Washington State university Spokane, WA, United States
Anil Singh, Farheen Sultan Shaikh and Salahuddin Ahmed, Washington State university, Spokane, WA
Background/Purpose: Terminal Uridyl Transferase (TUT) genes TUT4 and TUT7 mediate uridylation of mRNA and microRNAs to maintain cellular homeostasis via RNA turnover. Changes in the expression of TUTs may influence the RNA composition in the synovial microenvironment of RA patients. TNF-α upregulates the expression of TUT7 protein which potentiate the inflammatory function of TNF-α. However, TUT4 expression remains unchanged stoichiometrically in human RA synovial fibroblasts (RASFs). TUT-mediated RNA turnover within the synovial microenvironment and in RASFs are poorly understood. In this study, we evaluated the effect of TUT4 and TUT7 in TNF-α-induced inflammation in human RASFs.
Methods: The RNA-seq analysis of human RASFs treated with TNF-α (20 ng/ml) alone or in cells with knockdown of TUT4 or TUT7 using a validated siRNA for 48h was performed using Ion Torrent systems. Gene Ontology studies were performed using Ingenuity Pathway Analysis. ELISA, quantitative RT-PCR, and Western blot experiments were carried out to confirm inflammatory responses of TNF-α-stimulated RASFs under the influence of TUT4 or TUT7 knockdown. All the experiments were carried out at least in 3 RASFs donor lines. Statistical values of p< 0.05 were considered significant.
Results: Expression of TUT7 analyzed using Western blot confirms that RASFs express negligible amount of TUT7 which is significantly and selectively induced by TNF-α stimulation (p< 0.05; n=3); while the expression of TUT4 remains unchanged in RASFs with or without TNF-α stimulation. Knockdown of TUT7 using siRNA method showed a unique modulation of TNF-α-induced inflammatory signature such as IL-6, IL-8, MCP-1 and CXCL5 at mRNA and protein expression level. RNA-seq data identified 835 differentially expressed genes (DEGs) modulated by TUT7 knockdown affecting cellular process such as protein phosphorylation as one of the top enrichment candidates. TUT7 knockdown in RASFs by Western blot confirms the abrogation of two key phosphatase enzymes PP2 alpha and PP1 alpha and therefore, suggesting that TUT7 may have proinflammatory functions. TUT4 knockdown identified 289 DEGs (p< 0.05; n=4) and IPA analysis showed that it affects cellular processes by influencing catabolic process on protein metabolism as well as cell cycle including RNA splicing mechanism. The validation of key changes by TUT4 knockdown reveals that TUT4 inhibits TNF-α inflammatory properties by positively influencing methylation of Histone H3 (p< 0.05; n=3). Further validation TUT4 knockdown in RASFs revealed its anti-inflammatory function by the abrogation of inflammatory mediators IL-6, IL-8, CXCL5, CCL5, MMP-1 and MMP-3 in response to TNF-α (p< 0.05; n=3).
Conclusion: This study identifies a novel TNF-α-mediated RNA processing axis between TUT7 and TUT4 – key enzymes involved in RNA biogenesis and stability. Loss of TUT7 exacerbates pathogenesis of TNF-α mediated RA progression by positively influencing protein phosphorylation dynamics. However, silencing TUT4 resulted in significant decrease in inflammatory mediators, indicating selectivity in the pathological processes that drive inflammation in RA.
Disclosures: A. Singh, None; F. Shaikh, None; S. Ahmed, None.