Background/Purpose:   Myeloid cell leukemia (Mcl-1) phosphorylation at Ser64 position (p-Mcl-1^Ser64) has been shown to enhance Mcl-1’s stability and resistance to proteasomal degradation. However, the role of p-Mcl-1^Ser64 in rheumatoid arthritis synovial fibroblasts (RASFs) resistance to TNF-α-induced apoptosis and the influence of ubiquitin and de-ubiquitin ligases in regulating Mcl-1 resistance is not yet studied in RA. This study was undertaken to understand the posttranslational mechanism of Mcl-1 regulation by epigallocatechin-3-gallate (EGCG), a potent anti-inflammatory molecule found in green tea, in human RASFs.

Methods:   The expression of p-Mcl-1^Ser64 and of the Mcl-1-specific ubiquitin ligases (Mule/Huwe1 and β-TrCP), and de-ubiquitin ligase (USP9X) was determined in SFs from RA, osteoarthritis (OA), and non-diseased (NL) SFs. Efficacy of an acute EGCG concentration (50 μM) or physiologically-achievable chronic concentrations (0.1-1 μM) were tested in regulating these posttranslational modifications in RASFs using Western blotting and immunoprecipitation methods. P<0.05 was considered significant.

Results:   The levels of p-Mcl-1^Ser64, which enhances its anti-apoptotic property, was markedly upregulated in human RASFs compared to OASFs or NLSFs (p<0.05). Pretreatment of RASFs with EGCG (50 μM) for 24 h markedly inhibited p-Mcl-1^Ser64and total Mcl-1 expression and enhanced the expression of pro-apoptotic proteins (Bak and Bax) in TNF-α stimulated RASFs. Western blotting evaluation of the posttranslational processes showed a significant decrease in the expression of Mcl-1-specific ubiquitin ligases Mule/Huwe1 (~82%) and β-TrCP (~75%) in RASFs compared to NLSFs (p<0.05). However, the expression of Mcl-1-specific de-ubiquitin ligase, USP9X, was not significantly different between RASFs and NLSFs. Interestingly, Western blot analysis of samples immunoprecipitated with Mcl-1 antibody showed a reduced association with β-TrCP and an increased association with USP9X in RASFs compared to NLSFs. EGCG treatment significantly induced the expression of Mule/Huwe1 and β-TrCP in TNF-α-stimulated RASFs. In addition, Mcl-1 immunoprecipitation in the EGCG treated RASFs showed a marked decrease in Mcl-1-associated USP9X and an increase in Mcl-1-associated β-TrCP in TNF-α-stimulated RASFs compared to TNF-α alone group. The majority of beneficial effects of a single-dose EGCG (50 μM) were mimicked by the repeated RASF treatment for 7 days with EGCG (1 μM) to attain physiological concentrations of EGCG, if consumed in the form of green tea supplement.

Conclusion:   This study provides a novel evidence that EGCG induces Mule/Huwe1 and β-TrCP ubiquitin ligases and inhibits USP9X de-ubiquitin ligase that are Mcl-1-specific, thereby, facilitating Mcl-1 degradation in RASFs and possibly regulating tissue invasion and destruction in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/egcg-down-regulates-tnf-%ce%b1-induced-mcl-1-expression-by-modulating-mulehuwe1-%ce%b2-trcp-and-usp9x-ubiquitinde-ubiquitin-ligases-in-rheumatoid-arthritis-synovial-fibroblasts/