Background/Purpose: ZCCHC6, also known as TUT7 or TENT2, plays a crucial role in RNA processing and metabolism through its uridylyl transferase function. This function involves adding uridine residues to the 3′ ends of RNA molecules, including mRNA and microRNAs and is important for regulating RNA stability, decay, and turnover. However, its role in pro-inflammatory cytokines, such as IL-1β and TNF-α induced inflammatory function, remains elusive.

Methods: To investigate the role of ZCCHC6 in TNF-α-induced inflammation, RNA-seq analysis was performed in human rheumatoid arthritis synovial fibroblasts (RASFs) treated with TNF-α alone or with ZCCHC6 knockdown using siRNA method. Mass Spectrometry based untargeted phospho-proteomics analysis was conducted to determine changes in the global phosphorylation landscape of RASFs treated with ZCCHC6 siRNA in the presence of TNF-α stimulation. Additionally, Gene Ontology studies, ELISA, quantitative RT-PCR, and Western blot analyses were performed to determine the effect on inflammatory responses in TNF-α-activated RASFs with or without ZCCHC6 knockdown. The experiments were carried out using at least three RASF donor lines, with statistical significance set at p< 0.05.

Results: ZCCHC6 knockdown in human RASFs resulted in the differential regulation of 835 genes in the presence of TNF-α. Out of these genes, 384 genes were upregulated, whereas 451 genes were downregulated by at least 2-fold (N=3, p< 0.05). ZCCHC6 exhibited anti-inflammatory effects in TNF-α-stimulated RASFs, as its knockdown by targeted siRNA by enhanced the phosphorylation of signaling proteins important in MAPK-activated pathways, including ASK1, cJun, pERK, p38, pP65, and SAPK/JNK proteins (N=3, p< 0.05). The MS-based phospho-proteomic analysis confirmed that ZCCHC6 knockdown selectively influenced the phosphorylation of 263 unique proteins (N=3, p< 0.05). Gene Ontology studies revealed the upregulation of key pathways, such as mRNA metabolism, apoptosis, DNA damage, and cellular localization. Further analysis demonstrated critical changes in the cellular activity of phosphorylation events that are tightly regulated by Protein Phosphatase 2A (PP2A), a serine/threonine phosphatase. A loss of ZCCHC6 inhibited the TNF-α-induced expression of three regulatory subunits of PP2A, including the structural subunit (A), catalytic subunit (C), and regulatory subunit (B).

Conclusion: This study unveils a novel axis involving ZCCHC6 and TNF-α in the progression of RA. TNF-α utilizes ZCCHC6 to mediate internal phosphorylation signaling events, positively influencing Protein Phosphatase activity, and driving the pathological processes underlying inflammation in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/zcchc6-modulates-the-global-phosphorylation-landscape-in-tnf-a-induced-rheumatoid-arthritis-synovial-fibroblasts/