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Abstract Number: 1117

EGCG Down-Regulates TNF-α-Induced Mcl-1 Expression By Modulating Mule/Huwe1, β-TrCP, and USP9X Ubiquitin/De-Ubiquitin Ligases in Rheumatoid Arthritis Synovial Fibroblasts

Nahid Akhtar1, Sadiq Umar2, David Fox3 and Salahuddin Ahmed1, 1Department of Pharmaceutical Sciences, Washington State University, College of Pharmacy, Spokane, WA, 2Department of Pharmaceutical Science, Washington State University, College of Pharmacy, Spokane, WA, 3Department of Medicine [Division of Rheumatology], University of Michigan, Ann Arbor, MI

Meeting: 2016 ACR/ARHP Annual Meeting

Date of first publication: September 28, 2016

Keywords: Rheumatoid arthritis (RA), synovial cells, synovial fluid and tumor necrosis factor (TNF)

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Session Information

Date: Monday, November 14, 2016

Session Title: Cytokines, Mediators, Cell-Cell Adhesion, Cell Trafficking and Angiogenesis - Poster I

Session Type: ACR Poster Session B

Session Time: 9:00AM-11:00AM

Background/Purpose:   Myeloid cell leukemia (Mcl-1) phosphorylation at Ser64 position (p-Mcl-1Ser64) has been shown to enhance Mcl-1’s stability and resistance to proteasomal degradation. However, the role of p-Mcl-1Ser64 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-1Ser64 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-1Ser64, 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-1Ser64and 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.


Disclosure: N. Akhtar, None; S. Umar, None; D. Fox, None; S. Ahmed, None.

To cite this abstract in AMA style:

Akhtar N, Umar S, Fox D, Ahmed S. EGCG Down-Regulates TNF-α-Induced Mcl-1 Expression By Modulating Mule/Huwe1, β-TrCP, and USP9X Ubiquitin/De-Ubiquitin Ligases in Rheumatoid Arthritis Synovial Fibroblasts [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). 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/. Accessed January 31, 2023.
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