Background/Purpose:   Monosodium urate (MSU) crystals are known to activate inflammatory pathways that overlap with interleukin-1β (IL-1β) signaling. The present study was carried out to determine if MSU utilizes IL-1β signaling proteins to elicit inflammatory responses.

Methods:   THP-1 monocytes were converted to macrophages with phorbol myristate acetate (PMA) for 3 hours followed by stimulation with MSU crystals (25-200 µg/ml) for 0-24 hours. Whole cell extracts were prepared for the detection of cellular proteins by Western blotting method, while the conditioned media was collected for the quantitation of secreted IL-1β and TNF-α by ELISA. Molecular dynamics (MD) simulation was performed using protein preparation wizard of Schrodinger suit 2014.3 on TAK1-TAB1 protein with a tautomeric form of MSU as a ligand. Recombinant human TAK1 protein was used for in vitro kinase assay. For in vivo evaluation, C57BL/6J mice were administered (5Z)-7-Oxozeaenol (an inhibitor of TAK1; 5 mg/kg, p.o.) or febuxostat (5 mg/kg. p.o.) 2 hours prior to MSU injection (0.5 mg/20 µl) in the footpad. Changes in the paw circumferences were measured up to 48 hours of MSU injection.

Results:   Stimulation of THP-1 macrophages with MSU (25-200 µg/ml) resulted in a dose- and time-dependent increase in IL-1β and TNF-α production (p<0.05). At the cellular level, MSU selectively inhibited the global expression pattern of K⁶³-linked ubiquitination without affecting K⁴⁸-linked ubiquitination in activated THP-1 cells. Interestingly, a rapid phosphorylation of IRAK-4 (Thr³⁴⁵/Ser³⁴⁶), IRAK-1 (Thr³⁸⁷), IRAK-1 (Thr²⁰⁹), and TAK1 (Thr^184/187) in THP-1 macrophages was observed within 5 to 15 minutes of MSU stimulation. Blockade of IL-1β secretion by Brefeldin A in THP1 macrophages had no effect on MSU-induced TAK1 activation, suggesting TAK1 as a direct target of MSU. To evaluate the importance of IL-1β signaling proteins using chemical inhibitors, we found that the inhibition of TAK1, but not IRAK-1/4 or TRAF6, completely abrogated MSU-induced IL-1β and TNF-α production in THP-1 macrophages (p<0.01). Furthermore, in silico simulation of TAK1 revealed that MSU is capable of activating TAK1 and arresting its conformational position in active state, thereby, causing sustained TAK1 kinase activation. Furthermore, in vitro kinase assay results also showed that MSU (100-500 µg/ml) induces autophosphorylation of TAK1 protein in a dose-dependent manner (p<0.05). Findings from in vivo studies suggest that the pretreatment of mice with (5Z)-7-Oxozeaenol significantly inhibited MSU-induced paw circumferences in mice (p<0.01), which was comparable to febuxostat treatment.

Conclusion:   Our study shows that MSU is capable of inducing TAK1 activation in THP-1 macrophages independent of endogenous IL-1β, suggesting TAK1 as a potential therapeutic target for the treatment of gout.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/suppression-of-monosodium-urate-msu-crystals-induced-inflammatory-response-by-inhibiting-tgf-%ce%b2-activated-kinase-1-tak1/