Molecular mechanisms of TAK1 regulation in rheumatoid arthritis - PROJECT SUMMARY In rheumatoid arthritis (RA), activated synovial fibroblasts (SFs) transform into an invasive phenotype that recruits immune cells to cause tissue destruction. Recent findings show transforming growth factor β-activated kinase 1 (TAK1) as a pivotal mediator of IL-1β, TNF-α, and Toll-like receptor signaling cascades due to its central position upstream of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways. However, the molecular mechanism by which TAK1 regulates SF functions to promote RA pathogenesis remains unexplored. Using FAPα+Thy1+ RASFs, we showed that IL-1β-induced inflammatory signals are suppressed by inhibiting the TAK1 kinase domain (Thr184/187). Analysis of RNA sequencing data showed that the TAK1 kinase inhibitors type I (irreversible, 5Z-7-oxozeaenol, 5Z-7o) and type II (reversible, NG-25) significantly blocked the expression of inflammatory (IL6, CXCL9, CXCL1, NFKB1, and JUNB) and invasive (CDH11, PDPN, SOD2, PRG4, MMP1, and MMP3) genes in human RASFs. TAK1 inhibitors showed a marked inhibition of (IL-1β+TNF- α+IFN-γ)-induced IL-6, IL-8, MMP-1, and MMP-3 production and Cad-11, Cox-2, and PDPN expression. 5Z-7o and NG-25 triggered the nuclear translocation of TAK1 (Ser439) on the C-terminal domain, the function of which is not characterized. Immunoprecipitation of nuclear TAK1 followed by Western blot analysis showed its novel interaction with CBP/p300, PCAF, GCN5, and acetylated histone 3, suggesting a role for nuclear TAK1 in chromatin remodeling. In addition, K63-linked ubiquitination (K63-Ub), a post-translational mechanism that provides stability to signal proteins, was severely repressed in RASFs compared to non-diseased SFs (NLSFs) and inversely correlated with increased expression of CYLD, a deubiquitinase specific for hydrolyzing K63-Ub chains. Treatment of human RASFs with 5Z-7o or NG-25 enhanced K63-Ub proteins in a dose-dependent manner and transcriptionally inhibited IL-1β-induced CYLD expression. Administration of 5Z-7o or NG-25 (2 mg/kg, p.o. daily) for 10 days from the onset of the disease significantly ameliorated AIA in rats. However, several questions remain unanswered, including the nuclear function of the TAK1 C-terminal in chromatin remodeling, its contribution to the RASF invasive and inflammatory phenotype, and the molecular mechanisms of TAK1 regulation in vivo. Hence, we propose a hypothesis that TAK1 is central to RASF synovial inflammation, and targeting TAK1 functions may ameliorate RA. Hence, studies proposed in Aim 1 will determine the role of TAK1 in RASF functions and the effect of kinase inhibition on its nuclear translocation and interaction with chromatin remodelers. In Aim 2, we plan to understand the effect of TAK1 inhibitors on restoring K63-Ubiquitination to inhibit IL-1β signaling pathways in RASFs. In Aim 3, we plan to study the arthritogenic potential of TAK1 in RASFs and the mechanism of action of TAK1 inhibitors in collagen antibody-induced arthritis (CAIA) mice, and in fibroblast- specific TAK1 conditional knockout or overexpressing mice. These findings will provide a mechanism-based rationale for developing TAK1-targeted therapy for RA.
