Background/Purpose:

Myeloid cell leukemia (Mcl-1), a Bcl-2 family anti-apoptotic protein, is a critical determinant of rheumatoid arthritis synovial fibroblasts (RA-FLS) resistance to apoptosis. Phosphorylation of Mcl-1 at different sites may influence its stability and function, however, it’s implication on RA pathogenesis is not fully studied. In the present study, we evaluated the expression levels of phospho- and total Mcl-1 in FLS from RA, osteoarthritis (OA), and non-diseased (NL) donors and in the joints of rat adjuvant induced arthritis (AIA) model.

Methods:

FLS were obtained from RA (n=10) and OA (n=6) patients undergoing synovectomy or joint replacement surgery. NL samples (n=9) were obtained at the time of autopsy/amputation with no known history of OA or RA. Basal cell lysates (n=6) from NL-, OA-, and RA-FLS were prepared and analyzed for phosphorylated and total Mcl-1, and its endogenous regulators such as Noxa, Bak, and Bax using Western immunoblotting. Correlation between Noxa and Mcl-1 mRNA expression in NL-, OA-, and RA-FLS (n=6) and in synovial tissues (ST; n=5) was determined using qRT-PCR. A Bcl-2/Mcl-1 inhibitor (AT101; 1μM) was used as a control. Findings from human RA-FLS were further validated in rat AIA model. p<0.05 was considered significant.

Results:

Our results showed that Mcl-1 mRNA and protein expression was significantly higher, whereas Noxa expression was markedly reduced in OA- and RA-FLS compared to NL-FLS. In addition, a significantly higher Mcl-1/Noxa mRNA ratio (~20-fold) was also observed in RA-ST suggesting the resistance of RA-FLS to apoptosis compared to NL-ST. Studies in the rat AIA model showed an increase in Mcl-1 (~88%) and a decrease in Noxa (~55%) expression in the joints of AIA rats on day 18 compared to the naïve group. Studies suggest that the phosphorylation of Mcl-1 at Thr163 inhibits its anti-apoptotic activity and phosphorylation at Ser159 destabilizes Mcl-1. Our results showed a significantly lower phosphorylation state of Mcl-1 at Ser159/Thr163 (~63%) and specifically at Mcl-1 Ser159 (~60%) sites in RA-FLS indicating their pro-survival nature compared to NL-FLS. RA-FLS also showed an increase in p-Mcl-1 Ser64 protein expression which enhances its anti-apoptotic property. A similar decrease in p-Mcl-1 Ser159/Thr163 and p-Mcl-1 Ser159, and an increase in p-Mcl-1 Ser64 was observed in the joints of AIA rats compared to the naïve group. Pretreatment of RA-FLS with AT101 markedly inhibited TNF-α-induced Mcl-1 as well as pSer159/Thr163, pSer159 and pSer64 protein expression in RA-FLS. Moreover, this increase in Mcl-1 expression was in contrast to the paucity in the expression of pro-apoptotic proteins Bak and Bax in RA-FLS and in the joints of AIA rats compared to their respective controls.

Conclusion:

This study provides a novel evidence for higher Mcl-1/Noxa ratio and increased phosphorylation of Mcl-1 at Ser64 as a potential mechanisms of Mcl-1 stability and RA-FLS survival in RA pathogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/characterization-of-different-phosphorylation-sites-of-mcl-1-in-human-rheumatoid-arthritis-synovial-fibroblasts-and-their-correlation-with-ra-pathogenesis/