Background/Purpose: TP53, a tumor-suppressor protein known as the guardian of the genome, plays a critical role in regulating genomic stability and cellular function. When TP53 function is lost, it can lead to uncontrolled cell division and the development of tumors. Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and the destruction of joints. In the synovial microenvironment of RA, hyperplastic fibroblasts form a structure called pannus, which resembles a tumor. The downregulation of TP53 is influenced by TNF-α and IL-1β, two inflammatory molecules involved in RA.

Methods: We conducted RNA-seq analysis on human rheumatoid arthritis synovial fibroblasts (RASFs) to investigate the impact of TP53 loss in the synovial microenvironment. TP53 was transiently knocked down using siRNA. Additionally, we employed Gene Ontology studies, ELISA, and Western blot analyses to assess the effects of TP53 loss on inflammatory responses, specifically in the presence of TNF-α and IL-1β. These experiments utilized at least three donor lines of RASFs, with statistical significance set at p< 0.05.

Results: Our findings reveal that RASFs exhibit a significant ( >40%) decrease in endogenous TP53 levels compared to normal synovial fibroblasts (NSFs). Proinflammatory conditions, such as treatment with IL-1β and TNF-α, further reduce TP53 expression in RASFs by more than 35% within 30 minutes (N=3, p< 0.05). RASFs display higher endogenous expression of the MDM2 E3 ubiquitin ligase, responsible for TP53 degradation. Inhibition of p38 MAPK restores TP53 levels, suggesting the involvement of MAPK pathways in TP53 stability. Inhibition of TAK1 and the ubiquitin-proteasome system rescues TP53 loss in response to IL-1β stimulation (N=3, p< 0.05). Subcellular fractionation reveals TP53 loss in both the cytoplasmic and nuclear compartments. RNA sequencing analysis identifies 241 differentially regulated genes in TP53-knockdown RASFs (N=3, p< 0.05), including an upregulation of inflammatory gene signatures. Gene ontology studies confirm the inflammatory phenotype of RASFs following TP53 loss. These findings suggest that TP53 loss predisposes RASFs to inflammation and may contribute to the progression of rheumatoid arthritis.

Conclusion: This study provides insights into how RASFs are predisposed to proinflammatory reprogramming through selective loss or gain of TP53 functions, affecting resistance to apoptosis and proliferation in response to inflammation during the progression of rheumatoid arthritis. The identification of gene networks, protein factors, and epigenetic changes involved in this process will facilitate the development of novel targeted therapies, enabling a deeper understanding of fibroblast-directed molecular events with significant clinical implications in precision medicine.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-tp53-in-inflammatory-reprogramming-of-rheumatoid-arthritis-synovial-fibroblasts/