paw inflammation in C57BL/6 mice. THP-1 macrophage activation with MSU crystals (25–200 µg/ml) resulted in the rapid and sustained phosphorylation of interleukin-1 receptor-activated kinase 1 (IRAK1 Thr209) and TAK1 (Thr184/187) and their association with the E3 ubiquitin ligase TRAF6. At the cellular level, MSU inhibited the deubiquitinases A20 and UCHL2 and increased 20s proteasomal activity, leading to a global decrease in K63-linked ubiquitination and increase in K48-linked ubiquitination in THP-1 macrophages. While MSU did not stimulate cytokine production in NLSFs, it significantly amplified IL-1β-induced IL-6, IL-8, and ENA-78/CXCL5 production. Docking studies and MD simulations followed by TAK1 in vitro kinase assays revealed that uric acid molecules are capable of arresting TAK1 in an active-state conformation, resulting in sustained TAK1 kinase activation. Importantly, MSU-induced proinflammatory cytokine production was completely inhibited by 5Z-7-oxozeaenol but not IRAK1/4 or TRAF6 inhibitors. Administration of 5Z-7-oxozeaenol (5 or 15 mg/kg; orally) significantly inhibited MSU-induced paw inflammation in C57BL/6 mice. Our study identifies a novel post-translational mechanism of TAK1 activation by MSU and suggests the therapeutic potential of TAK1 in regulating MSU-induced inflammation.

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Acknowledgements

We thank the National Disease Research Interchange, Philadelphia, PA and the Cooperative Human Tissue Network (CHTN), Columbus, OH for providing synovial tissues. We would also like to thank Sadiq Umar and Bhanupriya Madarampalli for providing technical support in animal experiments. We also thank Ruby Siegel for critical reading of the manuscript. This study was supported by start-up funds from Washington State University.

Author information

A.K.S. and S.A. designed this study. A.K.S., K.O.S. and M.H. performed the experiments and participated in drafting the manuscript. S.A. participated in writing the manuscript and provided his support to the study. M.C. participated in writing the manuscript and conducted and analyzed docking and MD simulation experiments. M.O. performed histological analysis and interpreted the results. S.A. is the corresponding author of the manuscript.

Correspondence to Salahuddin Ahmed.

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Singh, A.K., Haque, M., O’Sullivan, K. et al. Suppression of monosodium urate crystal-induced inflammation by inhibiting TGF-β-activated kinase 1-dependent signaling: role of the ubiquitin proteasome system. Cell Mol Immunol (2019) doi:10.1038/s41423-019-0284-3

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Keywords

  • Monosodium urate crystals
  • Gout
  • TAK1
  • Ubiquitination
  • 5Z-7-oxozeaenol

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