EXAMINING TGF-β-ACTIVATED KINASE 1 AS A MASTER REGULATOR OF SYNOVIAL FIBROBLASTS DYSFUNCTION IN JUVENILE IDIOPATHIC ARTHRITIS

Meena Afroze Shanta

Juvenile idiopathic arthritis (JIA) is a chronic autoimmune condition marked by chronic and debilitating synovial inflammation. Despite the availability of several advanced biological therapies, a substantial fraction of the patients remains refractory to the treatments and continues to experience pain and joint destruction in adulthood, requiring more research to understand the disease process and devise effective therapeutic interventions. Given the critical position of transforming growth factor-β-activated kinase 1 (TAK1) downstream of several proinflammatory signaling pathways widely implicated in JIA pathogenesis, and TAK1’s highest expression in cultured fibroblasts, we hypothesize that TAK1 is central to aberrant gene expression in cytokine-activated JIA synovial fibroblasts (JIASFs) and that inhibition of TAK1 suppresses inflammation and tissue destruction caused by JIASFs. To characterize the potential of targeting TAK1 in JIA, RNA-sequencing, Western immunoblotting, enzyme-linked immunosorbent assays (ELISA), TAK1 knockdown study, immunofluorescence, real-time polymerase chain reaction (RT-PCR), CyQuant Cell Proliferation Assays, and subcellular fractionation studies were conducted to determine the overall impact of TAK1 inhibition in limiting the expression of inflammatory markers by JIASFs and their proliferation. An in vivo study was also conducted to investigate the anti-arthritogenic potential of TAK1 inhibition in IFN-γ knockout mice. The findings of this study revealed that TAK1 activation peaks between 5-15 minutes, and its downstream target between 15-30 minutes following cytokine stimulation. Among the four inhibitors tested, 5z-7-oxozeaenol (5Z) remained the most potent inhibitor of TAK1 activation, with an IC50 of 22.8 nM. The RNA-sequencing suggested that TAK1 inhibition restored cellular homeostasis by suppressing inflammation and upregulating genes involved in cell proliferation and cell-cycle regulation. In line with RNA-sequencing, in vitro inhibition of TAK1 with 5Z and NG-25 suppressed the expression of inflammatory markers such as cyclooxygenase-2 (COX-2), vascular cell adhesion molecule-1 (VCAM-1), interleukin-6 (IL-6), interleukin-8 (IL-8 or CXCL-8), matrix metalloproteinase-3 (MMP-3), and Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES or CCL5), significantly. The upregulation of p53 and p21 coincided with significantly reduced expression of proliferating cell nuclear antigen (PCNA) and reduced cell proliferation. TAK1 inhibition with 5Z also prevented the induction and severity of arthritis in IFN-γ knock-out mice, further validating our in vitro data. To elucidate the mechanism by which TAK1 promotes an inflammatory phenotype in JIASFs, p53 expression was knocked down. The results revealed that TAK1 suppressed p53, thereby upregulating inflammatory markers and proliferation in a cytokine-dependent manner. We also observed a significant reduction in p53 and elevated expression of inflammatory markers, including COX-2, cadherin-11, and CXCL-8/IL-8, following stimulation with synovial fluid derived from JIA patients, further highlighting the cytokine-driven phenotypic changes in JIASFs. Thus, this study provides a rationale for further research to understand the overall implications of TAK1 inhibition and its use in clinical settings.

EXAMINING TGF-β-ACTIVATED KINASE 1 AS A MASTER REGULATOR OF SYNOVIAL FIBROBLASTS DYSFUNCTION IN JUVENILE IDIOPATHIC ARTHRITIS

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